Research ArticleEffect of Acute Gamma Irradiation onCurcuma alismatifolia Varieties and Detection of DNAPolymorphism through SSR Marker
Sima Taheri1 Thohirah Lee Abdullah1 Zaiton Ahmad2
and Nur Ashikin Psyquay Abdullah1
1 Department of Crop Science Faculty of Agriculture University Putra Malaysia (UPM) 43400 Serdang Selangor Malaysia2 Agrotechnology and Biosciences Division Malaysian Nuclear Agency (Nuclear Malaysia) 43000 Bangi Selangor Malaysia
Correspondence should be addressed to Sima Taheri sima taheri65yahoocomandThohirah Lee Abdullah thohirahagriupmedumy
Received 13 July 2013 Revised 2 December 2013 Accepted 4 December 2013 Published 25 February 2014
Academic Editor Gjumrakch Aliev
Copyright copy 2014 Sima Taheri et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
The effects of eight different doses (0 10 20 25 35 40 60 and 100Gy) of acute gamma irradiation on 44 (three varieties ofCurcuma alismatifolia Chiang Mai Red Sweet Pink Kimono Pink and one Curcuma hybrid (Doi Tung 554) individual plantswere investigated Radiation sensitivity tests revealed that the LD
50values of the varieties were achieved at 21 Gy for Chiang Mai
Red 23Gy for Sweet Pink 25Gy for Kimono Pink and 28Gy for Doi Tung 554 From the analysis of variance (ANOVA) significantvariations were observed for vegetative traits flowering development and rhizome characteristics among the four varieties ofCurcuma alismatifolia and dose levels as well as the dose times variety interaction In irradiated plants the leaf length leaf widthinflorescence length the number of true flowers the number of pink bracts number of shoots plant height rhizome size numberof storage roots and number of new rhizomes decreased significantly (119875 lt 005) as the radiation dose increased The copheneticcorrelation coefficient (CCC) between genetic dissimilarity matrix estimated from the morphological characters and the UPGMAclusteringmethod was 119903 = 093 showing a proof fit In terms of genetic variation among the acutely irradiated samples the numberof presumed alleles revealed by simple sequence repeats ranged from two to seven alleles with a mean value of 31 45 and 53alleles per locus for radiation doses of 0 10 and 20Gy respectively The average values of the effective number of alleles Neirsquosgene diversity and Shannonrsquos information index were 25ndash32 051ndash066 and 09ndash13 respectively The constructed dendrogramgrouped the entities into seven clusters Principal component analysis (PCA) supported the clustering results Consequently it wasconcluded that irradiation with optimum doses of gamma rays efficiently induces mutations in Curcuma alismatifolia varieties
1 Introduction
The genus Curcuma is a member of the Zingiberaceae familythat has recently become popular for the use as floweringpot plants and cut flowers Most Curcuma species are usedas medicinal herbs or for culinary purposes However somepossess aesthetic value as ornamentals such as Curcumaalismatifolia which is a monocotyledonous perennial orig-inating from the tropical and subtropical areas of northernThailand and Cambodia It has great potential for use as cutflowers and flowering pot plants and as a garden plant fortropical landscaping in various regions [1] C alismatifolia
has flowering stems comprising of a showy inflorescencewith several apical bracts on a long peduncle Most basalbracts are green but the distal ones more numerous thanthe green ones are purplish pink prominent elliptical bractswhich determine the attractiveness of the flowering stemsBoth types of bracts bear two to seven small axillary flowerbuds Open flowers are small and have a purple flag petal [2]Few breeding programs have been carried out to improve thisspecies
Mutation induction and selection of mutants have beenpowerful tools for plant breeding as well as for physiologicaland molecular studies for the past 80 years X-ray gamma
Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 631813 18 pageshttpdxdoiorg1011552014631813
2 BioMed Research International
ray irradiation and chemical treatments have been used formutation breeding in a wide range of plants [3] Gammarays are a type of ionizing radiation which interacts withatoms or molecules to produce free radicals in cells whichdamage or modify important components of plant cellsand affect differently the morphology biochemistry andphysiology of plants Induced mutation is highly effective inenhancing natural genetic resources for the development ofimprovednew cultivars among vegetatively propagated cropsSome important ornamental plants for both cut flowers andpotted plants that have been used in mutation breeding arechrysanthemums [4 5] orchids [6] roses [7] pelargoniums[8] and canna [9]
The estimation of genetic variation on the basis of mor-phological traits alone which are the product of gene andenvironmental interactions does not determine the actuallevel of genetic variation among studied individuals [10] Sev-eral molecular markers such as random amplified polymor-phicDNA(RAPD) intersimple sequence repeats (ISSR) sim-ple sequence repeats (SSR) and amplified fragment lengthpolymorphism (AFLP) with different advantages and disad-vantages have been employed in genetic variation studies ofCurcuma species [10ndash15] RAPD markers are relatively easyto generate but may not be variable enough for some applica-tions or may have problems with reproducibility Among therobust class of molecular markers microsatellites or simplesequence repeats (SSRs) are extremely powerful tools forestimating genetic variation with high reproducibility in avariety of plant species These markers are characterized bythe presence of 1ndash6 nucleotide repeats within the codingand noncoding regions [16 17] of the genome which arecodominant hypervariable and multiallelic in nature [18]Genomic SSR markers have been developed in economicallyimportant spice crops such as Zingiber officinale [19] Vanillaplanifolia [20] and Piper nigrum [21] The development of17 EST-SSR and 17 genomic SSR markers has been recentlyreported in turmeric (Curcuma longa L) [13 22] Usinggenetic markers for internal quality control it is possibleto distinguish induced mutations from any nonmutationalgenetic variability and hence unequivocally demonstrate thatmutations induced by gamma irradiation were the majorsource of genetic variability [23 24] This study was designedto determine the optimumdose of acute gamma radiation forselected C alismatifolia varieties describe the morphologicalvariations as affected and developed from acute gammairradiation and elucidate the genetic variation among theirradiated C alismatifolia varieties using microsatellite DNAmarkers
2 Materials and Methods
21 Plant Materials The rhizomes of three C alismatifoliavarietiesmdashChiang Mai Red (SK 205112) Sweet Pink (SK205212) Kimono Pink (SK 205412) as well as one Curcumahybrid Doi Tung 554 (SK 205312)mdashwere provided from theCurcuma Nursery (Ubonrat) in Doisaket District ChiangMai 50220 Thailand (Table 1)
22 Gamma Irradiation for Radiation Sensitivity Test Irra-diation of the plant materials was conducted in the Facultyof Science and Technology University Kebangsaan Malaysia(UKM) using a Gammacell 220 Excel Irradiator (MDS Nor-dion Ottawa ON Canada) The source of gamma rays wasCobalt 60 Prepared rhizomes in the budding stage wereacutely irradiated with different doses of 10 (128 Sec) 20(256 Sec) 25 (311 Sec) 35 (436 Sec) 40 (512 Sec) 60(80 Sec) and 100Gy (130 Sec) In each variety 20 rhizomeswere treated for sensitivity testing at each dose After irra-diation the rhizomes were planted in 25 cm pots containinggrowthmedia consisting of topsoil cocopeat rice husk at theratio of 1 2 1 Radiation effect on test plants was recorded interms of the mortality rate () after exposure to the gammaradiation Number of mortal rhizomes were counted 40 daysafter planting (at each treatment) and expressed as percentageof the total number of rhizomes planted The experimentswere conducted in Green house number 1 Field 2 Facultyof Agriculture University Putra Malaysia (UPM) MalaysiaThe recorded data of mortality percentage were analyzed byPoloPlus (Probit and logic analysis) software version 2
23 Induction of Mutation with Selected Doses of GammaRadiation Based on LD
50and obtained confidence limits of
irradiation dose 20 rhizomes from each variety were irra-diated (March 2011) with gamma rays at doses of 0 (control)10 Gy and 20Gy The experiment was designed as 4 (variety)times 3 (dose) RCBD with five blocks and four replications foreach sample
24 Morphological Data Fourteen morphological traitsincluded vegetative traits flowering development and rhi-zome characteristics data were recorded duringMarch 2011 toSeptember 2011 for four varieties of C alismatifolia (Table 2)The traits included number of new shoots leaf length leafwidth leaf number plant height number of days to visiblebud inflorescence length number of days to anthesis num-ber of days to senescence number of true flowers number ofpink bracts number of rhizome rhizome size and number ofstorage roots
3 SSR Analysis
31 DNA Isolation Leaves of all mutants and control indi-viduals were stored at minus70∘C until used for DNA extractionDNA was isolated from leaves of selected 44 individualswith morphological variations using cetyltrimethylammo-nium bromide (CTAB) extraction buffer [25] The extractionbuffer comprised of 2 (wv) CTAB 14mM NaCl 100mMTris-HCL PH 80 20mMEDTA 2 (wv) PVP and 2 (vv)120573-mercaptoethanol The mixture was incubated at 65∘C for 1hour followed by two extractions with chloroformisoamylalcohol (24 1) Isopropanol was used to precipitate nucleicacids and the pellet obtained was washed with 70 ethanoldried and dissolved in a Tris-EDTA (TE) buffer (10mMTris-HCl pH = 80 and 1mM EDTA pH = 80) Copre-cipitated RNA was removed by digestion with RNAse Afterone hour incubation at 37∘C the concentration and purity
BioMed Research International 3
Table1Im
portantd
iscrim
inatingqu
alitativ
efeatureso
fstudied
Calism
atifolia
varie
tiesa
ndhybrid
varie
tiesfor
M1V
1generatio
n
Varie
tyFloralcharacters
Leafcharacters
Planttype
Spike
position
Color
ofcalyx
Color
ofcorolla
Inflo
rescence
lower
bractcolor
Inflo
rescence
comab
ractcolor
Leafhabit
Color
ofleaf
sheath
Leafmargin
Leafcolor
Leafmidrib
color
Leafshape
Chiang
MaiRe
dErect
Term
inal
White
Light
Purple-w
hite
Green
PurpleN78Alowast
Erect
Purple-green
Smoo
thDark
green
Purple
Long
narrow
and
stiff
DoiTu
ng554
Erect
Term
inal
White
Dark
Purple-w
hite
2014
Redpu
rple
N78Clowast
Erect
DarkPu
rple
Smoo
thDark
green
Purple
Long
narrow
and
stiff
SweetP
ink
Erect
Term
inal
White
Dark
Purple-w
hite
Green
Purple-violet
N80Dlowast
Erect
Purple-green
Lowwavy
Light
green
Purple
Widea
ndstiff
Kimon
oPink
Erect
Term
inal
White
Dark
Purple-w
hite
Green
Purple-violet
N80Clowast
Erect
Purple-green
Medium
wavy
Dark
green
Green
Long
and
narrow
lowastTh
eRoyalHortic
ulture
Society(RHS)
Lond
oncolorc
hart
4 BioMed Research International
Table 2 List of morphological traits and brief descriptions
Number Morphological traits
1Number of new shoots (number) total number ofproduced new shoots per rhizome
2Leaf length (cm) length of the fully opened firstleaf from the soil surface to leaf tip
3Leaf width (cm) breadth of the leaf at the widestpart of the leaf
4Number of leaves (number) number of fullyemerged leaves at the end of vegetative growthstage
5Plant height (cm) the height of the peduncle atthe top of the soil surface to the tip of theinflorescence
6
Number of days to visible bud appearance (days)number of days from the first day of planting toappearance of the first visible bud Buds appear atthe middle of two sheaths of leaves
4Inflorescence length (cm) the length between thelowest green bracts to tip of the upper pink bractsduring anthesis
8Number of days to anthesis (days) the number ofdays from planting to fully opened flower bud
9Number of days to senescence (days) the daysfrom first day of anthesis until end of the shelf lifeof the flower
10Number of true flowers (No) the number ofsmall axillary flower buds which develop insidebracts during anthesis
11
Number of pink bracts (No) inflorescence of Calismatifolia comprising several apical bractsMost basal bracts are green but the distal onesmore numerous than the green ones are purplishpink bracts which determine the attractiveness ofthe flowering stems The number of pink bractswas counted during anthesis
12Rhizome size (cm) the girth of the M0V0 andM1V1 rhizomes was measured with vernier caliperand mean was expressed in centimeter
13Number of new rhizomes (No) after harvestingthe total number of new rhizomes (M1V1) wasrecorded
14Number of storage roots (No) the total numberof storage roots of M0V0 and M1V1 rhizomes wasrecorded at harvesting time
of isolated DNA were determined using NanoDrop 2000(Thermo Fisher Scientific Inc) in the range of 250 to900 ng120583L which was adjusted to 70 ng120583L The quality wasverified by electrophoresis on 08 agarose gel
32 PCR Amplification and Product Electrophoresis Poly-merase chain reaction (PCR) was carried out for 17 SSRprimers whichwere developed forCurcuma longa in previousstudies [26] PCR was carried out in a 25 120583L reaction volumecontaining 70 ng120583L DNA and 2X DreamTaq Green PCRMasterMix (Fermentas International Inc USA)with 04120583Mprimer Amplification was performed in a thermal cycler(Bio-Rad Laboratories Inc USA) for a total of 40 cyclesAn initial denaturation of the template DNA at 94∘C for 3minutes was followed by 10 cycles of 94∘C for 40 secondsand a touch-down one-degree decrement for annealingtemperature starting with 7∘C above 119879
119898for each primer for
30 seconds and 72∘C for 1 minute This was then followed by30 cycles of 95∘C for 40 seconds a last annealing temperaturefor 30 seconds and 72∘C for 1 minute and a final extensionof 72∘C for 10 minutes The PCR products were separated on4 metaphor gel with 50 bp DNA ladder (N3231S BiolabsInc UK) The gel was stained with Midori green visualizedunder ultraviolet light and photographed by ChemilImagerGel Documentation imaging system (Alpha Innotech Corpo-ration CA USA)
4 Data Analysis
41 Morphological Data The recorded data (after normalityand homogeneity test) were subjected to analysis of variance(ANOVA) as per two-factor experiment with three irradia-tion treatments and four varieties arranged in a randomizedcomplete block design (RCBD) with four replications Theanalysis was carried out using the portable SAS 91 programand least significant differences (LSD) were used for compar-ison among treatment means at 119875 le 005 To evaluate therelationship among the different variables in the experimentcorrelation coefficients were used by SAS 9 1 3 portable Togroup the individuals based on morphological dissimilaritycluster analysis was conducted on the Euclidean distancematrix with the unweighted Pair-GroupMethod using Arith-metic average (UPGMA) using NTSYS software The sameprogram was used for principal components analysis (PCA)to define eigenvalues and eigenvectors and also for compar-ison of the mean of groups to define effective traits in sepa-ration of the groups Eigenvectors are the weights in a lineartransformation when computing principal component scoreswhile eigenvalues indicate the amount of variance explainedby each principal component The cophenetic correlationcoefficient (CCC) was used to measure the goodness of fit ofthe similarity matrices to their corresponding phenograms inmorphological data using PAST (PAleontological Statistics)software V 217 [27]
42 Molecular Data Allele size was measured with UVDoc9902 analysis software (UVI Tech Cambridge UK) by man-ual editing to increase accuracy This procedure was carriedout two times to exclude wrong scorings The PowerMarker325 software package [28]was used to produce a dendrogramusing UPGMAmethod Data were scored as ldquo1rdquo for presenceand ldquo0rdquo for absence The binary data matrix was entered intothe Numerical Taxonomy and Multivariate Analysis System
BioMed Research International 5
Table 3 The number of irradiated and mortal rhizomes of Curcuma alismatifolia varieties after acute irradiation with different doses ofgamma rays
Dose (Gray)Total number of
irradiated rhizomes ineach var
Number of mortal rhizomes after 40 days Mortality rate ()in each doseDoi Tung 554 Chiang Mai Red Sweet Pink Kimono Pink
(NTSYSpc 210e) [29] to generate Dicersquos similarity matrixThe software POPGENE32 Version 132 [29] was used tocalculate genetic variation parameters including observedheterozygosity (the proportion of heterozygous individualsin the population) (119867
119900) expected heterozygosity (119867
119890) [30]mdash
defined as the probability that two randomly chosen allelesfrom the population are different [31]mdashobserved numberof alleles (119899
119886) effective number of alleles (119899
119890) Neirsquos gene
diversity Shannonrsquos information index (119868) and percentageof polymorphic loci To compare the efficiency of primersand polymorphism information content (PIC) a measure ofallelic diversity at a locus was calculated using online PICcalculator software (httpwwwlivacuksimkempsjpichtml)using the following formula
PIC = 1 minus119899
sum
119894=1
1199011198942minus
119899minus1
sum
119894
119899
sum
119895=119894+1
211990111989421199011198952 (1)
where 119901119894is the frequency of the 119894th allele and 119899 is the
number of alleles Markers were classified as informativewhen PICwas ge05 Principal component analysis (PCA) wasalso generated for SSR data by NTSYS-pc 210e
5 Results and Discussions
51 Gamma Irradiation and Radiation Sensitivity Test Thesensitivity of C alismatifolia varieties to radiation was eval-uated by comparing the mortality rate () of irradiatedplants at 40 days after irradiation The plant mortality rateincreased with increasing irradiation dosage (Table 3) Thehybrid Doi Tung 554 was found to be least sensitive togamma irradiation than other varieties (51mortality) whileChiangMai Red variety showed the lowest survival rate (63mortality) Sweet Pink and Kimono Pink varieties showed58 and 55 mortality rate respectively At 50 survivalrate (LD
50) the gamma doses administered were 28 21 23
and 25Gy for Doi Tung 554 Chiang Mai Red Sweet Pink
and Kimono Pink respectively (Figure 1) Abdullah et al [32]had previously indicated that the LD
50for C alismatifolia
var Chiang Mai Pink was approximately at 25Gy The deathof plants is attributed to the interaction of radiation withother molecules in the cell particularly water to produce freeradicals (H OH) The free radicals could combine to formtoxic substances such as hydrogen peroxide (H
2O2) which
contribute to the destruction of cells This indirect effectis especially significant in vegetative cells the cytoplasmwhich contains about 80 water [33] However sensitivityof the plant material depends on the genetic constitutiondose-employed DNA amount moisture content and stageof development and genotype [34] The choice of the doseto be applied for the highest mutant rescue is often left tothe breederrsquos experience with the specific plant material itsgenetics and its physiology
52 Analysis of Variance (ANOVA) for Morphological Traitsof C alismatifolia in M1V1 Analysis of variance indicatedhighly significant differences among the varieties doses andtheir interaction for all traits in M
1V1generation (Table 4)
Some desired and undesired abnormalities such as dwarfismchlorophyll mutation (albinism) striata (yellow or whitelongitudinal bands altering with green colors) two-midribleaves split leaves double flower stalk in one plant doubleinflorescence marbled pink bracts two-tone pink-purplishbracts and two-flag petals were found in M
1V1plants
(Table 5)
53 Effect of Gamma Irradiation on Vegetative Traits in theM1V1 The growth of plants treated with 10 and 20Gy ofgamma rays was slower than that of the controls (Table 6)In irradiated plants the leaf length and leaf width decreasedsignificantly (119875 lt 005) as the radiation doses increasedThis trend is quite common in mutagenised populationsSuch effects are known to arise due to drastic chromoso-mal aberrations in addition to genetic mutations Similar
6 BioMed Research International
Table4Meansquareso
fanalysis
ofvaria
nce(ANOVA
)for
14morph
ologicaltraitsin
Calism
atifolia
(a)
Source
ofvaria
tion
Meansquares
dfGeneration
Num
bero
fsho
otLeafleng
thLeafwidth
Leafnu
mber
Daystovisib
lebu
dPlanth
eight
Block
4M
1V1
005
622
11014
9467
8358
Varie
ty3
M1V
1263lowast
2702lowast
3011lowast
500lowast
1455602lowast
253739lowast
Dose
2M
1V1
611lowast
373804lowast
4137lowast
1042lowast
1012001lowast
157076
6lowastVa
rlowastdo
se6
M1V
114
1lowast8555lowast
418lowast
167lowast
731271lowast
115934lowast
Error
44M
1V1
0094
2951
077
037
4903
Total
5984
93
CV(
)M
1V1
23183
176
206
91165
(b)
Source
ofvaria
tion
Meansquares
dfGeneration
Daysto
anthesis
Daysto
senescence
Noof
true
flow
Noof
pink
bract
Inflo
rescence
leng
thNoof
new
rhizom
esRh
izom
esiz
eNoof
storage
roots
Block
4M
1V1
12847
048
418
072
282
01
018
055
Varie
ty3
M1V
11608017
10091lowast
9166lowast
62543lowast
376lowast
163lowast
023
ns1726lowast
Dose
2M
1V1
1587261
128682lowast
58286lowast
35315lowast
56022lowast
1031lowast
531lowast
6481lowast
Varlowastdo
se6
M1V
1876957
3464lowast
2900lowast
977lowast
4004lowast
128lowast
025ns
332lowast
Error
44M
1V1
7752
322
392
226
445
011
011
121
Total
59CV
()
M1V
110
15229
169
165
2318
268
lowastSign
ificant
with
leastsqu
ared
ifference
test119875lt005
BioMed Research International 7
Dose (Gy)
0
10
10
20
20
30
30
40
40
50
50
60
60
70
70
80
80
90
90
100
100
Mor
talit
y (
)
Var Doi Tung554
LD50 = 28 Gy
(a)
0
10
10
20
20
30
30
40
40
50
50
60
60
70
70
80
80
90
90
100
100
Mor
talit
y (
)
Var Chiang Mai Red
Dose (Gy)
LD50 = 21Gy
(b)
Mor
talit
y (
)
Dose (Gy)
Var Sweet Pink
0
10
10
20
20
30
30
40
40
50
50
60
60
70
70
80
80
90
90
100
100
LD50 = 23Gy
(c)
0
10
10
20
20
30
30
40
40
50
50
60
60
70
70
80
80
90
90
100
100
Mor
talit
y (
)
Dose (Gy)
Var Kimono Pink
LD50 = 25Gy
(d)
Figure 1 PoloPlus plot of linear scale of dose versusmortality percent (a) Doi Tung 554 (b) ChiangMai Red (c) Sweet Pink and (d) KimonoPink
Table 5 Effect of acute gamma rays on vegetative and flowering traits of C alismatifolia in the M1V1 generation
Variety irradiated dose Observed variations Flower color variationChiang Mai Red
10Dwarfism no pink bracts inflorescence small inflorescencetwo-flag petal true flower double inflorescence undulate leafmargin and yellowwhite strip leaves
Light purple N78Clowast
20 Dwarfism narrow small leaves and yellowwhite strip leaves No flowerDoi Tung 554
20 Dwarfism fewer pink bracts and yellowwhite strip leavesSmall and narrow leave Light purple N80Dlowast
lowastRoyal British Society color chart (RHS)
8 BioMed Research International
Table 6 Effect of acute gamma rays on vegetative traits of C alismatifolia in M1V1 generation
Dose (Gray) Shoot number Leaf number Leaf length (cm) Leaf width (cm) Plant height (cm)CMR
0 20 plusmn 00a
30 plusmn 04a
556 plusmn 08a
71 plusmn 05a
1112 plusmn 25a
10 10 plusmn 00b
36 plusmn 05a
272 plusmn 25b
45 plusmn 05b
714 plusmn 96b
20 10 plusmn 00b
14 plusmn 05b
124 plusmn 120c
25 plusmn 24c
170 plusmn 80c
DT0 30 plusmn 00
a30 plusmn 00
a622 plusmn 08
a65 plusmn 05
a912 plusmn 19
a
10 16 plusmn 05b
28 plusmn 04a
280 plusmn 44b
46 plusmn 06b
542 plusmn 76b
20 12 plusmn 04b
24 plusmn 05a
242 plusmn 38b
38 plusmn 05c
512 plusmn 138b
SP0 16 plusmn 05
a32 plusmn 04
a461 plusmn 27
a101 plusmn 04
a746 plusmn 38
a
10 10 plusmn 00b
36 plusmn 05a
275 plusmn 34b
57 plusmn 02b
377 plusmn 137b
20 10 plusmn 00b
20 plusmn 10b
196 plusmn 74c
57 plusmn 04b
195 plusmn 73c
KP0 14 plusmn 00
a46 plusmn 05
a345 plusmn 29
a47 plusmn 04
a591 plusmn 28
a
10 10 + 05a
40 plusmn 05b
220 plusmn 24b
31 plusmn 04b
420 plusmn 54b
20 10 plusmn 00a
32 plusmn 04b
156 plusmn 43c
31 plusmn 11b
283 plusmn 174c
CV () 23 18 26 16 165Means with the same or common letter are not significantly different least square difference test 119875 lt 005
decreases in leaf size were reported by Pongchawee et al [35]and Tangpong et al [36] These results were in agreementwith an earlier study [4] which reported that the growthof chrysanthemum exposed to acute gamma rays was lessthan the control in the M
1V1generation All varieties doses
and interaction effects resulted in significant differences fornumber of leaves Among untreated plants Kimono Pinkvariety had higher number of leaves (46) than the otherthree (3 3 and 32) varieties In Chiang Mai Red and SweetPink varieties plants exposed to 10Gy showedhigher numberof leaves than untreated plants However at 20Gy therewas significant reduction in number of leaves for all studiedvarieties in comparison to control Similar stimulatory effectswere obtained at lower doses in ginger by Hegde [37] andGiridharan and Balakrishnan [38]
Progressive reduction in growth parameters can be inter-preted on interference in normal mitosis and frequent occur-rence of mitotic aberrations inhibition of rate of assimilationand consequent change in the nutrient level in the plantand inactivation of vital enzymes especially those associatedwith respiration [39] Dose-dependent negative effect wasalso detected for plant heightThe tallest plants were recordedfrom the untreated rhizomes (0Gy) with heights of 1112912 746 and 591 cm followed by the 10Gy irradiated plantswith heights of 714 542 377 and 420 cm and the 20Gyirradiated plants with corresponding heights of 170 512195 and 283 cm for Chiang Mai Red Doi Tung 554 SweetPink and Kimono Pink varieties respectively These resultsare in agreement with the findings of Abdullah et al [32]Reduction in growth parameters and dwarfism can be causedby interference of normal mitosis and frequent occurrenceof mitotic aberrations inhibition of assimilation rates andconsequent changes in nutrient levels in plants Additionallymutagenic effects such as auxin destruction inhibition of
auxin synthesis failure of assimilatory mechanism andchanges in the specific activity of enzymes can cause growthreductions [37] High doses of ionizing radiation have beenshown to damage macromolecular cellular components suchas cell walls membranes and DNA [40] The number ofshoots also decreased significantly as the radiation dosesincreased Radiation also affects organic molecules that areessential to the cell division process and thus causing celldivision to stop [36]
54 Effect of Gamma Irradiation on Flowering DevelopmentTraits in the M1V1 All control and 10Gy irradiated plantsproduced flowers while the Chiang Mai Red and SweetPink varieties which were exposed to 20Gy did not gointo the flowering stage Lamseejan et al [4] also showedthat flowering percentage decreases as gamma ray dosesare increased In the present study gamma rays caused lateflowering in all four varieties Days to appearance of firstvisible buds were also significantly different among the fourvarieties (Table 7) Gamma rays caused a noticeable delay inflowering of irradiated plants in comparison to the untreatedones First visible buds were observed at 652 and 874 days inthe control and 10Gy treatments respectively for the ChiangMai Red variety In Doi Tung 554 the first visible buds wereappeared at 474 656 and 840 days after planting at 0 10and 20Gy doses respectively In the Sweet Pink variety thenumber of days to visible bud appearance increased signif-icantly from 678 days in controls to 978 days in the 10Gyirradiated plants In comparison to other three varieties theKimono Pink variety needed the longest time to visible budappearance and same as other varieties there was a positivecorrelation between the number of days to first visible budand the gamma irradiation dose In the control and 10Gyand 20Gy irradiated individual plants flower buds were
BioMed Research International 9
Table 7 Effect of acute gamma rays on flower development characteristics of C alismatifolia in M1V1 generation
Dose (Gy) Days tovisible bud
Inflorescencelength (cm)
Days toanthesis
Number oftrue flowers
Number ofPink bracts
Days tosenescence
CMR0 652 plusmn 47
b162 plusmn 057
a742 plusmn 54
b132 plusmn 19
a104 plusmn 15
a21 plusmn 10
a
10 874 plusmn 95a
82 plusmn 397b
1020 plusmn 71a
56 plusmn 19b
50 plusmn 07b
10 plusmn 14b
20 00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
DT0 474 plusmn 43
c134 plusmn 054
a546 plusmn 54
c162 plusmn 13
a232 plusmn 04
a23 plusmn 00
a
10 656 plusmn 37b
94 plusmn 19b
780 plusmn 37b
112 plusmn 21b
166 plusmn 08b
122 plusmn 130b
20 840 plusmn 42a
76 plusmn 082c
994 plusmn 49a
88 plusmn 13b
156 plusmn 11b
106 plusmn 18b
SP0 678 plusmn 21
b146 plusmn 089
a756 plusmn 26
b162 plusmn 13
a100 plusmn 24
a188 plusmn 083
a
10 978 plusmn 181a
79 plusmn 20b
1098 plusmn 182a
68 plusmn 25b
52 plusmn 08b
92 plusmn 24b
20 00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
KP0 852 plusmn 23
c137 plusmn 044
a942 plusmn 19
c112 plusmn 12
a96 plusmn 21
a18 plusmn 18
a
10 1044 plusmn 65b
108 plusmn 108ab
1224 plusmn 100b
76 plusmn 27b
64 plusmn 16ab
102 plusmn 17b
20 1272 plusmn 65a
790 plusmn 49b
1426 plusmn 58a
60 plusmn 38b
42 plusmn 24b
66 plusmn 37b
CV () 10 23 9 22 169 15Means with the same or common letter are not significantly different least square difference test 119875 lt 005
visible at 852 1328 and 1374 days after planting respectivelyPrevious studies also showed that onset of flowering andformation of floral parts in mutants of Arabidopsis thalianamaize barely pea and tobacco involved growth regulators(or phytohormones) such as auxins cytokinins gibberellinsabscisic acid ethylene and brassinosteroids [41] There weresignificant differences among treatments for the length ofthe inflorescence In all varieties the longest inflorescencelength was observed in the untreated plants with 162134 146 and 137 cm lengths for Chiang Mai Red DoiTung 554 Sweet Pink and Kimono Pink respectively Thecorresponding inflorescence lengths were 82 94 79 and109 cm for the 10Gy irradiated plants The days to anthesisfor C alismatifolia varieties were significantly affected byvariety gamma irradiation doses and their interaction Thenumber of days to full bloom was noticeable earliest foruntreated plants at 742 546 756 and 942 days for ChiangMai Red Doi Tung 554 Sweet Pink and Kimono Pinkvarieties respectively This was then followed by plants at10Gy at 102 78 1098 and 1224 days The number of trueflowers or the secondary inflorescence developed in the axilof the primary bracts decreased as radiation dosage increasedIn the present study the gamma rays also decreased thedays to inflorescence senescence In this study there wasa strongly significantly and positively correlation (0919lowastlowast)(data not shown) between the number of true flowers andthe number of days to senescenceThe number of pink bractsalso decreasedwith increasing radiation dosageMost gammaray effects on senescence are considered as resulting from theaction of free radicals generated from water and oxygen bythe ionizing energy on the cellular components Membrane
deterioration is a general feature of natural senescence andstress-induced aging [42]
Irradiation induced some mutation spectrum of flowercolor variation that included colors such as purple palepurple rather pale purple white purple white (marbledpattern) and two-tone purple color
Mutation spectrum of flower shape variation includeddouble inflorescence within one stalk double stalk per plantinflorescence without bracts two-flag petal true flowersand chlorophyll mutation in the leaves which are generallycaused by induced gamma rays (Figure 2) Ionizing radiationincluding gamma rays induces fragment deletions or inser-tions that eventually lead to changes in amino acids and amodification of leaf and stem pigmentation [43] A mutationin the biosynthetic pathway of structural or regulatory genesmay cause a change in flower color [44] When the blockageoccurs at the early stages of anthocyanin synthesis whiteflowers will result while a blockage at later stages leads todifferent flower colors due to the accumulation of particularanthocyanins [45] Chloroplasts were extremely sensitive togamma radiation compared to other cell organelles [46]
55 Effect of Gamma Irradiation on Rhizome Characteristicsin Selected Doses in M1V1 The number of new rhizomes andthe number of storage roots per rhizome were significantlyaffected by varieties doses and their interaction (Table 8)The number of new rhizomes only in Kimono Pink varietydid not show any differences between untreated and treatedplants The rhizome size was influenced only by doses Asdose level increased the rhizome size decreased Amonguntreated plants the Sweet Pink rhizomes had the most
10 BioMed Research International
(a) (b) (c) (d)
(e)
(f)
Figure 2 Effect of acute gamma rays on flower traits in C alismatifolia (a) Untreated inflorescence bracts color Doi Tung 554 (b) Two tonebract color Doi Tung 554 (20Gy) (c) Marbled pattern of bract color Doi Tung 554 (20Gy) (d) Double inflorescence within one stalk varChiang Mai Red (10Gy) (e) True flower in nontreated plants (f) Two-flag petals true flowers (10Gy)
Figure 3 Dendrogram representing themorphological variation among 44 irradiated and nonirradiated individuals ofC alismatifolia across14 variables CMR = Chiang Mai Red DT = Doi Tung 554 SP = Sweet Pink Kp = Kimono Pink
number of storage roots (78) and the Kimono Pink rhizomeshad the least number of storage roots (40)This was followedby 10Gy irradiated rhizomes with 4 46 48 and 28 storageroot numbers for Chiang Mai Red Doi Tung 554 SweetPink and Kimono Pink respectively Irradiation with 20Gydecreased significantly the number of storage roots in allstudied varietiesTheplantC alismatifolia is propagated froma propagule (one rhizome + 5-6 t-roots) The rhizome sizedose matters in the growth of the plant Smaller rhizome sizeusually resulted in smaller plant size with narrow grass-likeleaves The storage roots play a very important role in thegrowth and flowering ofCurcumaThe t-rootsmake up about85 of the total fresh weight and 70 of the total dry weightof a typical propaguleThe storage organs act for plant growth
during dormancy and emergence [47] More storage root perpropagule resulted in faster flowering and higher yield plant[48] In this study also there was significant and negativecorrelation between number of storage roots and number ofdays to visible bud appearance (minus0525lowastlowast)
56 Cluster Analysis of C alismatifolia for MorphologicalTraits The morphological data were used to calculate thesimilarity between the treated and non-treated C alismat-ifolia samples and UPGMA dendrogram was constructed(Figure 3) The cophenetic correlation coefficient (CCC)value between the genetic dissimilaritymatrix estimated fromthe morphological characters and the UPGMA clusteringmethod was 119903 = 093 showing a proof fit This value was
BioMed Research International 11
Table 8 Effect of acute gamma rays on rhizome characteristics ofC alismatifolia in M1V1 generation
Dose (Gray) Rhizomesize
No of newrhizomes
No of storageroot
CMR0 23 plusmn 02
a26 plusmn 055
a46 plusmn 054
a
10 18 plusmn 005a12 plusmn 044
b40 plusmn 07
a
20 08 plusmn 07b
06 plusmn 054b
14 plusmn 13b
DT0 22 plusmn 01
a30 plusmn 00
a72 plusmn 109
a
10 18 plusmn 007b14 plusmn 054
b46 plusmn 13
b
20 16 plusmn 005b
10 plusmn 00b
28 plusmn 13c
SP0 22 plusmn 02
a24 plusmn 054
a78 plusmn 21
a
10 17 plusmn 007b
10 plusmn 00b
48 plusmn 044b
20 12 plusmn 02c
10 plusmn 00b
28 plusmn 083b
KP0 21 plusmn 005
a10 plusmn 00
a40 plusmn 00
a
10 16 plusmn 01ab
10 plusmn 00a
28 plusmn 044b
20 11 plusmn 06b
10 plusmn 00a
22 plusmn 083b
CV () 18 23 26Means with the same or common letter are not significantly different leastsquare difference test 119875 lt 005
higher than of studies on olive for morphological traits (119903 =069) [49] In this dendrogram the 44C alismatifolia samplesappeared to form three main clusters and five minor clustersat coefficient level 56 cluster I included three non-irradiatedmembers and cluster II included 10Gy irradiated individualplants of all four varieties and 20Gy irradiated samples ofDoi Tung 554 and Kimono Pink varieties except KP20-4 (noflowering) Main cluster III included the 20Gy irradiatedindividuals Chiang Mai Red and Sweet Pink varieties (asmentioned in morphological part these individuals didnot go to flowering stage) and KP20-4 Cluster II can bedivided to five subclusters The first subcluster included 10members (CMR10-1 CMR10-5 SP10-1 SP10-2 SP10-3 SP10-5 CMR10-2 CMR10-4 SP10-4 and CMR10-3) the secondone included nine members (KP10-1 KP10-2 KP10-3 KP10-4 KP10-5KP20-1 KP20-2 KP20-5 and KP20-3) KP0 wasthe lone member of the third sub-cluster The fourth sub-cluster included two members only (DT10-2 and DT10-3) The last sub-cluster main cluster II had eight membersincluded (DT10-1 DT10-4 DT10-5 DT20-1 DT20-2 DT20-3 DT20-4 and DT20-5) Mean value of groups for each traitis presented in Table 9 This table clearly shows the differentmean values of the three main clusters The minimum meanvalue referred to main cluster III while the maximum meanvalue for number of shoots leaf length leaf width plantheight number of true flowers inflorescence length rhizomesize number of new rhizomes and number of storage rootsbelonged to cluster I which was included non-treated plantsThese results showed that the gamma irradiation has inducedmorphological changes in C alismatifolia individuals of
four studied varieties so that they showed phenotypicallydifferences from their controls
57 Principal Component Analysis of C alismatifolia forMorphological Traits To assess the patterns of variation PCAwas done by considering all of the 14 variables The first threecomponents of PCA explained 828 of the total variation(Table 10) Only the first component which accounted for562 of the total variation was attributed to inflorescencelength plant height days to senescence number of trueflowers and rhizome size In PCA three-dimensional graphthe grouping of individuals confirmed the clustering results(Figure 4) The PCA graph proved that all irradiated indi-vidual plants of C alismatifolia varieties are phenotypicallydifferent from their non-irradiated individuals
6 Molecular Characterization
61 SSR Polymorphism Amplifications were successful forall the 17 SSR markers assayed This reflects a high levelof homology between SSR flanking regions in C longa andC alismatifolia Out of 17 primer pairs eight primer pairsresulted in polymorphic PCR products Table 11 summarizesthe results obtained based on the analysis of individuals of thefour studied varieties using the polymorphic SSR loci for 010 and 20Gy irradiated plants The number of alleles variedwidely among these loci A total of 25 36 and 41 alleles wereobserved among 0 10 and 20Gy irradiated individual plantsrespectively In the 10Gy treatment the number of allelesranged from three (clon09 and clon14) to seven (clon08) withan average value of 45 per locus In the 20Gy treatmentthe number of alleles varied from three (clon09 and clon14)to seven (clon08 and clon12) with an average value of 51per locus In the untreated individuals the number of allelesranged from two (clon04 clon09 clon11 and clon14) to five(clon12) with an average value of 31 The difference betweenthe average number of observed alleles and effective numberof alleles was due to the uneven frequency of each allele[50] For each of the eight SSR primers PIC values (whichmeasures allele diversity and frequency among varieties)varied from 019 (clon04) to 071 (clon01) in untreated plantsand from 025 (clon04) to 075 (clon08) in 10Gy acutelyirradiated plants In 20Gy irradiated individual plants thePIC value ranged from 042 (clon14) to 075 (clon08 andclon12) The mean PIC for all loci was 047 054 and 061for 0 10 and 20Gy irradiated plants respectively
The PIC value provides an estimate of the discriminatorypower of amarker by taking into account not only the numberof alleles at a locus but also the relative frequencies of thesealleles [51] Thus except for clon09 and clon14 which weremoderately polymorphic (025 lt PIC lt 05) all other lociwere highly polymorphic (05 lt PIC) while none of theloci showed low polymorphism The average discriminatingpower ofmicrosatellitemarkers (061) observed in the presentstudy ensures the future utility of microsatellite markersfor genetic variation studies in C alismatifolia varietiesOur results reflect similar findings as reported earlier inthe study of genetic variation in other species of Curcuma
12 BioMed Research International
Table9Clusterm
eans
for14charactersestim
ated
in44
individu
also
fCalismatifolia
Clusters
Shoo
tnu
mber
Leaf
leng
thLeaf
width
Leaf
number
Visib
lebu
d(days)
Inflo
rescence
leng
thPlant
height
Anthesis
(days)
Senescence
(days)
True
flow
number
Pink
bract
number
Rhizom
esiz
eNew
rhizom
enu
mber
Storage
root
number
I220
5464
736
300
6013
1473
9233
6926
2093
152
1453
229
266
653
II1
100
2735
512
360
9260
809
5457
1059
960
620
510
177
110
440
II2
122
1922
298
344
11422
1043
3910
1312
293
3755
588
156
100
266
II3
100
3452
416
460
8520
1370
5910
9420
180
120
960
206
100
400
II4
150
2500
45
250
6500
925
4950
7700
1150
950
1700
185
200
600
II5
137
2637
417
262
7725
840
5356
9162
1137
1012
1587
170
100
312
III
054
1500
465
181
000
000
000
000
000
000
000
090
081
200
BioMed Research International 13
Table 10 Eigenvectors eigenvalues and proportions of variabilityfor three principle components among 14 characters for 44 Calismatifolia
Variable PC1 PC2 PC3Number of new shoot 0270 minus0076 minus0173Leaf length 0269 minus0305 0033Leaf width 0096 minus0455 0430Leaf number 0210 0285 0490Days to visible bud 0199 0462 0181Plant height 0321 0027 minus0071Inflorescence length 0318 0166 minus0040Days to anthesis 0205 0460 0148Days to senescence 0342 0017 minus0131Number of true flower 0323 0029 minus0238Number of pink bracts 0266 minus0028 minus0546Rhizome size 0302 minus0101 0147Number of new rhizome 0250 minus0293 0028Number of storage roots 0264 minus0247 0298Eigenvalue 7877 2694 1032Proportion 56264 19246 7371Cumulative 56264 75511 82882
using SSR markers [13] The highest Neirsquos gene diversity(ℎ) was obtained with the 20Gy treatment (061) followedby the 10Gy treatment (06) and the untreated individuals(05) The mean Shannonrsquos information index (119868) was 092114 and 130 in the 0 10 and 20Gy irradiated plantsrespectively The high value of Shannonrsquos information indexrepresents the effectiveness of microsatellite loci to revealthe variation among these varieties at the different dosesused Overall genetic variability for the varieties studiedrepresented by the Shannon-Weiner index was relatively highin comparison to other studies involving C longa accessions[10 52] Additionally the 20Gy acutely irradiated individualsshowed a higher mean percentage of polymorphic loci (63)than the 10Gy (58) and untreated (022) ones This impliesthat irradiation with a dose of 20Gy induced more geneticvariation in the M
1V1generation
62 Cluster Analysis Thedendrogramwas constructed usingPowerMarker 323The 44 studied individuals were clusteredinto seven groups (G1ndashG7) Colors were applied accordingto our model-based cluster analysis results Group I iscomprised of nine individual plants of Chiang Mai Redvariety receiving 0 10 and 20Gy treatments (blue colorin Figure 5) One individual plant DT10-1 was assignedto group II (green color in Figure 5) All irradiated andnon-irradiated individuals of Doi Tung 554 variety wereassigned to group III (yellow color in Figure 5) DT20-1 andSP10-5 were the sole members of groups IV (red color inFigure 5) and V (gray color in Figure 5) respectively GroupVI included treated and untreated individuals of Sweet Pinkvariety (pink color in Figure 5) Lastly all Kimono Pinkindividuals were assigned to cluster VII (orange color in
Figure 5) The genetic similarity coefficient (data not shown)among the 44 individuals amplified using eight SSR primersvaried from 00 to 10 The highest value (10) correspondedto [(CMR10-3 CMR10-5 CMR and CMR20-2) (DT10-2and DT10-3) (DT and DT20-2) (KP20-3 KP-20-4 KP10-3andKP10-4)] individuals that generated identical fingerprintsacross the markers studied Among the studied varieties theSweet Pink individual plants showed the lowest similarity(00) withKimono Pink individuals which indicated that theywere relatively remote in relationship (Figure 5) An overviewof the clustering pattern indicates that the grouping of thestudied individual plants was based on different varieties andlargely independent of the doses of gamma irradiation Awide range in similarity values had also been observed indifferent species of Curcuma [12 13 52 53]
63 Principle Component Analysis (PCA) Thedata generatedfrom 44 C alismatifolia individual plants were subjected toprincipal component analysis (PCA) to visualize individualsin a multivariate space In the three-dimensional graphderived from the SSR analysis all studied individuals weregrouped into seven clusters (Figure 6) In PCA the firstthree principal components (PC) extracted a cumulative of6826 of the total variation among the 44 individuals of CalismatifoliaThe first three coordinates PC1 PC2 and PC3accounted for 4136 1578 1112 of the total variationrespectively The distribution of the individual plants in athree-dimensional plot using the first three principal com-ponents showed the genetic relationship between individualplants It was evident that both methods dendrogram andthree-dimensional plots of PCA were effective in illustratinggenetic relationships and the groups found were comparable[11 13] The PCA results were similar to those obtained fromcluster analysis where all individuals from different varietieswere assigned to different groups These graphical illus-trations enable the assessment of the relationshipdistancesamong all of the individuals in the study [50]
7 Conclusion
This is the first attempt to evaluate the effect of acutegamma irradiation on C alismatifolia varieties using bothmorphological characteristics and molecular markers Inplant breeding programs mutagenic treatments with lownegative physiological effects and strong genetic effects aredesirable Hence we used more effective doses of gammairradiation (10 and 20Gy) of which particularly 20Gy dosewas effective to influence morphological and molecularcharacteristics of studiedCurcuma alismatifolia varietiesThelower dose (10Gy) of radiation probably caused little damageto the plants genetic material so that the cells could repairthemselves in next generation As a result in this study 20Gyacute gamma irradiation resulted in a higher percentage ofmutation and getting desired mutants was more possibleBased on the LD
50values determined in this study it was
apparent that the varieties Chiang Mai Red and Sweet Pinkwere more sensitive to gamma rays compared to Doi Tung554 and Kimono Pink Our results show that the variety Doi
Figure 4 Two-dimensional graph of principal component analysis (PCA) for 14 morphological variables indicating relationships amongirradiated and nonirradiated four varieties of C alismatifolia
CMR10-1CMR10-5
SP10-1SP10-2SP10-3
SP10-5
CMR10-4
SP10-4
KP10-1KP10-3
KP20-1
KP20-2
KP10-5KP20-5
KP20-3
KP10-2
DT10-2
DT10-1
DT10-5
DT20-1
DT10-4DT20-4DT20-2
DT20-5DT20-3
CMR20-1
SP20-3SP20-4
SP20-5SP20-1
SP20-2
KP
SP
CMR 10-2
SP10-5
Figure 5 Unrooted neighbor joining tree showing genetic relationship among 44 irradiated and nonirradiated C alismatifolia using SSRmarkers The colors of the branches correspond to those of the same cluster
Figure 6Three-dimensional graph of principal component analysis (PCA) indicating relationships among irradiated and nonirradiated fourvarieties of C alismatifolia
Tung 554 (Curcuma hybrid) had the most morphologicalresponses to gamma rays The use of microsatellite markersas a codominant marker will facilitate the exploration ofgenetic variability among treated and non-treated plants andwill help to distinguish the plants showing differences inmorphological characters The overall effects on the M
1V1
generation revealed that acute gamma irradiation at optimumdoses has the potential for developing new varieties of Calismatifolia with improved commercial properties suitablefor the Malaysian flower industry
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work was funded by the Fundamental Research GrantScheme (FRGS) under the Ministry of Higher Education inMalaysia
References
[1] P Apavatjrut S Anuntalabhochai P Sirirugsa and C AlisildquoMolecularmarkers in the identification of some early floweringCurcuma L (Zingiberaceae) speciesrdquo Annals of Botany vol 84no 4 pp 529ndash534 1999
[2] K Bunya-Atichart S Ketsa andW G VanDoorn ldquoPostharvestphysiology of Curcuma alismatifolia flowersrdquo Postharvest Biol-ogy and Technology vol 34 no 2 pp 219ndash226 2004
[3] M Nakano J Amano Y Watanabe et al ldquoMorphologicalvariation in Tricyrtis hirta plants regenerated from heavy ionbeam-irradiated embryogenic callusesrdquo Plant Biotechnologyvol 27 no 2 pp 155ndash160 2010
[4] S Lamseejan P Jompuk A Wongpiyasatid A Deeseepan andP Kwanthammachart ldquoGamma-rays induced morphological
[5] G-J Lee S J Chung I S Park et al ldquoVariation in thephenotypic features and transcripts of colormutants of chrysan-themum (Dendranthema grandiflorum) derived from gammaraymutagenesisrdquo Journal of Plant Biology vol 51 no 6 pp 418ndash423 2008
[6] O K Kikuchi ldquoOrchid flowers tolerance to gamma-radiationrdquoRadiation Physics and Chemistry vol 57 no 3ndash6 pp 555ndash5572000
[7] N P Arnold N N Barthakur and M Tanguay ldquoMutageniceffects of acute gamma irradiation on miniature roses targettheory approachrdquo HortScience vol 33 no 1 pp 127ndash129 1998
[8] A A Youssef M S Aly and M S Hussein ldquoResponse ofgeranium (Pelargonium graveolenus L) to gamma irradiationand foliar application of Speed Growrdquo Egyptian Journal ofHorticulture vol 27 pp 41ndash53 2000
[9] N Chuantang and L Yazhi ldquoThe radiation inducedmutation ofcanna (Canna L)rdquo Acta Agricultute Nucleatae Sinica vol 2 pp33ndash39 1998
[10] M S Sigrist J B Pinheiro J A A Filho and M I ZucchildquoGenetic diversity of turmeric germplasm (Curcuma longaZingiberaceae) identified by microsatellite markersrdquo Geneticsand Molecular Research vol 10 no 1 pp 419ndash428 2011
[11] S Taheri T L Abdullah N A P Abdullah and Z AhmadldquoGenetic relationships among five varieties of Curcuma alis-matifolia (Zingiberaceae) based on ISSRmarkersrdquoGenetics andMolecular Research vol 11 pp 3069ndash3076 2012
[12] A Das V Kesari V M Satyanarayana A Parida and L Ran-gan ldquoGenetic relationship of Curcuma species from NortheastIndia using PCR-based markersrdquo Molecular Biotechnology vol49 no 1 pp 65ndash76 2011
[13] S Siju K Dhanya S Syamkumar et al ldquoDevelopment char-acterization and utilization of genomic microsatellite markersin turmeric (Curcuma longa L)rdquo Biochemical Systematics andEcology vol 38 no 4 pp 641ndash646 2010
[14] M K Panda S Mohanty E Subudhi L Acharya and SNayak ldquoAssessment of genetic stability of micropropagated
BioMed Research International 17
plants of Curcuma L by cytophotometery and RAPD analysisrdquoInternational Journal of Integrative Biology (IJIB) vol 1 pp 189ndash195 2007
[15] S Syamkumar and B Sasikumar ldquoMolecular marker basedgenetic diversity analysis of Curcuma species from IndiardquoScientia Horticulturae vol 112 no 2 pp 235ndash241 2007
[16] D Tautz and M Renz ldquoSimple sequences are ubiquitousrepetitive components of eukaryotic genomesrdquo Nucleic AcidsResearch vol 12 no 10 pp 4127ndash4138 1984
[17] G Toth Z Gaspari and J Jurka ldquoMicrosatellites in differenteukaryotic genomes surveys and analysisrdquo Genome Researchvol 10 no 7 pp 967ndash981 2000
[18] W Powell G C Machray and J Proven ldquoPolymorphismrevealed by simple sequence repeatsrdquo Trends in Plant Sciencevol 1 no 7 pp 215ndash222 1996
[19] S-Y Lee W K Fai M Zakaria et al ldquoCharacterizationof polymorphic microsatellite markers isolated from ginger(Zingiber officinale Rosc)rdquo Molecular Ecology Notes vol 7 no6 pp 1009ndash1011 2007
[20] S Bory D Da Silva A-M Risterucci M Grisoni P Besseand M-F Duval ldquoDevelopment of microsatellite markers incultivated vanilla polymorphism and transferability to othervanilla speciesrdquo Scientia Horticulturae vol 115 no 4 pp 420ndash425 2008
[21] I C Menezes F W Cidade A P Souza and I C SampaioldquoIsolation and characterization ofmicrosatellite loci in the blackpepper Piper Nigrum L (Piperaceae)rdquo Conservation GeneticsResources vol 1 no 1 pp 209ndash212 2009
[22] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[23] V Kumar Morphological and molecular characterization ofinduced mutants in Groundnut [PhD thesis] University ofAgricultural Sciences Dharwad India 2008
[24] J R Sharma Statistical and Biometrical Techniques in PlantBreeding New Age International New Delhi India 1998
[25] J J Doyle and J L Doyle ldquoA rapid DNA isolation procedure forsmall quantities of fresh leaf tissuerdquo Phytochemical Bulletin vol19 pp 11ndash15 1987
[26] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[27] Oslash Hammer D A T Harper and P D Ryan ldquoPast paleontolog-ical statistics software package for education and data analysisrdquoPalaeontologia Electronica vol 4 no 1 pp 4ndash9 2001
[28] K Liu and S V Muse ldquoPowerMaker an integrated analysisenvironment for genetic maker analysisrdquo Bioinformatics vol 21no 9 pp 2128ndash2129 2005
[29] F J RohlfNTSYS-Pc Numerical Taxonomy System Version 2 1Exeter Publishing Setauket New York NY USA 2002
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[31] S Kadkhodaei M Shahnazari M K Nekouei et al ldquoA compar-ative study of morphological and molecular diversity analysisamong cultivated almonds (Prunus dulcis)rdquo Australian Journalof Crop Science vol 5 no 1 pp 82ndash91 2011
[32] T L Abdullah J Endan and B M Nazir ldquoChanges in flowerdevelopment chlorophyll mutation and alteration in plant
morphology of Curcuma alismatifolia by gamma irradiationrdquoAmerican Journal of Applied Sciences vol 6 no 7 pp 1436ndash14392009
[33] E Kovacs and A Keresztes ldquoEffect of gamma and UV-BCradiation on plant cellsrdquoMicron vol 33 no 2 pp 199ndash210 2002
[34] H L Ramesh Y Murthy and V N Munirajappa ldquoEffect ofgamma radiation on morphological and growth parametersof Mulberry variety M5rdquo International Journal of Science andNature vol 3 pp 447ndash452 2010
[35] K Pongchawee R Pradissan andW Pipatcharoenchai ldquoInducemutation in Anubias spp through in vitro Irradiationrdquo ThaiFisheries Gazette vol 60 pp 493ndash497 2007
[36] P Tangpong T Taychasinpitak C Jompuk and P JompukldquoEffects of acute and chronic gamma irradiations on in vitroculture ofAnubias congensisNE BrownrdquoKasetsart Journal vol43 no 3 pp 449ndash457 2009
[37] R K Hegde Studies on induced mutagenesis and in vitroregeneration in turmeric (Curcuma longa L) [PhD thesis]University of Agricultural Sciences Dharwad India 2006
[38] M P Giridharan and S Balakrishnan ldquoGamma ray inducedvariability in vegetative and floral characters of ginger IndianCocoardquo Arecanut and Spices Journal vol 15 pp 68ndash672 1992
[39] A P Cesarett ldquoEffect of radiation on higher plants and plantcommunitiesrdquo inRadiation Biology United States Atomic EnergyCommission pp 284ndash309 Washington DC USA 1968
[40] G W Seung Y C Byung J-H Kim et al ldquoUltrastructuralchanges of cell organelles in Arabidopsis stems after gammairradationrdquo Journal of Plant Biology vol 48 no 2 pp 195ndash2002005
[41] B S Ahloowalia and M Maluszynski ldquoInduced mutationsmdashanew paradigm in plant breedingrdquo Euphytica vol 118 no 2 pp167ndash173 2001
[42] R Voisine L-P Vezina and C Willemot ldquoInduction ofsenescence-like deterioration of microsomal membranes fromcauliflower by free radicals generated during gamma irradia-tionrdquo Plant Physiology vol 97 no 2 pp 545ndash550 1991
[43] N Shikazono Y Yokota S Kitamura et al ldquoMutation rate andnovel tt mutants of Arabidopsis thaliana induced by carbonionsrdquo Genetics vol 163 no 4 pp 1449ndash1455 2003
[44] T Nakatsuka M Nishihara K Mishiba and S YamamuraldquoTwo different mutations are involved in the formation ofwhite-flowered gentian plantsrdquo Plant Science vol 169 no 5 pp949ndash958 2005
[45] M Mato T Onozaki Y Ozeki et al ldquoFlavonoid biosynthesisin white-flowered Sim carnations (Dianthus caryophyllus)rdquoScientia Horticulturae vol 84 no 3-4 pp 333ndash347 2000
[46] S G Wi B Y Chung J-S Kim et al ldquoEffects of gammairradiation on morphological changes and biological responsesin plantsrdquoMicron vol 38 no 6 pp 553ndash564 2007
[47] F Lee Chin Effects of light intensity and daylength on growth andflowering of siam tulip (Curcuma alismatifolia var Chiang MaiPink) [PhD thesis] University Putra Malaysia 2007
[48] A Hagiladi N Umiel and X H Yang ldquoCurcuma alismatifoliaII Effects of temperature and daylength on the development offlowers and propagulesrdquo Acta Horticulture vol 430 pp 755ndash761 1997
[49] MHagidimitriou A Katsiotis GMenexes C Pontikis andMLoukas ldquoGenetic diversity of major greek olive cultivars usingmolecular (AFLPs and RAPDs) markers and morphologicaltraitsrdquo Journal of the American Society for Horticultural Sciencevol 130 no 2 pp 211ndash217 2005
18 BioMed Research International
[50] N Babaei N A P Abdullah G Saleh and T L AbdullahldquoIsolation and characterization of microsatellite markers andanalysis of genetic variability in Curculigo latifolia DryandrdquoMolecular Biology Reports vol 39 pp 9869ndash9877 2012
[51] B Shiran N Amirbakhtiar S Kiani S Mohammadi B ESayed-Tabatabaei and H Moradi ldquoMolecular characterizationand genetic relationship among almond cultivars assessed byRAPD and SSR markersrdquo Scientia Horticulturae vol 111 no 3pp 280ndash292 2007
[52] S Singh M K Panda and S Nayak ldquoEvaluation of geneticdiversity in turmeric (Curcuma longa L) using RAPD and ISSRmarkersrdquo Industrial Crops and Products vol 37 no 1 pp 284ndash291 2012
[53] Y Paisooksantivatana S Kako and H Seko ldquoGenetic diversityof Curcuma alismatifolia Gagnep (Zingiberaceae) in Thailandas revealed by allozyme polymorphismrdquo Genetic Resources andCrop Evolution vol 48 no 5 pp 459ndash465 2001
ray irradiation and chemical treatments have been used formutation breeding in a wide range of plants [3] Gammarays are a type of ionizing radiation which interacts withatoms or molecules to produce free radicals in cells whichdamage or modify important components of plant cellsand affect differently the morphology biochemistry andphysiology of plants Induced mutation is highly effective inenhancing natural genetic resources for the development ofimprovednew cultivars among vegetatively propagated cropsSome important ornamental plants for both cut flowers andpotted plants that have been used in mutation breeding arechrysanthemums [4 5] orchids [6] roses [7] pelargoniums[8] and canna [9]
The estimation of genetic variation on the basis of mor-phological traits alone which are the product of gene andenvironmental interactions does not determine the actuallevel of genetic variation among studied individuals [10] Sev-eral molecular markers such as random amplified polymor-phicDNA(RAPD) intersimple sequence repeats (ISSR) sim-ple sequence repeats (SSR) and amplified fragment lengthpolymorphism (AFLP) with different advantages and disad-vantages have been employed in genetic variation studies ofCurcuma species [10ndash15] RAPD markers are relatively easyto generate but may not be variable enough for some applica-tions or may have problems with reproducibility Among therobust class of molecular markers microsatellites or simplesequence repeats (SSRs) are extremely powerful tools forestimating genetic variation with high reproducibility in avariety of plant species These markers are characterized bythe presence of 1ndash6 nucleotide repeats within the codingand noncoding regions [16 17] of the genome which arecodominant hypervariable and multiallelic in nature [18]Genomic SSR markers have been developed in economicallyimportant spice crops such as Zingiber officinale [19] Vanillaplanifolia [20] and Piper nigrum [21] The development of17 EST-SSR and 17 genomic SSR markers has been recentlyreported in turmeric (Curcuma longa L) [13 22] Usinggenetic markers for internal quality control it is possibleto distinguish induced mutations from any nonmutationalgenetic variability and hence unequivocally demonstrate thatmutations induced by gamma irradiation were the majorsource of genetic variability [23 24] This study was designedto determine the optimumdose of acute gamma radiation forselected C alismatifolia varieties describe the morphologicalvariations as affected and developed from acute gammairradiation and elucidate the genetic variation among theirradiated C alismatifolia varieties using microsatellite DNAmarkers
2 Materials and Methods
21 Plant Materials The rhizomes of three C alismatifoliavarietiesmdashChiang Mai Red (SK 205112) Sweet Pink (SK205212) Kimono Pink (SK 205412) as well as one Curcumahybrid Doi Tung 554 (SK 205312)mdashwere provided from theCurcuma Nursery (Ubonrat) in Doisaket District ChiangMai 50220 Thailand (Table 1)
22 Gamma Irradiation for Radiation Sensitivity Test Irra-diation of the plant materials was conducted in the Facultyof Science and Technology University Kebangsaan Malaysia(UKM) using a Gammacell 220 Excel Irradiator (MDS Nor-dion Ottawa ON Canada) The source of gamma rays wasCobalt 60 Prepared rhizomes in the budding stage wereacutely irradiated with different doses of 10 (128 Sec) 20(256 Sec) 25 (311 Sec) 35 (436 Sec) 40 (512 Sec) 60(80 Sec) and 100Gy (130 Sec) In each variety 20 rhizomeswere treated for sensitivity testing at each dose After irra-diation the rhizomes were planted in 25 cm pots containinggrowthmedia consisting of topsoil cocopeat rice husk at theratio of 1 2 1 Radiation effect on test plants was recorded interms of the mortality rate () after exposure to the gammaradiation Number of mortal rhizomes were counted 40 daysafter planting (at each treatment) and expressed as percentageof the total number of rhizomes planted The experimentswere conducted in Green house number 1 Field 2 Facultyof Agriculture University Putra Malaysia (UPM) MalaysiaThe recorded data of mortality percentage were analyzed byPoloPlus (Probit and logic analysis) software version 2
23 Induction of Mutation with Selected Doses of GammaRadiation Based on LD
50and obtained confidence limits of
irradiation dose 20 rhizomes from each variety were irra-diated (March 2011) with gamma rays at doses of 0 (control)10 Gy and 20Gy The experiment was designed as 4 (variety)times 3 (dose) RCBD with five blocks and four replications foreach sample
24 Morphological Data Fourteen morphological traitsincluded vegetative traits flowering development and rhi-zome characteristics data were recorded duringMarch 2011 toSeptember 2011 for four varieties of C alismatifolia (Table 2)The traits included number of new shoots leaf length leafwidth leaf number plant height number of days to visiblebud inflorescence length number of days to anthesis num-ber of days to senescence number of true flowers number ofpink bracts number of rhizome rhizome size and number ofstorage roots
3 SSR Analysis
31 DNA Isolation Leaves of all mutants and control indi-viduals were stored at minus70∘C until used for DNA extractionDNA was isolated from leaves of selected 44 individualswith morphological variations using cetyltrimethylammo-nium bromide (CTAB) extraction buffer [25] The extractionbuffer comprised of 2 (wv) CTAB 14mM NaCl 100mMTris-HCL PH 80 20mMEDTA 2 (wv) PVP and 2 (vv)120573-mercaptoethanol The mixture was incubated at 65∘C for 1hour followed by two extractions with chloroformisoamylalcohol (24 1) Isopropanol was used to precipitate nucleicacids and the pellet obtained was washed with 70 ethanoldried and dissolved in a Tris-EDTA (TE) buffer (10mMTris-HCl pH = 80 and 1mM EDTA pH = 80) Copre-cipitated RNA was removed by digestion with RNAse Afterone hour incubation at 37∘C the concentration and purity
BioMed Research International 3
Table1Im
portantd
iscrim
inatingqu
alitativ
efeatureso
fstudied
Calism
atifolia
varie
tiesa
ndhybrid
varie
tiesfor
M1V
1generatio
n
Varie
tyFloralcharacters
Leafcharacters
Planttype
Spike
position
Color
ofcalyx
Color
ofcorolla
Inflo
rescence
lower
bractcolor
Inflo
rescence
comab
ractcolor
Leafhabit
Color
ofleaf
sheath
Leafmargin
Leafcolor
Leafmidrib
color
Leafshape
Chiang
MaiRe
dErect
Term
inal
White
Light
Purple-w
hite
Green
PurpleN78Alowast
Erect
Purple-green
Smoo
thDark
green
Purple
Long
narrow
and
stiff
DoiTu
ng554
Erect
Term
inal
White
Dark
Purple-w
hite
2014
Redpu
rple
N78Clowast
Erect
DarkPu
rple
Smoo
thDark
green
Purple
Long
narrow
and
stiff
SweetP
ink
Erect
Term
inal
White
Dark
Purple-w
hite
Green
Purple-violet
N80Dlowast
Erect
Purple-green
Lowwavy
Light
green
Purple
Widea
ndstiff
Kimon
oPink
Erect
Term
inal
White
Dark
Purple-w
hite
Green
Purple-violet
N80Clowast
Erect
Purple-green
Medium
wavy
Dark
green
Green
Long
and
narrow
lowastTh
eRoyalHortic
ulture
Society(RHS)
Lond
oncolorc
hart
4 BioMed Research International
Table 2 List of morphological traits and brief descriptions
Number Morphological traits
1Number of new shoots (number) total number ofproduced new shoots per rhizome
2Leaf length (cm) length of the fully opened firstleaf from the soil surface to leaf tip
3Leaf width (cm) breadth of the leaf at the widestpart of the leaf
4Number of leaves (number) number of fullyemerged leaves at the end of vegetative growthstage
5Plant height (cm) the height of the peduncle atthe top of the soil surface to the tip of theinflorescence
6
Number of days to visible bud appearance (days)number of days from the first day of planting toappearance of the first visible bud Buds appear atthe middle of two sheaths of leaves
4Inflorescence length (cm) the length between thelowest green bracts to tip of the upper pink bractsduring anthesis
8Number of days to anthesis (days) the number ofdays from planting to fully opened flower bud
9Number of days to senescence (days) the daysfrom first day of anthesis until end of the shelf lifeof the flower
10Number of true flowers (No) the number ofsmall axillary flower buds which develop insidebracts during anthesis
11
Number of pink bracts (No) inflorescence of Calismatifolia comprising several apical bractsMost basal bracts are green but the distal onesmore numerous than the green ones are purplishpink bracts which determine the attractiveness ofthe flowering stems The number of pink bractswas counted during anthesis
12Rhizome size (cm) the girth of the M0V0 andM1V1 rhizomes was measured with vernier caliperand mean was expressed in centimeter
13Number of new rhizomes (No) after harvestingthe total number of new rhizomes (M1V1) wasrecorded
14Number of storage roots (No) the total numberof storage roots of M0V0 and M1V1 rhizomes wasrecorded at harvesting time
of isolated DNA were determined using NanoDrop 2000(Thermo Fisher Scientific Inc) in the range of 250 to900 ng120583L which was adjusted to 70 ng120583L The quality wasverified by electrophoresis on 08 agarose gel
32 PCR Amplification and Product Electrophoresis Poly-merase chain reaction (PCR) was carried out for 17 SSRprimers whichwere developed forCurcuma longa in previousstudies [26] PCR was carried out in a 25 120583L reaction volumecontaining 70 ng120583L DNA and 2X DreamTaq Green PCRMasterMix (Fermentas International Inc USA)with 04120583Mprimer Amplification was performed in a thermal cycler(Bio-Rad Laboratories Inc USA) for a total of 40 cyclesAn initial denaturation of the template DNA at 94∘C for 3minutes was followed by 10 cycles of 94∘C for 40 secondsand a touch-down one-degree decrement for annealingtemperature starting with 7∘C above 119879
119898for each primer for
30 seconds and 72∘C for 1 minute This was then followed by30 cycles of 95∘C for 40 seconds a last annealing temperaturefor 30 seconds and 72∘C for 1 minute and a final extensionof 72∘C for 10 minutes The PCR products were separated on4 metaphor gel with 50 bp DNA ladder (N3231S BiolabsInc UK) The gel was stained with Midori green visualizedunder ultraviolet light and photographed by ChemilImagerGel Documentation imaging system (Alpha Innotech Corpo-ration CA USA)
4 Data Analysis
41 Morphological Data The recorded data (after normalityand homogeneity test) were subjected to analysis of variance(ANOVA) as per two-factor experiment with three irradia-tion treatments and four varieties arranged in a randomizedcomplete block design (RCBD) with four replications Theanalysis was carried out using the portable SAS 91 programand least significant differences (LSD) were used for compar-ison among treatment means at 119875 le 005 To evaluate therelationship among the different variables in the experimentcorrelation coefficients were used by SAS 9 1 3 portable Togroup the individuals based on morphological dissimilaritycluster analysis was conducted on the Euclidean distancematrix with the unweighted Pair-GroupMethod using Arith-metic average (UPGMA) using NTSYS software The sameprogram was used for principal components analysis (PCA)to define eigenvalues and eigenvectors and also for compar-ison of the mean of groups to define effective traits in sepa-ration of the groups Eigenvectors are the weights in a lineartransformation when computing principal component scoreswhile eigenvalues indicate the amount of variance explainedby each principal component The cophenetic correlationcoefficient (CCC) was used to measure the goodness of fit ofthe similarity matrices to their corresponding phenograms inmorphological data using PAST (PAleontological Statistics)software V 217 [27]
42 Molecular Data Allele size was measured with UVDoc9902 analysis software (UVI Tech Cambridge UK) by man-ual editing to increase accuracy This procedure was carriedout two times to exclude wrong scorings The PowerMarker325 software package [28]was used to produce a dendrogramusing UPGMAmethod Data were scored as ldquo1rdquo for presenceand ldquo0rdquo for absence The binary data matrix was entered intothe Numerical Taxonomy and Multivariate Analysis System
BioMed Research International 5
Table 3 The number of irradiated and mortal rhizomes of Curcuma alismatifolia varieties after acute irradiation with different doses ofgamma rays
Dose (Gray)Total number of
irradiated rhizomes ineach var
Number of mortal rhizomes after 40 days Mortality rate ()in each doseDoi Tung 554 Chiang Mai Red Sweet Pink Kimono Pink
(NTSYSpc 210e) [29] to generate Dicersquos similarity matrixThe software POPGENE32 Version 132 [29] was used tocalculate genetic variation parameters including observedheterozygosity (the proportion of heterozygous individualsin the population) (119867
119900) expected heterozygosity (119867
119890) [30]mdash
defined as the probability that two randomly chosen allelesfrom the population are different [31]mdashobserved numberof alleles (119899
119886) effective number of alleles (119899
119890) Neirsquos gene
diversity Shannonrsquos information index (119868) and percentageof polymorphic loci To compare the efficiency of primersand polymorphism information content (PIC) a measure ofallelic diversity at a locus was calculated using online PICcalculator software (httpwwwlivacuksimkempsjpichtml)using the following formula
PIC = 1 minus119899
sum
119894=1
1199011198942minus
119899minus1
sum
119894
119899
sum
119895=119894+1
211990111989421199011198952 (1)
where 119901119894is the frequency of the 119894th allele and 119899 is the
number of alleles Markers were classified as informativewhen PICwas ge05 Principal component analysis (PCA) wasalso generated for SSR data by NTSYS-pc 210e
5 Results and Discussions
51 Gamma Irradiation and Radiation Sensitivity Test Thesensitivity of C alismatifolia varieties to radiation was eval-uated by comparing the mortality rate () of irradiatedplants at 40 days after irradiation The plant mortality rateincreased with increasing irradiation dosage (Table 3) Thehybrid Doi Tung 554 was found to be least sensitive togamma irradiation than other varieties (51mortality) whileChiangMai Red variety showed the lowest survival rate (63mortality) Sweet Pink and Kimono Pink varieties showed58 and 55 mortality rate respectively At 50 survivalrate (LD
50) the gamma doses administered were 28 21 23
and 25Gy for Doi Tung 554 Chiang Mai Red Sweet Pink
and Kimono Pink respectively (Figure 1) Abdullah et al [32]had previously indicated that the LD
50for C alismatifolia
var Chiang Mai Pink was approximately at 25Gy The deathof plants is attributed to the interaction of radiation withother molecules in the cell particularly water to produce freeradicals (H OH) The free radicals could combine to formtoxic substances such as hydrogen peroxide (H
2O2) which
contribute to the destruction of cells This indirect effectis especially significant in vegetative cells the cytoplasmwhich contains about 80 water [33] However sensitivityof the plant material depends on the genetic constitutiondose-employed DNA amount moisture content and stageof development and genotype [34] The choice of the doseto be applied for the highest mutant rescue is often left tothe breederrsquos experience with the specific plant material itsgenetics and its physiology
52 Analysis of Variance (ANOVA) for Morphological Traitsof C alismatifolia in M1V1 Analysis of variance indicatedhighly significant differences among the varieties doses andtheir interaction for all traits in M
1V1generation (Table 4)
Some desired and undesired abnormalities such as dwarfismchlorophyll mutation (albinism) striata (yellow or whitelongitudinal bands altering with green colors) two-midribleaves split leaves double flower stalk in one plant doubleinflorescence marbled pink bracts two-tone pink-purplishbracts and two-flag petals were found in M
1V1plants
(Table 5)
53 Effect of Gamma Irradiation on Vegetative Traits in theM1V1 The growth of plants treated with 10 and 20Gy ofgamma rays was slower than that of the controls (Table 6)In irradiated plants the leaf length and leaf width decreasedsignificantly (119875 lt 005) as the radiation doses increasedThis trend is quite common in mutagenised populationsSuch effects are known to arise due to drastic chromoso-mal aberrations in addition to genetic mutations Similar
6 BioMed Research International
Table4Meansquareso
fanalysis
ofvaria
nce(ANOVA
)for
14morph
ologicaltraitsin
Calism
atifolia
(a)
Source
ofvaria
tion
Meansquares
dfGeneration
Num
bero
fsho
otLeafleng
thLeafwidth
Leafnu
mber
Daystovisib
lebu
dPlanth
eight
Block
4M
1V1
005
622
11014
9467
8358
Varie
ty3
M1V
1263lowast
2702lowast
3011lowast
500lowast
1455602lowast
253739lowast
Dose
2M
1V1
611lowast
373804lowast
4137lowast
1042lowast
1012001lowast
157076
6lowastVa
rlowastdo
se6
M1V
114
1lowast8555lowast
418lowast
167lowast
731271lowast
115934lowast
Error
44M
1V1
0094
2951
077
037
4903
Total
5984
93
CV(
)M
1V1
23183
176
206
91165
(b)
Source
ofvaria
tion
Meansquares
dfGeneration
Daysto
anthesis
Daysto
senescence
Noof
true
flow
Noof
pink
bract
Inflo
rescence
leng
thNoof
new
rhizom
esRh
izom
esiz
eNoof
storage
roots
Block
4M
1V1
12847
048
418
072
282
01
018
055
Varie
ty3
M1V
11608017
10091lowast
9166lowast
62543lowast
376lowast
163lowast
023
ns1726lowast
Dose
2M
1V1
1587261
128682lowast
58286lowast
35315lowast
56022lowast
1031lowast
531lowast
6481lowast
Varlowastdo
se6
M1V
1876957
3464lowast
2900lowast
977lowast
4004lowast
128lowast
025ns
332lowast
Error
44M
1V1
7752
322
392
226
445
011
011
121
Total
59CV
()
M1V
110
15229
169
165
2318
268
lowastSign
ificant
with
leastsqu
ared
ifference
test119875lt005
BioMed Research International 7
Dose (Gy)
0
10
10
20
20
30
30
40
40
50
50
60
60
70
70
80
80
90
90
100
100
Mor
talit
y (
)
Var Doi Tung554
LD50 = 28 Gy
(a)
0
10
10
20
20
30
30
40
40
50
50
60
60
70
70
80
80
90
90
100
100
Mor
talit
y (
)
Var Chiang Mai Red
Dose (Gy)
LD50 = 21Gy
(b)
Mor
talit
y (
)
Dose (Gy)
Var Sweet Pink
0
10
10
20
20
30
30
40
40
50
50
60
60
70
70
80
80
90
90
100
100
LD50 = 23Gy
(c)
0
10
10
20
20
30
30
40
40
50
50
60
60
70
70
80
80
90
90
100
100
Mor
talit
y (
)
Dose (Gy)
Var Kimono Pink
LD50 = 25Gy
(d)
Figure 1 PoloPlus plot of linear scale of dose versusmortality percent (a) Doi Tung 554 (b) ChiangMai Red (c) Sweet Pink and (d) KimonoPink
Table 5 Effect of acute gamma rays on vegetative and flowering traits of C alismatifolia in the M1V1 generation
Variety irradiated dose Observed variations Flower color variationChiang Mai Red
10Dwarfism no pink bracts inflorescence small inflorescencetwo-flag petal true flower double inflorescence undulate leafmargin and yellowwhite strip leaves
Light purple N78Clowast
20 Dwarfism narrow small leaves and yellowwhite strip leaves No flowerDoi Tung 554
20 Dwarfism fewer pink bracts and yellowwhite strip leavesSmall and narrow leave Light purple N80Dlowast
lowastRoyal British Society color chart (RHS)
8 BioMed Research International
Table 6 Effect of acute gamma rays on vegetative traits of C alismatifolia in M1V1 generation
Dose (Gray) Shoot number Leaf number Leaf length (cm) Leaf width (cm) Plant height (cm)CMR
0 20 plusmn 00a
30 plusmn 04a
556 plusmn 08a
71 plusmn 05a
1112 plusmn 25a
10 10 plusmn 00b
36 plusmn 05a
272 plusmn 25b
45 plusmn 05b
714 plusmn 96b
20 10 plusmn 00b
14 plusmn 05b
124 plusmn 120c
25 plusmn 24c
170 plusmn 80c
DT0 30 plusmn 00
a30 plusmn 00
a622 plusmn 08
a65 plusmn 05
a912 plusmn 19
a
10 16 plusmn 05b
28 plusmn 04a
280 plusmn 44b
46 plusmn 06b
542 plusmn 76b
20 12 plusmn 04b
24 plusmn 05a
242 plusmn 38b
38 plusmn 05c
512 plusmn 138b
SP0 16 plusmn 05
a32 plusmn 04
a461 plusmn 27
a101 plusmn 04
a746 plusmn 38
a
10 10 plusmn 00b
36 plusmn 05a
275 plusmn 34b
57 plusmn 02b
377 plusmn 137b
20 10 plusmn 00b
20 plusmn 10b
196 plusmn 74c
57 plusmn 04b
195 plusmn 73c
KP0 14 plusmn 00
a46 plusmn 05
a345 plusmn 29
a47 plusmn 04
a591 plusmn 28
a
10 10 + 05a
40 plusmn 05b
220 plusmn 24b
31 plusmn 04b
420 plusmn 54b
20 10 plusmn 00a
32 plusmn 04b
156 plusmn 43c
31 plusmn 11b
283 plusmn 174c
CV () 23 18 26 16 165Means with the same or common letter are not significantly different least square difference test 119875 lt 005
decreases in leaf size were reported by Pongchawee et al [35]and Tangpong et al [36] These results were in agreementwith an earlier study [4] which reported that the growthof chrysanthemum exposed to acute gamma rays was lessthan the control in the M
1V1generation All varieties doses
and interaction effects resulted in significant differences fornumber of leaves Among untreated plants Kimono Pinkvariety had higher number of leaves (46) than the otherthree (3 3 and 32) varieties In Chiang Mai Red and SweetPink varieties plants exposed to 10Gy showedhigher numberof leaves than untreated plants However at 20Gy therewas significant reduction in number of leaves for all studiedvarieties in comparison to control Similar stimulatory effectswere obtained at lower doses in ginger by Hegde [37] andGiridharan and Balakrishnan [38]
Progressive reduction in growth parameters can be inter-preted on interference in normal mitosis and frequent occur-rence of mitotic aberrations inhibition of rate of assimilationand consequent change in the nutrient level in the plantand inactivation of vital enzymes especially those associatedwith respiration [39] Dose-dependent negative effect wasalso detected for plant heightThe tallest plants were recordedfrom the untreated rhizomes (0Gy) with heights of 1112912 746 and 591 cm followed by the 10Gy irradiated plantswith heights of 714 542 377 and 420 cm and the 20Gyirradiated plants with corresponding heights of 170 512195 and 283 cm for Chiang Mai Red Doi Tung 554 SweetPink and Kimono Pink varieties respectively These resultsare in agreement with the findings of Abdullah et al [32]Reduction in growth parameters and dwarfism can be causedby interference of normal mitosis and frequent occurrenceof mitotic aberrations inhibition of assimilation rates andconsequent changes in nutrient levels in plants Additionallymutagenic effects such as auxin destruction inhibition of
auxin synthesis failure of assimilatory mechanism andchanges in the specific activity of enzymes can cause growthreductions [37] High doses of ionizing radiation have beenshown to damage macromolecular cellular components suchas cell walls membranes and DNA [40] The number ofshoots also decreased significantly as the radiation dosesincreased Radiation also affects organic molecules that areessential to the cell division process and thus causing celldivision to stop [36]
54 Effect of Gamma Irradiation on Flowering DevelopmentTraits in the M1V1 All control and 10Gy irradiated plantsproduced flowers while the Chiang Mai Red and SweetPink varieties which were exposed to 20Gy did not gointo the flowering stage Lamseejan et al [4] also showedthat flowering percentage decreases as gamma ray dosesare increased In the present study gamma rays caused lateflowering in all four varieties Days to appearance of firstvisible buds were also significantly different among the fourvarieties (Table 7) Gamma rays caused a noticeable delay inflowering of irradiated plants in comparison to the untreatedones First visible buds were observed at 652 and 874 days inthe control and 10Gy treatments respectively for the ChiangMai Red variety In Doi Tung 554 the first visible buds wereappeared at 474 656 and 840 days after planting at 0 10and 20Gy doses respectively In the Sweet Pink variety thenumber of days to visible bud appearance increased signif-icantly from 678 days in controls to 978 days in the 10Gyirradiated plants In comparison to other three varieties theKimono Pink variety needed the longest time to visible budappearance and same as other varieties there was a positivecorrelation between the number of days to first visible budand the gamma irradiation dose In the control and 10Gyand 20Gy irradiated individual plants flower buds were
BioMed Research International 9
Table 7 Effect of acute gamma rays on flower development characteristics of C alismatifolia in M1V1 generation
Dose (Gy) Days tovisible bud
Inflorescencelength (cm)
Days toanthesis
Number oftrue flowers
Number ofPink bracts
Days tosenescence
CMR0 652 plusmn 47
b162 plusmn 057
a742 plusmn 54
b132 plusmn 19
a104 plusmn 15
a21 plusmn 10
a
10 874 plusmn 95a
82 plusmn 397b
1020 plusmn 71a
56 plusmn 19b
50 plusmn 07b
10 plusmn 14b
20 00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
DT0 474 plusmn 43
c134 plusmn 054
a546 plusmn 54
c162 plusmn 13
a232 plusmn 04
a23 plusmn 00
a
10 656 plusmn 37b
94 plusmn 19b
780 plusmn 37b
112 plusmn 21b
166 plusmn 08b
122 plusmn 130b
20 840 plusmn 42a
76 plusmn 082c
994 plusmn 49a
88 plusmn 13b
156 plusmn 11b
106 plusmn 18b
SP0 678 plusmn 21
b146 plusmn 089
a756 plusmn 26
b162 plusmn 13
a100 plusmn 24
a188 plusmn 083
a
10 978 plusmn 181a
79 plusmn 20b
1098 plusmn 182a
68 plusmn 25b
52 plusmn 08b
92 plusmn 24b
20 00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
KP0 852 plusmn 23
c137 plusmn 044
a942 plusmn 19
c112 plusmn 12
a96 plusmn 21
a18 plusmn 18
a
10 1044 plusmn 65b
108 plusmn 108ab
1224 plusmn 100b
76 plusmn 27b
64 plusmn 16ab
102 plusmn 17b
20 1272 plusmn 65a
790 plusmn 49b
1426 plusmn 58a
60 plusmn 38b
42 plusmn 24b
66 plusmn 37b
CV () 10 23 9 22 169 15Means with the same or common letter are not significantly different least square difference test 119875 lt 005
visible at 852 1328 and 1374 days after planting respectivelyPrevious studies also showed that onset of flowering andformation of floral parts in mutants of Arabidopsis thalianamaize barely pea and tobacco involved growth regulators(or phytohormones) such as auxins cytokinins gibberellinsabscisic acid ethylene and brassinosteroids [41] There weresignificant differences among treatments for the length ofthe inflorescence In all varieties the longest inflorescencelength was observed in the untreated plants with 162134 146 and 137 cm lengths for Chiang Mai Red DoiTung 554 Sweet Pink and Kimono Pink respectively Thecorresponding inflorescence lengths were 82 94 79 and109 cm for the 10Gy irradiated plants The days to anthesisfor C alismatifolia varieties were significantly affected byvariety gamma irradiation doses and their interaction Thenumber of days to full bloom was noticeable earliest foruntreated plants at 742 546 756 and 942 days for ChiangMai Red Doi Tung 554 Sweet Pink and Kimono Pinkvarieties respectively This was then followed by plants at10Gy at 102 78 1098 and 1224 days The number of trueflowers or the secondary inflorescence developed in the axilof the primary bracts decreased as radiation dosage increasedIn the present study the gamma rays also decreased thedays to inflorescence senescence In this study there wasa strongly significantly and positively correlation (0919lowastlowast)(data not shown) between the number of true flowers andthe number of days to senescenceThe number of pink bractsalso decreasedwith increasing radiation dosageMost gammaray effects on senescence are considered as resulting from theaction of free radicals generated from water and oxygen bythe ionizing energy on the cellular components Membrane
deterioration is a general feature of natural senescence andstress-induced aging [42]
Irradiation induced some mutation spectrum of flowercolor variation that included colors such as purple palepurple rather pale purple white purple white (marbledpattern) and two-tone purple color
Mutation spectrum of flower shape variation includeddouble inflorescence within one stalk double stalk per plantinflorescence without bracts two-flag petal true flowersand chlorophyll mutation in the leaves which are generallycaused by induced gamma rays (Figure 2) Ionizing radiationincluding gamma rays induces fragment deletions or inser-tions that eventually lead to changes in amino acids and amodification of leaf and stem pigmentation [43] A mutationin the biosynthetic pathway of structural or regulatory genesmay cause a change in flower color [44] When the blockageoccurs at the early stages of anthocyanin synthesis whiteflowers will result while a blockage at later stages leads todifferent flower colors due to the accumulation of particularanthocyanins [45] Chloroplasts were extremely sensitive togamma radiation compared to other cell organelles [46]
55 Effect of Gamma Irradiation on Rhizome Characteristicsin Selected Doses in M1V1 The number of new rhizomes andthe number of storage roots per rhizome were significantlyaffected by varieties doses and their interaction (Table 8)The number of new rhizomes only in Kimono Pink varietydid not show any differences between untreated and treatedplants The rhizome size was influenced only by doses Asdose level increased the rhizome size decreased Amonguntreated plants the Sweet Pink rhizomes had the most
10 BioMed Research International
(a) (b) (c) (d)
(e)
(f)
Figure 2 Effect of acute gamma rays on flower traits in C alismatifolia (a) Untreated inflorescence bracts color Doi Tung 554 (b) Two tonebract color Doi Tung 554 (20Gy) (c) Marbled pattern of bract color Doi Tung 554 (20Gy) (d) Double inflorescence within one stalk varChiang Mai Red (10Gy) (e) True flower in nontreated plants (f) Two-flag petals true flowers (10Gy)
Figure 3 Dendrogram representing themorphological variation among 44 irradiated and nonirradiated individuals ofC alismatifolia across14 variables CMR = Chiang Mai Red DT = Doi Tung 554 SP = Sweet Pink Kp = Kimono Pink
number of storage roots (78) and the Kimono Pink rhizomeshad the least number of storage roots (40)This was followedby 10Gy irradiated rhizomes with 4 46 48 and 28 storageroot numbers for Chiang Mai Red Doi Tung 554 SweetPink and Kimono Pink respectively Irradiation with 20Gydecreased significantly the number of storage roots in allstudied varietiesTheplantC alismatifolia is propagated froma propagule (one rhizome + 5-6 t-roots) The rhizome sizedose matters in the growth of the plant Smaller rhizome sizeusually resulted in smaller plant size with narrow grass-likeleaves The storage roots play a very important role in thegrowth and flowering ofCurcumaThe t-rootsmake up about85 of the total fresh weight and 70 of the total dry weightof a typical propaguleThe storage organs act for plant growth
during dormancy and emergence [47] More storage root perpropagule resulted in faster flowering and higher yield plant[48] In this study also there was significant and negativecorrelation between number of storage roots and number ofdays to visible bud appearance (minus0525lowastlowast)
56 Cluster Analysis of C alismatifolia for MorphologicalTraits The morphological data were used to calculate thesimilarity between the treated and non-treated C alismat-ifolia samples and UPGMA dendrogram was constructed(Figure 3) The cophenetic correlation coefficient (CCC)value between the genetic dissimilaritymatrix estimated fromthe morphological characters and the UPGMA clusteringmethod was 119903 = 093 showing a proof fit This value was
BioMed Research International 11
Table 8 Effect of acute gamma rays on rhizome characteristics ofC alismatifolia in M1V1 generation
Dose (Gray) Rhizomesize
No of newrhizomes
No of storageroot
CMR0 23 plusmn 02
a26 plusmn 055
a46 plusmn 054
a
10 18 plusmn 005a12 plusmn 044
b40 plusmn 07
a
20 08 plusmn 07b
06 plusmn 054b
14 plusmn 13b
DT0 22 plusmn 01
a30 plusmn 00
a72 plusmn 109
a
10 18 plusmn 007b14 plusmn 054
b46 plusmn 13
b
20 16 plusmn 005b
10 plusmn 00b
28 plusmn 13c
SP0 22 plusmn 02
a24 plusmn 054
a78 plusmn 21
a
10 17 plusmn 007b
10 plusmn 00b
48 plusmn 044b
20 12 plusmn 02c
10 plusmn 00b
28 plusmn 083b
KP0 21 plusmn 005
a10 plusmn 00
a40 plusmn 00
a
10 16 plusmn 01ab
10 plusmn 00a
28 plusmn 044b
20 11 plusmn 06b
10 plusmn 00a
22 plusmn 083b
CV () 18 23 26Means with the same or common letter are not significantly different leastsquare difference test 119875 lt 005
higher than of studies on olive for morphological traits (119903 =069) [49] In this dendrogram the 44C alismatifolia samplesappeared to form three main clusters and five minor clustersat coefficient level 56 cluster I included three non-irradiatedmembers and cluster II included 10Gy irradiated individualplants of all four varieties and 20Gy irradiated samples ofDoi Tung 554 and Kimono Pink varieties except KP20-4 (noflowering) Main cluster III included the 20Gy irradiatedindividuals Chiang Mai Red and Sweet Pink varieties (asmentioned in morphological part these individuals didnot go to flowering stage) and KP20-4 Cluster II can bedivided to five subclusters The first subcluster included 10members (CMR10-1 CMR10-5 SP10-1 SP10-2 SP10-3 SP10-5 CMR10-2 CMR10-4 SP10-4 and CMR10-3) the secondone included nine members (KP10-1 KP10-2 KP10-3 KP10-4 KP10-5KP20-1 KP20-2 KP20-5 and KP20-3) KP0 wasthe lone member of the third sub-cluster The fourth sub-cluster included two members only (DT10-2 and DT10-3) The last sub-cluster main cluster II had eight membersincluded (DT10-1 DT10-4 DT10-5 DT20-1 DT20-2 DT20-3 DT20-4 and DT20-5) Mean value of groups for each traitis presented in Table 9 This table clearly shows the differentmean values of the three main clusters The minimum meanvalue referred to main cluster III while the maximum meanvalue for number of shoots leaf length leaf width plantheight number of true flowers inflorescence length rhizomesize number of new rhizomes and number of storage rootsbelonged to cluster I which was included non-treated plantsThese results showed that the gamma irradiation has inducedmorphological changes in C alismatifolia individuals of
four studied varieties so that they showed phenotypicallydifferences from their controls
57 Principal Component Analysis of C alismatifolia forMorphological Traits To assess the patterns of variation PCAwas done by considering all of the 14 variables The first threecomponents of PCA explained 828 of the total variation(Table 10) Only the first component which accounted for562 of the total variation was attributed to inflorescencelength plant height days to senescence number of trueflowers and rhizome size In PCA three-dimensional graphthe grouping of individuals confirmed the clustering results(Figure 4) The PCA graph proved that all irradiated indi-vidual plants of C alismatifolia varieties are phenotypicallydifferent from their non-irradiated individuals
6 Molecular Characterization
61 SSR Polymorphism Amplifications were successful forall the 17 SSR markers assayed This reflects a high levelof homology between SSR flanking regions in C longa andC alismatifolia Out of 17 primer pairs eight primer pairsresulted in polymorphic PCR products Table 11 summarizesthe results obtained based on the analysis of individuals of thefour studied varieties using the polymorphic SSR loci for 010 and 20Gy irradiated plants The number of alleles variedwidely among these loci A total of 25 36 and 41 alleles wereobserved among 0 10 and 20Gy irradiated individual plantsrespectively In the 10Gy treatment the number of allelesranged from three (clon09 and clon14) to seven (clon08) withan average value of 45 per locus In the 20Gy treatmentthe number of alleles varied from three (clon09 and clon14)to seven (clon08 and clon12) with an average value of 51per locus In the untreated individuals the number of allelesranged from two (clon04 clon09 clon11 and clon14) to five(clon12) with an average value of 31 The difference betweenthe average number of observed alleles and effective numberof alleles was due to the uneven frequency of each allele[50] For each of the eight SSR primers PIC values (whichmeasures allele diversity and frequency among varieties)varied from 019 (clon04) to 071 (clon01) in untreated plantsand from 025 (clon04) to 075 (clon08) in 10Gy acutelyirradiated plants In 20Gy irradiated individual plants thePIC value ranged from 042 (clon14) to 075 (clon08 andclon12) The mean PIC for all loci was 047 054 and 061for 0 10 and 20Gy irradiated plants respectively
The PIC value provides an estimate of the discriminatorypower of amarker by taking into account not only the numberof alleles at a locus but also the relative frequencies of thesealleles [51] Thus except for clon09 and clon14 which weremoderately polymorphic (025 lt PIC lt 05) all other lociwere highly polymorphic (05 lt PIC) while none of theloci showed low polymorphism The average discriminatingpower ofmicrosatellitemarkers (061) observed in the presentstudy ensures the future utility of microsatellite markersfor genetic variation studies in C alismatifolia varietiesOur results reflect similar findings as reported earlier inthe study of genetic variation in other species of Curcuma
12 BioMed Research International
Table9Clusterm
eans
for14charactersestim
ated
in44
individu
also
fCalismatifolia
Clusters
Shoo
tnu
mber
Leaf
leng
thLeaf
width
Leaf
number
Visib
lebu
d(days)
Inflo
rescence
leng
thPlant
height
Anthesis
(days)
Senescence
(days)
True
flow
number
Pink
bract
number
Rhizom
esiz
eNew
rhizom
enu
mber
Storage
root
number
I220
5464
736
300
6013
1473
9233
6926
2093
152
1453
229
266
653
II1
100
2735
512
360
9260
809
5457
1059
960
620
510
177
110
440
II2
122
1922
298
344
11422
1043
3910
1312
293
3755
588
156
100
266
II3
100
3452
416
460
8520
1370
5910
9420
180
120
960
206
100
400
II4
150
2500
45
250
6500
925
4950
7700
1150
950
1700
185
200
600
II5
137
2637
417
262
7725
840
5356
9162
1137
1012
1587
170
100
312
III
054
1500
465
181
000
000
000
000
000
000
000
090
081
200
BioMed Research International 13
Table 10 Eigenvectors eigenvalues and proportions of variabilityfor three principle components among 14 characters for 44 Calismatifolia
Variable PC1 PC2 PC3Number of new shoot 0270 minus0076 minus0173Leaf length 0269 minus0305 0033Leaf width 0096 minus0455 0430Leaf number 0210 0285 0490Days to visible bud 0199 0462 0181Plant height 0321 0027 minus0071Inflorescence length 0318 0166 minus0040Days to anthesis 0205 0460 0148Days to senescence 0342 0017 minus0131Number of true flower 0323 0029 minus0238Number of pink bracts 0266 minus0028 minus0546Rhizome size 0302 minus0101 0147Number of new rhizome 0250 minus0293 0028Number of storage roots 0264 minus0247 0298Eigenvalue 7877 2694 1032Proportion 56264 19246 7371Cumulative 56264 75511 82882
using SSR markers [13] The highest Neirsquos gene diversity(ℎ) was obtained with the 20Gy treatment (061) followedby the 10Gy treatment (06) and the untreated individuals(05) The mean Shannonrsquos information index (119868) was 092114 and 130 in the 0 10 and 20Gy irradiated plantsrespectively The high value of Shannonrsquos information indexrepresents the effectiveness of microsatellite loci to revealthe variation among these varieties at the different dosesused Overall genetic variability for the varieties studiedrepresented by the Shannon-Weiner index was relatively highin comparison to other studies involving C longa accessions[10 52] Additionally the 20Gy acutely irradiated individualsshowed a higher mean percentage of polymorphic loci (63)than the 10Gy (58) and untreated (022) ones This impliesthat irradiation with a dose of 20Gy induced more geneticvariation in the M
1V1generation
62 Cluster Analysis Thedendrogramwas constructed usingPowerMarker 323The 44 studied individuals were clusteredinto seven groups (G1ndashG7) Colors were applied accordingto our model-based cluster analysis results Group I iscomprised of nine individual plants of Chiang Mai Redvariety receiving 0 10 and 20Gy treatments (blue colorin Figure 5) One individual plant DT10-1 was assignedto group II (green color in Figure 5) All irradiated andnon-irradiated individuals of Doi Tung 554 variety wereassigned to group III (yellow color in Figure 5) DT20-1 andSP10-5 were the sole members of groups IV (red color inFigure 5) and V (gray color in Figure 5) respectively GroupVI included treated and untreated individuals of Sweet Pinkvariety (pink color in Figure 5) Lastly all Kimono Pinkindividuals were assigned to cluster VII (orange color in
Figure 5) The genetic similarity coefficient (data not shown)among the 44 individuals amplified using eight SSR primersvaried from 00 to 10 The highest value (10) correspondedto [(CMR10-3 CMR10-5 CMR and CMR20-2) (DT10-2and DT10-3) (DT and DT20-2) (KP20-3 KP-20-4 KP10-3andKP10-4)] individuals that generated identical fingerprintsacross the markers studied Among the studied varieties theSweet Pink individual plants showed the lowest similarity(00) withKimono Pink individuals which indicated that theywere relatively remote in relationship (Figure 5) An overviewof the clustering pattern indicates that the grouping of thestudied individual plants was based on different varieties andlargely independent of the doses of gamma irradiation Awide range in similarity values had also been observed indifferent species of Curcuma [12 13 52 53]
63 Principle Component Analysis (PCA) Thedata generatedfrom 44 C alismatifolia individual plants were subjected toprincipal component analysis (PCA) to visualize individualsin a multivariate space In the three-dimensional graphderived from the SSR analysis all studied individuals weregrouped into seven clusters (Figure 6) In PCA the firstthree principal components (PC) extracted a cumulative of6826 of the total variation among the 44 individuals of CalismatifoliaThe first three coordinates PC1 PC2 and PC3accounted for 4136 1578 1112 of the total variationrespectively The distribution of the individual plants in athree-dimensional plot using the first three principal com-ponents showed the genetic relationship between individualplants It was evident that both methods dendrogram andthree-dimensional plots of PCA were effective in illustratinggenetic relationships and the groups found were comparable[11 13] The PCA results were similar to those obtained fromcluster analysis where all individuals from different varietieswere assigned to different groups These graphical illus-trations enable the assessment of the relationshipdistancesamong all of the individuals in the study [50]
7 Conclusion
This is the first attempt to evaluate the effect of acutegamma irradiation on C alismatifolia varieties using bothmorphological characteristics and molecular markers Inplant breeding programs mutagenic treatments with lownegative physiological effects and strong genetic effects aredesirable Hence we used more effective doses of gammairradiation (10 and 20Gy) of which particularly 20Gy dosewas effective to influence morphological and molecularcharacteristics of studiedCurcuma alismatifolia varietiesThelower dose (10Gy) of radiation probably caused little damageto the plants genetic material so that the cells could repairthemselves in next generation As a result in this study 20Gyacute gamma irradiation resulted in a higher percentage ofmutation and getting desired mutants was more possibleBased on the LD
50values determined in this study it was
apparent that the varieties Chiang Mai Red and Sweet Pinkwere more sensitive to gamma rays compared to Doi Tung554 and Kimono Pink Our results show that the variety Doi
Figure 4 Two-dimensional graph of principal component analysis (PCA) for 14 morphological variables indicating relationships amongirradiated and nonirradiated four varieties of C alismatifolia
CMR10-1CMR10-5
SP10-1SP10-2SP10-3
SP10-5
CMR10-4
SP10-4
KP10-1KP10-3
KP20-1
KP20-2
KP10-5KP20-5
KP20-3
KP10-2
DT10-2
DT10-1
DT10-5
DT20-1
DT10-4DT20-4DT20-2
DT20-5DT20-3
CMR20-1
SP20-3SP20-4
SP20-5SP20-1
SP20-2
KP
SP
CMR 10-2
SP10-5
Figure 5 Unrooted neighbor joining tree showing genetic relationship among 44 irradiated and nonirradiated C alismatifolia using SSRmarkers The colors of the branches correspond to those of the same cluster
Figure 6Three-dimensional graph of principal component analysis (PCA) indicating relationships among irradiated and nonirradiated fourvarieties of C alismatifolia
Tung 554 (Curcuma hybrid) had the most morphologicalresponses to gamma rays The use of microsatellite markersas a codominant marker will facilitate the exploration ofgenetic variability among treated and non-treated plants andwill help to distinguish the plants showing differences inmorphological characters The overall effects on the M
1V1
generation revealed that acute gamma irradiation at optimumdoses has the potential for developing new varieties of Calismatifolia with improved commercial properties suitablefor the Malaysian flower industry
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work was funded by the Fundamental Research GrantScheme (FRGS) under the Ministry of Higher Education inMalaysia
References
[1] P Apavatjrut S Anuntalabhochai P Sirirugsa and C AlisildquoMolecularmarkers in the identification of some early floweringCurcuma L (Zingiberaceae) speciesrdquo Annals of Botany vol 84no 4 pp 529ndash534 1999
[2] K Bunya-Atichart S Ketsa andW G VanDoorn ldquoPostharvestphysiology of Curcuma alismatifolia flowersrdquo Postharvest Biol-ogy and Technology vol 34 no 2 pp 219ndash226 2004
[3] M Nakano J Amano Y Watanabe et al ldquoMorphologicalvariation in Tricyrtis hirta plants regenerated from heavy ionbeam-irradiated embryogenic callusesrdquo Plant Biotechnologyvol 27 no 2 pp 155ndash160 2010
[4] S Lamseejan P Jompuk A Wongpiyasatid A Deeseepan andP Kwanthammachart ldquoGamma-rays induced morphological
[5] G-J Lee S J Chung I S Park et al ldquoVariation in thephenotypic features and transcripts of colormutants of chrysan-themum (Dendranthema grandiflorum) derived from gammaraymutagenesisrdquo Journal of Plant Biology vol 51 no 6 pp 418ndash423 2008
[6] O K Kikuchi ldquoOrchid flowers tolerance to gamma-radiationrdquoRadiation Physics and Chemistry vol 57 no 3ndash6 pp 555ndash5572000
[7] N P Arnold N N Barthakur and M Tanguay ldquoMutageniceffects of acute gamma irradiation on miniature roses targettheory approachrdquo HortScience vol 33 no 1 pp 127ndash129 1998
[8] A A Youssef M S Aly and M S Hussein ldquoResponse ofgeranium (Pelargonium graveolenus L) to gamma irradiationand foliar application of Speed Growrdquo Egyptian Journal ofHorticulture vol 27 pp 41ndash53 2000
[9] N Chuantang and L Yazhi ldquoThe radiation inducedmutation ofcanna (Canna L)rdquo Acta Agricultute Nucleatae Sinica vol 2 pp33ndash39 1998
[10] M S Sigrist J B Pinheiro J A A Filho and M I ZucchildquoGenetic diversity of turmeric germplasm (Curcuma longaZingiberaceae) identified by microsatellite markersrdquo Geneticsand Molecular Research vol 10 no 1 pp 419ndash428 2011
[11] S Taheri T L Abdullah N A P Abdullah and Z AhmadldquoGenetic relationships among five varieties of Curcuma alis-matifolia (Zingiberaceae) based on ISSRmarkersrdquoGenetics andMolecular Research vol 11 pp 3069ndash3076 2012
[12] A Das V Kesari V M Satyanarayana A Parida and L Ran-gan ldquoGenetic relationship of Curcuma species from NortheastIndia using PCR-based markersrdquo Molecular Biotechnology vol49 no 1 pp 65ndash76 2011
[13] S Siju K Dhanya S Syamkumar et al ldquoDevelopment char-acterization and utilization of genomic microsatellite markersin turmeric (Curcuma longa L)rdquo Biochemical Systematics andEcology vol 38 no 4 pp 641ndash646 2010
[14] M K Panda S Mohanty E Subudhi L Acharya and SNayak ldquoAssessment of genetic stability of micropropagated
BioMed Research International 17
plants of Curcuma L by cytophotometery and RAPD analysisrdquoInternational Journal of Integrative Biology (IJIB) vol 1 pp 189ndash195 2007
[15] S Syamkumar and B Sasikumar ldquoMolecular marker basedgenetic diversity analysis of Curcuma species from IndiardquoScientia Horticulturae vol 112 no 2 pp 235ndash241 2007
[16] D Tautz and M Renz ldquoSimple sequences are ubiquitousrepetitive components of eukaryotic genomesrdquo Nucleic AcidsResearch vol 12 no 10 pp 4127ndash4138 1984
[17] G Toth Z Gaspari and J Jurka ldquoMicrosatellites in differenteukaryotic genomes surveys and analysisrdquo Genome Researchvol 10 no 7 pp 967ndash981 2000
[18] W Powell G C Machray and J Proven ldquoPolymorphismrevealed by simple sequence repeatsrdquo Trends in Plant Sciencevol 1 no 7 pp 215ndash222 1996
[19] S-Y Lee W K Fai M Zakaria et al ldquoCharacterizationof polymorphic microsatellite markers isolated from ginger(Zingiber officinale Rosc)rdquo Molecular Ecology Notes vol 7 no6 pp 1009ndash1011 2007
[20] S Bory D Da Silva A-M Risterucci M Grisoni P Besseand M-F Duval ldquoDevelopment of microsatellite markers incultivated vanilla polymorphism and transferability to othervanilla speciesrdquo Scientia Horticulturae vol 115 no 4 pp 420ndash425 2008
[21] I C Menezes F W Cidade A P Souza and I C SampaioldquoIsolation and characterization ofmicrosatellite loci in the blackpepper Piper Nigrum L (Piperaceae)rdquo Conservation GeneticsResources vol 1 no 1 pp 209ndash212 2009
[22] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[23] V Kumar Morphological and molecular characterization ofinduced mutants in Groundnut [PhD thesis] University ofAgricultural Sciences Dharwad India 2008
[24] J R Sharma Statistical and Biometrical Techniques in PlantBreeding New Age International New Delhi India 1998
[25] J J Doyle and J L Doyle ldquoA rapid DNA isolation procedure forsmall quantities of fresh leaf tissuerdquo Phytochemical Bulletin vol19 pp 11ndash15 1987
[26] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[27] Oslash Hammer D A T Harper and P D Ryan ldquoPast paleontolog-ical statistics software package for education and data analysisrdquoPalaeontologia Electronica vol 4 no 1 pp 4ndash9 2001
[28] K Liu and S V Muse ldquoPowerMaker an integrated analysisenvironment for genetic maker analysisrdquo Bioinformatics vol 21no 9 pp 2128ndash2129 2005
[29] F J RohlfNTSYS-Pc Numerical Taxonomy System Version 2 1Exeter Publishing Setauket New York NY USA 2002
[30] M Nei ldquoAnalysis of gene diversity in subdivided populationsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 70 no 12 1973
[31] S Kadkhodaei M Shahnazari M K Nekouei et al ldquoA compar-ative study of morphological and molecular diversity analysisamong cultivated almonds (Prunus dulcis)rdquo Australian Journalof Crop Science vol 5 no 1 pp 82ndash91 2011
[32] T L Abdullah J Endan and B M Nazir ldquoChanges in flowerdevelopment chlorophyll mutation and alteration in plant
morphology of Curcuma alismatifolia by gamma irradiationrdquoAmerican Journal of Applied Sciences vol 6 no 7 pp 1436ndash14392009
[33] E Kovacs and A Keresztes ldquoEffect of gamma and UV-BCradiation on plant cellsrdquoMicron vol 33 no 2 pp 199ndash210 2002
[34] H L Ramesh Y Murthy and V N Munirajappa ldquoEffect ofgamma radiation on morphological and growth parametersof Mulberry variety M5rdquo International Journal of Science andNature vol 3 pp 447ndash452 2010
[35] K Pongchawee R Pradissan andW Pipatcharoenchai ldquoInducemutation in Anubias spp through in vitro Irradiationrdquo ThaiFisheries Gazette vol 60 pp 493ndash497 2007
[36] P Tangpong T Taychasinpitak C Jompuk and P JompukldquoEffects of acute and chronic gamma irradiations on in vitroculture ofAnubias congensisNE BrownrdquoKasetsart Journal vol43 no 3 pp 449ndash457 2009
[37] R K Hegde Studies on induced mutagenesis and in vitroregeneration in turmeric (Curcuma longa L) [PhD thesis]University of Agricultural Sciences Dharwad India 2006
[38] M P Giridharan and S Balakrishnan ldquoGamma ray inducedvariability in vegetative and floral characters of ginger IndianCocoardquo Arecanut and Spices Journal vol 15 pp 68ndash672 1992
[39] A P Cesarett ldquoEffect of radiation on higher plants and plantcommunitiesrdquo inRadiation Biology United States Atomic EnergyCommission pp 284ndash309 Washington DC USA 1968
[40] G W Seung Y C Byung J-H Kim et al ldquoUltrastructuralchanges of cell organelles in Arabidopsis stems after gammairradationrdquo Journal of Plant Biology vol 48 no 2 pp 195ndash2002005
[41] B S Ahloowalia and M Maluszynski ldquoInduced mutationsmdashanew paradigm in plant breedingrdquo Euphytica vol 118 no 2 pp167ndash173 2001
[42] R Voisine L-P Vezina and C Willemot ldquoInduction ofsenescence-like deterioration of microsomal membranes fromcauliflower by free radicals generated during gamma irradia-tionrdquo Plant Physiology vol 97 no 2 pp 545ndash550 1991
[43] N Shikazono Y Yokota S Kitamura et al ldquoMutation rate andnovel tt mutants of Arabidopsis thaliana induced by carbonionsrdquo Genetics vol 163 no 4 pp 1449ndash1455 2003
[44] T Nakatsuka M Nishihara K Mishiba and S YamamuraldquoTwo different mutations are involved in the formation ofwhite-flowered gentian plantsrdquo Plant Science vol 169 no 5 pp949ndash958 2005
[45] M Mato T Onozaki Y Ozeki et al ldquoFlavonoid biosynthesisin white-flowered Sim carnations (Dianthus caryophyllus)rdquoScientia Horticulturae vol 84 no 3-4 pp 333ndash347 2000
[46] S G Wi B Y Chung J-S Kim et al ldquoEffects of gammairradiation on morphological changes and biological responsesin plantsrdquoMicron vol 38 no 6 pp 553ndash564 2007
[47] F Lee Chin Effects of light intensity and daylength on growth andflowering of siam tulip (Curcuma alismatifolia var Chiang MaiPink) [PhD thesis] University Putra Malaysia 2007
[48] A Hagiladi N Umiel and X H Yang ldquoCurcuma alismatifoliaII Effects of temperature and daylength on the development offlowers and propagulesrdquo Acta Horticulture vol 430 pp 755ndash761 1997
[49] MHagidimitriou A Katsiotis GMenexes C Pontikis andMLoukas ldquoGenetic diversity of major greek olive cultivars usingmolecular (AFLPs and RAPDs) markers and morphologicaltraitsrdquo Journal of the American Society for Horticultural Sciencevol 130 no 2 pp 211ndash217 2005
18 BioMed Research International
[50] N Babaei N A P Abdullah G Saleh and T L AbdullahldquoIsolation and characterization of microsatellite markers andanalysis of genetic variability in Curculigo latifolia DryandrdquoMolecular Biology Reports vol 39 pp 9869ndash9877 2012
[51] B Shiran N Amirbakhtiar S Kiani S Mohammadi B ESayed-Tabatabaei and H Moradi ldquoMolecular characterizationand genetic relationship among almond cultivars assessed byRAPD and SSR markersrdquo Scientia Horticulturae vol 111 no 3pp 280ndash292 2007
[52] S Singh M K Panda and S Nayak ldquoEvaluation of geneticdiversity in turmeric (Curcuma longa L) using RAPD and ISSRmarkersrdquo Industrial Crops and Products vol 37 no 1 pp 284ndash291 2012
[53] Y Paisooksantivatana S Kako and H Seko ldquoGenetic diversityof Curcuma alismatifolia Gagnep (Zingiberaceae) in Thailandas revealed by allozyme polymorphismrdquo Genetic Resources andCrop Evolution vol 48 no 5 pp 459ndash465 2001
Table 2 List of morphological traits and brief descriptions
Number Morphological traits
1Number of new shoots (number) total number ofproduced new shoots per rhizome
2Leaf length (cm) length of the fully opened firstleaf from the soil surface to leaf tip
3Leaf width (cm) breadth of the leaf at the widestpart of the leaf
4Number of leaves (number) number of fullyemerged leaves at the end of vegetative growthstage
5Plant height (cm) the height of the peduncle atthe top of the soil surface to the tip of theinflorescence
6
Number of days to visible bud appearance (days)number of days from the first day of planting toappearance of the first visible bud Buds appear atthe middle of two sheaths of leaves
4Inflorescence length (cm) the length between thelowest green bracts to tip of the upper pink bractsduring anthesis
8Number of days to anthesis (days) the number ofdays from planting to fully opened flower bud
9Number of days to senescence (days) the daysfrom first day of anthesis until end of the shelf lifeof the flower
10Number of true flowers (No) the number ofsmall axillary flower buds which develop insidebracts during anthesis
11
Number of pink bracts (No) inflorescence of Calismatifolia comprising several apical bractsMost basal bracts are green but the distal onesmore numerous than the green ones are purplishpink bracts which determine the attractiveness ofthe flowering stems The number of pink bractswas counted during anthesis
12Rhizome size (cm) the girth of the M0V0 andM1V1 rhizomes was measured with vernier caliperand mean was expressed in centimeter
13Number of new rhizomes (No) after harvestingthe total number of new rhizomes (M1V1) wasrecorded
14Number of storage roots (No) the total numberof storage roots of M0V0 and M1V1 rhizomes wasrecorded at harvesting time
of isolated DNA were determined using NanoDrop 2000(Thermo Fisher Scientific Inc) in the range of 250 to900 ng120583L which was adjusted to 70 ng120583L The quality wasverified by electrophoresis on 08 agarose gel
32 PCR Amplification and Product Electrophoresis Poly-merase chain reaction (PCR) was carried out for 17 SSRprimers whichwere developed forCurcuma longa in previousstudies [26] PCR was carried out in a 25 120583L reaction volumecontaining 70 ng120583L DNA and 2X DreamTaq Green PCRMasterMix (Fermentas International Inc USA)with 04120583Mprimer Amplification was performed in a thermal cycler(Bio-Rad Laboratories Inc USA) for a total of 40 cyclesAn initial denaturation of the template DNA at 94∘C for 3minutes was followed by 10 cycles of 94∘C for 40 secondsand a touch-down one-degree decrement for annealingtemperature starting with 7∘C above 119879
119898for each primer for
30 seconds and 72∘C for 1 minute This was then followed by30 cycles of 95∘C for 40 seconds a last annealing temperaturefor 30 seconds and 72∘C for 1 minute and a final extensionof 72∘C for 10 minutes The PCR products were separated on4 metaphor gel with 50 bp DNA ladder (N3231S BiolabsInc UK) The gel was stained with Midori green visualizedunder ultraviolet light and photographed by ChemilImagerGel Documentation imaging system (Alpha Innotech Corpo-ration CA USA)
4 Data Analysis
41 Morphological Data The recorded data (after normalityand homogeneity test) were subjected to analysis of variance(ANOVA) as per two-factor experiment with three irradia-tion treatments and four varieties arranged in a randomizedcomplete block design (RCBD) with four replications Theanalysis was carried out using the portable SAS 91 programand least significant differences (LSD) were used for compar-ison among treatment means at 119875 le 005 To evaluate therelationship among the different variables in the experimentcorrelation coefficients were used by SAS 9 1 3 portable Togroup the individuals based on morphological dissimilaritycluster analysis was conducted on the Euclidean distancematrix with the unweighted Pair-GroupMethod using Arith-metic average (UPGMA) using NTSYS software The sameprogram was used for principal components analysis (PCA)to define eigenvalues and eigenvectors and also for compar-ison of the mean of groups to define effective traits in sepa-ration of the groups Eigenvectors are the weights in a lineartransformation when computing principal component scoreswhile eigenvalues indicate the amount of variance explainedby each principal component The cophenetic correlationcoefficient (CCC) was used to measure the goodness of fit ofthe similarity matrices to their corresponding phenograms inmorphological data using PAST (PAleontological Statistics)software V 217 [27]
42 Molecular Data Allele size was measured with UVDoc9902 analysis software (UVI Tech Cambridge UK) by man-ual editing to increase accuracy This procedure was carriedout two times to exclude wrong scorings The PowerMarker325 software package [28]was used to produce a dendrogramusing UPGMAmethod Data were scored as ldquo1rdquo for presenceand ldquo0rdquo for absence The binary data matrix was entered intothe Numerical Taxonomy and Multivariate Analysis System
BioMed Research International 5
Table 3 The number of irradiated and mortal rhizomes of Curcuma alismatifolia varieties after acute irradiation with different doses ofgamma rays
Dose (Gray)Total number of
irradiated rhizomes ineach var
Number of mortal rhizomes after 40 days Mortality rate ()in each doseDoi Tung 554 Chiang Mai Red Sweet Pink Kimono Pink
(NTSYSpc 210e) [29] to generate Dicersquos similarity matrixThe software POPGENE32 Version 132 [29] was used tocalculate genetic variation parameters including observedheterozygosity (the proportion of heterozygous individualsin the population) (119867
119900) expected heterozygosity (119867
119890) [30]mdash
defined as the probability that two randomly chosen allelesfrom the population are different [31]mdashobserved numberof alleles (119899
119886) effective number of alleles (119899
119890) Neirsquos gene
diversity Shannonrsquos information index (119868) and percentageof polymorphic loci To compare the efficiency of primersand polymorphism information content (PIC) a measure ofallelic diversity at a locus was calculated using online PICcalculator software (httpwwwlivacuksimkempsjpichtml)using the following formula
PIC = 1 minus119899
sum
119894=1
1199011198942minus
119899minus1
sum
119894
119899
sum
119895=119894+1
211990111989421199011198952 (1)
where 119901119894is the frequency of the 119894th allele and 119899 is the
number of alleles Markers were classified as informativewhen PICwas ge05 Principal component analysis (PCA) wasalso generated for SSR data by NTSYS-pc 210e
5 Results and Discussions
51 Gamma Irradiation and Radiation Sensitivity Test Thesensitivity of C alismatifolia varieties to radiation was eval-uated by comparing the mortality rate () of irradiatedplants at 40 days after irradiation The plant mortality rateincreased with increasing irradiation dosage (Table 3) Thehybrid Doi Tung 554 was found to be least sensitive togamma irradiation than other varieties (51mortality) whileChiangMai Red variety showed the lowest survival rate (63mortality) Sweet Pink and Kimono Pink varieties showed58 and 55 mortality rate respectively At 50 survivalrate (LD
50) the gamma doses administered were 28 21 23
and 25Gy for Doi Tung 554 Chiang Mai Red Sweet Pink
and Kimono Pink respectively (Figure 1) Abdullah et al [32]had previously indicated that the LD
50for C alismatifolia
var Chiang Mai Pink was approximately at 25Gy The deathof plants is attributed to the interaction of radiation withother molecules in the cell particularly water to produce freeradicals (H OH) The free radicals could combine to formtoxic substances such as hydrogen peroxide (H
2O2) which
contribute to the destruction of cells This indirect effectis especially significant in vegetative cells the cytoplasmwhich contains about 80 water [33] However sensitivityof the plant material depends on the genetic constitutiondose-employed DNA amount moisture content and stageof development and genotype [34] The choice of the doseto be applied for the highest mutant rescue is often left tothe breederrsquos experience with the specific plant material itsgenetics and its physiology
52 Analysis of Variance (ANOVA) for Morphological Traitsof C alismatifolia in M1V1 Analysis of variance indicatedhighly significant differences among the varieties doses andtheir interaction for all traits in M
1V1generation (Table 4)
Some desired and undesired abnormalities such as dwarfismchlorophyll mutation (albinism) striata (yellow or whitelongitudinal bands altering with green colors) two-midribleaves split leaves double flower stalk in one plant doubleinflorescence marbled pink bracts two-tone pink-purplishbracts and two-flag petals were found in M
1V1plants
(Table 5)
53 Effect of Gamma Irradiation on Vegetative Traits in theM1V1 The growth of plants treated with 10 and 20Gy ofgamma rays was slower than that of the controls (Table 6)In irradiated plants the leaf length and leaf width decreasedsignificantly (119875 lt 005) as the radiation doses increasedThis trend is quite common in mutagenised populationsSuch effects are known to arise due to drastic chromoso-mal aberrations in addition to genetic mutations Similar
6 BioMed Research International
Table4Meansquareso
fanalysis
ofvaria
nce(ANOVA
)for
14morph
ologicaltraitsin
Calism
atifolia
(a)
Source
ofvaria
tion
Meansquares
dfGeneration
Num
bero
fsho
otLeafleng
thLeafwidth
Leafnu
mber
Daystovisib
lebu
dPlanth
eight
Block
4M
1V1
005
622
11014
9467
8358
Varie
ty3
M1V
1263lowast
2702lowast
3011lowast
500lowast
1455602lowast
253739lowast
Dose
2M
1V1
611lowast
373804lowast
4137lowast
1042lowast
1012001lowast
157076
6lowastVa
rlowastdo
se6
M1V
114
1lowast8555lowast
418lowast
167lowast
731271lowast
115934lowast
Error
44M
1V1
0094
2951
077
037
4903
Total
5984
93
CV(
)M
1V1
23183
176
206
91165
(b)
Source
ofvaria
tion
Meansquares
dfGeneration
Daysto
anthesis
Daysto
senescence
Noof
true
flow
Noof
pink
bract
Inflo
rescence
leng
thNoof
new
rhizom
esRh
izom
esiz
eNoof
storage
roots
Block
4M
1V1
12847
048
418
072
282
01
018
055
Varie
ty3
M1V
11608017
10091lowast
9166lowast
62543lowast
376lowast
163lowast
023
ns1726lowast
Dose
2M
1V1
1587261
128682lowast
58286lowast
35315lowast
56022lowast
1031lowast
531lowast
6481lowast
Varlowastdo
se6
M1V
1876957
3464lowast
2900lowast
977lowast
4004lowast
128lowast
025ns
332lowast
Error
44M
1V1
7752
322
392
226
445
011
011
121
Total
59CV
()
M1V
110
15229
169
165
2318
268
lowastSign
ificant
with
leastsqu
ared
ifference
test119875lt005
BioMed Research International 7
Dose (Gy)
0
10
10
20
20
30
30
40
40
50
50
60
60
70
70
80
80
90
90
100
100
Mor
talit
y (
)
Var Doi Tung554
LD50 = 28 Gy
(a)
0
10
10
20
20
30
30
40
40
50
50
60
60
70
70
80
80
90
90
100
100
Mor
talit
y (
)
Var Chiang Mai Red
Dose (Gy)
LD50 = 21Gy
(b)
Mor
talit
y (
)
Dose (Gy)
Var Sweet Pink
0
10
10
20
20
30
30
40
40
50
50
60
60
70
70
80
80
90
90
100
100
LD50 = 23Gy
(c)
0
10
10
20
20
30
30
40
40
50
50
60
60
70
70
80
80
90
90
100
100
Mor
talit
y (
)
Dose (Gy)
Var Kimono Pink
LD50 = 25Gy
(d)
Figure 1 PoloPlus plot of linear scale of dose versusmortality percent (a) Doi Tung 554 (b) ChiangMai Red (c) Sweet Pink and (d) KimonoPink
Table 5 Effect of acute gamma rays on vegetative and flowering traits of C alismatifolia in the M1V1 generation
Variety irradiated dose Observed variations Flower color variationChiang Mai Red
10Dwarfism no pink bracts inflorescence small inflorescencetwo-flag petal true flower double inflorescence undulate leafmargin and yellowwhite strip leaves
Light purple N78Clowast
20 Dwarfism narrow small leaves and yellowwhite strip leaves No flowerDoi Tung 554
20 Dwarfism fewer pink bracts and yellowwhite strip leavesSmall and narrow leave Light purple N80Dlowast
lowastRoyal British Society color chart (RHS)
8 BioMed Research International
Table 6 Effect of acute gamma rays on vegetative traits of C alismatifolia in M1V1 generation
Dose (Gray) Shoot number Leaf number Leaf length (cm) Leaf width (cm) Plant height (cm)CMR
0 20 plusmn 00a
30 plusmn 04a
556 plusmn 08a
71 plusmn 05a
1112 plusmn 25a
10 10 plusmn 00b
36 plusmn 05a
272 plusmn 25b
45 plusmn 05b
714 plusmn 96b
20 10 plusmn 00b
14 plusmn 05b
124 plusmn 120c
25 plusmn 24c
170 plusmn 80c
DT0 30 plusmn 00
a30 plusmn 00
a622 plusmn 08
a65 plusmn 05
a912 plusmn 19
a
10 16 plusmn 05b
28 plusmn 04a
280 plusmn 44b
46 plusmn 06b
542 plusmn 76b
20 12 plusmn 04b
24 plusmn 05a
242 plusmn 38b
38 plusmn 05c
512 plusmn 138b
SP0 16 plusmn 05
a32 plusmn 04
a461 plusmn 27
a101 plusmn 04
a746 plusmn 38
a
10 10 plusmn 00b
36 plusmn 05a
275 plusmn 34b
57 plusmn 02b
377 plusmn 137b
20 10 plusmn 00b
20 plusmn 10b
196 plusmn 74c
57 plusmn 04b
195 plusmn 73c
KP0 14 plusmn 00
a46 plusmn 05
a345 plusmn 29
a47 plusmn 04
a591 plusmn 28
a
10 10 + 05a
40 plusmn 05b
220 plusmn 24b
31 plusmn 04b
420 plusmn 54b
20 10 plusmn 00a
32 plusmn 04b
156 plusmn 43c
31 plusmn 11b
283 plusmn 174c
CV () 23 18 26 16 165Means with the same or common letter are not significantly different least square difference test 119875 lt 005
decreases in leaf size were reported by Pongchawee et al [35]and Tangpong et al [36] These results were in agreementwith an earlier study [4] which reported that the growthof chrysanthemum exposed to acute gamma rays was lessthan the control in the M
1V1generation All varieties doses
and interaction effects resulted in significant differences fornumber of leaves Among untreated plants Kimono Pinkvariety had higher number of leaves (46) than the otherthree (3 3 and 32) varieties In Chiang Mai Red and SweetPink varieties plants exposed to 10Gy showedhigher numberof leaves than untreated plants However at 20Gy therewas significant reduction in number of leaves for all studiedvarieties in comparison to control Similar stimulatory effectswere obtained at lower doses in ginger by Hegde [37] andGiridharan and Balakrishnan [38]
Progressive reduction in growth parameters can be inter-preted on interference in normal mitosis and frequent occur-rence of mitotic aberrations inhibition of rate of assimilationand consequent change in the nutrient level in the plantand inactivation of vital enzymes especially those associatedwith respiration [39] Dose-dependent negative effect wasalso detected for plant heightThe tallest plants were recordedfrom the untreated rhizomes (0Gy) with heights of 1112912 746 and 591 cm followed by the 10Gy irradiated plantswith heights of 714 542 377 and 420 cm and the 20Gyirradiated plants with corresponding heights of 170 512195 and 283 cm for Chiang Mai Red Doi Tung 554 SweetPink and Kimono Pink varieties respectively These resultsare in agreement with the findings of Abdullah et al [32]Reduction in growth parameters and dwarfism can be causedby interference of normal mitosis and frequent occurrenceof mitotic aberrations inhibition of assimilation rates andconsequent changes in nutrient levels in plants Additionallymutagenic effects such as auxin destruction inhibition of
auxin synthesis failure of assimilatory mechanism andchanges in the specific activity of enzymes can cause growthreductions [37] High doses of ionizing radiation have beenshown to damage macromolecular cellular components suchas cell walls membranes and DNA [40] The number ofshoots also decreased significantly as the radiation dosesincreased Radiation also affects organic molecules that areessential to the cell division process and thus causing celldivision to stop [36]
54 Effect of Gamma Irradiation on Flowering DevelopmentTraits in the M1V1 All control and 10Gy irradiated plantsproduced flowers while the Chiang Mai Red and SweetPink varieties which were exposed to 20Gy did not gointo the flowering stage Lamseejan et al [4] also showedthat flowering percentage decreases as gamma ray dosesare increased In the present study gamma rays caused lateflowering in all four varieties Days to appearance of firstvisible buds were also significantly different among the fourvarieties (Table 7) Gamma rays caused a noticeable delay inflowering of irradiated plants in comparison to the untreatedones First visible buds were observed at 652 and 874 days inthe control and 10Gy treatments respectively for the ChiangMai Red variety In Doi Tung 554 the first visible buds wereappeared at 474 656 and 840 days after planting at 0 10and 20Gy doses respectively In the Sweet Pink variety thenumber of days to visible bud appearance increased signif-icantly from 678 days in controls to 978 days in the 10Gyirradiated plants In comparison to other three varieties theKimono Pink variety needed the longest time to visible budappearance and same as other varieties there was a positivecorrelation between the number of days to first visible budand the gamma irradiation dose In the control and 10Gyand 20Gy irradiated individual plants flower buds were
BioMed Research International 9
Table 7 Effect of acute gamma rays on flower development characteristics of C alismatifolia in M1V1 generation
Dose (Gy) Days tovisible bud
Inflorescencelength (cm)
Days toanthesis
Number oftrue flowers
Number ofPink bracts
Days tosenescence
CMR0 652 plusmn 47
b162 plusmn 057
a742 plusmn 54
b132 plusmn 19
a104 plusmn 15
a21 plusmn 10
a
10 874 plusmn 95a
82 plusmn 397b
1020 plusmn 71a
56 plusmn 19b
50 plusmn 07b
10 plusmn 14b
20 00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
DT0 474 plusmn 43
c134 plusmn 054
a546 plusmn 54
c162 plusmn 13
a232 plusmn 04
a23 plusmn 00
a
10 656 plusmn 37b
94 plusmn 19b
780 plusmn 37b
112 plusmn 21b
166 plusmn 08b
122 plusmn 130b
20 840 plusmn 42a
76 plusmn 082c
994 plusmn 49a
88 plusmn 13b
156 plusmn 11b
106 plusmn 18b
SP0 678 plusmn 21
b146 plusmn 089
a756 plusmn 26
b162 plusmn 13
a100 plusmn 24
a188 plusmn 083
a
10 978 plusmn 181a
79 plusmn 20b
1098 plusmn 182a
68 plusmn 25b
52 plusmn 08b
92 plusmn 24b
20 00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
KP0 852 plusmn 23
c137 plusmn 044
a942 plusmn 19
c112 plusmn 12
a96 plusmn 21
a18 plusmn 18
a
10 1044 plusmn 65b
108 plusmn 108ab
1224 plusmn 100b
76 plusmn 27b
64 plusmn 16ab
102 plusmn 17b
20 1272 plusmn 65a
790 plusmn 49b
1426 plusmn 58a
60 plusmn 38b
42 plusmn 24b
66 plusmn 37b
CV () 10 23 9 22 169 15Means with the same or common letter are not significantly different least square difference test 119875 lt 005
visible at 852 1328 and 1374 days after planting respectivelyPrevious studies also showed that onset of flowering andformation of floral parts in mutants of Arabidopsis thalianamaize barely pea and tobacco involved growth regulators(or phytohormones) such as auxins cytokinins gibberellinsabscisic acid ethylene and brassinosteroids [41] There weresignificant differences among treatments for the length ofthe inflorescence In all varieties the longest inflorescencelength was observed in the untreated plants with 162134 146 and 137 cm lengths for Chiang Mai Red DoiTung 554 Sweet Pink and Kimono Pink respectively Thecorresponding inflorescence lengths were 82 94 79 and109 cm for the 10Gy irradiated plants The days to anthesisfor C alismatifolia varieties were significantly affected byvariety gamma irradiation doses and their interaction Thenumber of days to full bloom was noticeable earliest foruntreated plants at 742 546 756 and 942 days for ChiangMai Red Doi Tung 554 Sweet Pink and Kimono Pinkvarieties respectively This was then followed by plants at10Gy at 102 78 1098 and 1224 days The number of trueflowers or the secondary inflorescence developed in the axilof the primary bracts decreased as radiation dosage increasedIn the present study the gamma rays also decreased thedays to inflorescence senescence In this study there wasa strongly significantly and positively correlation (0919lowastlowast)(data not shown) between the number of true flowers andthe number of days to senescenceThe number of pink bractsalso decreasedwith increasing radiation dosageMost gammaray effects on senescence are considered as resulting from theaction of free radicals generated from water and oxygen bythe ionizing energy on the cellular components Membrane
deterioration is a general feature of natural senescence andstress-induced aging [42]
Irradiation induced some mutation spectrum of flowercolor variation that included colors such as purple palepurple rather pale purple white purple white (marbledpattern) and two-tone purple color
Mutation spectrum of flower shape variation includeddouble inflorescence within one stalk double stalk per plantinflorescence without bracts two-flag petal true flowersand chlorophyll mutation in the leaves which are generallycaused by induced gamma rays (Figure 2) Ionizing radiationincluding gamma rays induces fragment deletions or inser-tions that eventually lead to changes in amino acids and amodification of leaf and stem pigmentation [43] A mutationin the biosynthetic pathway of structural or regulatory genesmay cause a change in flower color [44] When the blockageoccurs at the early stages of anthocyanin synthesis whiteflowers will result while a blockage at later stages leads todifferent flower colors due to the accumulation of particularanthocyanins [45] Chloroplasts were extremely sensitive togamma radiation compared to other cell organelles [46]
55 Effect of Gamma Irradiation on Rhizome Characteristicsin Selected Doses in M1V1 The number of new rhizomes andthe number of storage roots per rhizome were significantlyaffected by varieties doses and their interaction (Table 8)The number of new rhizomes only in Kimono Pink varietydid not show any differences between untreated and treatedplants The rhizome size was influenced only by doses Asdose level increased the rhizome size decreased Amonguntreated plants the Sweet Pink rhizomes had the most
10 BioMed Research International
(a) (b) (c) (d)
(e)
(f)
Figure 2 Effect of acute gamma rays on flower traits in C alismatifolia (a) Untreated inflorescence bracts color Doi Tung 554 (b) Two tonebract color Doi Tung 554 (20Gy) (c) Marbled pattern of bract color Doi Tung 554 (20Gy) (d) Double inflorescence within one stalk varChiang Mai Red (10Gy) (e) True flower in nontreated plants (f) Two-flag petals true flowers (10Gy)
Figure 3 Dendrogram representing themorphological variation among 44 irradiated and nonirradiated individuals ofC alismatifolia across14 variables CMR = Chiang Mai Red DT = Doi Tung 554 SP = Sweet Pink Kp = Kimono Pink
number of storage roots (78) and the Kimono Pink rhizomeshad the least number of storage roots (40)This was followedby 10Gy irradiated rhizomes with 4 46 48 and 28 storageroot numbers for Chiang Mai Red Doi Tung 554 SweetPink and Kimono Pink respectively Irradiation with 20Gydecreased significantly the number of storage roots in allstudied varietiesTheplantC alismatifolia is propagated froma propagule (one rhizome + 5-6 t-roots) The rhizome sizedose matters in the growth of the plant Smaller rhizome sizeusually resulted in smaller plant size with narrow grass-likeleaves The storage roots play a very important role in thegrowth and flowering ofCurcumaThe t-rootsmake up about85 of the total fresh weight and 70 of the total dry weightof a typical propaguleThe storage organs act for plant growth
during dormancy and emergence [47] More storage root perpropagule resulted in faster flowering and higher yield plant[48] In this study also there was significant and negativecorrelation between number of storage roots and number ofdays to visible bud appearance (minus0525lowastlowast)
56 Cluster Analysis of C alismatifolia for MorphologicalTraits The morphological data were used to calculate thesimilarity between the treated and non-treated C alismat-ifolia samples and UPGMA dendrogram was constructed(Figure 3) The cophenetic correlation coefficient (CCC)value between the genetic dissimilaritymatrix estimated fromthe morphological characters and the UPGMA clusteringmethod was 119903 = 093 showing a proof fit This value was
BioMed Research International 11
Table 8 Effect of acute gamma rays on rhizome characteristics ofC alismatifolia in M1V1 generation
Dose (Gray) Rhizomesize
No of newrhizomes
No of storageroot
CMR0 23 plusmn 02
a26 plusmn 055
a46 plusmn 054
a
10 18 plusmn 005a12 plusmn 044
b40 plusmn 07
a
20 08 plusmn 07b
06 plusmn 054b
14 plusmn 13b
DT0 22 plusmn 01
a30 plusmn 00
a72 plusmn 109
a
10 18 plusmn 007b14 plusmn 054
b46 plusmn 13
b
20 16 plusmn 005b
10 plusmn 00b
28 plusmn 13c
SP0 22 plusmn 02
a24 plusmn 054
a78 plusmn 21
a
10 17 plusmn 007b
10 plusmn 00b
48 plusmn 044b
20 12 plusmn 02c
10 plusmn 00b
28 plusmn 083b
KP0 21 plusmn 005
a10 plusmn 00
a40 plusmn 00
a
10 16 plusmn 01ab
10 plusmn 00a
28 plusmn 044b
20 11 plusmn 06b
10 plusmn 00a
22 plusmn 083b
CV () 18 23 26Means with the same or common letter are not significantly different leastsquare difference test 119875 lt 005
higher than of studies on olive for morphological traits (119903 =069) [49] In this dendrogram the 44C alismatifolia samplesappeared to form three main clusters and five minor clustersat coefficient level 56 cluster I included three non-irradiatedmembers and cluster II included 10Gy irradiated individualplants of all four varieties and 20Gy irradiated samples ofDoi Tung 554 and Kimono Pink varieties except KP20-4 (noflowering) Main cluster III included the 20Gy irradiatedindividuals Chiang Mai Red and Sweet Pink varieties (asmentioned in morphological part these individuals didnot go to flowering stage) and KP20-4 Cluster II can bedivided to five subclusters The first subcluster included 10members (CMR10-1 CMR10-5 SP10-1 SP10-2 SP10-3 SP10-5 CMR10-2 CMR10-4 SP10-4 and CMR10-3) the secondone included nine members (KP10-1 KP10-2 KP10-3 KP10-4 KP10-5KP20-1 KP20-2 KP20-5 and KP20-3) KP0 wasthe lone member of the third sub-cluster The fourth sub-cluster included two members only (DT10-2 and DT10-3) The last sub-cluster main cluster II had eight membersincluded (DT10-1 DT10-4 DT10-5 DT20-1 DT20-2 DT20-3 DT20-4 and DT20-5) Mean value of groups for each traitis presented in Table 9 This table clearly shows the differentmean values of the three main clusters The minimum meanvalue referred to main cluster III while the maximum meanvalue for number of shoots leaf length leaf width plantheight number of true flowers inflorescence length rhizomesize number of new rhizomes and number of storage rootsbelonged to cluster I which was included non-treated plantsThese results showed that the gamma irradiation has inducedmorphological changes in C alismatifolia individuals of
four studied varieties so that they showed phenotypicallydifferences from their controls
57 Principal Component Analysis of C alismatifolia forMorphological Traits To assess the patterns of variation PCAwas done by considering all of the 14 variables The first threecomponents of PCA explained 828 of the total variation(Table 10) Only the first component which accounted for562 of the total variation was attributed to inflorescencelength plant height days to senescence number of trueflowers and rhizome size In PCA three-dimensional graphthe grouping of individuals confirmed the clustering results(Figure 4) The PCA graph proved that all irradiated indi-vidual plants of C alismatifolia varieties are phenotypicallydifferent from their non-irradiated individuals
6 Molecular Characterization
61 SSR Polymorphism Amplifications were successful forall the 17 SSR markers assayed This reflects a high levelof homology between SSR flanking regions in C longa andC alismatifolia Out of 17 primer pairs eight primer pairsresulted in polymorphic PCR products Table 11 summarizesthe results obtained based on the analysis of individuals of thefour studied varieties using the polymorphic SSR loci for 010 and 20Gy irradiated plants The number of alleles variedwidely among these loci A total of 25 36 and 41 alleles wereobserved among 0 10 and 20Gy irradiated individual plantsrespectively In the 10Gy treatment the number of allelesranged from three (clon09 and clon14) to seven (clon08) withan average value of 45 per locus In the 20Gy treatmentthe number of alleles varied from three (clon09 and clon14)to seven (clon08 and clon12) with an average value of 51per locus In the untreated individuals the number of allelesranged from two (clon04 clon09 clon11 and clon14) to five(clon12) with an average value of 31 The difference betweenthe average number of observed alleles and effective numberof alleles was due to the uneven frequency of each allele[50] For each of the eight SSR primers PIC values (whichmeasures allele diversity and frequency among varieties)varied from 019 (clon04) to 071 (clon01) in untreated plantsand from 025 (clon04) to 075 (clon08) in 10Gy acutelyirradiated plants In 20Gy irradiated individual plants thePIC value ranged from 042 (clon14) to 075 (clon08 andclon12) The mean PIC for all loci was 047 054 and 061for 0 10 and 20Gy irradiated plants respectively
The PIC value provides an estimate of the discriminatorypower of amarker by taking into account not only the numberof alleles at a locus but also the relative frequencies of thesealleles [51] Thus except for clon09 and clon14 which weremoderately polymorphic (025 lt PIC lt 05) all other lociwere highly polymorphic (05 lt PIC) while none of theloci showed low polymorphism The average discriminatingpower ofmicrosatellitemarkers (061) observed in the presentstudy ensures the future utility of microsatellite markersfor genetic variation studies in C alismatifolia varietiesOur results reflect similar findings as reported earlier inthe study of genetic variation in other species of Curcuma
12 BioMed Research International
Table9Clusterm
eans
for14charactersestim
ated
in44
individu
also
fCalismatifolia
Clusters
Shoo
tnu
mber
Leaf
leng
thLeaf
width
Leaf
number
Visib
lebu
d(days)
Inflo
rescence
leng
thPlant
height
Anthesis
(days)
Senescence
(days)
True
flow
number
Pink
bract
number
Rhizom
esiz
eNew
rhizom
enu
mber
Storage
root
number
I220
5464
736
300
6013
1473
9233
6926
2093
152
1453
229
266
653
II1
100
2735
512
360
9260
809
5457
1059
960
620
510
177
110
440
II2
122
1922
298
344
11422
1043
3910
1312
293
3755
588
156
100
266
II3
100
3452
416
460
8520
1370
5910
9420
180
120
960
206
100
400
II4
150
2500
45
250
6500
925
4950
7700
1150
950
1700
185
200
600
II5
137
2637
417
262
7725
840
5356
9162
1137
1012
1587
170
100
312
III
054
1500
465
181
000
000
000
000
000
000
000
090
081
200
BioMed Research International 13
Table 10 Eigenvectors eigenvalues and proportions of variabilityfor three principle components among 14 characters for 44 Calismatifolia
Variable PC1 PC2 PC3Number of new shoot 0270 minus0076 minus0173Leaf length 0269 minus0305 0033Leaf width 0096 minus0455 0430Leaf number 0210 0285 0490Days to visible bud 0199 0462 0181Plant height 0321 0027 minus0071Inflorescence length 0318 0166 minus0040Days to anthesis 0205 0460 0148Days to senescence 0342 0017 minus0131Number of true flower 0323 0029 minus0238Number of pink bracts 0266 minus0028 minus0546Rhizome size 0302 minus0101 0147Number of new rhizome 0250 minus0293 0028Number of storage roots 0264 minus0247 0298Eigenvalue 7877 2694 1032Proportion 56264 19246 7371Cumulative 56264 75511 82882
using SSR markers [13] The highest Neirsquos gene diversity(ℎ) was obtained with the 20Gy treatment (061) followedby the 10Gy treatment (06) and the untreated individuals(05) The mean Shannonrsquos information index (119868) was 092114 and 130 in the 0 10 and 20Gy irradiated plantsrespectively The high value of Shannonrsquos information indexrepresents the effectiveness of microsatellite loci to revealthe variation among these varieties at the different dosesused Overall genetic variability for the varieties studiedrepresented by the Shannon-Weiner index was relatively highin comparison to other studies involving C longa accessions[10 52] Additionally the 20Gy acutely irradiated individualsshowed a higher mean percentage of polymorphic loci (63)than the 10Gy (58) and untreated (022) ones This impliesthat irradiation with a dose of 20Gy induced more geneticvariation in the M
1V1generation
62 Cluster Analysis Thedendrogramwas constructed usingPowerMarker 323The 44 studied individuals were clusteredinto seven groups (G1ndashG7) Colors were applied accordingto our model-based cluster analysis results Group I iscomprised of nine individual plants of Chiang Mai Redvariety receiving 0 10 and 20Gy treatments (blue colorin Figure 5) One individual plant DT10-1 was assignedto group II (green color in Figure 5) All irradiated andnon-irradiated individuals of Doi Tung 554 variety wereassigned to group III (yellow color in Figure 5) DT20-1 andSP10-5 were the sole members of groups IV (red color inFigure 5) and V (gray color in Figure 5) respectively GroupVI included treated and untreated individuals of Sweet Pinkvariety (pink color in Figure 5) Lastly all Kimono Pinkindividuals were assigned to cluster VII (orange color in
Figure 5) The genetic similarity coefficient (data not shown)among the 44 individuals amplified using eight SSR primersvaried from 00 to 10 The highest value (10) correspondedto [(CMR10-3 CMR10-5 CMR and CMR20-2) (DT10-2and DT10-3) (DT and DT20-2) (KP20-3 KP-20-4 KP10-3andKP10-4)] individuals that generated identical fingerprintsacross the markers studied Among the studied varieties theSweet Pink individual plants showed the lowest similarity(00) withKimono Pink individuals which indicated that theywere relatively remote in relationship (Figure 5) An overviewof the clustering pattern indicates that the grouping of thestudied individual plants was based on different varieties andlargely independent of the doses of gamma irradiation Awide range in similarity values had also been observed indifferent species of Curcuma [12 13 52 53]
63 Principle Component Analysis (PCA) Thedata generatedfrom 44 C alismatifolia individual plants were subjected toprincipal component analysis (PCA) to visualize individualsin a multivariate space In the three-dimensional graphderived from the SSR analysis all studied individuals weregrouped into seven clusters (Figure 6) In PCA the firstthree principal components (PC) extracted a cumulative of6826 of the total variation among the 44 individuals of CalismatifoliaThe first three coordinates PC1 PC2 and PC3accounted for 4136 1578 1112 of the total variationrespectively The distribution of the individual plants in athree-dimensional plot using the first three principal com-ponents showed the genetic relationship between individualplants It was evident that both methods dendrogram andthree-dimensional plots of PCA were effective in illustratinggenetic relationships and the groups found were comparable[11 13] The PCA results were similar to those obtained fromcluster analysis where all individuals from different varietieswere assigned to different groups These graphical illus-trations enable the assessment of the relationshipdistancesamong all of the individuals in the study [50]
7 Conclusion
This is the first attempt to evaluate the effect of acutegamma irradiation on C alismatifolia varieties using bothmorphological characteristics and molecular markers Inplant breeding programs mutagenic treatments with lownegative physiological effects and strong genetic effects aredesirable Hence we used more effective doses of gammairradiation (10 and 20Gy) of which particularly 20Gy dosewas effective to influence morphological and molecularcharacteristics of studiedCurcuma alismatifolia varietiesThelower dose (10Gy) of radiation probably caused little damageto the plants genetic material so that the cells could repairthemselves in next generation As a result in this study 20Gyacute gamma irradiation resulted in a higher percentage ofmutation and getting desired mutants was more possibleBased on the LD
50values determined in this study it was
apparent that the varieties Chiang Mai Red and Sweet Pinkwere more sensitive to gamma rays compared to Doi Tung554 and Kimono Pink Our results show that the variety Doi
Figure 4 Two-dimensional graph of principal component analysis (PCA) for 14 morphological variables indicating relationships amongirradiated and nonirradiated four varieties of C alismatifolia
CMR10-1CMR10-5
SP10-1SP10-2SP10-3
SP10-5
CMR10-4
SP10-4
KP10-1KP10-3
KP20-1
KP20-2
KP10-5KP20-5
KP20-3
KP10-2
DT10-2
DT10-1
DT10-5
DT20-1
DT10-4DT20-4DT20-2
DT20-5DT20-3
CMR20-1
SP20-3SP20-4
SP20-5SP20-1
SP20-2
KP
SP
CMR 10-2
SP10-5
Figure 5 Unrooted neighbor joining tree showing genetic relationship among 44 irradiated and nonirradiated C alismatifolia using SSRmarkers The colors of the branches correspond to those of the same cluster
Figure 6Three-dimensional graph of principal component analysis (PCA) indicating relationships among irradiated and nonirradiated fourvarieties of C alismatifolia
Tung 554 (Curcuma hybrid) had the most morphologicalresponses to gamma rays The use of microsatellite markersas a codominant marker will facilitate the exploration ofgenetic variability among treated and non-treated plants andwill help to distinguish the plants showing differences inmorphological characters The overall effects on the M
1V1
generation revealed that acute gamma irradiation at optimumdoses has the potential for developing new varieties of Calismatifolia with improved commercial properties suitablefor the Malaysian flower industry
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work was funded by the Fundamental Research GrantScheme (FRGS) under the Ministry of Higher Education inMalaysia
References
[1] P Apavatjrut S Anuntalabhochai P Sirirugsa and C AlisildquoMolecularmarkers in the identification of some early floweringCurcuma L (Zingiberaceae) speciesrdquo Annals of Botany vol 84no 4 pp 529ndash534 1999
[2] K Bunya-Atichart S Ketsa andW G VanDoorn ldquoPostharvestphysiology of Curcuma alismatifolia flowersrdquo Postharvest Biol-ogy and Technology vol 34 no 2 pp 219ndash226 2004
[3] M Nakano J Amano Y Watanabe et al ldquoMorphologicalvariation in Tricyrtis hirta plants regenerated from heavy ionbeam-irradiated embryogenic callusesrdquo Plant Biotechnologyvol 27 no 2 pp 155ndash160 2010
[4] S Lamseejan P Jompuk A Wongpiyasatid A Deeseepan andP Kwanthammachart ldquoGamma-rays induced morphological
[5] G-J Lee S J Chung I S Park et al ldquoVariation in thephenotypic features and transcripts of colormutants of chrysan-themum (Dendranthema grandiflorum) derived from gammaraymutagenesisrdquo Journal of Plant Biology vol 51 no 6 pp 418ndash423 2008
[6] O K Kikuchi ldquoOrchid flowers tolerance to gamma-radiationrdquoRadiation Physics and Chemistry vol 57 no 3ndash6 pp 555ndash5572000
[7] N P Arnold N N Barthakur and M Tanguay ldquoMutageniceffects of acute gamma irradiation on miniature roses targettheory approachrdquo HortScience vol 33 no 1 pp 127ndash129 1998
[8] A A Youssef M S Aly and M S Hussein ldquoResponse ofgeranium (Pelargonium graveolenus L) to gamma irradiationand foliar application of Speed Growrdquo Egyptian Journal ofHorticulture vol 27 pp 41ndash53 2000
[9] N Chuantang and L Yazhi ldquoThe radiation inducedmutation ofcanna (Canna L)rdquo Acta Agricultute Nucleatae Sinica vol 2 pp33ndash39 1998
[10] M S Sigrist J B Pinheiro J A A Filho and M I ZucchildquoGenetic diversity of turmeric germplasm (Curcuma longaZingiberaceae) identified by microsatellite markersrdquo Geneticsand Molecular Research vol 10 no 1 pp 419ndash428 2011
[11] S Taheri T L Abdullah N A P Abdullah and Z AhmadldquoGenetic relationships among five varieties of Curcuma alis-matifolia (Zingiberaceae) based on ISSRmarkersrdquoGenetics andMolecular Research vol 11 pp 3069ndash3076 2012
[12] A Das V Kesari V M Satyanarayana A Parida and L Ran-gan ldquoGenetic relationship of Curcuma species from NortheastIndia using PCR-based markersrdquo Molecular Biotechnology vol49 no 1 pp 65ndash76 2011
[13] S Siju K Dhanya S Syamkumar et al ldquoDevelopment char-acterization and utilization of genomic microsatellite markersin turmeric (Curcuma longa L)rdquo Biochemical Systematics andEcology vol 38 no 4 pp 641ndash646 2010
[14] M K Panda S Mohanty E Subudhi L Acharya and SNayak ldquoAssessment of genetic stability of micropropagated
BioMed Research International 17
plants of Curcuma L by cytophotometery and RAPD analysisrdquoInternational Journal of Integrative Biology (IJIB) vol 1 pp 189ndash195 2007
[15] S Syamkumar and B Sasikumar ldquoMolecular marker basedgenetic diversity analysis of Curcuma species from IndiardquoScientia Horticulturae vol 112 no 2 pp 235ndash241 2007
[16] D Tautz and M Renz ldquoSimple sequences are ubiquitousrepetitive components of eukaryotic genomesrdquo Nucleic AcidsResearch vol 12 no 10 pp 4127ndash4138 1984
[17] G Toth Z Gaspari and J Jurka ldquoMicrosatellites in differenteukaryotic genomes surveys and analysisrdquo Genome Researchvol 10 no 7 pp 967ndash981 2000
[18] W Powell G C Machray and J Proven ldquoPolymorphismrevealed by simple sequence repeatsrdquo Trends in Plant Sciencevol 1 no 7 pp 215ndash222 1996
[19] S-Y Lee W K Fai M Zakaria et al ldquoCharacterizationof polymorphic microsatellite markers isolated from ginger(Zingiber officinale Rosc)rdquo Molecular Ecology Notes vol 7 no6 pp 1009ndash1011 2007
[20] S Bory D Da Silva A-M Risterucci M Grisoni P Besseand M-F Duval ldquoDevelopment of microsatellite markers incultivated vanilla polymorphism and transferability to othervanilla speciesrdquo Scientia Horticulturae vol 115 no 4 pp 420ndash425 2008
[21] I C Menezes F W Cidade A P Souza and I C SampaioldquoIsolation and characterization ofmicrosatellite loci in the blackpepper Piper Nigrum L (Piperaceae)rdquo Conservation GeneticsResources vol 1 no 1 pp 209ndash212 2009
[22] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[23] V Kumar Morphological and molecular characterization ofinduced mutants in Groundnut [PhD thesis] University ofAgricultural Sciences Dharwad India 2008
[24] J R Sharma Statistical and Biometrical Techniques in PlantBreeding New Age International New Delhi India 1998
[25] J J Doyle and J L Doyle ldquoA rapid DNA isolation procedure forsmall quantities of fresh leaf tissuerdquo Phytochemical Bulletin vol19 pp 11ndash15 1987
[26] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[27] Oslash Hammer D A T Harper and P D Ryan ldquoPast paleontolog-ical statistics software package for education and data analysisrdquoPalaeontologia Electronica vol 4 no 1 pp 4ndash9 2001
[28] K Liu and S V Muse ldquoPowerMaker an integrated analysisenvironment for genetic maker analysisrdquo Bioinformatics vol 21no 9 pp 2128ndash2129 2005
[29] F J RohlfNTSYS-Pc Numerical Taxonomy System Version 2 1Exeter Publishing Setauket New York NY USA 2002
[30] M Nei ldquoAnalysis of gene diversity in subdivided populationsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 70 no 12 1973
[31] S Kadkhodaei M Shahnazari M K Nekouei et al ldquoA compar-ative study of morphological and molecular diversity analysisamong cultivated almonds (Prunus dulcis)rdquo Australian Journalof Crop Science vol 5 no 1 pp 82ndash91 2011
[32] T L Abdullah J Endan and B M Nazir ldquoChanges in flowerdevelopment chlorophyll mutation and alteration in plant
morphology of Curcuma alismatifolia by gamma irradiationrdquoAmerican Journal of Applied Sciences vol 6 no 7 pp 1436ndash14392009
[33] E Kovacs and A Keresztes ldquoEffect of gamma and UV-BCradiation on plant cellsrdquoMicron vol 33 no 2 pp 199ndash210 2002
[34] H L Ramesh Y Murthy and V N Munirajappa ldquoEffect ofgamma radiation on morphological and growth parametersof Mulberry variety M5rdquo International Journal of Science andNature vol 3 pp 447ndash452 2010
[35] K Pongchawee R Pradissan andW Pipatcharoenchai ldquoInducemutation in Anubias spp through in vitro Irradiationrdquo ThaiFisheries Gazette vol 60 pp 493ndash497 2007
[36] P Tangpong T Taychasinpitak C Jompuk and P JompukldquoEffects of acute and chronic gamma irradiations on in vitroculture ofAnubias congensisNE BrownrdquoKasetsart Journal vol43 no 3 pp 449ndash457 2009
[37] R K Hegde Studies on induced mutagenesis and in vitroregeneration in turmeric (Curcuma longa L) [PhD thesis]University of Agricultural Sciences Dharwad India 2006
[38] M P Giridharan and S Balakrishnan ldquoGamma ray inducedvariability in vegetative and floral characters of ginger IndianCocoardquo Arecanut and Spices Journal vol 15 pp 68ndash672 1992
[39] A P Cesarett ldquoEffect of radiation on higher plants and plantcommunitiesrdquo inRadiation Biology United States Atomic EnergyCommission pp 284ndash309 Washington DC USA 1968
[40] G W Seung Y C Byung J-H Kim et al ldquoUltrastructuralchanges of cell organelles in Arabidopsis stems after gammairradationrdquo Journal of Plant Biology vol 48 no 2 pp 195ndash2002005
[41] B S Ahloowalia and M Maluszynski ldquoInduced mutationsmdashanew paradigm in plant breedingrdquo Euphytica vol 118 no 2 pp167ndash173 2001
[42] R Voisine L-P Vezina and C Willemot ldquoInduction ofsenescence-like deterioration of microsomal membranes fromcauliflower by free radicals generated during gamma irradia-tionrdquo Plant Physiology vol 97 no 2 pp 545ndash550 1991
[43] N Shikazono Y Yokota S Kitamura et al ldquoMutation rate andnovel tt mutants of Arabidopsis thaliana induced by carbonionsrdquo Genetics vol 163 no 4 pp 1449ndash1455 2003
[44] T Nakatsuka M Nishihara K Mishiba and S YamamuraldquoTwo different mutations are involved in the formation ofwhite-flowered gentian plantsrdquo Plant Science vol 169 no 5 pp949ndash958 2005
[45] M Mato T Onozaki Y Ozeki et al ldquoFlavonoid biosynthesisin white-flowered Sim carnations (Dianthus caryophyllus)rdquoScientia Horticulturae vol 84 no 3-4 pp 333ndash347 2000
[46] S G Wi B Y Chung J-S Kim et al ldquoEffects of gammairradiation on morphological changes and biological responsesin plantsrdquoMicron vol 38 no 6 pp 553ndash564 2007
[47] F Lee Chin Effects of light intensity and daylength on growth andflowering of siam tulip (Curcuma alismatifolia var Chiang MaiPink) [PhD thesis] University Putra Malaysia 2007
[48] A Hagiladi N Umiel and X H Yang ldquoCurcuma alismatifoliaII Effects of temperature and daylength on the development offlowers and propagulesrdquo Acta Horticulture vol 430 pp 755ndash761 1997
[49] MHagidimitriou A Katsiotis GMenexes C Pontikis andMLoukas ldquoGenetic diversity of major greek olive cultivars usingmolecular (AFLPs and RAPDs) markers and morphologicaltraitsrdquo Journal of the American Society for Horticultural Sciencevol 130 no 2 pp 211ndash217 2005
18 BioMed Research International
[50] N Babaei N A P Abdullah G Saleh and T L AbdullahldquoIsolation and characterization of microsatellite markers andanalysis of genetic variability in Curculigo latifolia DryandrdquoMolecular Biology Reports vol 39 pp 9869ndash9877 2012
[51] B Shiran N Amirbakhtiar S Kiani S Mohammadi B ESayed-Tabatabaei and H Moradi ldquoMolecular characterizationand genetic relationship among almond cultivars assessed byRAPD and SSR markersrdquo Scientia Horticulturae vol 111 no 3pp 280ndash292 2007
[52] S Singh M K Panda and S Nayak ldquoEvaluation of geneticdiversity in turmeric (Curcuma longa L) using RAPD and ISSRmarkersrdquo Industrial Crops and Products vol 37 no 1 pp 284ndash291 2012
[53] Y Paisooksantivatana S Kako and H Seko ldquoGenetic diversityof Curcuma alismatifolia Gagnep (Zingiberaceae) in Thailandas revealed by allozyme polymorphismrdquo Genetic Resources andCrop Evolution vol 48 no 5 pp 459ndash465 2001
Table 2 List of morphological traits and brief descriptions
Number Morphological traits
1Number of new shoots (number) total number ofproduced new shoots per rhizome
2Leaf length (cm) length of the fully opened firstleaf from the soil surface to leaf tip
3Leaf width (cm) breadth of the leaf at the widestpart of the leaf
4Number of leaves (number) number of fullyemerged leaves at the end of vegetative growthstage
5Plant height (cm) the height of the peduncle atthe top of the soil surface to the tip of theinflorescence
6
Number of days to visible bud appearance (days)number of days from the first day of planting toappearance of the first visible bud Buds appear atthe middle of two sheaths of leaves
4Inflorescence length (cm) the length between thelowest green bracts to tip of the upper pink bractsduring anthesis
8Number of days to anthesis (days) the number ofdays from planting to fully opened flower bud
9Number of days to senescence (days) the daysfrom first day of anthesis until end of the shelf lifeof the flower
10Number of true flowers (No) the number ofsmall axillary flower buds which develop insidebracts during anthesis
11
Number of pink bracts (No) inflorescence of Calismatifolia comprising several apical bractsMost basal bracts are green but the distal onesmore numerous than the green ones are purplishpink bracts which determine the attractiveness ofthe flowering stems The number of pink bractswas counted during anthesis
12Rhizome size (cm) the girth of the M0V0 andM1V1 rhizomes was measured with vernier caliperand mean was expressed in centimeter
13Number of new rhizomes (No) after harvestingthe total number of new rhizomes (M1V1) wasrecorded
14Number of storage roots (No) the total numberof storage roots of M0V0 and M1V1 rhizomes wasrecorded at harvesting time
of isolated DNA were determined using NanoDrop 2000(Thermo Fisher Scientific Inc) in the range of 250 to900 ng120583L which was adjusted to 70 ng120583L The quality wasverified by electrophoresis on 08 agarose gel
32 PCR Amplification and Product Electrophoresis Poly-merase chain reaction (PCR) was carried out for 17 SSRprimers whichwere developed forCurcuma longa in previousstudies [26] PCR was carried out in a 25 120583L reaction volumecontaining 70 ng120583L DNA and 2X DreamTaq Green PCRMasterMix (Fermentas International Inc USA)with 04120583Mprimer Amplification was performed in a thermal cycler(Bio-Rad Laboratories Inc USA) for a total of 40 cyclesAn initial denaturation of the template DNA at 94∘C for 3minutes was followed by 10 cycles of 94∘C for 40 secondsand a touch-down one-degree decrement for annealingtemperature starting with 7∘C above 119879
119898for each primer for
30 seconds and 72∘C for 1 minute This was then followed by30 cycles of 95∘C for 40 seconds a last annealing temperaturefor 30 seconds and 72∘C for 1 minute and a final extensionof 72∘C for 10 minutes The PCR products were separated on4 metaphor gel with 50 bp DNA ladder (N3231S BiolabsInc UK) The gel was stained with Midori green visualizedunder ultraviolet light and photographed by ChemilImagerGel Documentation imaging system (Alpha Innotech Corpo-ration CA USA)
4 Data Analysis
41 Morphological Data The recorded data (after normalityand homogeneity test) were subjected to analysis of variance(ANOVA) as per two-factor experiment with three irradia-tion treatments and four varieties arranged in a randomizedcomplete block design (RCBD) with four replications Theanalysis was carried out using the portable SAS 91 programand least significant differences (LSD) were used for compar-ison among treatment means at 119875 le 005 To evaluate therelationship among the different variables in the experimentcorrelation coefficients were used by SAS 9 1 3 portable Togroup the individuals based on morphological dissimilaritycluster analysis was conducted on the Euclidean distancematrix with the unweighted Pair-GroupMethod using Arith-metic average (UPGMA) using NTSYS software The sameprogram was used for principal components analysis (PCA)to define eigenvalues and eigenvectors and also for compar-ison of the mean of groups to define effective traits in sepa-ration of the groups Eigenvectors are the weights in a lineartransformation when computing principal component scoreswhile eigenvalues indicate the amount of variance explainedby each principal component The cophenetic correlationcoefficient (CCC) was used to measure the goodness of fit ofthe similarity matrices to their corresponding phenograms inmorphological data using PAST (PAleontological Statistics)software V 217 [27]
42 Molecular Data Allele size was measured with UVDoc9902 analysis software (UVI Tech Cambridge UK) by man-ual editing to increase accuracy This procedure was carriedout two times to exclude wrong scorings The PowerMarker325 software package [28]was used to produce a dendrogramusing UPGMAmethod Data were scored as ldquo1rdquo for presenceand ldquo0rdquo for absence The binary data matrix was entered intothe Numerical Taxonomy and Multivariate Analysis System
BioMed Research International 5
Table 3 The number of irradiated and mortal rhizomes of Curcuma alismatifolia varieties after acute irradiation with different doses ofgamma rays
Dose (Gray)Total number of
irradiated rhizomes ineach var
Number of mortal rhizomes after 40 days Mortality rate ()in each doseDoi Tung 554 Chiang Mai Red Sweet Pink Kimono Pink
(NTSYSpc 210e) [29] to generate Dicersquos similarity matrixThe software POPGENE32 Version 132 [29] was used tocalculate genetic variation parameters including observedheterozygosity (the proportion of heterozygous individualsin the population) (119867
119900) expected heterozygosity (119867
119890) [30]mdash
defined as the probability that two randomly chosen allelesfrom the population are different [31]mdashobserved numberof alleles (119899
119886) effective number of alleles (119899
119890) Neirsquos gene
diversity Shannonrsquos information index (119868) and percentageof polymorphic loci To compare the efficiency of primersand polymorphism information content (PIC) a measure ofallelic diversity at a locus was calculated using online PICcalculator software (httpwwwlivacuksimkempsjpichtml)using the following formula
PIC = 1 minus119899
sum
119894=1
1199011198942minus
119899minus1
sum
119894
119899
sum
119895=119894+1
211990111989421199011198952 (1)
where 119901119894is the frequency of the 119894th allele and 119899 is the
number of alleles Markers were classified as informativewhen PICwas ge05 Principal component analysis (PCA) wasalso generated for SSR data by NTSYS-pc 210e
5 Results and Discussions
51 Gamma Irradiation and Radiation Sensitivity Test Thesensitivity of C alismatifolia varieties to radiation was eval-uated by comparing the mortality rate () of irradiatedplants at 40 days after irradiation The plant mortality rateincreased with increasing irradiation dosage (Table 3) Thehybrid Doi Tung 554 was found to be least sensitive togamma irradiation than other varieties (51mortality) whileChiangMai Red variety showed the lowest survival rate (63mortality) Sweet Pink and Kimono Pink varieties showed58 and 55 mortality rate respectively At 50 survivalrate (LD
50) the gamma doses administered were 28 21 23
and 25Gy for Doi Tung 554 Chiang Mai Red Sweet Pink
and Kimono Pink respectively (Figure 1) Abdullah et al [32]had previously indicated that the LD
50for C alismatifolia
var Chiang Mai Pink was approximately at 25Gy The deathof plants is attributed to the interaction of radiation withother molecules in the cell particularly water to produce freeradicals (H OH) The free radicals could combine to formtoxic substances such as hydrogen peroxide (H
2O2) which
contribute to the destruction of cells This indirect effectis especially significant in vegetative cells the cytoplasmwhich contains about 80 water [33] However sensitivityof the plant material depends on the genetic constitutiondose-employed DNA amount moisture content and stageof development and genotype [34] The choice of the doseto be applied for the highest mutant rescue is often left tothe breederrsquos experience with the specific plant material itsgenetics and its physiology
52 Analysis of Variance (ANOVA) for Morphological Traitsof C alismatifolia in M1V1 Analysis of variance indicatedhighly significant differences among the varieties doses andtheir interaction for all traits in M
1V1generation (Table 4)
Some desired and undesired abnormalities such as dwarfismchlorophyll mutation (albinism) striata (yellow or whitelongitudinal bands altering with green colors) two-midribleaves split leaves double flower stalk in one plant doubleinflorescence marbled pink bracts two-tone pink-purplishbracts and two-flag petals were found in M
1V1plants
(Table 5)
53 Effect of Gamma Irradiation on Vegetative Traits in theM1V1 The growth of plants treated with 10 and 20Gy ofgamma rays was slower than that of the controls (Table 6)In irradiated plants the leaf length and leaf width decreasedsignificantly (119875 lt 005) as the radiation doses increasedThis trend is quite common in mutagenised populationsSuch effects are known to arise due to drastic chromoso-mal aberrations in addition to genetic mutations Similar
6 BioMed Research International
Table4Meansquareso
fanalysis
ofvaria
nce(ANOVA
)for
14morph
ologicaltraitsin
Calism
atifolia
(a)
Source
ofvaria
tion
Meansquares
dfGeneration
Num
bero
fsho
otLeafleng
thLeafwidth
Leafnu
mber
Daystovisib
lebu
dPlanth
eight
Block
4M
1V1
005
622
11014
9467
8358
Varie
ty3
M1V
1263lowast
2702lowast
3011lowast
500lowast
1455602lowast
253739lowast
Dose
2M
1V1
611lowast
373804lowast
4137lowast
1042lowast
1012001lowast
157076
6lowastVa
rlowastdo
se6
M1V
114
1lowast8555lowast
418lowast
167lowast
731271lowast
115934lowast
Error
44M
1V1
0094
2951
077
037
4903
Total
5984
93
CV(
)M
1V1
23183
176
206
91165
(b)
Source
ofvaria
tion
Meansquares
dfGeneration
Daysto
anthesis
Daysto
senescence
Noof
true
flow
Noof
pink
bract
Inflo
rescence
leng
thNoof
new
rhizom
esRh
izom
esiz
eNoof
storage
roots
Block
4M
1V1
12847
048
418
072
282
01
018
055
Varie
ty3
M1V
11608017
10091lowast
9166lowast
62543lowast
376lowast
163lowast
023
ns1726lowast
Dose
2M
1V1
1587261
128682lowast
58286lowast
35315lowast
56022lowast
1031lowast
531lowast
6481lowast
Varlowastdo
se6
M1V
1876957
3464lowast
2900lowast
977lowast
4004lowast
128lowast
025ns
332lowast
Error
44M
1V1
7752
322
392
226
445
011
011
121
Total
59CV
()
M1V
110
15229
169
165
2318
268
lowastSign
ificant
with
leastsqu
ared
ifference
test119875lt005
BioMed Research International 7
Dose (Gy)
0
10
10
20
20
30
30
40
40
50
50
60
60
70
70
80
80
90
90
100
100
Mor
talit
y (
)
Var Doi Tung554
LD50 = 28 Gy
(a)
0
10
10
20
20
30
30
40
40
50
50
60
60
70
70
80
80
90
90
100
100
Mor
talit
y (
)
Var Chiang Mai Red
Dose (Gy)
LD50 = 21Gy
(b)
Mor
talit
y (
)
Dose (Gy)
Var Sweet Pink
0
10
10
20
20
30
30
40
40
50
50
60
60
70
70
80
80
90
90
100
100
LD50 = 23Gy
(c)
0
10
10
20
20
30
30
40
40
50
50
60
60
70
70
80
80
90
90
100
100
Mor
talit
y (
)
Dose (Gy)
Var Kimono Pink
LD50 = 25Gy
(d)
Figure 1 PoloPlus plot of linear scale of dose versusmortality percent (a) Doi Tung 554 (b) ChiangMai Red (c) Sweet Pink and (d) KimonoPink
Table 5 Effect of acute gamma rays on vegetative and flowering traits of C alismatifolia in the M1V1 generation
Variety irradiated dose Observed variations Flower color variationChiang Mai Red
10Dwarfism no pink bracts inflorescence small inflorescencetwo-flag petal true flower double inflorescence undulate leafmargin and yellowwhite strip leaves
Light purple N78Clowast
20 Dwarfism narrow small leaves and yellowwhite strip leaves No flowerDoi Tung 554
20 Dwarfism fewer pink bracts and yellowwhite strip leavesSmall and narrow leave Light purple N80Dlowast
lowastRoyal British Society color chart (RHS)
8 BioMed Research International
Table 6 Effect of acute gamma rays on vegetative traits of C alismatifolia in M1V1 generation
Dose (Gray) Shoot number Leaf number Leaf length (cm) Leaf width (cm) Plant height (cm)CMR
0 20 plusmn 00a
30 plusmn 04a
556 plusmn 08a
71 plusmn 05a
1112 plusmn 25a
10 10 plusmn 00b
36 plusmn 05a
272 plusmn 25b
45 plusmn 05b
714 plusmn 96b
20 10 plusmn 00b
14 plusmn 05b
124 plusmn 120c
25 plusmn 24c
170 plusmn 80c
DT0 30 plusmn 00
a30 plusmn 00
a622 plusmn 08
a65 plusmn 05
a912 plusmn 19
a
10 16 plusmn 05b
28 plusmn 04a
280 plusmn 44b
46 plusmn 06b
542 plusmn 76b
20 12 plusmn 04b
24 plusmn 05a
242 plusmn 38b
38 plusmn 05c
512 plusmn 138b
SP0 16 plusmn 05
a32 plusmn 04
a461 plusmn 27
a101 plusmn 04
a746 plusmn 38
a
10 10 plusmn 00b
36 plusmn 05a
275 plusmn 34b
57 plusmn 02b
377 plusmn 137b
20 10 plusmn 00b
20 plusmn 10b
196 plusmn 74c
57 plusmn 04b
195 plusmn 73c
KP0 14 plusmn 00
a46 plusmn 05
a345 plusmn 29
a47 plusmn 04
a591 plusmn 28
a
10 10 + 05a
40 plusmn 05b
220 plusmn 24b
31 plusmn 04b
420 plusmn 54b
20 10 plusmn 00a
32 plusmn 04b
156 plusmn 43c
31 plusmn 11b
283 plusmn 174c
CV () 23 18 26 16 165Means with the same or common letter are not significantly different least square difference test 119875 lt 005
decreases in leaf size were reported by Pongchawee et al [35]and Tangpong et al [36] These results were in agreementwith an earlier study [4] which reported that the growthof chrysanthemum exposed to acute gamma rays was lessthan the control in the M
1V1generation All varieties doses
and interaction effects resulted in significant differences fornumber of leaves Among untreated plants Kimono Pinkvariety had higher number of leaves (46) than the otherthree (3 3 and 32) varieties In Chiang Mai Red and SweetPink varieties plants exposed to 10Gy showedhigher numberof leaves than untreated plants However at 20Gy therewas significant reduction in number of leaves for all studiedvarieties in comparison to control Similar stimulatory effectswere obtained at lower doses in ginger by Hegde [37] andGiridharan and Balakrishnan [38]
Progressive reduction in growth parameters can be inter-preted on interference in normal mitosis and frequent occur-rence of mitotic aberrations inhibition of rate of assimilationand consequent change in the nutrient level in the plantand inactivation of vital enzymes especially those associatedwith respiration [39] Dose-dependent negative effect wasalso detected for plant heightThe tallest plants were recordedfrom the untreated rhizomes (0Gy) with heights of 1112912 746 and 591 cm followed by the 10Gy irradiated plantswith heights of 714 542 377 and 420 cm and the 20Gyirradiated plants with corresponding heights of 170 512195 and 283 cm for Chiang Mai Red Doi Tung 554 SweetPink and Kimono Pink varieties respectively These resultsare in agreement with the findings of Abdullah et al [32]Reduction in growth parameters and dwarfism can be causedby interference of normal mitosis and frequent occurrenceof mitotic aberrations inhibition of assimilation rates andconsequent changes in nutrient levels in plants Additionallymutagenic effects such as auxin destruction inhibition of
auxin synthesis failure of assimilatory mechanism andchanges in the specific activity of enzymes can cause growthreductions [37] High doses of ionizing radiation have beenshown to damage macromolecular cellular components suchas cell walls membranes and DNA [40] The number ofshoots also decreased significantly as the radiation dosesincreased Radiation also affects organic molecules that areessential to the cell division process and thus causing celldivision to stop [36]
54 Effect of Gamma Irradiation on Flowering DevelopmentTraits in the M1V1 All control and 10Gy irradiated plantsproduced flowers while the Chiang Mai Red and SweetPink varieties which were exposed to 20Gy did not gointo the flowering stage Lamseejan et al [4] also showedthat flowering percentage decreases as gamma ray dosesare increased In the present study gamma rays caused lateflowering in all four varieties Days to appearance of firstvisible buds were also significantly different among the fourvarieties (Table 7) Gamma rays caused a noticeable delay inflowering of irradiated plants in comparison to the untreatedones First visible buds were observed at 652 and 874 days inthe control and 10Gy treatments respectively for the ChiangMai Red variety In Doi Tung 554 the first visible buds wereappeared at 474 656 and 840 days after planting at 0 10and 20Gy doses respectively In the Sweet Pink variety thenumber of days to visible bud appearance increased signif-icantly from 678 days in controls to 978 days in the 10Gyirradiated plants In comparison to other three varieties theKimono Pink variety needed the longest time to visible budappearance and same as other varieties there was a positivecorrelation between the number of days to first visible budand the gamma irradiation dose In the control and 10Gyand 20Gy irradiated individual plants flower buds were
BioMed Research International 9
Table 7 Effect of acute gamma rays on flower development characteristics of C alismatifolia in M1V1 generation
Dose (Gy) Days tovisible bud
Inflorescencelength (cm)
Days toanthesis
Number oftrue flowers
Number ofPink bracts
Days tosenescence
CMR0 652 plusmn 47
b162 plusmn 057
a742 plusmn 54
b132 plusmn 19
a104 plusmn 15
a21 plusmn 10
a
10 874 plusmn 95a
82 plusmn 397b
1020 plusmn 71a
56 plusmn 19b
50 plusmn 07b
10 plusmn 14b
20 00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
DT0 474 plusmn 43
c134 plusmn 054
a546 plusmn 54
c162 plusmn 13
a232 plusmn 04
a23 plusmn 00
a
10 656 plusmn 37b
94 plusmn 19b
780 plusmn 37b
112 plusmn 21b
166 plusmn 08b
122 plusmn 130b
20 840 plusmn 42a
76 plusmn 082c
994 plusmn 49a
88 plusmn 13b
156 plusmn 11b
106 plusmn 18b
SP0 678 plusmn 21
b146 plusmn 089
a756 plusmn 26
b162 plusmn 13
a100 plusmn 24
a188 plusmn 083
a
10 978 plusmn 181a
79 plusmn 20b
1098 plusmn 182a
68 plusmn 25b
52 plusmn 08b
92 plusmn 24b
20 00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
KP0 852 plusmn 23
c137 plusmn 044
a942 plusmn 19
c112 plusmn 12
a96 plusmn 21
a18 plusmn 18
a
10 1044 plusmn 65b
108 plusmn 108ab
1224 plusmn 100b
76 plusmn 27b
64 plusmn 16ab
102 plusmn 17b
20 1272 plusmn 65a
790 plusmn 49b
1426 plusmn 58a
60 plusmn 38b
42 plusmn 24b
66 plusmn 37b
CV () 10 23 9 22 169 15Means with the same or common letter are not significantly different least square difference test 119875 lt 005
visible at 852 1328 and 1374 days after planting respectivelyPrevious studies also showed that onset of flowering andformation of floral parts in mutants of Arabidopsis thalianamaize barely pea and tobacco involved growth regulators(or phytohormones) such as auxins cytokinins gibberellinsabscisic acid ethylene and brassinosteroids [41] There weresignificant differences among treatments for the length ofthe inflorescence In all varieties the longest inflorescencelength was observed in the untreated plants with 162134 146 and 137 cm lengths for Chiang Mai Red DoiTung 554 Sweet Pink and Kimono Pink respectively Thecorresponding inflorescence lengths were 82 94 79 and109 cm for the 10Gy irradiated plants The days to anthesisfor C alismatifolia varieties were significantly affected byvariety gamma irradiation doses and their interaction Thenumber of days to full bloom was noticeable earliest foruntreated plants at 742 546 756 and 942 days for ChiangMai Red Doi Tung 554 Sweet Pink and Kimono Pinkvarieties respectively This was then followed by plants at10Gy at 102 78 1098 and 1224 days The number of trueflowers or the secondary inflorescence developed in the axilof the primary bracts decreased as radiation dosage increasedIn the present study the gamma rays also decreased thedays to inflorescence senescence In this study there wasa strongly significantly and positively correlation (0919lowastlowast)(data not shown) between the number of true flowers andthe number of days to senescenceThe number of pink bractsalso decreasedwith increasing radiation dosageMost gammaray effects on senescence are considered as resulting from theaction of free radicals generated from water and oxygen bythe ionizing energy on the cellular components Membrane
deterioration is a general feature of natural senescence andstress-induced aging [42]
Irradiation induced some mutation spectrum of flowercolor variation that included colors such as purple palepurple rather pale purple white purple white (marbledpattern) and two-tone purple color
Mutation spectrum of flower shape variation includeddouble inflorescence within one stalk double stalk per plantinflorescence without bracts two-flag petal true flowersand chlorophyll mutation in the leaves which are generallycaused by induced gamma rays (Figure 2) Ionizing radiationincluding gamma rays induces fragment deletions or inser-tions that eventually lead to changes in amino acids and amodification of leaf and stem pigmentation [43] A mutationin the biosynthetic pathway of structural or regulatory genesmay cause a change in flower color [44] When the blockageoccurs at the early stages of anthocyanin synthesis whiteflowers will result while a blockage at later stages leads todifferent flower colors due to the accumulation of particularanthocyanins [45] Chloroplasts were extremely sensitive togamma radiation compared to other cell organelles [46]
55 Effect of Gamma Irradiation on Rhizome Characteristicsin Selected Doses in M1V1 The number of new rhizomes andthe number of storage roots per rhizome were significantlyaffected by varieties doses and their interaction (Table 8)The number of new rhizomes only in Kimono Pink varietydid not show any differences between untreated and treatedplants The rhizome size was influenced only by doses Asdose level increased the rhizome size decreased Amonguntreated plants the Sweet Pink rhizomes had the most
10 BioMed Research International
(a) (b) (c) (d)
(e)
(f)
Figure 2 Effect of acute gamma rays on flower traits in C alismatifolia (a) Untreated inflorescence bracts color Doi Tung 554 (b) Two tonebract color Doi Tung 554 (20Gy) (c) Marbled pattern of bract color Doi Tung 554 (20Gy) (d) Double inflorescence within one stalk varChiang Mai Red (10Gy) (e) True flower in nontreated plants (f) Two-flag petals true flowers (10Gy)
Figure 3 Dendrogram representing themorphological variation among 44 irradiated and nonirradiated individuals ofC alismatifolia across14 variables CMR = Chiang Mai Red DT = Doi Tung 554 SP = Sweet Pink Kp = Kimono Pink
number of storage roots (78) and the Kimono Pink rhizomeshad the least number of storage roots (40)This was followedby 10Gy irradiated rhizomes with 4 46 48 and 28 storageroot numbers for Chiang Mai Red Doi Tung 554 SweetPink and Kimono Pink respectively Irradiation with 20Gydecreased significantly the number of storage roots in allstudied varietiesTheplantC alismatifolia is propagated froma propagule (one rhizome + 5-6 t-roots) The rhizome sizedose matters in the growth of the plant Smaller rhizome sizeusually resulted in smaller plant size with narrow grass-likeleaves The storage roots play a very important role in thegrowth and flowering ofCurcumaThe t-rootsmake up about85 of the total fresh weight and 70 of the total dry weightof a typical propaguleThe storage organs act for plant growth
during dormancy and emergence [47] More storage root perpropagule resulted in faster flowering and higher yield plant[48] In this study also there was significant and negativecorrelation between number of storage roots and number ofdays to visible bud appearance (minus0525lowastlowast)
56 Cluster Analysis of C alismatifolia for MorphologicalTraits The morphological data were used to calculate thesimilarity between the treated and non-treated C alismat-ifolia samples and UPGMA dendrogram was constructed(Figure 3) The cophenetic correlation coefficient (CCC)value between the genetic dissimilaritymatrix estimated fromthe morphological characters and the UPGMA clusteringmethod was 119903 = 093 showing a proof fit This value was
BioMed Research International 11
Table 8 Effect of acute gamma rays on rhizome characteristics ofC alismatifolia in M1V1 generation
Dose (Gray) Rhizomesize
No of newrhizomes
No of storageroot
CMR0 23 plusmn 02
a26 plusmn 055
a46 plusmn 054
a
10 18 plusmn 005a12 plusmn 044
b40 plusmn 07
a
20 08 plusmn 07b
06 plusmn 054b
14 plusmn 13b
DT0 22 plusmn 01
a30 plusmn 00
a72 plusmn 109
a
10 18 plusmn 007b14 plusmn 054
b46 plusmn 13
b
20 16 plusmn 005b
10 plusmn 00b
28 plusmn 13c
SP0 22 plusmn 02
a24 plusmn 054
a78 plusmn 21
a
10 17 plusmn 007b
10 plusmn 00b
48 plusmn 044b
20 12 plusmn 02c
10 plusmn 00b
28 plusmn 083b
KP0 21 plusmn 005
a10 plusmn 00
a40 plusmn 00
a
10 16 plusmn 01ab
10 plusmn 00a
28 plusmn 044b
20 11 plusmn 06b
10 plusmn 00a
22 plusmn 083b
CV () 18 23 26Means with the same or common letter are not significantly different leastsquare difference test 119875 lt 005
higher than of studies on olive for morphological traits (119903 =069) [49] In this dendrogram the 44C alismatifolia samplesappeared to form three main clusters and five minor clustersat coefficient level 56 cluster I included three non-irradiatedmembers and cluster II included 10Gy irradiated individualplants of all four varieties and 20Gy irradiated samples ofDoi Tung 554 and Kimono Pink varieties except KP20-4 (noflowering) Main cluster III included the 20Gy irradiatedindividuals Chiang Mai Red and Sweet Pink varieties (asmentioned in morphological part these individuals didnot go to flowering stage) and KP20-4 Cluster II can bedivided to five subclusters The first subcluster included 10members (CMR10-1 CMR10-5 SP10-1 SP10-2 SP10-3 SP10-5 CMR10-2 CMR10-4 SP10-4 and CMR10-3) the secondone included nine members (KP10-1 KP10-2 KP10-3 KP10-4 KP10-5KP20-1 KP20-2 KP20-5 and KP20-3) KP0 wasthe lone member of the third sub-cluster The fourth sub-cluster included two members only (DT10-2 and DT10-3) The last sub-cluster main cluster II had eight membersincluded (DT10-1 DT10-4 DT10-5 DT20-1 DT20-2 DT20-3 DT20-4 and DT20-5) Mean value of groups for each traitis presented in Table 9 This table clearly shows the differentmean values of the three main clusters The minimum meanvalue referred to main cluster III while the maximum meanvalue for number of shoots leaf length leaf width plantheight number of true flowers inflorescence length rhizomesize number of new rhizomes and number of storage rootsbelonged to cluster I which was included non-treated plantsThese results showed that the gamma irradiation has inducedmorphological changes in C alismatifolia individuals of
four studied varieties so that they showed phenotypicallydifferences from their controls
57 Principal Component Analysis of C alismatifolia forMorphological Traits To assess the patterns of variation PCAwas done by considering all of the 14 variables The first threecomponents of PCA explained 828 of the total variation(Table 10) Only the first component which accounted for562 of the total variation was attributed to inflorescencelength plant height days to senescence number of trueflowers and rhizome size In PCA three-dimensional graphthe grouping of individuals confirmed the clustering results(Figure 4) The PCA graph proved that all irradiated indi-vidual plants of C alismatifolia varieties are phenotypicallydifferent from their non-irradiated individuals
6 Molecular Characterization
61 SSR Polymorphism Amplifications were successful forall the 17 SSR markers assayed This reflects a high levelof homology between SSR flanking regions in C longa andC alismatifolia Out of 17 primer pairs eight primer pairsresulted in polymorphic PCR products Table 11 summarizesthe results obtained based on the analysis of individuals of thefour studied varieties using the polymorphic SSR loci for 010 and 20Gy irradiated plants The number of alleles variedwidely among these loci A total of 25 36 and 41 alleles wereobserved among 0 10 and 20Gy irradiated individual plantsrespectively In the 10Gy treatment the number of allelesranged from three (clon09 and clon14) to seven (clon08) withan average value of 45 per locus In the 20Gy treatmentthe number of alleles varied from three (clon09 and clon14)to seven (clon08 and clon12) with an average value of 51per locus In the untreated individuals the number of allelesranged from two (clon04 clon09 clon11 and clon14) to five(clon12) with an average value of 31 The difference betweenthe average number of observed alleles and effective numberof alleles was due to the uneven frequency of each allele[50] For each of the eight SSR primers PIC values (whichmeasures allele diversity and frequency among varieties)varied from 019 (clon04) to 071 (clon01) in untreated plantsand from 025 (clon04) to 075 (clon08) in 10Gy acutelyirradiated plants In 20Gy irradiated individual plants thePIC value ranged from 042 (clon14) to 075 (clon08 andclon12) The mean PIC for all loci was 047 054 and 061for 0 10 and 20Gy irradiated plants respectively
The PIC value provides an estimate of the discriminatorypower of amarker by taking into account not only the numberof alleles at a locus but also the relative frequencies of thesealleles [51] Thus except for clon09 and clon14 which weremoderately polymorphic (025 lt PIC lt 05) all other lociwere highly polymorphic (05 lt PIC) while none of theloci showed low polymorphism The average discriminatingpower ofmicrosatellitemarkers (061) observed in the presentstudy ensures the future utility of microsatellite markersfor genetic variation studies in C alismatifolia varietiesOur results reflect similar findings as reported earlier inthe study of genetic variation in other species of Curcuma
12 BioMed Research International
Table9Clusterm
eans
for14charactersestim
ated
in44
individu
also
fCalismatifolia
Clusters
Shoo
tnu
mber
Leaf
leng
thLeaf
width
Leaf
number
Visib
lebu
d(days)
Inflo
rescence
leng
thPlant
height
Anthesis
(days)
Senescence
(days)
True
flow
number
Pink
bract
number
Rhizom
esiz
eNew
rhizom
enu
mber
Storage
root
number
I220
5464
736
300
6013
1473
9233
6926
2093
152
1453
229
266
653
II1
100
2735
512
360
9260
809
5457
1059
960
620
510
177
110
440
II2
122
1922
298
344
11422
1043
3910
1312
293
3755
588
156
100
266
II3
100
3452
416
460
8520
1370
5910
9420
180
120
960
206
100
400
II4
150
2500
45
250
6500
925
4950
7700
1150
950
1700
185
200
600
II5
137
2637
417
262
7725
840
5356
9162
1137
1012
1587
170
100
312
III
054
1500
465
181
000
000
000
000
000
000
000
090
081
200
BioMed Research International 13
Table 10 Eigenvectors eigenvalues and proportions of variabilityfor three principle components among 14 characters for 44 Calismatifolia
Variable PC1 PC2 PC3Number of new shoot 0270 minus0076 minus0173Leaf length 0269 minus0305 0033Leaf width 0096 minus0455 0430Leaf number 0210 0285 0490Days to visible bud 0199 0462 0181Plant height 0321 0027 minus0071Inflorescence length 0318 0166 minus0040Days to anthesis 0205 0460 0148Days to senescence 0342 0017 minus0131Number of true flower 0323 0029 minus0238Number of pink bracts 0266 minus0028 minus0546Rhizome size 0302 minus0101 0147Number of new rhizome 0250 minus0293 0028Number of storage roots 0264 minus0247 0298Eigenvalue 7877 2694 1032Proportion 56264 19246 7371Cumulative 56264 75511 82882
using SSR markers [13] The highest Neirsquos gene diversity(ℎ) was obtained with the 20Gy treatment (061) followedby the 10Gy treatment (06) and the untreated individuals(05) The mean Shannonrsquos information index (119868) was 092114 and 130 in the 0 10 and 20Gy irradiated plantsrespectively The high value of Shannonrsquos information indexrepresents the effectiveness of microsatellite loci to revealthe variation among these varieties at the different dosesused Overall genetic variability for the varieties studiedrepresented by the Shannon-Weiner index was relatively highin comparison to other studies involving C longa accessions[10 52] Additionally the 20Gy acutely irradiated individualsshowed a higher mean percentage of polymorphic loci (63)than the 10Gy (58) and untreated (022) ones This impliesthat irradiation with a dose of 20Gy induced more geneticvariation in the M
1V1generation
62 Cluster Analysis Thedendrogramwas constructed usingPowerMarker 323The 44 studied individuals were clusteredinto seven groups (G1ndashG7) Colors were applied accordingto our model-based cluster analysis results Group I iscomprised of nine individual plants of Chiang Mai Redvariety receiving 0 10 and 20Gy treatments (blue colorin Figure 5) One individual plant DT10-1 was assignedto group II (green color in Figure 5) All irradiated andnon-irradiated individuals of Doi Tung 554 variety wereassigned to group III (yellow color in Figure 5) DT20-1 andSP10-5 were the sole members of groups IV (red color inFigure 5) and V (gray color in Figure 5) respectively GroupVI included treated and untreated individuals of Sweet Pinkvariety (pink color in Figure 5) Lastly all Kimono Pinkindividuals were assigned to cluster VII (orange color in
Figure 5) The genetic similarity coefficient (data not shown)among the 44 individuals amplified using eight SSR primersvaried from 00 to 10 The highest value (10) correspondedto [(CMR10-3 CMR10-5 CMR and CMR20-2) (DT10-2and DT10-3) (DT and DT20-2) (KP20-3 KP-20-4 KP10-3andKP10-4)] individuals that generated identical fingerprintsacross the markers studied Among the studied varieties theSweet Pink individual plants showed the lowest similarity(00) withKimono Pink individuals which indicated that theywere relatively remote in relationship (Figure 5) An overviewof the clustering pattern indicates that the grouping of thestudied individual plants was based on different varieties andlargely independent of the doses of gamma irradiation Awide range in similarity values had also been observed indifferent species of Curcuma [12 13 52 53]
63 Principle Component Analysis (PCA) Thedata generatedfrom 44 C alismatifolia individual plants were subjected toprincipal component analysis (PCA) to visualize individualsin a multivariate space In the three-dimensional graphderived from the SSR analysis all studied individuals weregrouped into seven clusters (Figure 6) In PCA the firstthree principal components (PC) extracted a cumulative of6826 of the total variation among the 44 individuals of CalismatifoliaThe first three coordinates PC1 PC2 and PC3accounted for 4136 1578 1112 of the total variationrespectively The distribution of the individual plants in athree-dimensional plot using the first three principal com-ponents showed the genetic relationship between individualplants It was evident that both methods dendrogram andthree-dimensional plots of PCA were effective in illustratinggenetic relationships and the groups found were comparable[11 13] The PCA results were similar to those obtained fromcluster analysis where all individuals from different varietieswere assigned to different groups These graphical illus-trations enable the assessment of the relationshipdistancesamong all of the individuals in the study [50]
7 Conclusion
This is the first attempt to evaluate the effect of acutegamma irradiation on C alismatifolia varieties using bothmorphological characteristics and molecular markers Inplant breeding programs mutagenic treatments with lownegative physiological effects and strong genetic effects aredesirable Hence we used more effective doses of gammairradiation (10 and 20Gy) of which particularly 20Gy dosewas effective to influence morphological and molecularcharacteristics of studiedCurcuma alismatifolia varietiesThelower dose (10Gy) of radiation probably caused little damageto the plants genetic material so that the cells could repairthemselves in next generation As a result in this study 20Gyacute gamma irradiation resulted in a higher percentage ofmutation and getting desired mutants was more possibleBased on the LD
50values determined in this study it was
apparent that the varieties Chiang Mai Red and Sweet Pinkwere more sensitive to gamma rays compared to Doi Tung554 and Kimono Pink Our results show that the variety Doi
Figure 4 Two-dimensional graph of principal component analysis (PCA) for 14 morphological variables indicating relationships amongirradiated and nonirradiated four varieties of C alismatifolia
CMR10-1CMR10-5
SP10-1SP10-2SP10-3
SP10-5
CMR10-4
SP10-4
KP10-1KP10-3
KP20-1
KP20-2
KP10-5KP20-5
KP20-3
KP10-2
DT10-2
DT10-1
DT10-5
DT20-1
DT10-4DT20-4DT20-2
DT20-5DT20-3
CMR20-1
SP20-3SP20-4
SP20-5SP20-1
SP20-2
KP
SP
CMR 10-2
SP10-5
Figure 5 Unrooted neighbor joining tree showing genetic relationship among 44 irradiated and nonirradiated C alismatifolia using SSRmarkers The colors of the branches correspond to those of the same cluster
Figure 6Three-dimensional graph of principal component analysis (PCA) indicating relationships among irradiated and nonirradiated fourvarieties of C alismatifolia
Tung 554 (Curcuma hybrid) had the most morphologicalresponses to gamma rays The use of microsatellite markersas a codominant marker will facilitate the exploration ofgenetic variability among treated and non-treated plants andwill help to distinguish the plants showing differences inmorphological characters The overall effects on the M
1V1
generation revealed that acute gamma irradiation at optimumdoses has the potential for developing new varieties of Calismatifolia with improved commercial properties suitablefor the Malaysian flower industry
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work was funded by the Fundamental Research GrantScheme (FRGS) under the Ministry of Higher Education inMalaysia
References
[1] P Apavatjrut S Anuntalabhochai P Sirirugsa and C AlisildquoMolecularmarkers in the identification of some early floweringCurcuma L (Zingiberaceae) speciesrdquo Annals of Botany vol 84no 4 pp 529ndash534 1999
[2] K Bunya-Atichart S Ketsa andW G VanDoorn ldquoPostharvestphysiology of Curcuma alismatifolia flowersrdquo Postharvest Biol-ogy and Technology vol 34 no 2 pp 219ndash226 2004
[3] M Nakano J Amano Y Watanabe et al ldquoMorphologicalvariation in Tricyrtis hirta plants regenerated from heavy ionbeam-irradiated embryogenic callusesrdquo Plant Biotechnologyvol 27 no 2 pp 155ndash160 2010
[4] S Lamseejan P Jompuk A Wongpiyasatid A Deeseepan andP Kwanthammachart ldquoGamma-rays induced morphological
[5] G-J Lee S J Chung I S Park et al ldquoVariation in thephenotypic features and transcripts of colormutants of chrysan-themum (Dendranthema grandiflorum) derived from gammaraymutagenesisrdquo Journal of Plant Biology vol 51 no 6 pp 418ndash423 2008
[6] O K Kikuchi ldquoOrchid flowers tolerance to gamma-radiationrdquoRadiation Physics and Chemistry vol 57 no 3ndash6 pp 555ndash5572000
[7] N P Arnold N N Barthakur and M Tanguay ldquoMutageniceffects of acute gamma irradiation on miniature roses targettheory approachrdquo HortScience vol 33 no 1 pp 127ndash129 1998
[8] A A Youssef M S Aly and M S Hussein ldquoResponse ofgeranium (Pelargonium graveolenus L) to gamma irradiationand foliar application of Speed Growrdquo Egyptian Journal ofHorticulture vol 27 pp 41ndash53 2000
[9] N Chuantang and L Yazhi ldquoThe radiation inducedmutation ofcanna (Canna L)rdquo Acta Agricultute Nucleatae Sinica vol 2 pp33ndash39 1998
[10] M S Sigrist J B Pinheiro J A A Filho and M I ZucchildquoGenetic diversity of turmeric germplasm (Curcuma longaZingiberaceae) identified by microsatellite markersrdquo Geneticsand Molecular Research vol 10 no 1 pp 419ndash428 2011
[11] S Taheri T L Abdullah N A P Abdullah and Z AhmadldquoGenetic relationships among five varieties of Curcuma alis-matifolia (Zingiberaceae) based on ISSRmarkersrdquoGenetics andMolecular Research vol 11 pp 3069ndash3076 2012
[12] A Das V Kesari V M Satyanarayana A Parida and L Ran-gan ldquoGenetic relationship of Curcuma species from NortheastIndia using PCR-based markersrdquo Molecular Biotechnology vol49 no 1 pp 65ndash76 2011
[13] S Siju K Dhanya S Syamkumar et al ldquoDevelopment char-acterization and utilization of genomic microsatellite markersin turmeric (Curcuma longa L)rdquo Biochemical Systematics andEcology vol 38 no 4 pp 641ndash646 2010
[14] M K Panda S Mohanty E Subudhi L Acharya and SNayak ldquoAssessment of genetic stability of micropropagated
BioMed Research International 17
plants of Curcuma L by cytophotometery and RAPD analysisrdquoInternational Journal of Integrative Biology (IJIB) vol 1 pp 189ndash195 2007
[15] S Syamkumar and B Sasikumar ldquoMolecular marker basedgenetic diversity analysis of Curcuma species from IndiardquoScientia Horticulturae vol 112 no 2 pp 235ndash241 2007
[16] D Tautz and M Renz ldquoSimple sequences are ubiquitousrepetitive components of eukaryotic genomesrdquo Nucleic AcidsResearch vol 12 no 10 pp 4127ndash4138 1984
[17] G Toth Z Gaspari and J Jurka ldquoMicrosatellites in differenteukaryotic genomes surveys and analysisrdquo Genome Researchvol 10 no 7 pp 967ndash981 2000
[18] W Powell G C Machray and J Proven ldquoPolymorphismrevealed by simple sequence repeatsrdquo Trends in Plant Sciencevol 1 no 7 pp 215ndash222 1996
[19] S-Y Lee W K Fai M Zakaria et al ldquoCharacterizationof polymorphic microsatellite markers isolated from ginger(Zingiber officinale Rosc)rdquo Molecular Ecology Notes vol 7 no6 pp 1009ndash1011 2007
[20] S Bory D Da Silva A-M Risterucci M Grisoni P Besseand M-F Duval ldquoDevelopment of microsatellite markers incultivated vanilla polymorphism and transferability to othervanilla speciesrdquo Scientia Horticulturae vol 115 no 4 pp 420ndash425 2008
[21] I C Menezes F W Cidade A P Souza and I C SampaioldquoIsolation and characterization ofmicrosatellite loci in the blackpepper Piper Nigrum L (Piperaceae)rdquo Conservation GeneticsResources vol 1 no 1 pp 209ndash212 2009
[22] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[23] V Kumar Morphological and molecular characterization ofinduced mutants in Groundnut [PhD thesis] University ofAgricultural Sciences Dharwad India 2008
[24] J R Sharma Statistical and Biometrical Techniques in PlantBreeding New Age International New Delhi India 1998
[25] J J Doyle and J L Doyle ldquoA rapid DNA isolation procedure forsmall quantities of fresh leaf tissuerdquo Phytochemical Bulletin vol19 pp 11ndash15 1987
[26] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[27] Oslash Hammer D A T Harper and P D Ryan ldquoPast paleontolog-ical statistics software package for education and data analysisrdquoPalaeontologia Electronica vol 4 no 1 pp 4ndash9 2001
[28] K Liu and S V Muse ldquoPowerMaker an integrated analysisenvironment for genetic maker analysisrdquo Bioinformatics vol 21no 9 pp 2128ndash2129 2005
[29] F J RohlfNTSYS-Pc Numerical Taxonomy System Version 2 1Exeter Publishing Setauket New York NY USA 2002
[30] M Nei ldquoAnalysis of gene diversity in subdivided populationsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 70 no 12 1973
[31] S Kadkhodaei M Shahnazari M K Nekouei et al ldquoA compar-ative study of morphological and molecular diversity analysisamong cultivated almonds (Prunus dulcis)rdquo Australian Journalof Crop Science vol 5 no 1 pp 82ndash91 2011
[32] T L Abdullah J Endan and B M Nazir ldquoChanges in flowerdevelopment chlorophyll mutation and alteration in plant
morphology of Curcuma alismatifolia by gamma irradiationrdquoAmerican Journal of Applied Sciences vol 6 no 7 pp 1436ndash14392009
[33] E Kovacs and A Keresztes ldquoEffect of gamma and UV-BCradiation on plant cellsrdquoMicron vol 33 no 2 pp 199ndash210 2002
[34] H L Ramesh Y Murthy and V N Munirajappa ldquoEffect ofgamma radiation on morphological and growth parametersof Mulberry variety M5rdquo International Journal of Science andNature vol 3 pp 447ndash452 2010
[35] K Pongchawee R Pradissan andW Pipatcharoenchai ldquoInducemutation in Anubias spp through in vitro Irradiationrdquo ThaiFisheries Gazette vol 60 pp 493ndash497 2007
[36] P Tangpong T Taychasinpitak C Jompuk and P JompukldquoEffects of acute and chronic gamma irradiations on in vitroculture ofAnubias congensisNE BrownrdquoKasetsart Journal vol43 no 3 pp 449ndash457 2009
[37] R K Hegde Studies on induced mutagenesis and in vitroregeneration in turmeric (Curcuma longa L) [PhD thesis]University of Agricultural Sciences Dharwad India 2006
[38] M P Giridharan and S Balakrishnan ldquoGamma ray inducedvariability in vegetative and floral characters of ginger IndianCocoardquo Arecanut and Spices Journal vol 15 pp 68ndash672 1992
[39] A P Cesarett ldquoEffect of radiation on higher plants and plantcommunitiesrdquo inRadiation Biology United States Atomic EnergyCommission pp 284ndash309 Washington DC USA 1968
[40] G W Seung Y C Byung J-H Kim et al ldquoUltrastructuralchanges of cell organelles in Arabidopsis stems after gammairradationrdquo Journal of Plant Biology vol 48 no 2 pp 195ndash2002005
[41] B S Ahloowalia and M Maluszynski ldquoInduced mutationsmdashanew paradigm in plant breedingrdquo Euphytica vol 118 no 2 pp167ndash173 2001
[42] R Voisine L-P Vezina and C Willemot ldquoInduction ofsenescence-like deterioration of microsomal membranes fromcauliflower by free radicals generated during gamma irradia-tionrdquo Plant Physiology vol 97 no 2 pp 545ndash550 1991
[43] N Shikazono Y Yokota S Kitamura et al ldquoMutation rate andnovel tt mutants of Arabidopsis thaliana induced by carbonionsrdquo Genetics vol 163 no 4 pp 1449ndash1455 2003
[44] T Nakatsuka M Nishihara K Mishiba and S YamamuraldquoTwo different mutations are involved in the formation ofwhite-flowered gentian plantsrdquo Plant Science vol 169 no 5 pp949ndash958 2005
[45] M Mato T Onozaki Y Ozeki et al ldquoFlavonoid biosynthesisin white-flowered Sim carnations (Dianthus caryophyllus)rdquoScientia Horticulturae vol 84 no 3-4 pp 333ndash347 2000
[46] S G Wi B Y Chung J-S Kim et al ldquoEffects of gammairradiation on morphological changes and biological responsesin plantsrdquoMicron vol 38 no 6 pp 553ndash564 2007
[47] F Lee Chin Effects of light intensity and daylength on growth andflowering of siam tulip (Curcuma alismatifolia var Chiang MaiPink) [PhD thesis] University Putra Malaysia 2007
[48] A Hagiladi N Umiel and X H Yang ldquoCurcuma alismatifoliaII Effects of temperature and daylength on the development offlowers and propagulesrdquo Acta Horticulture vol 430 pp 755ndash761 1997
[49] MHagidimitriou A Katsiotis GMenexes C Pontikis andMLoukas ldquoGenetic diversity of major greek olive cultivars usingmolecular (AFLPs and RAPDs) markers and morphologicaltraitsrdquo Journal of the American Society for Horticultural Sciencevol 130 no 2 pp 211ndash217 2005
18 BioMed Research International
[50] N Babaei N A P Abdullah G Saleh and T L AbdullahldquoIsolation and characterization of microsatellite markers andanalysis of genetic variability in Curculigo latifolia DryandrdquoMolecular Biology Reports vol 39 pp 9869ndash9877 2012
[51] B Shiran N Amirbakhtiar S Kiani S Mohammadi B ESayed-Tabatabaei and H Moradi ldquoMolecular characterizationand genetic relationship among almond cultivars assessed byRAPD and SSR markersrdquo Scientia Horticulturae vol 111 no 3pp 280ndash292 2007
[52] S Singh M K Panda and S Nayak ldquoEvaluation of geneticdiversity in turmeric (Curcuma longa L) using RAPD and ISSRmarkersrdquo Industrial Crops and Products vol 37 no 1 pp 284ndash291 2012
[53] Y Paisooksantivatana S Kako and H Seko ldquoGenetic diversityof Curcuma alismatifolia Gagnep (Zingiberaceae) in Thailandas revealed by allozyme polymorphismrdquo Genetic Resources andCrop Evolution vol 48 no 5 pp 459ndash465 2001
(NTSYSpc 210e) [29] to generate Dicersquos similarity matrixThe software POPGENE32 Version 132 [29] was used tocalculate genetic variation parameters including observedheterozygosity (the proportion of heterozygous individualsin the population) (119867
119900) expected heterozygosity (119867
119890) [30]mdash
defined as the probability that two randomly chosen allelesfrom the population are different [31]mdashobserved numberof alleles (119899
119886) effective number of alleles (119899
119890) Neirsquos gene
diversity Shannonrsquos information index (119868) and percentageof polymorphic loci To compare the efficiency of primersand polymorphism information content (PIC) a measure ofallelic diversity at a locus was calculated using online PICcalculator software (httpwwwlivacuksimkempsjpichtml)using the following formula
PIC = 1 minus119899
sum
119894=1
1199011198942minus
119899minus1
sum
119894
119899
sum
119895=119894+1
211990111989421199011198952 (1)
where 119901119894is the frequency of the 119894th allele and 119899 is the
number of alleles Markers were classified as informativewhen PICwas ge05 Principal component analysis (PCA) wasalso generated for SSR data by NTSYS-pc 210e
5 Results and Discussions
51 Gamma Irradiation and Radiation Sensitivity Test Thesensitivity of C alismatifolia varieties to radiation was eval-uated by comparing the mortality rate () of irradiatedplants at 40 days after irradiation The plant mortality rateincreased with increasing irradiation dosage (Table 3) Thehybrid Doi Tung 554 was found to be least sensitive togamma irradiation than other varieties (51mortality) whileChiangMai Red variety showed the lowest survival rate (63mortality) Sweet Pink and Kimono Pink varieties showed58 and 55 mortality rate respectively At 50 survivalrate (LD
50) the gamma doses administered were 28 21 23
and 25Gy for Doi Tung 554 Chiang Mai Red Sweet Pink
and Kimono Pink respectively (Figure 1) Abdullah et al [32]had previously indicated that the LD
50for C alismatifolia
var Chiang Mai Pink was approximately at 25Gy The deathof plants is attributed to the interaction of radiation withother molecules in the cell particularly water to produce freeradicals (H OH) The free radicals could combine to formtoxic substances such as hydrogen peroxide (H
2O2) which
contribute to the destruction of cells This indirect effectis especially significant in vegetative cells the cytoplasmwhich contains about 80 water [33] However sensitivityof the plant material depends on the genetic constitutiondose-employed DNA amount moisture content and stageof development and genotype [34] The choice of the doseto be applied for the highest mutant rescue is often left tothe breederrsquos experience with the specific plant material itsgenetics and its physiology
52 Analysis of Variance (ANOVA) for Morphological Traitsof C alismatifolia in M1V1 Analysis of variance indicatedhighly significant differences among the varieties doses andtheir interaction for all traits in M
1V1generation (Table 4)
Some desired and undesired abnormalities such as dwarfismchlorophyll mutation (albinism) striata (yellow or whitelongitudinal bands altering with green colors) two-midribleaves split leaves double flower stalk in one plant doubleinflorescence marbled pink bracts two-tone pink-purplishbracts and two-flag petals were found in M
1V1plants
(Table 5)
53 Effect of Gamma Irradiation on Vegetative Traits in theM1V1 The growth of plants treated with 10 and 20Gy ofgamma rays was slower than that of the controls (Table 6)In irradiated plants the leaf length and leaf width decreasedsignificantly (119875 lt 005) as the radiation doses increasedThis trend is quite common in mutagenised populationsSuch effects are known to arise due to drastic chromoso-mal aberrations in addition to genetic mutations Similar
6 BioMed Research International
Table4Meansquareso
fanalysis
ofvaria
nce(ANOVA
)for
14morph
ologicaltraitsin
Calism
atifolia
(a)
Source
ofvaria
tion
Meansquares
dfGeneration
Num
bero
fsho
otLeafleng
thLeafwidth
Leafnu
mber
Daystovisib
lebu
dPlanth
eight
Block
4M
1V1
005
622
11014
9467
8358
Varie
ty3
M1V
1263lowast
2702lowast
3011lowast
500lowast
1455602lowast
253739lowast
Dose
2M
1V1
611lowast
373804lowast
4137lowast
1042lowast
1012001lowast
157076
6lowastVa
rlowastdo
se6
M1V
114
1lowast8555lowast
418lowast
167lowast
731271lowast
115934lowast
Error
44M
1V1
0094
2951
077
037
4903
Total
5984
93
CV(
)M
1V1
23183
176
206
91165
(b)
Source
ofvaria
tion
Meansquares
dfGeneration
Daysto
anthesis
Daysto
senescence
Noof
true
flow
Noof
pink
bract
Inflo
rescence
leng
thNoof
new
rhizom
esRh
izom
esiz
eNoof
storage
roots
Block
4M
1V1
12847
048
418
072
282
01
018
055
Varie
ty3
M1V
11608017
10091lowast
9166lowast
62543lowast
376lowast
163lowast
023
ns1726lowast
Dose
2M
1V1
1587261
128682lowast
58286lowast
35315lowast
56022lowast
1031lowast
531lowast
6481lowast
Varlowastdo
se6
M1V
1876957
3464lowast
2900lowast
977lowast
4004lowast
128lowast
025ns
332lowast
Error
44M
1V1
7752
322
392
226
445
011
011
121
Total
59CV
()
M1V
110
15229
169
165
2318
268
lowastSign
ificant
with
leastsqu
ared
ifference
test119875lt005
BioMed Research International 7
Dose (Gy)
0
10
10
20
20
30
30
40
40
50
50
60
60
70
70
80
80
90
90
100
100
Mor
talit
y (
)
Var Doi Tung554
LD50 = 28 Gy
(a)
0
10
10
20
20
30
30
40
40
50
50
60
60
70
70
80
80
90
90
100
100
Mor
talit
y (
)
Var Chiang Mai Red
Dose (Gy)
LD50 = 21Gy
(b)
Mor
talit
y (
)
Dose (Gy)
Var Sweet Pink
0
10
10
20
20
30
30
40
40
50
50
60
60
70
70
80
80
90
90
100
100
LD50 = 23Gy
(c)
0
10
10
20
20
30
30
40
40
50
50
60
60
70
70
80
80
90
90
100
100
Mor
talit
y (
)
Dose (Gy)
Var Kimono Pink
LD50 = 25Gy
(d)
Figure 1 PoloPlus plot of linear scale of dose versusmortality percent (a) Doi Tung 554 (b) ChiangMai Red (c) Sweet Pink and (d) KimonoPink
Table 5 Effect of acute gamma rays on vegetative and flowering traits of C alismatifolia in the M1V1 generation
Variety irradiated dose Observed variations Flower color variationChiang Mai Red
10Dwarfism no pink bracts inflorescence small inflorescencetwo-flag petal true flower double inflorescence undulate leafmargin and yellowwhite strip leaves
Light purple N78Clowast
20 Dwarfism narrow small leaves and yellowwhite strip leaves No flowerDoi Tung 554
20 Dwarfism fewer pink bracts and yellowwhite strip leavesSmall and narrow leave Light purple N80Dlowast
lowastRoyal British Society color chart (RHS)
8 BioMed Research International
Table 6 Effect of acute gamma rays on vegetative traits of C alismatifolia in M1V1 generation
Dose (Gray) Shoot number Leaf number Leaf length (cm) Leaf width (cm) Plant height (cm)CMR
0 20 plusmn 00a
30 plusmn 04a
556 plusmn 08a
71 plusmn 05a
1112 plusmn 25a
10 10 plusmn 00b
36 plusmn 05a
272 plusmn 25b
45 plusmn 05b
714 plusmn 96b
20 10 plusmn 00b
14 plusmn 05b
124 plusmn 120c
25 plusmn 24c
170 plusmn 80c
DT0 30 plusmn 00
a30 plusmn 00
a622 plusmn 08
a65 plusmn 05
a912 plusmn 19
a
10 16 plusmn 05b
28 plusmn 04a
280 plusmn 44b
46 plusmn 06b
542 plusmn 76b
20 12 plusmn 04b
24 plusmn 05a
242 plusmn 38b
38 plusmn 05c
512 plusmn 138b
SP0 16 plusmn 05
a32 plusmn 04
a461 plusmn 27
a101 plusmn 04
a746 plusmn 38
a
10 10 plusmn 00b
36 plusmn 05a
275 plusmn 34b
57 plusmn 02b
377 plusmn 137b
20 10 plusmn 00b
20 plusmn 10b
196 plusmn 74c
57 plusmn 04b
195 plusmn 73c
KP0 14 plusmn 00
a46 plusmn 05
a345 plusmn 29
a47 plusmn 04
a591 plusmn 28
a
10 10 + 05a
40 plusmn 05b
220 plusmn 24b
31 plusmn 04b
420 plusmn 54b
20 10 plusmn 00a
32 plusmn 04b
156 plusmn 43c
31 plusmn 11b
283 plusmn 174c
CV () 23 18 26 16 165Means with the same or common letter are not significantly different least square difference test 119875 lt 005
decreases in leaf size were reported by Pongchawee et al [35]and Tangpong et al [36] These results were in agreementwith an earlier study [4] which reported that the growthof chrysanthemum exposed to acute gamma rays was lessthan the control in the M
1V1generation All varieties doses
and interaction effects resulted in significant differences fornumber of leaves Among untreated plants Kimono Pinkvariety had higher number of leaves (46) than the otherthree (3 3 and 32) varieties In Chiang Mai Red and SweetPink varieties plants exposed to 10Gy showedhigher numberof leaves than untreated plants However at 20Gy therewas significant reduction in number of leaves for all studiedvarieties in comparison to control Similar stimulatory effectswere obtained at lower doses in ginger by Hegde [37] andGiridharan and Balakrishnan [38]
Progressive reduction in growth parameters can be inter-preted on interference in normal mitosis and frequent occur-rence of mitotic aberrations inhibition of rate of assimilationand consequent change in the nutrient level in the plantand inactivation of vital enzymes especially those associatedwith respiration [39] Dose-dependent negative effect wasalso detected for plant heightThe tallest plants were recordedfrom the untreated rhizomes (0Gy) with heights of 1112912 746 and 591 cm followed by the 10Gy irradiated plantswith heights of 714 542 377 and 420 cm and the 20Gyirradiated plants with corresponding heights of 170 512195 and 283 cm for Chiang Mai Red Doi Tung 554 SweetPink and Kimono Pink varieties respectively These resultsare in agreement with the findings of Abdullah et al [32]Reduction in growth parameters and dwarfism can be causedby interference of normal mitosis and frequent occurrenceof mitotic aberrations inhibition of assimilation rates andconsequent changes in nutrient levels in plants Additionallymutagenic effects such as auxin destruction inhibition of
auxin synthesis failure of assimilatory mechanism andchanges in the specific activity of enzymes can cause growthreductions [37] High doses of ionizing radiation have beenshown to damage macromolecular cellular components suchas cell walls membranes and DNA [40] The number ofshoots also decreased significantly as the radiation dosesincreased Radiation also affects organic molecules that areessential to the cell division process and thus causing celldivision to stop [36]
54 Effect of Gamma Irradiation on Flowering DevelopmentTraits in the M1V1 All control and 10Gy irradiated plantsproduced flowers while the Chiang Mai Red and SweetPink varieties which were exposed to 20Gy did not gointo the flowering stage Lamseejan et al [4] also showedthat flowering percentage decreases as gamma ray dosesare increased In the present study gamma rays caused lateflowering in all four varieties Days to appearance of firstvisible buds were also significantly different among the fourvarieties (Table 7) Gamma rays caused a noticeable delay inflowering of irradiated plants in comparison to the untreatedones First visible buds were observed at 652 and 874 days inthe control and 10Gy treatments respectively for the ChiangMai Red variety In Doi Tung 554 the first visible buds wereappeared at 474 656 and 840 days after planting at 0 10and 20Gy doses respectively In the Sweet Pink variety thenumber of days to visible bud appearance increased signif-icantly from 678 days in controls to 978 days in the 10Gyirradiated plants In comparison to other three varieties theKimono Pink variety needed the longest time to visible budappearance and same as other varieties there was a positivecorrelation between the number of days to first visible budand the gamma irradiation dose In the control and 10Gyand 20Gy irradiated individual plants flower buds were
BioMed Research International 9
Table 7 Effect of acute gamma rays on flower development characteristics of C alismatifolia in M1V1 generation
Dose (Gy) Days tovisible bud
Inflorescencelength (cm)
Days toanthesis
Number oftrue flowers
Number ofPink bracts
Days tosenescence
CMR0 652 plusmn 47
b162 plusmn 057
a742 plusmn 54
b132 plusmn 19
a104 plusmn 15
a21 plusmn 10
a
10 874 plusmn 95a
82 plusmn 397b
1020 plusmn 71a
56 plusmn 19b
50 plusmn 07b
10 plusmn 14b
20 00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
DT0 474 plusmn 43
c134 plusmn 054
a546 plusmn 54
c162 plusmn 13
a232 plusmn 04
a23 plusmn 00
a
10 656 plusmn 37b
94 plusmn 19b
780 plusmn 37b
112 plusmn 21b
166 plusmn 08b
122 plusmn 130b
20 840 plusmn 42a
76 plusmn 082c
994 plusmn 49a
88 plusmn 13b
156 plusmn 11b
106 plusmn 18b
SP0 678 plusmn 21
b146 plusmn 089
a756 plusmn 26
b162 plusmn 13
a100 plusmn 24
a188 plusmn 083
a
10 978 plusmn 181a
79 plusmn 20b
1098 plusmn 182a
68 plusmn 25b
52 plusmn 08b
92 plusmn 24b
20 00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
KP0 852 plusmn 23
c137 plusmn 044
a942 plusmn 19
c112 plusmn 12
a96 plusmn 21
a18 plusmn 18
a
10 1044 plusmn 65b
108 plusmn 108ab
1224 plusmn 100b
76 plusmn 27b
64 plusmn 16ab
102 plusmn 17b
20 1272 plusmn 65a
790 plusmn 49b
1426 plusmn 58a
60 plusmn 38b
42 plusmn 24b
66 plusmn 37b
CV () 10 23 9 22 169 15Means with the same or common letter are not significantly different least square difference test 119875 lt 005
visible at 852 1328 and 1374 days after planting respectivelyPrevious studies also showed that onset of flowering andformation of floral parts in mutants of Arabidopsis thalianamaize barely pea and tobacco involved growth regulators(or phytohormones) such as auxins cytokinins gibberellinsabscisic acid ethylene and brassinosteroids [41] There weresignificant differences among treatments for the length ofthe inflorescence In all varieties the longest inflorescencelength was observed in the untreated plants with 162134 146 and 137 cm lengths for Chiang Mai Red DoiTung 554 Sweet Pink and Kimono Pink respectively Thecorresponding inflorescence lengths were 82 94 79 and109 cm for the 10Gy irradiated plants The days to anthesisfor C alismatifolia varieties were significantly affected byvariety gamma irradiation doses and their interaction Thenumber of days to full bloom was noticeable earliest foruntreated plants at 742 546 756 and 942 days for ChiangMai Red Doi Tung 554 Sweet Pink and Kimono Pinkvarieties respectively This was then followed by plants at10Gy at 102 78 1098 and 1224 days The number of trueflowers or the secondary inflorescence developed in the axilof the primary bracts decreased as radiation dosage increasedIn the present study the gamma rays also decreased thedays to inflorescence senescence In this study there wasa strongly significantly and positively correlation (0919lowastlowast)(data not shown) between the number of true flowers andthe number of days to senescenceThe number of pink bractsalso decreasedwith increasing radiation dosageMost gammaray effects on senescence are considered as resulting from theaction of free radicals generated from water and oxygen bythe ionizing energy on the cellular components Membrane
deterioration is a general feature of natural senescence andstress-induced aging [42]
Irradiation induced some mutation spectrum of flowercolor variation that included colors such as purple palepurple rather pale purple white purple white (marbledpattern) and two-tone purple color
Mutation spectrum of flower shape variation includeddouble inflorescence within one stalk double stalk per plantinflorescence without bracts two-flag petal true flowersand chlorophyll mutation in the leaves which are generallycaused by induced gamma rays (Figure 2) Ionizing radiationincluding gamma rays induces fragment deletions or inser-tions that eventually lead to changes in amino acids and amodification of leaf and stem pigmentation [43] A mutationin the biosynthetic pathway of structural or regulatory genesmay cause a change in flower color [44] When the blockageoccurs at the early stages of anthocyanin synthesis whiteflowers will result while a blockage at later stages leads todifferent flower colors due to the accumulation of particularanthocyanins [45] Chloroplasts were extremely sensitive togamma radiation compared to other cell organelles [46]
55 Effect of Gamma Irradiation on Rhizome Characteristicsin Selected Doses in M1V1 The number of new rhizomes andthe number of storage roots per rhizome were significantlyaffected by varieties doses and their interaction (Table 8)The number of new rhizomes only in Kimono Pink varietydid not show any differences between untreated and treatedplants The rhizome size was influenced only by doses Asdose level increased the rhizome size decreased Amonguntreated plants the Sweet Pink rhizomes had the most
10 BioMed Research International
(a) (b) (c) (d)
(e)
(f)
Figure 2 Effect of acute gamma rays on flower traits in C alismatifolia (a) Untreated inflorescence bracts color Doi Tung 554 (b) Two tonebract color Doi Tung 554 (20Gy) (c) Marbled pattern of bract color Doi Tung 554 (20Gy) (d) Double inflorescence within one stalk varChiang Mai Red (10Gy) (e) True flower in nontreated plants (f) Two-flag petals true flowers (10Gy)
Figure 3 Dendrogram representing themorphological variation among 44 irradiated and nonirradiated individuals ofC alismatifolia across14 variables CMR = Chiang Mai Red DT = Doi Tung 554 SP = Sweet Pink Kp = Kimono Pink
number of storage roots (78) and the Kimono Pink rhizomeshad the least number of storage roots (40)This was followedby 10Gy irradiated rhizomes with 4 46 48 and 28 storageroot numbers for Chiang Mai Red Doi Tung 554 SweetPink and Kimono Pink respectively Irradiation with 20Gydecreased significantly the number of storage roots in allstudied varietiesTheplantC alismatifolia is propagated froma propagule (one rhizome + 5-6 t-roots) The rhizome sizedose matters in the growth of the plant Smaller rhizome sizeusually resulted in smaller plant size with narrow grass-likeleaves The storage roots play a very important role in thegrowth and flowering ofCurcumaThe t-rootsmake up about85 of the total fresh weight and 70 of the total dry weightof a typical propaguleThe storage organs act for plant growth
during dormancy and emergence [47] More storage root perpropagule resulted in faster flowering and higher yield plant[48] In this study also there was significant and negativecorrelation between number of storage roots and number ofdays to visible bud appearance (minus0525lowastlowast)
56 Cluster Analysis of C alismatifolia for MorphologicalTraits The morphological data were used to calculate thesimilarity between the treated and non-treated C alismat-ifolia samples and UPGMA dendrogram was constructed(Figure 3) The cophenetic correlation coefficient (CCC)value between the genetic dissimilaritymatrix estimated fromthe morphological characters and the UPGMA clusteringmethod was 119903 = 093 showing a proof fit This value was
BioMed Research International 11
Table 8 Effect of acute gamma rays on rhizome characteristics ofC alismatifolia in M1V1 generation
Dose (Gray) Rhizomesize
No of newrhizomes
No of storageroot
CMR0 23 plusmn 02
a26 plusmn 055
a46 plusmn 054
a
10 18 plusmn 005a12 plusmn 044
b40 plusmn 07
a
20 08 plusmn 07b
06 plusmn 054b
14 plusmn 13b
DT0 22 plusmn 01
a30 plusmn 00
a72 plusmn 109
a
10 18 plusmn 007b14 plusmn 054
b46 plusmn 13
b
20 16 plusmn 005b
10 plusmn 00b
28 plusmn 13c
SP0 22 plusmn 02
a24 plusmn 054
a78 plusmn 21
a
10 17 plusmn 007b
10 plusmn 00b
48 plusmn 044b
20 12 plusmn 02c
10 plusmn 00b
28 plusmn 083b
KP0 21 plusmn 005
a10 plusmn 00
a40 plusmn 00
a
10 16 plusmn 01ab
10 plusmn 00a
28 plusmn 044b
20 11 plusmn 06b
10 plusmn 00a
22 plusmn 083b
CV () 18 23 26Means with the same or common letter are not significantly different leastsquare difference test 119875 lt 005
higher than of studies on olive for morphological traits (119903 =069) [49] In this dendrogram the 44C alismatifolia samplesappeared to form three main clusters and five minor clustersat coefficient level 56 cluster I included three non-irradiatedmembers and cluster II included 10Gy irradiated individualplants of all four varieties and 20Gy irradiated samples ofDoi Tung 554 and Kimono Pink varieties except KP20-4 (noflowering) Main cluster III included the 20Gy irradiatedindividuals Chiang Mai Red and Sweet Pink varieties (asmentioned in morphological part these individuals didnot go to flowering stage) and KP20-4 Cluster II can bedivided to five subclusters The first subcluster included 10members (CMR10-1 CMR10-5 SP10-1 SP10-2 SP10-3 SP10-5 CMR10-2 CMR10-4 SP10-4 and CMR10-3) the secondone included nine members (KP10-1 KP10-2 KP10-3 KP10-4 KP10-5KP20-1 KP20-2 KP20-5 and KP20-3) KP0 wasthe lone member of the third sub-cluster The fourth sub-cluster included two members only (DT10-2 and DT10-3) The last sub-cluster main cluster II had eight membersincluded (DT10-1 DT10-4 DT10-5 DT20-1 DT20-2 DT20-3 DT20-4 and DT20-5) Mean value of groups for each traitis presented in Table 9 This table clearly shows the differentmean values of the three main clusters The minimum meanvalue referred to main cluster III while the maximum meanvalue for number of shoots leaf length leaf width plantheight number of true flowers inflorescence length rhizomesize number of new rhizomes and number of storage rootsbelonged to cluster I which was included non-treated plantsThese results showed that the gamma irradiation has inducedmorphological changes in C alismatifolia individuals of
four studied varieties so that they showed phenotypicallydifferences from their controls
57 Principal Component Analysis of C alismatifolia forMorphological Traits To assess the patterns of variation PCAwas done by considering all of the 14 variables The first threecomponents of PCA explained 828 of the total variation(Table 10) Only the first component which accounted for562 of the total variation was attributed to inflorescencelength plant height days to senescence number of trueflowers and rhizome size In PCA three-dimensional graphthe grouping of individuals confirmed the clustering results(Figure 4) The PCA graph proved that all irradiated indi-vidual plants of C alismatifolia varieties are phenotypicallydifferent from their non-irradiated individuals
6 Molecular Characterization
61 SSR Polymorphism Amplifications were successful forall the 17 SSR markers assayed This reflects a high levelof homology between SSR flanking regions in C longa andC alismatifolia Out of 17 primer pairs eight primer pairsresulted in polymorphic PCR products Table 11 summarizesthe results obtained based on the analysis of individuals of thefour studied varieties using the polymorphic SSR loci for 010 and 20Gy irradiated plants The number of alleles variedwidely among these loci A total of 25 36 and 41 alleles wereobserved among 0 10 and 20Gy irradiated individual plantsrespectively In the 10Gy treatment the number of allelesranged from three (clon09 and clon14) to seven (clon08) withan average value of 45 per locus In the 20Gy treatmentthe number of alleles varied from three (clon09 and clon14)to seven (clon08 and clon12) with an average value of 51per locus In the untreated individuals the number of allelesranged from two (clon04 clon09 clon11 and clon14) to five(clon12) with an average value of 31 The difference betweenthe average number of observed alleles and effective numberof alleles was due to the uneven frequency of each allele[50] For each of the eight SSR primers PIC values (whichmeasures allele diversity and frequency among varieties)varied from 019 (clon04) to 071 (clon01) in untreated plantsand from 025 (clon04) to 075 (clon08) in 10Gy acutelyirradiated plants In 20Gy irradiated individual plants thePIC value ranged from 042 (clon14) to 075 (clon08 andclon12) The mean PIC for all loci was 047 054 and 061for 0 10 and 20Gy irradiated plants respectively
The PIC value provides an estimate of the discriminatorypower of amarker by taking into account not only the numberof alleles at a locus but also the relative frequencies of thesealleles [51] Thus except for clon09 and clon14 which weremoderately polymorphic (025 lt PIC lt 05) all other lociwere highly polymorphic (05 lt PIC) while none of theloci showed low polymorphism The average discriminatingpower ofmicrosatellitemarkers (061) observed in the presentstudy ensures the future utility of microsatellite markersfor genetic variation studies in C alismatifolia varietiesOur results reflect similar findings as reported earlier inthe study of genetic variation in other species of Curcuma
12 BioMed Research International
Table9Clusterm
eans
for14charactersestim
ated
in44
individu
also
fCalismatifolia
Clusters
Shoo
tnu
mber
Leaf
leng
thLeaf
width
Leaf
number
Visib
lebu
d(days)
Inflo
rescence
leng
thPlant
height
Anthesis
(days)
Senescence
(days)
True
flow
number
Pink
bract
number
Rhizom
esiz
eNew
rhizom
enu
mber
Storage
root
number
I220
5464
736
300
6013
1473
9233
6926
2093
152
1453
229
266
653
II1
100
2735
512
360
9260
809
5457
1059
960
620
510
177
110
440
II2
122
1922
298
344
11422
1043
3910
1312
293
3755
588
156
100
266
II3
100
3452
416
460
8520
1370
5910
9420
180
120
960
206
100
400
II4
150
2500
45
250
6500
925
4950
7700
1150
950
1700
185
200
600
II5
137
2637
417
262
7725
840
5356
9162
1137
1012
1587
170
100
312
III
054
1500
465
181
000
000
000
000
000
000
000
090
081
200
BioMed Research International 13
Table 10 Eigenvectors eigenvalues and proportions of variabilityfor three principle components among 14 characters for 44 Calismatifolia
Variable PC1 PC2 PC3Number of new shoot 0270 minus0076 minus0173Leaf length 0269 minus0305 0033Leaf width 0096 minus0455 0430Leaf number 0210 0285 0490Days to visible bud 0199 0462 0181Plant height 0321 0027 minus0071Inflorescence length 0318 0166 minus0040Days to anthesis 0205 0460 0148Days to senescence 0342 0017 minus0131Number of true flower 0323 0029 minus0238Number of pink bracts 0266 minus0028 minus0546Rhizome size 0302 minus0101 0147Number of new rhizome 0250 minus0293 0028Number of storage roots 0264 minus0247 0298Eigenvalue 7877 2694 1032Proportion 56264 19246 7371Cumulative 56264 75511 82882
using SSR markers [13] The highest Neirsquos gene diversity(ℎ) was obtained with the 20Gy treatment (061) followedby the 10Gy treatment (06) and the untreated individuals(05) The mean Shannonrsquos information index (119868) was 092114 and 130 in the 0 10 and 20Gy irradiated plantsrespectively The high value of Shannonrsquos information indexrepresents the effectiveness of microsatellite loci to revealthe variation among these varieties at the different dosesused Overall genetic variability for the varieties studiedrepresented by the Shannon-Weiner index was relatively highin comparison to other studies involving C longa accessions[10 52] Additionally the 20Gy acutely irradiated individualsshowed a higher mean percentage of polymorphic loci (63)than the 10Gy (58) and untreated (022) ones This impliesthat irradiation with a dose of 20Gy induced more geneticvariation in the M
1V1generation
62 Cluster Analysis Thedendrogramwas constructed usingPowerMarker 323The 44 studied individuals were clusteredinto seven groups (G1ndashG7) Colors were applied accordingto our model-based cluster analysis results Group I iscomprised of nine individual plants of Chiang Mai Redvariety receiving 0 10 and 20Gy treatments (blue colorin Figure 5) One individual plant DT10-1 was assignedto group II (green color in Figure 5) All irradiated andnon-irradiated individuals of Doi Tung 554 variety wereassigned to group III (yellow color in Figure 5) DT20-1 andSP10-5 were the sole members of groups IV (red color inFigure 5) and V (gray color in Figure 5) respectively GroupVI included treated and untreated individuals of Sweet Pinkvariety (pink color in Figure 5) Lastly all Kimono Pinkindividuals were assigned to cluster VII (orange color in
Figure 5) The genetic similarity coefficient (data not shown)among the 44 individuals amplified using eight SSR primersvaried from 00 to 10 The highest value (10) correspondedto [(CMR10-3 CMR10-5 CMR and CMR20-2) (DT10-2and DT10-3) (DT and DT20-2) (KP20-3 KP-20-4 KP10-3andKP10-4)] individuals that generated identical fingerprintsacross the markers studied Among the studied varieties theSweet Pink individual plants showed the lowest similarity(00) withKimono Pink individuals which indicated that theywere relatively remote in relationship (Figure 5) An overviewof the clustering pattern indicates that the grouping of thestudied individual plants was based on different varieties andlargely independent of the doses of gamma irradiation Awide range in similarity values had also been observed indifferent species of Curcuma [12 13 52 53]
63 Principle Component Analysis (PCA) Thedata generatedfrom 44 C alismatifolia individual plants were subjected toprincipal component analysis (PCA) to visualize individualsin a multivariate space In the three-dimensional graphderived from the SSR analysis all studied individuals weregrouped into seven clusters (Figure 6) In PCA the firstthree principal components (PC) extracted a cumulative of6826 of the total variation among the 44 individuals of CalismatifoliaThe first three coordinates PC1 PC2 and PC3accounted for 4136 1578 1112 of the total variationrespectively The distribution of the individual plants in athree-dimensional plot using the first three principal com-ponents showed the genetic relationship between individualplants It was evident that both methods dendrogram andthree-dimensional plots of PCA were effective in illustratinggenetic relationships and the groups found were comparable[11 13] The PCA results were similar to those obtained fromcluster analysis where all individuals from different varietieswere assigned to different groups These graphical illus-trations enable the assessment of the relationshipdistancesamong all of the individuals in the study [50]
7 Conclusion
This is the first attempt to evaluate the effect of acutegamma irradiation on C alismatifolia varieties using bothmorphological characteristics and molecular markers Inplant breeding programs mutagenic treatments with lownegative physiological effects and strong genetic effects aredesirable Hence we used more effective doses of gammairradiation (10 and 20Gy) of which particularly 20Gy dosewas effective to influence morphological and molecularcharacteristics of studiedCurcuma alismatifolia varietiesThelower dose (10Gy) of radiation probably caused little damageto the plants genetic material so that the cells could repairthemselves in next generation As a result in this study 20Gyacute gamma irradiation resulted in a higher percentage ofmutation and getting desired mutants was more possibleBased on the LD
50values determined in this study it was
apparent that the varieties Chiang Mai Red and Sweet Pinkwere more sensitive to gamma rays compared to Doi Tung554 and Kimono Pink Our results show that the variety Doi
Figure 4 Two-dimensional graph of principal component analysis (PCA) for 14 morphological variables indicating relationships amongirradiated and nonirradiated four varieties of C alismatifolia
CMR10-1CMR10-5
SP10-1SP10-2SP10-3
SP10-5
CMR10-4
SP10-4
KP10-1KP10-3
KP20-1
KP20-2
KP10-5KP20-5
KP20-3
KP10-2
DT10-2
DT10-1
DT10-5
DT20-1
DT10-4DT20-4DT20-2
DT20-5DT20-3
CMR20-1
SP20-3SP20-4
SP20-5SP20-1
SP20-2
KP
SP
CMR 10-2
SP10-5
Figure 5 Unrooted neighbor joining tree showing genetic relationship among 44 irradiated and nonirradiated C alismatifolia using SSRmarkers The colors of the branches correspond to those of the same cluster
Figure 6Three-dimensional graph of principal component analysis (PCA) indicating relationships among irradiated and nonirradiated fourvarieties of C alismatifolia
Tung 554 (Curcuma hybrid) had the most morphologicalresponses to gamma rays The use of microsatellite markersas a codominant marker will facilitate the exploration ofgenetic variability among treated and non-treated plants andwill help to distinguish the plants showing differences inmorphological characters The overall effects on the M
1V1
generation revealed that acute gamma irradiation at optimumdoses has the potential for developing new varieties of Calismatifolia with improved commercial properties suitablefor the Malaysian flower industry
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work was funded by the Fundamental Research GrantScheme (FRGS) under the Ministry of Higher Education inMalaysia
References
[1] P Apavatjrut S Anuntalabhochai P Sirirugsa and C AlisildquoMolecularmarkers in the identification of some early floweringCurcuma L (Zingiberaceae) speciesrdquo Annals of Botany vol 84no 4 pp 529ndash534 1999
[2] K Bunya-Atichart S Ketsa andW G VanDoorn ldquoPostharvestphysiology of Curcuma alismatifolia flowersrdquo Postharvest Biol-ogy and Technology vol 34 no 2 pp 219ndash226 2004
[3] M Nakano J Amano Y Watanabe et al ldquoMorphologicalvariation in Tricyrtis hirta plants regenerated from heavy ionbeam-irradiated embryogenic callusesrdquo Plant Biotechnologyvol 27 no 2 pp 155ndash160 2010
[4] S Lamseejan P Jompuk A Wongpiyasatid A Deeseepan andP Kwanthammachart ldquoGamma-rays induced morphological
[5] G-J Lee S J Chung I S Park et al ldquoVariation in thephenotypic features and transcripts of colormutants of chrysan-themum (Dendranthema grandiflorum) derived from gammaraymutagenesisrdquo Journal of Plant Biology vol 51 no 6 pp 418ndash423 2008
[6] O K Kikuchi ldquoOrchid flowers tolerance to gamma-radiationrdquoRadiation Physics and Chemistry vol 57 no 3ndash6 pp 555ndash5572000
[7] N P Arnold N N Barthakur and M Tanguay ldquoMutageniceffects of acute gamma irradiation on miniature roses targettheory approachrdquo HortScience vol 33 no 1 pp 127ndash129 1998
[8] A A Youssef M S Aly and M S Hussein ldquoResponse ofgeranium (Pelargonium graveolenus L) to gamma irradiationand foliar application of Speed Growrdquo Egyptian Journal ofHorticulture vol 27 pp 41ndash53 2000
[9] N Chuantang and L Yazhi ldquoThe radiation inducedmutation ofcanna (Canna L)rdquo Acta Agricultute Nucleatae Sinica vol 2 pp33ndash39 1998
[10] M S Sigrist J B Pinheiro J A A Filho and M I ZucchildquoGenetic diversity of turmeric germplasm (Curcuma longaZingiberaceae) identified by microsatellite markersrdquo Geneticsand Molecular Research vol 10 no 1 pp 419ndash428 2011
[11] S Taheri T L Abdullah N A P Abdullah and Z AhmadldquoGenetic relationships among five varieties of Curcuma alis-matifolia (Zingiberaceae) based on ISSRmarkersrdquoGenetics andMolecular Research vol 11 pp 3069ndash3076 2012
[12] A Das V Kesari V M Satyanarayana A Parida and L Ran-gan ldquoGenetic relationship of Curcuma species from NortheastIndia using PCR-based markersrdquo Molecular Biotechnology vol49 no 1 pp 65ndash76 2011
[13] S Siju K Dhanya S Syamkumar et al ldquoDevelopment char-acterization and utilization of genomic microsatellite markersin turmeric (Curcuma longa L)rdquo Biochemical Systematics andEcology vol 38 no 4 pp 641ndash646 2010
[14] M K Panda S Mohanty E Subudhi L Acharya and SNayak ldquoAssessment of genetic stability of micropropagated
BioMed Research International 17
plants of Curcuma L by cytophotometery and RAPD analysisrdquoInternational Journal of Integrative Biology (IJIB) vol 1 pp 189ndash195 2007
[15] S Syamkumar and B Sasikumar ldquoMolecular marker basedgenetic diversity analysis of Curcuma species from IndiardquoScientia Horticulturae vol 112 no 2 pp 235ndash241 2007
[16] D Tautz and M Renz ldquoSimple sequences are ubiquitousrepetitive components of eukaryotic genomesrdquo Nucleic AcidsResearch vol 12 no 10 pp 4127ndash4138 1984
[17] G Toth Z Gaspari and J Jurka ldquoMicrosatellites in differenteukaryotic genomes surveys and analysisrdquo Genome Researchvol 10 no 7 pp 967ndash981 2000
[18] W Powell G C Machray and J Proven ldquoPolymorphismrevealed by simple sequence repeatsrdquo Trends in Plant Sciencevol 1 no 7 pp 215ndash222 1996
[19] S-Y Lee W K Fai M Zakaria et al ldquoCharacterizationof polymorphic microsatellite markers isolated from ginger(Zingiber officinale Rosc)rdquo Molecular Ecology Notes vol 7 no6 pp 1009ndash1011 2007
[20] S Bory D Da Silva A-M Risterucci M Grisoni P Besseand M-F Duval ldquoDevelopment of microsatellite markers incultivated vanilla polymorphism and transferability to othervanilla speciesrdquo Scientia Horticulturae vol 115 no 4 pp 420ndash425 2008
[21] I C Menezes F W Cidade A P Souza and I C SampaioldquoIsolation and characterization ofmicrosatellite loci in the blackpepper Piper Nigrum L (Piperaceae)rdquo Conservation GeneticsResources vol 1 no 1 pp 209ndash212 2009
[22] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[23] V Kumar Morphological and molecular characterization ofinduced mutants in Groundnut [PhD thesis] University ofAgricultural Sciences Dharwad India 2008
[24] J R Sharma Statistical and Biometrical Techniques in PlantBreeding New Age International New Delhi India 1998
[25] J J Doyle and J L Doyle ldquoA rapid DNA isolation procedure forsmall quantities of fresh leaf tissuerdquo Phytochemical Bulletin vol19 pp 11ndash15 1987
[26] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[27] Oslash Hammer D A T Harper and P D Ryan ldquoPast paleontolog-ical statistics software package for education and data analysisrdquoPalaeontologia Electronica vol 4 no 1 pp 4ndash9 2001
[28] K Liu and S V Muse ldquoPowerMaker an integrated analysisenvironment for genetic maker analysisrdquo Bioinformatics vol 21no 9 pp 2128ndash2129 2005
[29] F J RohlfNTSYS-Pc Numerical Taxonomy System Version 2 1Exeter Publishing Setauket New York NY USA 2002
[30] M Nei ldquoAnalysis of gene diversity in subdivided populationsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 70 no 12 1973
[31] S Kadkhodaei M Shahnazari M K Nekouei et al ldquoA compar-ative study of morphological and molecular diversity analysisamong cultivated almonds (Prunus dulcis)rdquo Australian Journalof Crop Science vol 5 no 1 pp 82ndash91 2011
[32] T L Abdullah J Endan and B M Nazir ldquoChanges in flowerdevelopment chlorophyll mutation and alteration in plant
morphology of Curcuma alismatifolia by gamma irradiationrdquoAmerican Journal of Applied Sciences vol 6 no 7 pp 1436ndash14392009
[33] E Kovacs and A Keresztes ldquoEffect of gamma and UV-BCradiation on plant cellsrdquoMicron vol 33 no 2 pp 199ndash210 2002
[34] H L Ramesh Y Murthy and V N Munirajappa ldquoEffect ofgamma radiation on morphological and growth parametersof Mulberry variety M5rdquo International Journal of Science andNature vol 3 pp 447ndash452 2010
[35] K Pongchawee R Pradissan andW Pipatcharoenchai ldquoInducemutation in Anubias spp through in vitro Irradiationrdquo ThaiFisheries Gazette vol 60 pp 493ndash497 2007
[36] P Tangpong T Taychasinpitak C Jompuk and P JompukldquoEffects of acute and chronic gamma irradiations on in vitroculture ofAnubias congensisNE BrownrdquoKasetsart Journal vol43 no 3 pp 449ndash457 2009
[37] R K Hegde Studies on induced mutagenesis and in vitroregeneration in turmeric (Curcuma longa L) [PhD thesis]University of Agricultural Sciences Dharwad India 2006
[38] M P Giridharan and S Balakrishnan ldquoGamma ray inducedvariability in vegetative and floral characters of ginger IndianCocoardquo Arecanut and Spices Journal vol 15 pp 68ndash672 1992
[39] A P Cesarett ldquoEffect of radiation on higher plants and plantcommunitiesrdquo inRadiation Biology United States Atomic EnergyCommission pp 284ndash309 Washington DC USA 1968
[40] G W Seung Y C Byung J-H Kim et al ldquoUltrastructuralchanges of cell organelles in Arabidopsis stems after gammairradationrdquo Journal of Plant Biology vol 48 no 2 pp 195ndash2002005
[41] B S Ahloowalia and M Maluszynski ldquoInduced mutationsmdashanew paradigm in plant breedingrdquo Euphytica vol 118 no 2 pp167ndash173 2001
[42] R Voisine L-P Vezina and C Willemot ldquoInduction ofsenescence-like deterioration of microsomal membranes fromcauliflower by free radicals generated during gamma irradia-tionrdquo Plant Physiology vol 97 no 2 pp 545ndash550 1991
[43] N Shikazono Y Yokota S Kitamura et al ldquoMutation rate andnovel tt mutants of Arabidopsis thaliana induced by carbonionsrdquo Genetics vol 163 no 4 pp 1449ndash1455 2003
[44] T Nakatsuka M Nishihara K Mishiba and S YamamuraldquoTwo different mutations are involved in the formation ofwhite-flowered gentian plantsrdquo Plant Science vol 169 no 5 pp949ndash958 2005
[45] M Mato T Onozaki Y Ozeki et al ldquoFlavonoid biosynthesisin white-flowered Sim carnations (Dianthus caryophyllus)rdquoScientia Horticulturae vol 84 no 3-4 pp 333ndash347 2000
[46] S G Wi B Y Chung J-S Kim et al ldquoEffects of gammairradiation on morphological changes and biological responsesin plantsrdquoMicron vol 38 no 6 pp 553ndash564 2007
[47] F Lee Chin Effects of light intensity and daylength on growth andflowering of siam tulip (Curcuma alismatifolia var Chiang MaiPink) [PhD thesis] University Putra Malaysia 2007
[48] A Hagiladi N Umiel and X H Yang ldquoCurcuma alismatifoliaII Effects of temperature and daylength on the development offlowers and propagulesrdquo Acta Horticulture vol 430 pp 755ndash761 1997
[49] MHagidimitriou A Katsiotis GMenexes C Pontikis andMLoukas ldquoGenetic diversity of major greek olive cultivars usingmolecular (AFLPs and RAPDs) markers and morphologicaltraitsrdquo Journal of the American Society for Horticultural Sciencevol 130 no 2 pp 211ndash217 2005
18 BioMed Research International
[50] N Babaei N A P Abdullah G Saleh and T L AbdullahldquoIsolation and characterization of microsatellite markers andanalysis of genetic variability in Curculigo latifolia DryandrdquoMolecular Biology Reports vol 39 pp 9869ndash9877 2012
[51] B Shiran N Amirbakhtiar S Kiani S Mohammadi B ESayed-Tabatabaei and H Moradi ldquoMolecular characterizationand genetic relationship among almond cultivars assessed byRAPD and SSR markersrdquo Scientia Horticulturae vol 111 no 3pp 280ndash292 2007
[52] S Singh M K Panda and S Nayak ldquoEvaluation of geneticdiversity in turmeric (Curcuma longa L) using RAPD and ISSRmarkersrdquo Industrial Crops and Products vol 37 no 1 pp 284ndash291 2012
[53] Y Paisooksantivatana S Kako and H Seko ldquoGenetic diversityof Curcuma alismatifolia Gagnep (Zingiberaceae) in Thailandas revealed by allozyme polymorphismrdquo Genetic Resources andCrop Evolution vol 48 no 5 pp 459ndash465 2001
20 Dwarfism fewer pink bracts and yellowwhite strip leavesSmall and narrow leave Light purple N80Dlowast
lowastRoyal British Society color chart (RHS)
8 BioMed Research International
Table 6 Effect of acute gamma rays on vegetative traits of C alismatifolia in M1V1 generation
Dose (Gray) Shoot number Leaf number Leaf length (cm) Leaf width (cm) Plant height (cm)CMR
0 20 plusmn 00a
30 plusmn 04a
556 plusmn 08a
71 plusmn 05a
1112 plusmn 25a
10 10 plusmn 00b
36 plusmn 05a
272 plusmn 25b
45 plusmn 05b
714 plusmn 96b
20 10 plusmn 00b
14 plusmn 05b
124 plusmn 120c
25 plusmn 24c
170 plusmn 80c
DT0 30 plusmn 00
a30 plusmn 00
a622 plusmn 08
a65 plusmn 05
a912 plusmn 19
a
10 16 plusmn 05b
28 plusmn 04a
280 plusmn 44b
46 plusmn 06b
542 plusmn 76b
20 12 plusmn 04b
24 plusmn 05a
242 plusmn 38b
38 plusmn 05c
512 plusmn 138b
SP0 16 plusmn 05
a32 plusmn 04
a461 plusmn 27
a101 plusmn 04
a746 plusmn 38
a
10 10 plusmn 00b
36 plusmn 05a
275 plusmn 34b
57 plusmn 02b
377 plusmn 137b
20 10 plusmn 00b
20 plusmn 10b
196 plusmn 74c
57 plusmn 04b
195 plusmn 73c
KP0 14 plusmn 00
a46 plusmn 05
a345 plusmn 29
a47 plusmn 04
a591 plusmn 28
a
10 10 + 05a
40 plusmn 05b
220 plusmn 24b
31 plusmn 04b
420 plusmn 54b
20 10 plusmn 00a
32 plusmn 04b
156 plusmn 43c
31 plusmn 11b
283 plusmn 174c
CV () 23 18 26 16 165Means with the same or common letter are not significantly different least square difference test 119875 lt 005
decreases in leaf size were reported by Pongchawee et al [35]and Tangpong et al [36] These results were in agreementwith an earlier study [4] which reported that the growthof chrysanthemum exposed to acute gamma rays was lessthan the control in the M
1V1generation All varieties doses
and interaction effects resulted in significant differences fornumber of leaves Among untreated plants Kimono Pinkvariety had higher number of leaves (46) than the otherthree (3 3 and 32) varieties In Chiang Mai Red and SweetPink varieties plants exposed to 10Gy showedhigher numberof leaves than untreated plants However at 20Gy therewas significant reduction in number of leaves for all studiedvarieties in comparison to control Similar stimulatory effectswere obtained at lower doses in ginger by Hegde [37] andGiridharan and Balakrishnan [38]
Progressive reduction in growth parameters can be inter-preted on interference in normal mitosis and frequent occur-rence of mitotic aberrations inhibition of rate of assimilationand consequent change in the nutrient level in the plantand inactivation of vital enzymes especially those associatedwith respiration [39] Dose-dependent negative effect wasalso detected for plant heightThe tallest plants were recordedfrom the untreated rhizomes (0Gy) with heights of 1112912 746 and 591 cm followed by the 10Gy irradiated plantswith heights of 714 542 377 and 420 cm and the 20Gyirradiated plants with corresponding heights of 170 512195 and 283 cm for Chiang Mai Red Doi Tung 554 SweetPink and Kimono Pink varieties respectively These resultsare in agreement with the findings of Abdullah et al [32]Reduction in growth parameters and dwarfism can be causedby interference of normal mitosis and frequent occurrenceof mitotic aberrations inhibition of assimilation rates andconsequent changes in nutrient levels in plants Additionallymutagenic effects such as auxin destruction inhibition of
auxin synthesis failure of assimilatory mechanism andchanges in the specific activity of enzymes can cause growthreductions [37] High doses of ionizing radiation have beenshown to damage macromolecular cellular components suchas cell walls membranes and DNA [40] The number ofshoots also decreased significantly as the radiation dosesincreased Radiation also affects organic molecules that areessential to the cell division process and thus causing celldivision to stop [36]
54 Effect of Gamma Irradiation on Flowering DevelopmentTraits in the M1V1 All control and 10Gy irradiated plantsproduced flowers while the Chiang Mai Red and SweetPink varieties which were exposed to 20Gy did not gointo the flowering stage Lamseejan et al [4] also showedthat flowering percentage decreases as gamma ray dosesare increased In the present study gamma rays caused lateflowering in all four varieties Days to appearance of firstvisible buds were also significantly different among the fourvarieties (Table 7) Gamma rays caused a noticeable delay inflowering of irradiated plants in comparison to the untreatedones First visible buds were observed at 652 and 874 days inthe control and 10Gy treatments respectively for the ChiangMai Red variety In Doi Tung 554 the first visible buds wereappeared at 474 656 and 840 days after planting at 0 10and 20Gy doses respectively In the Sweet Pink variety thenumber of days to visible bud appearance increased signif-icantly from 678 days in controls to 978 days in the 10Gyirradiated plants In comparison to other three varieties theKimono Pink variety needed the longest time to visible budappearance and same as other varieties there was a positivecorrelation between the number of days to first visible budand the gamma irradiation dose In the control and 10Gyand 20Gy irradiated individual plants flower buds were
BioMed Research International 9
Table 7 Effect of acute gamma rays on flower development characteristics of C alismatifolia in M1V1 generation
Dose (Gy) Days tovisible bud
Inflorescencelength (cm)
Days toanthesis
Number oftrue flowers
Number ofPink bracts
Days tosenescence
CMR0 652 plusmn 47
b162 plusmn 057
a742 plusmn 54
b132 plusmn 19
a104 plusmn 15
a21 plusmn 10
a
10 874 plusmn 95a
82 plusmn 397b
1020 plusmn 71a
56 plusmn 19b
50 plusmn 07b
10 plusmn 14b
20 00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
DT0 474 plusmn 43
c134 plusmn 054
a546 plusmn 54
c162 plusmn 13
a232 plusmn 04
a23 plusmn 00
a
10 656 plusmn 37b
94 plusmn 19b
780 plusmn 37b
112 plusmn 21b
166 plusmn 08b
122 plusmn 130b
20 840 plusmn 42a
76 plusmn 082c
994 plusmn 49a
88 plusmn 13b
156 plusmn 11b
106 plusmn 18b
SP0 678 plusmn 21
b146 plusmn 089
a756 plusmn 26
b162 plusmn 13
a100 plusmn 24
a188 plusmn 083
a
10 978 plusmn 181a
79 plusmn 20b
1098 plusmn 182a
68 plusmn 25b
52 plusmn 08b
92 plusmn 24b
20 00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
KP0 852 plusmn 23
c137 plusmn 044
a942 plusmn 19
c112 plusmn 12
a96 plusmn 21
a18 plusmn 18
a
10 1044 plusmn 65b
108 plusmn 108ab
1224 plusmn 100b
76 plusmn 27b
64 plusmn 16ab
102 plusmn 17b
20 1272 plusmn 65a
790 plusmn 49b
1426 plusmn 58a
60 plusmn 38b
42 plusmn 24b
66 plusmn 37b
CV () 10 23 9 22 169 15Means with the same or common letter are not significantly different least square difference test 119875 lt 005
visible at 852 1328 and 1374 days after planting respectivelyPrevious studies also showed that onset of flowering andformation of floral parts in mutants of Arabidopsis thalianamaize barely pea and tobacco involved growth regulators(or phytohormones) such as auxins cytokinins gibberellinsabscisic acid ethylene and brassinosteroids [41] There weresignificant differences among treatments for the length ofthe inflorescence In all varieties the longest inflorescencelength was observed in the untreated plants with 162134 146 and 137 cm lengths for Chiang Mai Red DoiTung 554 Sweet Pink and Kimono Pink respectively Thecorresponding inflorescence lengths were 82 94 79 and109 cm for the 10Gy irradiated plants The days to anthesisfor C alismatifolia varieties were significantly affected byvariety gamma irradiation doses and their interaction Thenumber of days to full bloom was noticeable earliest foruntreated plants at 742 546 756 and 942 days for ChiangMai Red Doi Tung 554 Sweet Pink and Kimono Pinkvarieties respectively This was then followed by plants at10Gy at 102 78 1098 and 1224 days The number of trueflowers or the secondary inflorescence developed in the axilof the primary bracts decreased as radiation dosage increasedIn the present study the gamma rays also decreased thedays to inflorescence senescence In this study there wasa strongly significantly and positively correlation (0919lowastlowast)(data not shown) between the number of true flowers andthe number of days to senescenceThe number of pink bractsalso decreasedwith increasing radiation dosageMost gammaray effects on senescence are considered as resulting from theaction of free radicals generated from water and oxygen bythe ionizing energy on the cellular components Membrane
deterioration is a general feature of natural senescence andstress-induced aging [42]
Irradiation induced some mutation spectrum of flowercolor variation that included colors such as purple palepurple rather pale purple white purple white (marbledpattern) and two-tone purple color
Mutation spectrum of flower shape variation includeddouble inflorescence within one stalk double stalk per plantinflorescence without bracts two-flag petal true flowersand chlorophyll mutation in the leaves which are generallycaused by induced gamma rays (Figure 2) Ionizing radiationincluding gamma rays induces fragment deletions or inser-tions that eventually lead to changes in amino acids and amodification of leaf and stem pigmentation [43] A mutationin the biosynthetic pathway of structural or regulatory genesmay cause a change in flower color [44] When the blockageoccurs at the early stages of anthocyanin synthesis whiteflowers will result while a blockage at later stages leads todifferent flower colors due to the accumulation of particularanthocyanins [45] Chloroplasts were extremely sensitive togamma radiation compared to other cell organelles [46]
55 Effect of Gamma Irradiation on Rhizome Characteristicsin Selected Doses in M1V1 The number of new rhizomes andthe number of storage roots per rhizome were significantlyaffected by varieties doses and their interaction (Table 8)The number of new rhizomes only in Kimono Pink varietydid not show any differences between untreated and treatedplants The rhizome size was influenced only by doses Asdose level increased the rhizome size decreased Amonguntreated plants the Sweet Pink rhizomes had the most
10 BioMed Research International
(a) (b) (c) (d)
(e)
(f)
Figure 2 Effect of acute gamma rays on flower traits in C alismatifolia (a) Untreated inflorescence bracts color Doi Tung 554 (b) Two tonebract color Doi Tung 554 (20Gy) (c) Marbled pattern of bract color Doi Tung 554 (20Gy) (d) Double inflorescence within one stalk varChiang Mai Red (10Gy) (e) True flower in nontreated plants (f) Two-flag petals true flowers (10Gy)
Figure 3 Dendrogram representing themorphological variation among 44 irradiated and nonirradiated individuals ofC alismatifolia across14 variables CMR = Chiang Mai Red DT = Doi Tung 554 SP = Sweet Pink Kp = Kimono Pink
number of storage roots (78) and the Kimono Pink rhizomeshad the least number of storage roots (40)This was followedby 10Gy irradiated rhizomes with 4 46 48 and 28 storageroot numbers for Chiang Mai Red Doi Tung 554 SweetPink and Kimono Pink respectively Irradiation with 20Gydecreased significantly the number of storage roots in allstudied varietiesTheplantC alismatifolia is propagated froma propagule (one rhizome + 5-6 t-roots) The rhizome sizedose matters in the growth of the plant Smaller rhizome sizeusually resulted in smaller plant size with narrow grass-likeleaves The storage roots play a very important role in thegrowth and flowering ofCurcumaThe t-rootsmake up about85 of the total fresh weight and 70 of the total dry weightof a typical propaguleThe storage organs act for plant growth
during dormancy and emergence [47] More storage root perpropagule resulted in faster flowering and higher yield plant[48] In this study also there was significant and negativecorrelation between number of storage roots and number ofdays to visible bud appearance (minus0525lowastlowast)
56 Cluster Analysis of C alismatifolia for MorphologicalTraits The morphological data were used to calculate thesimilarity between the treated and non-treated C alismat-ifolia samples and UPGMA dendrogram was constructed(Figure 3) The cophenetic correlation coefficient (CCC)value between the genetic dissimilaritymatrix estimated fromthe morphological characters and the UPGMA clusteringmethod was 119903 = 093 showing a proof fit This value was
BioMed Research International 11
Table 8 Effect of acute gamma rays on rhizome characteristics ofC alismatifolia in M1V1 generation
Dose (Gray) Rhizomesize
No of newrhizomes
No of storageroot
CMR0 23 plusmn 02
a26 plusmn 055
a46 plusmn 054
a
10 18 plusmn 005a12 plusmn 044
b40 plusmn 07
a
20 08 plusmn 07b
06 plusmn 054b
14 plusmn 13b
DT0 22 plusmn 01
a30 plusmn 00
a72 plusmn 109
a
10 18 plusmn 007b14 plusmn 054
b46 plusmn 13
b
20 16 plusmn 005b
10 plusmn 00b
28 plusmn 13c
SP0 22 plusmn 02
a24 plusmn 054
a78 plusmn 21
a
10 17 plusmn 007b
10 plusmn 00b
48 plusmn 044b
20 12 plusmn 02c
10 plusmn 00b
28 plusmn 083b
KP0 21 plusmn 005
a10 plusmn 00
a40 plusmn 00
a
10 16 plusmn 01ab
10 plusmn 00a
28 plusmn 044b
20 11 plusmn 06b
10 plusmn 00a
22 plusmn 083b
CV () 18 23 26Means with the same or common letter are not significantly different leastsquare difference test 119875 lt 005
higher than of studies on olive for morphological traits (119903 =069) [49] In this dendrogram the 44C alismatifolia samplesappeared to form three main clusters and five minor clustersat coefficient level 56 cluster I included three non-irradiatedmembers and cluster II included 10Gy irradiated individualplants of all four varieties and 20Gy irradiated samples ofDoi Tung 554 and Kimono Pink varieties except KP20-4 (noflowering) Main cluster III included the 20Gy irradiatedindividuals Chiang Mai Red and Sweet Pink varieties (asmentioned in morphological part these individuals didnot go to flowering stage) and KP20-4 Cluster II can bedivided to five subclusters The first subcluster included 10members (CMR10-1 CMR10-5 SP10-1 SP10-2 SP10-3 SP10-5 CMR10-2 CMR10-4 SP10-4 and CMR10-3) the secondone included nine members (KP10-1 KP10-2 KP10-3 KP10-4 KP10-5KP20-1 KP20-2 KP20-5 and KP20-3) KP0 wasthe lone member of the third sub-cluster The fourth sub-cluster included two members only (DT10-2 and DT10-3) The last sub-cluster main cluster II had eight membersincluded (DT10-1 DT10-4 DT10-5 DT20-1 DT20-2 DT20-3 DT20-4 and DT20-5) Mean value of groups for each traitis presented in Table 9 This table clearly shows the differentmean values of the three main clusters The minimum meanvalue referred to main cluster III while the maximum meanvalue for number of shoots leaf length leaf width plantheight number of true flowers inflorescence length rhizomesize number of new rhizomes and number of storage rootsbelonged to cluster I which was included non-treated plantsThese results showed that the gamma irradiation has inducedmorphological changes in C alismatifolia individuals of
four studied varieties so that they showed phenotypicallydifferences from their controls
57 Principal Component Analysis of C alismatifolia forMorphological Traits To assess the patterns of variation PCAwas done by considering all of the 14 variables The first threecomponents of PCA explained 828 of the total variation(Table 10) Only the first component which accounted for562 of the total variation was attributed to inflorescencelength plant height days to senescence number of trueflowers and rhizome size In PCA three-dimensional graphthe grouping of individuals confirmed the clustering results(Figure 4) The PCA graph proved that all irradiated indi-vidual plants of C alismatifolia varieties are phenotypicallydifferent from their non-irradiated individuals
6 Molecular Characterization
61 SSR Polymorphism Amplifications were successful forall the 17 SSR markers assayed This reflects a high levelof homology between SSR flanking regions in C longa andC alismatifolia Out of 17 primer pairs eight primer pairsresulted in polymorphic PCR products Table 11 summarizesthe results obtained based on the analysis of individuals of thefour studied varieties using the polymorphic SSR loci for 010 and 20Gy irradiated plants The number of alleles variedwidely among these loci A total of 25 36 and 41 alleles wereobserved among 0 10 and 20Gy irradiated individual plantsrespectively In the 10Gy treatment the number of allelesranged from three (clon09 and clon14) to seven (clon08) withan average value of 45 per locus In the 20Gy treatmentthe number of alleles varied from three (clon09 and clon14)to seven (clon08 and clon12) with an average value of 51per locus In the untreated individuals the number of allelesranged from two (clon04 clon09 clon11 and clon14) to five(clon12) with an average value of 31 The difference betweenthe average number of observed alleles and effective numberof alleles was due to the uneven frequency of each allele[50] For each of the eight SSR primers PIC values (whichmeasures allele diversity and frequency among varieties)varied from 019 (clon04) to 071 (clon01) in untreated plantsand from 025 (clon04) to 075 (clon08) in 10Gy acutelyirradiated plants In 20Gy irradiated individual plants thePIC value ranged from 042 (clon14) to 075 (clon08 andclon12) The mean PIC for all loci was 047 054 and 061for 0 10 and 20Gy irradiated plants respectively
The PIC value provides an estimate of the discriminatorypower of amarker by taking into account not only the numberof alleles at a locus but also the relative frequencies of thesealleles [51] Thus except for clon09 and clon14 which weremoderately polymorphic (025 lt PIC lt 05) all other lociwere highly polymorphic (05 lt PIC) while none of theloci showed low polymorphism The average discriminatingpower ofmicrosatellitemarkers (061) observed in the presentstudy ensures the future utility of microsatellite markersfor genetic variation studies in C alismatifolia varietiesOur results reflect similar findings as reported earlier inthe study of genetic variation in other species of Curcuma
12 BioMed Research International
Table9Clusterm
eans
for14charactersestim
ated
in44
individu
also
fCalismatifolia
Clusters
Shoo
tnu
mber
Leaf
leng
thLeaf
width
Leaf
number
Visib
lebu
d(days)
Inflo
rescence
leng
thPlant
height
Anthesis
(days)
Senescence
(days)
True
flow
number
Pink
bract
number
Rhizom
esiz
eNew
rhizom
enu
mber
Storage
root
number
I220
5464
736
300
6013
1473
9233
6926
2093
152
1453
229
266
653
II1
100
2735
512
360
9260
809
5457
1059
960
620
510
177
110
440
II2
122
1922
298
344
11422
1043
3910
1312
293
3755
588
156
100
266
II3
100
3452
416
460
8520
1370
5910
9420
180
120
960
206
100
400
II4
150
2500
45
250
6500
925
4950
7700
1150
950
1700
185
200
600
II5
137
2637
417
262
7725
840
5356
9162
1137
1012
1587
170
100
312
III
054
1500
465
181
000
000
000
000
000
000
000
090
081
200
BioMed Research International 13
Table 10 Eigenvectors eigenvalues and proportions of variabilityfor three principle components among 14 characters for 44 Calismatifolia
Variable PC1 PC2 PC3Number of new shoot 0270 minus0076 minus0173Leaf length 0269 minus0305 0033Leaf width 0096 minus0455 0430Leaf number 0210 0285 0490Days to visible bud 0199 0462 0181Plant height 0321 0027 minus0071Inflorescence length 0318 0166 minus0040Days to anthesis 0205 0460 0148Days to senescence 0342 0017 minus0131Number of true flower 0323 0029 minus0238Number of pink bracts 0266 minus0028 minus0546Rhizome size 0302 minus0101 0147Number of new rhizome 0250 minus0293 0028Number of storage roots 0264 minus0247 0298Eigenvalue 7877 2694 1032Proportion 56264 19246 7371Cumulative 56264 75511 82882
using SSR markers [13] The highest Neirsquos gene diversity(ℎ) was obtained with the 20Gy treatment (061) followedby the 10Gy treatment (06) and the untreated individuals(05) The mean Shannonrsquos information index (119868) was 092114 and 130 in the 0 10 and 20Gy irradiated plantsrespectively The high value of Shannonrsquos information indexrepresents the effectiveness of microsatellite loci to revealthe variation among these varieties at the different dosesused Overall genetic variability for the varieties studiedrepresented by the Shannon-Weiner index was relatively highin comparison to other studies involving C longa accessions[10 52] Additionally the 20Gy acutely irradiated individualsshowed a higher mean percentage of polymorphic loci (63)than the 10Gy (58) and untreated (022) ones This impliesthat irradiation with a dose of 20Gy induced more geneticvariation in the M
1V1generation
62 Cluster Analysis Thedendrogramwas constructed usingPowerMarker 323The 44 studied individuals were clusteredinto seven groups (G1ndashG7) Colors were applied accordingto our model-based cluster analysis results Group I iscomprised of nine individual plants of Chiang Mai Redvariety receiving 0 10 and 20Gy treatments (blue colorin Figure 5) One individual plant DT10-1 was assignedto group II (green color in Figure 5) All irradiated andnon-irradiated individuals of Doi Tung 554 variety wereassigned to group III (yellow color in Figure 5) DT20-1 andSP10-5 were the sole members of groups IV (red color inFigure 5) and V (gray color in Figure 5) respectively GroupVI included treated and untreated individuals of Sweet Pinkvariety (pink color in Figure 5) Lastly all Kimono Pinkindividuals were assigned to cluster VII (orange color in
Figure 5) The genetic similarity coefficient (data not shown)among the 44 individuals amplified using eight SSR primersvaried from 00 to 10 The highest value (10) correspondedto [(CMR10-3 CMR10-5 CMR and CMR20-2) (DT10-2and DT10-3) (DT and DT20-2) (KP20-3 KP-20-4 KP10-3andKP10-4)] individuals that generated identical fingerprintsacross the markers studied Among the studied varieties theSweet Pink individual plants showed the lowest similarity(00) withKimono Pink individuals which indicated that theywere relatively remote in relationship (Figure 5) An overviewof the clustering pattern indicates that the grouping of thestudied individual plants was based on different varieties andlargely independent of the doses of gamma irradiation Awide range in similarity values had also been observed indifferent species of Curcuma [12 13 52 53]
63 Principle Component Analysis (PCA) Thedata generatedfrom 44 C alismatifolia individual plants were subjected toprincipal component analysis (PCA) to visualize individualsin a multivariate space In the three-dimensional graphderived from the SSR analysis all studied individuals weregrouped into seven clusters (Figure 6) In PCA the firstthree principal components (PC) extracted a cumulative of6826 of the total variation among the 44 individuals of CalismatifoliaThe first three coordinates PC1 PC2 and PC3accounted for 4136 1578 1112 of the total variationrespectively The distribution of the individual plants in athree-dimensional plot using the first three principal com-ponents showed the genetic relationship between individualplants It was evident that both methods dendrogram andthree-dimensional plots of PCA were effective in illustratinggenetic relationships and the groups found were comparable[11 13] The PCA results were similar to those obtained fromcluster analysis where all individuals from different varietieswere assigned to different groups These graphical illus-trations enable the assessment of the relationshipdistancesamong all of the individuals in the study [50]
7 Conclusion
This is the first attempt to evaluate the effect of acutegamma irradiation on C alismatifolia varieties using bothmorphological characteristics and molecular markers Inplant breeding programs mutagenic treatments with lownegative physiological effects and strong genetic effects aredesirable Hence we used more effective doses of gammairradiation (10 and 20Gy) of which particularly 20Gy dosewas effective to influence morphological and molecularcharacteristics of studiedCurcuma alismatifolia varietiesThelower dose (10Gy) of radiation probably caused little damageto the plants genetic material so that the cells could repairthemselves in next generation As a result in this study 20Gyacute gamma irradiation resulted in a higher percentage ofmutation and getting desired mutants was more possibleBased on the LD
50values determined in this study it was
apparent that the varieties Chiang Mai Red and Sweet Pinkwere more sensitive to gamma rays compared to Doi Tung554 and Kimono Pink Our results show that the variety Doi
Figure 4 Two-dimensional graph of principal component analysis (PCA) for 14 morphological variables indicating relationships amongirradiated and nonirradiated four varieties of C alismatifolia
CMR10-1CMR10-5
SP10-1SP10-2SP10-3
SP10-5
CMR10-4
SP10-4
KP10-1KP10-3
KP20-1
KP20-2
KP10-5KP20-5
KP20-3
KP10-2
DT10-2
DT10-1
DT10-5
DT20-1
DT10-4DT20-4DT20-2
DT20-5DT20-3
CMR20-1
SP20-3SP20-4
SP20-5SP20-1
SP20-2
KP
SP
CMR 10-2
SP10-5
Figure 5 Unrooted neighbor joining tree showing genetic relationship among 44 irradiated and nonirradiated C alismatifolia using SSRmarkers The colors of the branches correspond to those of the same cluster
Figure 6Three-dimensional graph of principal component analysis (PCA) indicating relationships among irradiated and nonirradiated fourvarieties of C alismatifolia
Tung 554 (Curcuma hybrid) had the most morphologicalresponses to gamma rays The use of microsatellite markersas a codominant marker will facilitate the exploration ofgenetic variability among treated and non-treated plants andwill help to distinguish the plants showing differences inmorphological characters The overall effects on the M
1V1
generation revealed that acute gamma irradiation at optimumdoses has the potential for developing new varieties of Calismatifolia with improved commercial properties suitablefor the Malaysian flower industry
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work was funded by the Fundamental Research GrantScheme (FRGS) under the Ministry of Higher Education inMalaysia
References
[1] P Apavatjrut S Anuntalabhochai P Sirirugsa and C AlisildquoMolecularmarkers in the identification of some early floweringCurcuma L (Zingiberaceae) speciesrdquo Annals of Botany vol 84no 4 pp 529ndash534 1999
[2] K Bunya-Atichart S Ketsa andW G VanDoorn ldquoPostharvestphysiology of Curcuma alismatifolia flowersrdquo Postharvest Biol-ogy and Technology vol 34 no 2 pp 219ndash226 2004
[3] M Nakano J Amano Y Watanabe et al ldquoMorphologicalvariation in Tricyrtis hirta plants regenerated from heavy ionbeam-irradiated embryogenic callusesrdquo Plant Biotechnologyvol 27 no 2 pp 155ndash160 2010
[4] S Lamseejan P Jompuk A Wongpiyasatid A Deeseepan andP Kwanthammachart ldquoGamma-rays induced morphological
[5] G-J Lee S J Chung I S Park et al ldquoVariation in thephenotypic features and transcripts of colormutants of chrysan-themum (Dendranthema grandiflorum) derived from gammaraymutagenesisrdquo Journal of Plant Biology vol 51 no 6 pp 418ndash423 2008
[6] O K Kikuchi ldquoOrchid flowers tolerance to gamma-radiationrdquoRadiation Physics and Chemistry vol 57 no 3ndash6 pp 555ndash5572000
[7] N P Arnold N N Barthakur and M Tanguay ldquoMutageniceffects of acute gamma irradiation on miniature roses targettheory approachrdquo HortScience vol 33 no 1 pp 127ndash129 1998
[8] A A Youssef M S Aly and M S Hussein ldquoResponse ofgeranium (Pelargonium graveolenus L) to gamma irradiationand foliar application of Speed Growrdquo Egyptian Journal ofHorticulture vol 27 pp 41ndash53 2000
[9] N Chuantang and L Yazhi ldquoThe radiation inducedmutation ofcanna (Canna L)rdquo Acta Agricultute Nucleatae Sinica vol 2 pp33ndash39 1998
[10] M S Sigrist J B Pinheiro J A A Filho and M I ZucchildquoGenetic diversity of turmeric germplasm (Curcuma longaZingiberaceae) identified by microsatellite markersrdquo Geneticsand Molecular Research vol 10 no 1 pp 419ndash428 2011
[11] S Taheri T L Abdullah N A P Abdullah and Z AhmadldquoGenetic relationships among five varieties of Curcuma alis-matifolia (Zingiberaceae) based on ISSRmarkersrdquoGenetics andMolecular Research vol 11 pp 3069ndash3076 2012
[12] A Das V Kesari V M Satyanarayana A Parida and L Ran-gan ldquoGenetic relationship of Curcuma species from NortheastIndia using PCR-based markersrdquo Molecular Biotechnology vol49 no 1 pp 65ndash76 2011
[13] S Siju K Dhanya S Syamkumar et al ldquoDevelopment char-acterization and utilization of genomic microsatellite markersin turmeric (Curcuma longa L)rdquo Biochemical Systematics andEcology vol 38 no 4 pp 641ndash646 2010
[14] M K Panda S Mohanty E Subudhi L Acharya and SNayak ldquoAssessment of genetic stability of micropropagated
BioMed Research International 17
plants of Curcuma L by cytophotometery and RAPD analysisrdquoInternational Journal of Integrative Biology (IJIB) vol 1 pp 189ndash195 2007
[15] S Syamkumar and B Sasikumar ldquoMolecular marker basedgenetic diversity analysis of Curcuma species from IndiardquoScientia Horticulturae vol 112 no 2 pp 235ndash241 2007
[16] D Tautz and M Renz ldquoSimple sequences are ubiquitousrepetitive components of eukaryotic genomesrdquo Nucleic AcidsResearch vol 12 no 10 pp 4127ndash4138 1984
[17] G Toth Z Gaspari and J Jurka ldquoMicrosatellites in differenteukaryotic genomes surveys and analysisrdquo Genome Researchvol 10 no 7 pp 967ndash981 2000
[18] W Powell G C Machray and J Proven ldquoPolymorphismrevealed by simple sequence repeatsrdquo Trends in Plant Sciencevol 1 no 7 pp 215ndash222 1996
[19] S-Y Lee W K Fai M Zakaria et al ldquoCharacterizationof polymorphic microsatellite markers isolated from ginger(Zingiber officinale Rosc)rdquo Molecular Ecology Notes vol 7 no6 pp 1009ndash1011 2007
[20] S Bory D Da Silva A-M Risterucci M Grisoni P Besseand M-F Duval ldquoDevelopment of microsatellite markers incultivated vanilla polymorphism and transferability to othervanilla speciesrdquo Scientia Horticulturae vol 115 no 4 pp 420ndash425 2008
[21] I C Menezes F W Cidade A P Souza and I C SampaioldquoIsolation and characterization ofmicrosatellite loci in the blackpepper Piper Nigrum L (Piperaceae)rdquo Conservation GeneticsResources vol 1 no 1 pp 209ndash212 2009
[22] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[23] V Kumar Morphological and molecular characterization ofinduced mutants in Groundnut [PhD thesis] University ofAgricultural Sciences Dharwad India 2008
[24] J R Sharma Statistical and Biometrical Techniques in PlantBreeding New Age International New Delhi India 1998
[25] J J Doyle and J L Doyle ldquoA rapid DNA isolation procedure forsmall quantities of fresh leaf tissuerdquo Phytochemical Bulletin vol19 pp 11ndash15 1987
[26] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[27] Oslash Hammer D A T Harper and P D Ryan ldquoPast paleontolog-ical statistics software package for education and data analysisrdquoPalaeontologia Electronica vol 4 no 1 pp 4ndash9 2001
[28] K Liu and S V Muse ldquoPowerMaker an integrated analysisenvironment for genetic maker analysisrdquo Bioinformatics vol 21no 9 pp 2128ndash2129 2005
[29] F J RohlfNTSYS-Pc Numerical Taxonomy System Version 2 1Exeter Publishing Setauket New York NY USA 2002
[30] M Nei ldquoAnalysis of gene diversity in subdivided populationsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 70 no 12 1973
[31] S Kadkhodaei M Shahnazari M K Nekouei et al ldquoA compar-ative study of morphological and molecular diversity analysisamong cultivated almonds (Prunus dulcis)rdquo Australian Journalof Crop Science vol 5 no 1 pp 82ndash91 2011
[32] T L Abdullah J Endan and B M Nazir ldquoChanges in flowerdevelopment chlorophyll mutation and alteration in plant
morphology of Curcuma alismatifolia by gamma irradiationrdquoAmerican Journal of Applied Sciences vol 6 no 7 pp 1436ndash14392009
[33] E Kovacs and A Keresztes ldquoEffect of gamma and UV-BCradiation on plant cellsrdquoMicron vol 33 no 2 pp 199ndash210 2002
[34] H L Ramesh Y Murthy and V N Munirajappa ldquoEffect ofgamma radiation on morphological and growth parametersof Mulberry variety M5rdquo International Journal of Science andNature vol 3 pp 447ndash452 2010
[35] K Pongchawee R Pradissan andW Pipatcharoenchai ldquoInducemutation in Anubias spp through in vitro Irradiationrdquo ThaiFisheries Gazette vol 60 pp 493ndash497 2007
[36] P Tangpong T Taychasinpitak C Jompuk and P JompukldquoEffects of acute and chronic gamma irradiations on in vitroculture ofAnubias congensisNE BrownrdquoKasetsart Journal vol43 no 3 pp 449ndash457 2009
[37] R K Hegde Studies on induced mutagenesis and in vitroregeneration in turmeric (Curcuma longa L) [PhD thesis]University of Agricultural Sciences Dharwad India 2006
[38] M P Giridharan and S Balakrishnan ldquoGamma ray inducedvariability in vegetative and floral characters of ginger IndianCocoardquo Arecanut and Spices Journal vol 15 pp 68ndash672 1992
[39] A P Cesarett ldquoEffect of radiation on higher plants and plantcommunitiesrdquo inRadiation Biology United States Atomic EnergyCommission pp 284ndash309 Washington DC USA 1968
[40] G W Seung Y C Byung J-H Kim et al ldquoUltrastructuralchanges of cell organelles in Arabidopsis stems after gammairradationrdquo Journal of Plant Biology vol 48 no 2 pp 195ndash2002005
[41] B S Ahloowalia and M Maluszynski ldquoInduced mutationsmdashanew paradigm in plant breedingrdquo Euphytica vol 118 no 2 pp167ndash173 2001
[42] R Voisine L-P Vezina and C Willemot ldquoInduction ofsenescence-like deterioration of microsomal membranes fromcauliflower by free radicals generated during gamma irradia-tionrdquo Plant Physiology vol 97 no 2 pp 545ndash550 1991
[43] N Shikazono Y Yokota S Kitamura et al ldquoMutation rate andnovel tt mutants of Arabidopsis thaliana induced by carbonionsrdquo Genetics vol 163 no 4 pp 1449ndash1455 2003
[44] T Nakatsuka M Nishihara K Mishiba and S YamamuraldquoTwo different mutations are involved in the formation ofwhite-flowered gentian plantsrdquo Plant Science vol 169 no 5 pp949ndash958 2005
[45] M Mato T Onozaki Y Ozeki et al ldquoFlavonoid biosynthesisin white-flowered Sim carnations (Dianthus caryophyllus)rdquoScientia Horticulturae vol 84 no 3-4 pp 333ndash347 2000
[46] S G Wi B Y Chung J-S Kim et al ldquoEffects of gammairradiation on morphological changes and biological responsesin plantsrdquoMicron vol 38 no 6 pp 553ndash564 2007
[47] F Lee Chin Effects of light intensity and daylength on growth andflowering of siam tulip (Curcuma alismatifolia var Chiang MaiPink) [PhD thesis] University Putra Malaysia 2007
[48] A Hagiladi N Umiel and X H Yang ldquoCurcuma alismatifoliaII Effects of temperature and daylength on the development offlowers and propagulesrdquo Acta Horticulture vol 430 pp 755ndash761 1997
[49] MHagidimitriou A Katsiotis GMenexes C Pontikis andMLoukas ldquoGenetic diversity of major greek olive cultivars usingmolecular (AFLPs and RAPDs) markers and morphologicaltraitsrdquo Journal of the American Society for Horticultural Sciencevol 130 no 2 pp 211ndash217 2005
18 BioMed Research International
[50] N Babaei N A P Abdullah G Saleh and T L AbdullahldquoIsolation and characterization of microsatellite markers andanalysis of genetic variability in Curculigo latifolia DryandrdquoMolecular Biology Reports vol 39 pp 9869ndash9877 2012
[51] B Shiran N Amirbakhtiar S Kiani S Mohammadi B ESayed-Tabatabaei and H Moradi ldquoMolecular characterizationand genetic relationship among almond cultivars assessed byRAPD and SSR markersrdquo Scientia Horticulturae vol 111 no 3pp 280ndash292 2007
[52] S Singh M K Panda and S Nayak ldquoEvaluation of geneticdiversity in turmeric (Curcuma longa L) using RAPD and ISSRmarkersrdquo Industrial Crops and Products vol 37 no 1 pp 284ndash291 2012
[53] Y Paisooksantivatana S Kako and H Seko ldquoGenetic diversityof Curcuma alismatifolia Gagnep (Zingiberaceae) in Thailandas revealed by allozyme polymorphismrdquo Genetic Resources andCrop Evolution vol 48 no 5 pp 459ndash465 2001
20 Dwarfism fewer pink bracts and yellowwhite strip leavesSmall and narrow leave Light purple N80Dlowast
lowastRoyal British Society color chart (RHS)
8 BioMed Research International
Table 6 Effect of acute gamma rays on vegetative traits of C alismatifolia in M1V1 generation
Dose (Gray) Shoot number Leaf number Leaf length (cm) Leaf width (cm) Plant height (cm)CMR
0 20 plusmn 00a
30 plusmn 04a
556 plusmn 08a
71 plusmn 05a
1112 plusmn 25a
10 10 plusmn 00b
36 plusmn 05a
272 plusmn 25b
45 plusmn 05b
714 plusmn 96b
20 10 plusmn 00b
14 plusmn 05b
124 plusmn 120c
25 plusmn 24c
170 plusmn 80c
DT0 30 plusmn 00
a30 plusmn 00
a622 plusmn 08
a65 plusmn 05
a912 plusmn 19
a
10 16 plusmn 05b
28 plusmn 04a
280 plusmn 44b
46 plusmn 06b
542 plusmn 76b
20 12 plusmn 04b
24 plusmn 05a
242 plusmn 38b
38 plusmn 05c
512 plusmn 138b
SP0 16 plusmn 05
a32 plusmn 04
a461 plusmn 27
a101 plusmn 04
a746 plusmn 38
a
10 10 plusmn 00b
36 plusmn 05a
275 plusmn 34b
57 plusmn 02b
377 plusmn 137b
20 10 plusmn 00b
20 plusmn 10b
196 plusmn 74c
57 plusmn 04b
195 plusmn 73c
KP0 14 plusmn 00
a46 plusmn 05
a345 plusmn 29
a47 plusmn 04
a591 plusmn 28
a
10 10 + 05a
40 plusmn 05b
220 plusmn 24b
31 plusmn 04b
420 plusmn 54b
20 10 plusmn 00a
32 plusmn 04b
156 plusmn 43c
31 plusmn 11b
283 plusmn 174c
CV () 23 18 26 16 165Means with the same or common letter are not significantly different least square difference test 119875 lt 005
decreases in leaf size were reported by Pongchawee et al [35]and Tangpong et al [36] These results were in agreementwith an earlier study [4] which reported that the growthof chrysanthemum exposed to acute gamma rays was lessthan the control in the M
1V1generation All varieties doses
and interaction effects resulted in significant differences fornumber of leaves Among untreated plants Kimono Pinkvariety had higher number of leaves (46) than the otherthree (3 3 and 32) varieties In Chiang Mai Red and SweetPink varieties plants exposed to 10Gy showedhigher numberof leaves than untreated plants However at 20Gy therewas significant reduction in number of leaves for all studiedvarieties in comparison to control Similar stimulatory effectswere obtained at lower doses in ginger by Hegde [37] andGiridharan and Balakrishnan [38]
Progressive reduction in growth parameters can be inter-preted on interference in normal mitosis and frequent occur-rence of mitotic aberrations inhibition of rate of assimilationand consequent change in the nutrient level in the plantand inactivation of vital enzymes especially those associatedwith respiration [39] Dose-dependent negative effect wasalso detected for plant heightThe tallest plants were recordedfrom the untreated rhizomes (0Gy) with heights of 1112912 746 and 591 cm followed by the 10Gy irradiated plantswith heights of 714 542 377 and 420 cm and the 20Gyirradiated plants with corresponding heights of 170 512195 and 283 cm for Chiang Mai Red Doi Tung 554 SweetPink and Kimono Pink varieties respectively These resultsare in agreement with the findings of Abdullah et al [32]Reduction in growth parameters and dwarfism can be causedby interference of normal mitosis and frequent occurrenceof mitotic aberrations inhibition of assimilation rates andconsequent changes in nutrient levels in plants Additionallymutagenic effects such as auxin destruction inhibition of
auxin synthesis failure of assimilatory mechanism andchanges in the specific activity of enzymes can cause growthreductions [37] High doses of ionizing radiation have beenshown to damage macromolecular cellular components suchas cell walls membranes and DNA [40] The number ofshoots also decreased significantly as the radiation dosesincreased Radiation also affects organic molecules that areessential to the cell division process and thus causing celldivision to stop [36]
54 Effect of Gamma Irradiation on Flowering DevelopmentTraits in the M1V1 All control and 10Gy irradiated plantsproduced flowers while the Chiang Mai Red and SweetPink varieties which were exposed to 20Gy did not gointo the flowering stage Lamseejan et al [4] also showedthat flowering percentage decreases as gamma ray dosesare increased In the present study gamma rays caused lateflowering in all four varieties Days to appearance of firstvisible buds were also significantly different among the fourvarieties (Table 7) Gamma rays caused a noticeable delay inflowering of irradiated plants in comparison to the untreatedones First visible buds were observed at 652 and 874 days inthe control and 10Gy treatments respectively for the ChiangMai Red variety In Doi Tung 554 the first visible buds wereappeared at 474 656 and 840 days after planting at 0 10and 20Gy doses respectively In the Sweet Pink variety thenumber of days to visible bud appearance increased signif-icantly from 678 days in controls to 978 days in the 10Gyirradiated plants In comparison to other three varieties theKimono Pink variety needed the longest time to visible budappearance and same as other varieties there was a positivecorrelation between the number of days to first visible budand the gamma irradiation dose In the control and 10Gyand 20Gy irradiated individual plants flower buds were
BioMed Research International 9
Table 7 Effect of acute gamma rays on flower development characteristics of C alismatifolia in M1V1 generation
Dose (Gy) Days tovisible bud
Inflorescencelength (cm)
Days toanthesis
Number oftrue flowers
Number ofPink bracts
Days tosenescence
CMR0 652 plusmn 47
b162 plusmn 057
a742 plusmn 54
b132 plusmn 19
a104 plusmn 15
a21 plusmn 10
a
10 874 plusmn 95a
82 plusmn 397b
1020 plusmn 71a
56 plusmn 19b
50 plusmn 07b
10 plusmn 14b
20 00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
DT0 474 plusmn 43
c134 plusmn 054
a546 plusmn 54
c162 plusmn 13
a232 plusmn 04
a23 plusmn 00
a
10 656 plusmn 37b
94 plusmn 19b
780 plusmn 37b
112 plusmn 21b
166 plusmn 08b
122 plusmn 130b
20 840 plusmn 42a
76 plusmn 082c
994 plusmn 49a
88 plusmn 13b
156 plusmn 11b
106 plusmn 18b
SP0 678 plusmn 21
b146 plusmn 089
a756 plusmn 26
b162 plusmn 13
a100 plusmn 24
a188 plusmn 083
a
10 978 plusmn 181a
79 plusmn 20b
1098 plusmn 182a
68 plusmn 25b
52 plusmn 08b
92 plusmn 24b
20 00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
KP0 852 plusmn 23
c137 plusmn 044
a942 plusmn 19
c112 plusmn 12
a96 plusmn 21
a18 plusmn 18
a
10 1044 plusmn 65b
108 plusmn 108ab
1224 plusmn 100b
76 plusmn 27b
64 plusmn 16ab
102 plusmn 17b
20 1272 plusmn 65a
790 plusmn 49b
1426 plusmn 58a
60 plusmn 38b
42 plusmn 24b
66 plusmn 37b
CV () 10 23 9 22 169 15Means with the same or common letter are not significantly different least square difference test 119875 lt 005
visible at 852 1328 and 1374 days after planting respectivelyPrevious studies also showed that onset of flowering andformation of floral parts in mutants of Arabidopsis thalianamaize barely pea and tobacco involved growth regulators(or phytohormones) such as auxins cytokinins gibberellinsabscisic acid ethylene and brassinosteroids [41] There weresignificant differences among treatments for the length ofthe inflorescence In all varieties the longest inflorescencelength was observed in the untreated plants with 162134 146 and 137 cm lengths for Chiang Mai Red DoiTung 554 Sweet Pink and Kimono Pink respectively Thecorresponding inflorescence lengths were 82 94 79 and109 cm for the 10Gy irradiated plants The days to anthesisfor C alismatifolia varieties were significantly affected byvariety gamma irradiation doses and their interaction Thenumber of days to full bloom was noticeable earliest foruntreated plants at 742 546 756 and 942 days for ChiangMai Red Doi Tung 554 Sweet Pink and Kimono Pinkvarieties respectively This was then followed by plants at10Gy at 102 78 1098 and 1224 days The number of trueflowers or the secondary inflorescence developed in the axilof the primary bracts decreased as radiation dosage increasedIn the present study the gamma rays also decreased thedays to inflorescence senescence In this study there wasa strongly significantly and positively correlation (0919lowastlowast)(data not shown) between the number of true flowers andthe number of days to senescenceThe number of pink bractsalso decreasedwith increasing radiation dosageMost gammaray effects on senescence are considered as resulting from theaction of free radicals generated from water and oxygen bythe ionizing energy on the cellular components Membrane
deterioration is a general feature of natural senescence andstress-induced aging [42]
Irradiation induced some mutation spectrum of flowercolor variation that included colors such as purple palepurple rather pale purple white purple white (marbledpattern) and two-tone purple color
Mutation spectrum of flower shape variation includeddouble inflorescence within one stalk double stalk per plantinflorescence without bracts two-flag petal true flowersand chlorophyll mutation in the leaves which are generallycaused by induced gamma rays (Figure 2) Ionizing radiationincluding gamma rays induces fragment deletions or inser-tions that eventually lead to changes in amino acids and amodification of leaf and stem pigmentation [43] A mutationin the biosynthetic pathway of structural or regulatory genesmay cause a change in flower color [44] When the blockageoccurs at the early stages of anthocyanin synthesis whiteflowers will result while a blockage at later stages leads todifferent flower colors due to the accumulation of particularanthocyanins [45] Chloroplasts were extremely sensitive togamma radiation compared to other cell organelles [46]
55 Effect of Gamma Irradiation on Rhizome Characteristicsin Selected Doses in M1V1 The number of new rhizomes andthe number of storage roots per rhizome were significantlyaffected by varieties doses and their interaction (Table 8)The number of new rhizomes only in Kimono Pink varietydid not show any differences between untreated and treatedplants The rhizome size was influenced only by doses Asdose level increased the rhizome size decreased Amonguntreated plants the Sweet Pink rhizomes had the most
10 BioMed Research International
(a) (b) (c) (d)
(e)
(f)
Figure 2 Effect of acute gamma rays on flower traits in C alismatifolia (a) Untreated inflorescence bracts color Doi Tung 554 (b) Two tonebract color Doi Tung 554 (20Gy) (c) Marbled pattern of bract color Doi Tung 554 (20Gy) (d) Double inflorescence within one stalk varChiang Mai Red (10Gy) (e) True flower in nontreated plants (f) Two-flag petals true flowers (10Gy)
Figure 3 Dendrogram representing themorphological variation among 44 irradiated and nonirradiated individuals ofC alismatifolia across14 variables CMR = Chiang Mai Red DT = Doi Tung 554 SP = Sweet Pink Kp = Kimono Pink
number of storage roots (78) and the Kimono Pink rhizomeshad the least number of storage roots (40)This was followedby 10Gy irradiated rhizomes with 4 46 48 and 28 storageroot numbers for Chiang Mai Red Doi Tung 554 SweetPink and Kimono Pink respectively Irradiation with 20Gydecreased significantly the number of storage roots in allstudied varietiesTheplantC alismatifolia is propagated froma propagule (one rhizome + 5-6 t-roots) The rhizome sizedose matters in the growth of the plant Smaller rhizome sizeusually resulted in smaller plant size with narrow grass-likeleaves The storage roots play a very important role in thegrowth and flowering ofCurcumaThe t-rootsmake up about85 of the total fresh weight and 70 of the total dry weightof a typical propaguleThe storage organs act for plant growth
during dormancy and emergence [47] More storage root perpropagule resulted in faster flowering and higher yield plant[48] In this study also there was significant and negativecorrelation between number of storage roots and number ofdays to visible bud appearance (minus0525lowastlowast)
56 Cluster Analysis of C alismatifolia for MorphologicalTraits The morphological data were used to calculate thesimilarity between the treated and non-treated C alismat-ifolia samples and UPGMA dendrogram was constructed(Figure 3) The cophenetic correlation coefficient (CCC)value between the genetic dissimilaritymatrix estimated fromthe morphological characters and the UPGMA clusteringmethod was 119903 = 093 showing a proof fit This value was
BioMed Research International 11
Table 8 Effect of acute gamma rays on rhizome characteristics ofC alismatifolia in M1V1 generation
Dose (Gray) Rhizomesize
No of newrhizomes
No of storageroot
CMR0 23 plusmn 02
a26 plusmn 055
a46 plusmn 054
a
10 18 plusmn 005a12 plusmn 044
b40 plusmn 07
a
20 08 plusmn 07b
06 plusmn 054b
14 plusmn 13b
DT0 22 plusmn 01
a30 plusmn 00
a72 plusmn 109
a
10 18 plusmn 007b14 plusmn 054
b46 plusmn 13
b
20 16 plusmn 005b
10 plusmn 00b
28 plusmn 13c
SP0 22 plusmn 02
a24 plusmn 054
a78 plusmn 21
a
10 17 plusmn 007b
10 plusmn 00b
48 plusmn 044b
20 12 plusmn 02c
10 plusmn 00b
28 plusmn 083b
KP0 21 plusmn 005
a10 plusmn 00
a40 plusmn 00
a
10 16 plusmn 01ab
10 plusmn 00a
28 plusmn 044b
20 11 plusmn 06b
10 plusmn 00a
22 plusmn 083b
CV () 18 23 26Means with the same or common letter are not significantly different leastsquare difference test 119875 lt 005
higher than of studies on olive for morphological traits (119903 =069) [49] In this dendrogram the 44C alismatifolia samplesappeared to form three main clusters and five minor clustersat coefficient level 56 cluster I included three non-irradiatedmembers and cluster II included 10Gy irradiated individualplants of all four varieties and 20Gy irradiated samples ofDoi Tung 554 and Kimono Pink varieties except KP20-4 (noflowering) Main cluster III included the 20Gy irradiatedindividuals Chiang Mai Red and Sweet Pink varieties (asmentioned in morphological part these individuals didnot go to flowering stage) and KP20-4 Cluster II can bedivided to five subclusters The first subcluster included 10members (CMR10-1 CMR10-5 SP10-1 SP10-2 SP10-3 SP10-5 CMR10-2 CMR10-4 SP10-4 and CMR10-3) the secondone included nine members (KP10-1 KP10-2 KP10-3 KP10-4 KP10-5KP20-1 KP20-2 KP20-5 and KP20-3) KP0 wasthe lone member of the third sub-cluster The fourth sub-cluster included two members only (DT10-2 and DT10-3) The last sub-cluster main cluster II had eight membersincluded (DT10-1 DT10-4 DT10-5 DT20-1 DT20-2 DT20-3 DT20-4 and DT20-5) Mean value of groups for each traitis presented in Table 9 This table clearly shows the differentmean values of the three main clusters The minimum meanvalue referred to main cluster III while the maximum meanvalue for number of shoots leaf length leaf width plantheight number of true flowers inflorescence length rhizomesize number of new rhizomes and number of storage rootsbelonged to cluster I which was included non-treated plantsThese results showed that the gamma irradiation has inducedmorphological changes in C alismatifolia individuals of
four studied varieties so that they showed phenotypicallydifferences from their controls
57 Principal Component Analysis of C alismatifolia forMorphological Traits To assess the patterns of variation PCAwas done by considering all of the 14 variables The first threecomponents of PCA explained 828 of the total variation(Table 10) Only the first component which accounted for562 of the total variation was attributed to inflorescencelength plant height days to senescence number of trueflowers and rhizome size In PCA three-dimensional graphthe grouping of individuals confirmed the clustering results(Figure 4) The PCA graph proved that all irradiated indi-vidual plants of C alismatifolia varieties are phenotypicallydifferent from their non-irradiated individuals
6 Molecular Characterization
61 SSR Polymorphism Amplifications were successful forall the 17 SSR markers assayed This reflects a high levelof homology between SSR flanking regions in C longa andC alismatifolia Out of 17 primer pairs eight primer pairsresulted in polymorphic PCR products Table 11 summarizesthe results obtained based on the analysis of individuals of thefour studied varieties using the polymorphic SSR loci for 010 and 20Gy irradiated plants The number of alleles variedwidely among these loci A total of 25 36 and 41 alleles wereobserved among 0 10 and 20Gy irradiated individual plantsrespectively In the 10Gy treatment the number of allelesranged from three (clon09 and clon14) to seven (clon08) withan average value of 45 per locus In the 20Gy treatmentthe number of alleles varied from three (clon09 and clon14)to seven (clon08 and clon12) with an average value of 51per locus In the untreated individuals the number of allelesranged from two (clon04 clon09 clon11 and clon14) to five(clon12) with an average value of 31 The difference betweenthe average number of observed alleles and effective numberof alleles was due to the uneven frequency of each allele[50] For each of the eight SSR primers PIC values (whichmeasures allele diversity and frequency among varieties)varied from 019 (clon04) to 071 (clon01) in untreated plantsand from 025 (clon04) to 075 (clon08) in 10Gy acutelyirradiated plants In 20Gy irradiated individual plants thePIC value ranged from 042 (clon14) to 075 (clon08 andclon12) The mean PIC for all loci was 047 054 and 061for 0 10 and 20Gy irradiated plants respectively
The PIC value provides an estimate of the discriminatorypower of amarker by taking into account not only the numberof alleles at a locus but also the relative frequencies of thesealleles [51] Thus except for clon09 and clon14 which weremoderately polymorphic (025 lt PIC lt 05) all other lociwere highly polymorphic (05 lt PIC) while none of theloci showed low polymorphism The average discriminatingpower ofmicrosatellitemarkers (061) observed in the presentstudy ensures the future utility of microsatellite markersfor genetic variation studies in C alismatifolia varietiesOur results reflect similar findings as reported earlier inthe study of genetic variation in other species of Curcuma
12 BioMed Research International
Table9Clusterm
eans
for14charactersestim
ated
in44
individu
also
fCalismatifolia
Clusters
Shoo
tnu
mber
Leaf
leng
thLeaf
width
Leaf
number
Visib
lebu
d(days)
Inflo
rescence
leng
thPlant
height
Anthesis
(days)
Senescence
(days)
True
flow
number
Pink
bract
number
Rhizom
esiz
eNew
rhizom
enu
mber
Storage
root
number
I220
5464
736
300
6013
1473
9233
6926
2093
152
1453
229
266
653
II1
100
2735
512
360
9260
809
5457
1059
960
620
510
177
110
440
II2
122
1922
298
344
11422
1043
3910
1312
293
3755
588
156
100
266
II3
100
3452
416
460
8520
1370
5910
9420
180
120
960
206
100
400
II4
150
2500
45
250
6500
925
4950
7700
1150
950
1700
185
200
600
II5
137
2637
417
262
7725
840
5356
9162
1137
1012
1587
170
100
312
III
054
1500
465
181
000
000
000
000
000
000
000
090
081
200
BioMed Research International 13
Table 10 Eigenvectors eigenvalues and proportions of variabilityfor three principle components among 14 characters for 44 Calismatifolia
Variable PC1 PC2 PC3Number of new shoot 0270 minus0076 minus0173Leaf length 0269 minus0305 0033Leaf width 0096 minus0455 0430Leaf number 0210 0285 0490Days to visible bud 0199 0462 0181Plant height 0321 0027 minus0071Inflorescence length 0318 0166 minus0040Days to anthesis 0205 0460 0148Days to senescence 0342 0017 minus0131Number of true flower 0323 0029 minus0238Number of pink bracts 0266 minus0028 minus0546Rhizome size 0302 minus0101 0147Number of new rhizome 0250 minus0293 0028Number of storage roots 0264 minus0247 0298Eigenvalue 7877 2694 1032Proportion 56264 19246 7371Cumulative 56264 75511 82882
using SSR markers [13] The highest Neirsquos gene diversity(ℎ) was obtained with the 20Gy treatment (061) followedby the 10Gy treatment (06) and the untreated individuals(05) The mean Shannonrsquos information index (119868) was 092114 and 130 in the 0 10 and 20Gy irradiated plantsrespectively The high value of Shannonrsquos information indexrepresents the effectiveness of microsatellite loci to revealthe variation among these varieties at the different dosesused Overall genetic variability for the varieties studiedrepresented by the Shannon-Weiner index was relatively highin comparison to other studies involving C longa accessions[10 52] Additionally the 20Gy acutely irradiated individualsshowed a higher mean percentage of polymorphic loci (63)than the 10Gy (58) and untreated (022) ones This impliesthat irradiation with a dose of 20Gy induced more geneticvariation in the M
1V1generation
62 Cluster Analysis Thedendrogramwas constructed usingPowerMarker 323The 44 studied individuals were clusteredinto seven groups (G1ndashG7) Colors were applied accordingto our model-based cluster analysis results Group I iscomprised of nine individual plants of Chiang Mai Redvariety receiving 0 10 and 20Gy treatments (blue colorin Figure 5) One individual plant DT10-1 was assignedto group II (green color in Figure 5) All irradiated andnon-irradiated individuals of Doi Tung 554 variety wereassigned to group III (yellow color in Figure 5) DT20-1 andSP10-5 were the sole members of groups IV (red color inFigure 5) and V (gray color in Figure 5) respectively GroupVI included treated and untreated individuals of Sweet Pinkvariety (pink color in Figure 5) Lastly all Kimono Pinkindividuals were assigned to cluster VII (orange color in
Figure 5) The genetic similarity coefficient (data not shown)among the 44 individuals amplified using eight SSR primersvaried from 00 to 10 The highest value (10) correspondedto [(CMR10-3 CMR10-5 CMR and CMR20-2) (DT10-2and DT10-3) (DT and DT20-2) (KP20-3 KP-20-4 KP10-3andKP10-4)] individuals that generated identical fingerprintsacross the markers studied Among the studied varieties theSweet Pink individual plants showed the lowest similarity(00) withKimono Pink individuals which indicated that theywere relatively remote in relationship (Figure 5) An overviewof the clustering pattern indicates that the grouping of thestudied individual plants was based on different varieties andlargely independent of the doses of gamma irradiation Awide range in similarity values had also been observed indifferent species of Curcuma [12 13 52 53]
63 Principle Component Analysis (PCA) Thedata generatedfrom 44 C alismatifolia individual plants were subjected toprincipal component analysis (PCA) to visualize individualsin a multivariate space In the three-dimensional graphderived from the SSR analysis all studied individuals weregrouped into seven clusters (Figure 6) In PCA the firstthree principal components (PC) extracted a cumulative of6826 of the total variation among the 44 individuals of CalismatifoliaThe first three coordinates PC1 PC2 and PC3accounted for 4136 1578 1112 of the total variationrespectively The distribution of the individual plants in athree-dimensional plot using the first three principal com-ponents showed the genetic relationship between individualplants It was evident that both methods dendrogram andthree-dimensional plots of PCA were effective in illustratinggenetic relationships and the groups found were comparable[11 13] The PCA results were similar to those obtained fromcluster analysis where all individuals from different varietieswere assigned to different groups These graphical illus-trations enable the assessment of the relationshipdistancesamong all of the individuals in the study [50]
7 Conclusion
This is the first attempt to evaluate the effect of acutegamma irradiation on C alismatifolia varieties using bothmorphological characteristics and molecular markers Inplant breeding programs mutagenic treatments with lownegative physiological effects and strong genetic effects aredesirable Hence we used more effective doses of gammairradiation (10 and 20Gy) of which particularly 20Gy dosewas effective to influence morphological and molecularcharacteristics of studiedCurcuma alismatifolia varietiesThelower dose (10Gy) of radiation probably caused little damageto the plants genetic material so that the cells could repairthemselves in next generation As a result in this study 20Gyacute gamma irradiation resulted in a higher percentage ofmutation and getting desired mutants was more possibleBased on the LD
50values determined in this study it was
apparent that the varieties Chiang Mai Red and Sweet Pinkwere more sensitive to gamma rays compared to Doi Tung554 and Kimono Pink Our results show that the variety Doi
Figure 4 Two-dimensional graph of principal component analysis (PCA) for 14 morphological variables indicating relationships amongirradiated and nonirradiated four varieties of C alismatifolia
CMR10-1CMR10-5
SP10-1SP10-2SP10-3
SP10-5
CMR10-4
SP10-4
KP10-1KP10-3
KP20-1
KP20-2
KP10-5KP20-5
KP20-3
KP10-2
DT10-2
DT10-1
DT10-5
DT20-1
DT10-4DT20-4DT20-2
DT20-5DT20-3
CMR20-1
SP20-3SP20-4
SP20-5SP20-1
SP20-2
KP
SP
CMR 10-2
SP10-5
Figure 5 Unrooted neighbor joining tree showing genetic relationship among 44 irradiated and nonirradiated C alismatifolia using SSRmarkers The colors of the branches correspond to those of the same cluster
Figure 6Three-dimensional graph of principal component analysis (PCA) indicating relationships among irradiated and nonirradiated fourvarieties of C alismatifolia
Tung 554 (Curcuma hybrid) had the most morphologicalresponses to gamma rays The use of microsatellite markersas a codominant marker will facilitate the exploration ofgenetic variability among treated and non-treated plants andwill help to distinguish the plants showing differences inmorphological characters The overall effects on the M
1V1
generation revealed that acute gamma irradiation at optimumdoses has the potential for developing new varieties of Calismatifolia with improved commercial properties suitablefor the Malaysian flower industry
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work was funded by the Fundamental Research GrantScheme (FRGS) under the Ministry of Higher Education inMalaysia
References
[1] P Apavatjrut S Anuntalabhochai P Sirirugsa and C AlisildquoMolecularmarkers in the identification of some early floweringCurcuma L (Zingiberaceae) speciesrdquo Annals of Botany vol 84no 4 pp 529ndash534 1999
[2] K Bunya-Atichart S Ketsa andW G VanDoorn ldquoPostharvestphysiology of Curcuma alismatifolia flowersrdquo Postharvest Biol-ogy and Technology vol 34 no 2 pp 219ndash226 2004
[3] M Nakano J Amano Y Watanabe et al ldquoMorphologicalvariation in Tricyrtis hirta plants regenerated from heavy ionbeam-irradiated embryogenic callusesrdquo Plant Biotechnologyvol 27 no 2 pp 155ndash160 2010
[4] S Lamseejan P Jompuk A Wongpiyasatid A Deeseepan andP Kwanthammachart ldquoGamma-rays induced morphological
[5] G-J Lee S J Chung I S Park et al ldquoVariation in thephenotypic features and transcripts of colormutants of chrysan-themum (Dendranthema grandiflorum) derived from gammaraymutagenesisrdquo Journal of Plant Biology vol 51 no 6 pp 418ndash423 2008
[6] O K Kikuchi ldquoOrchid flowers tolerance to gamma-radiationrdquoRadiation Physics and Chemistry vol 57 no 3ndash6 pp 555ndash5572000
[7] N P Arnold N N Barthakur and M Tanguay ldquoMutageniceffects of acute gamma irradiation on miniature roses targettheory approachrdquo HortScience vol 33 no 1 pp 127ndash129 1998
[8] A A Youssef M S Aly and M S Hussein ldquoResponse ofgeranium (Pelargonium graveolenus L) to gamma irradiationand foliar application of Speed Growrdquo Egyptian Journal ofHorticulture vol 27 pp 41ndash53 2000
[9] N Chuantang and L Yazhi ldquoThe radiation inducedmutation ofcanna (Canna L)rdquo Acta Agricultute Nucleatae Sinica vol 2 pp33ndash39 1998
[10] M S Sigrist J B Pinheiro J A A Filho and M I ZucchildquoGenetic diversity of turmeric germplasm (Curcuma longaZingiberaceae) identified by microsatellite markersrdquo Geneticsand Molecular Research vol 10 no 1 pp 419ndash428 2011
[11] S Taheri T L Abdullah N A P Abdullah and Z AhmadldquoGenetic relationships among five varieties of Curcuma alis-matifolia (Zingiberaceae) based on ISSRmarkersrdquoGenetics andMolecular Research vol 11 pp 3069ndash3076 2012
[12] A Das V Kesari V M Satyanarayana A Parida and L Ran-gan ldquoGenetic relationship of Curcuma species from NortheastIndia using PCR-based markersrdquo Molecular Biotechnology vol49 no 1 pp 65ndash76 2011
[13] S Siju K Dhanya S Syamkumar et al ldquoDevelopment char-acterization and utilization of genomic microsatellite markersin turmeric (Curcuma longa L)rdquo Biochemical Systematics andEcology vol 38 no 4 pp 641ndash646 2010
[14] M K Panda S Mohanty E Subudhi L Acharya and SNayak ldquoAssessment of genetic stability of micropropagated
BioMed Research International 17
plants of Curcuma L by cytophotometery and RAPD analysisrdquoInternational Journal of Integrative Biology (IJIB) vol 1 pp 189ndash195 2007
[15] S Syamkumar and B Sasikumar ldquoMolecular marker basedgenetic diversity analysis of Curcuma species from IndiardquoScientia Horticulturae vol 112 no 2 pp 235ndash241 2007
[16] D Tautz and M Renz ldquoSimple sequences are ubiquitousrepetitive components of eukaryotic genomesrdquo Nucleic AcidsResearch vol 12 no 10 pp 4127ndash4138 1984
[17] G Toth Z Gaspari and J Jurka ldquoMicrosatellites in differenteukaryotic genomes surveys and analysisrdquo Genome Researchvol 10 no 7 pp 967ndash981 2000
[18] W Powell G C Machray and J Proven ldquoPolymorphismrevealed by simple sequence repeatsrdquo Trends in Plant Sciencevol 1 no 7 pp 215ndash222 1996
[19] S-Y Lee W K Fai M Zakaria et al ldquoCharacterizationof polymorphic microsatellite markers isolated from ginger(Zingiber officinale Rosc)rdquo Molecular Ecology Notes vol 7 no6 pp 1009ndash1011 2007
[20] S Bory D Da Silva A-M Risterucci M Grisoni P Besseand M-F Duval ldquoDevelopment of microsatellite markers incultivated vanilla polymorphism and transferability to othervanilla speciesrdquo Scientia Horticulturae vol 115 no 4 pp 420ndash425 2008
[21] I C Menezes F W Cidade A P Souza and I C SampaioldquoIsolation and characterization ofmicrosatellite loci in the blackpepper Piper Nigrum L (Piperaceae)rdquo Conservation GeneticsResources vol 1 no 1 pp 209ndash212 2009
[22] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[23] V Kumar Morphological and molecular characterization ofinduced mutants in Groundnut [PhD thesis] University ofAgricultural Sciences Dharwad India 2008
[24] J R Sharma Statistical and Biometrical Techniques in PlantBreeding New Age International New Delhi India 1998
[25] J J Doyle and J L Doyle ldquoA rapid DNA isolation procedure forsmall quantities of fresh leaf tissuerdquo Phytochemical Bulletin vol19 pp 11ndash15 1987
[26] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[27] Oslash Hammer D A T Harper and P D Ryan ldquoPast paleontolog-ical statistics software package for education and data analysisrdquoPalaeontologia Electronica vol 4 no 1 pp 4ndash9 2001
[28] K Liu and S V Muse ldquoPowerMaker an integrated analysisenvironment for genetic maker analysisrdquo Bioinformatics vol 21no 9 pp 2128ndash2129 2005
[29] F J RohlfNTSYS-Pc Numerical Taxonomy System Version 2 1Exeter Publishing Setauket New York NY USA 2002
[30] M Nei ldquoAnalysis of gene diversity in subdivided populationsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 70 no 12 1973
[31] S Kadkhodaei M Shahnazari M K Nekouei et al ldquoA compar-ative study of morphological and molecular diversity analysisamong cultivated almonds (Prunus dulcis)rdquo Australian Journalof Crop Science vol 5 no 1 pp 82ndash91 2011
[32] T L Abdullah J Endan and B M Nazir ldquoChanges in flowerdevelopment chlorophyll mutation and alteration in plant
morphology of Curcuma alismatifolia by gamma irradiationrdquoAmerican Journal of Applied Sciences vol 6 no 7 pp 1436ndash14392009
[33] E Kovacs and A Keresztes ldquoEffect of gamma and UV-BCradiation on plant cellsrdquoMicron vol 33 no 2 pp 199ndash210 2002
[34] H L Ramesh Y Murthy and V N Munirajappa ldquoEffect ofgamma radiation on morphological and growth parametersof Mulberry variety M5rdquo International Journal of Science andNature vol 3 pp 447ndash452 2010
[35] K Pongchawee R Pradissan andW Pipatcharoenchai ldquoInducemutation in Anubias spp through in vitro Irradiationrdquo ThaiFisheries Gazette vol 60 pp 493ndash497 2007
[36] P Tangpong T Taychasinpitak C Jompuk and P JompukldquoEffects of acute and chronic gamma irradiations on in vitroculture ofAnubias congensisNE BrownrdquoKasetsart Journal vol43 no 3 pp 449ndash457 2009
[37] R K Hegde Studies on induced mutagenesis and in vitroregeneration in turmeric (Curcuma longa L) [PhD thesis]University of Agricultural Sciences Dharwad India 2006
[38] M P Giridharan and S Balakrishnan ldquoGamma ray inducedvariability in vegetative and floral characters of ginger IndianCocoardquo Arecanut and Spices Journal vol 15 pp 68ndash672 1992
[39] A P Cesarett ldquoEffect of radiation on higher plants and plantcommunitiesrdquo inRadiation Biology United States Atomic EnergyCommission pp 284ndash309 Washington DC USA 1968
[40] G W Seung Y C Byung J-H Kim et al ldquoUltrastructuralchanges of cell organelles in Arabidopsis stems after gammairradationrdquo Journal of Plant Biology vol 48 no 2 pp 195ndash2002005
[41] B S Ahloowalia and M Maluszynski ldquoInduced mutationsmdashanew paradigm in plant breedingrdquo Euphytica vol 118 no 2 pp167ndash173 2001
[42] R Voisine L-P Vezina and C Willemot ldquoInduction ofsenescence-like deterioration of microsomal membranes fromcauliflower by free radicals generated during gamma irradia-tionrdquo Plant Physiology vol 97 no 2 pp 545ndash550 1991
[43] N Shikazono Y Yokota S Kitamura et al ldquoMutation rate andnovel tt mutants of Arabidopsis thaliana induced by carbonionsrdquo Genetics vol 163 no 4 pp 1449ndash1455 2003
[44] T Nakatsuka M Nishihara K Mishiba and S YamamuraldquoTwo different mutations are involved in the formation ofwhite-flowered gentian plantsrdquo Plant Science vol 169 no 5 pp949ndash958 2005
[45] M Mato T Onozaki Y Ozeki et al ldquoFlavonoid biosynthesisin white-flowered Sim carnations (Dianthus caryophyllus)rdquoScientia Horticulturae vol 84 no 3-4 pp 333ndash347 2000
[46] S G Wi B Y Chung J-S Kim et al ldquoEffects of gammairradiation on morphological changes and biological responsesin plantsrdquoMicron vol 38 no 6 pp 553ndash564 2007
[47] F Lee Chin Effects of light intensity and daylength on growth andflowering of siam tulip (Curcuma alismatifolia var Chiang MaiPink) [PhD thesis] University Putra Malaysia 2007
[48] A Hagiladi N Umiel and X H Yang ldquoCurcuma alismatifoliaII Effects of temperature and daylength on the development offlowers and propagulesrdquo Acta Horticulture vol 430 pp 755ndash761 1997
[49] MHagidimitriou A Katsiotis GMenexes C Pontikis andMLoukas ldquoGenetic diversity of major greek olive cultivars usingmolecular (AFLPs and RAPDs) markers and morphologicaltraitsrdquo Journal of the American Society for Horticultural Sciencevol 130 no 2 pp 211ndash217 2005
18 BioMed Research International
[50] N Babaei N A P Abdullah G Saleh and T L AbdullahldquoIsolation and characterization of microsatellite markers andanalysis of genetic variability in Curculigo latifolia DryandrdquoMolecular Biology Reports vol 39 pp 9869ndash9877 2012
[51] B Shiran N Amirbakhtiar S Kiani S Mohammadi B ESayed-Tabatabaei and H Moradi ldquoMolecular characterizationand genetic relationship among almond cultivars assessed byRAPD and SSR markersrdquo Scientia Horticulturae vol 111 no 3pp 280ndash292 2007
[52] S Singh M K Panda and S Nayak ldquoEvaluation of geneticdiversity in turmeric (Curcuma longa L) using RAPD and ISSRmarkersrdquo Industrial Crops and Products vol 37 no 1 pp 284ndash291 2012
[53] Y Paisooksantivatana S Kako and H Seko ldquoGenetic diversityof Curcuma alismatifolia Gagnep (Zingiberaceae) in Thailandas revealed by allozyme polymorphismrdquo Genetic Resources andCrop Evolution vol 48 no 5 pp 459ndash465 2001
Table 6 Effect of acute gamma rays on vegetative traits of C alismatifolia in M1V1 generation
Dose (Gray) Shoot number Leaf number Leaf length (cm) Leaf width (cm) Plant height (cm)CMR
0 20 plusmn 00a
30 plusmn 04a
556 plusmn 08a
71 plusmn 05a
1112 plusmn 25a
10 10 plusmn 00b
36 plusmn 05a
272 plusmn 25b
45 plusmn 05b
714 plusmn 96b
20 10 plusmn 00b
14 plusmn 05b
124 plusmn 120c
25 plusmn 24c
170 plusmn 80c
DT0 30 plusmn 00
a30 plusmn 00
a622 plusmn 08
a65 plusmn 05
a912 plusmn 19
a
10 16 plusmn 05b
28 plusmn 04a
280 plusmn 44b
46 plusmn 06b
542 plusmn 76b
20 12 plusmn 04b
24 plusmn 05a
242 plusmn 38b
38 plusmn 05c
512 plusmn 138b
SP0 16 plusmn 05
a32 plusmn 04
a461 plusmn 27
a101 plusmn 04
a746 plusmn 38
a
10 10 plusmn 00b
36 plusmn 05a
275 plusmn 34b
57 plusmn 02b
377 plusmn 137b
20 10 plusmn 00b
20 plusmn 10b
196 plusmn 74c
57 plusmn 04b
195 plusmn 73c
KP0 14 plusmn 00
a46 plusmn 05
a345 plusmn 29
a47 plusmn 04
a591 plusmn 28
a
10 10 + 05a
40 plusmn 05b
220 plusmn 24b
31 plusmn 04b
420 plusmn 54b
20 10 plusmn 00a
32 plusmn 04b
156 plusmn 43c
31 plusmn 11b
283 plusmn 174c
CV () 23 18 26 16 165Means with the same or common letter are not significantly different least square difference test 119875 lt 005
decreases in leaf size were reported by Pongchawee et al [35]and Tangpong et al [36] These results were in agreementwith an earlier study [4] which reported that the growthof chrysanthemum exposed to acute gamma rays was lessthan the control in the M
1V1generation All varieties doses
and interaction effects resulted in significant differences fornumber of leaves Among untreated plants Kimono Pinkvariety had higher number of leaves (46) than the otherthree (3 3 and 32) varieties In Chiang Mai Red and SweetPink varieties plants exposed to 10Gy showedhigher numberof leaves than untreated plants However at 20Gy therewas significant reduction in number of leaves for all studiedvarieties in comparison to control Similar stimulatory effectswere obtained at lower doses in ginger by Hegde [37] andGiridharan and Balakrishnan [38]
Progressive reduction in growth parameters can be inter-preted on interference in normal mitosis and frequent occur-rence of mitotic aberrations inhibition of rate of assimilationand consequent change in the nutrient level in the plantand inactivation of vital enzymes especially those associatedwith respiration [39] Dose-dependent negative effect wasalso detected for plant heightThe tallest plants were recordedfrom the untreated rhizomes (0Gy) with heights of 1112912 746 and 591 cm followed by the 10Gy irradiated plantswith heights of 714 542 377 and 420 cm and the 20Gyirradiated plants with corresponding heights of 170 512195 and 283 cm for Chiang Mai Red Doi Tung 554 SweetPink and Kimono Pink varieties respectively These resultsare in agreement with the findings of Abdullah et al [32]Reduction in growth parameters and dwarfism can be causedby interference of normal mitosis and frequent occurrenceof mitotic aberrations inhibition of assimilation rates andconsequent changes in nutrient levels in plants Additionallymutagenic effects such as auxin destruction inhibition of
auxin synthesis failure of assimilatory mechanism andchanges in the specific activity of enzymes can cause growthreductions [37] High doses of ionizing radiation have beenshown to damage macromolecular cellular components suchas cell walls membranes and DNA [40] The number ofshoots also decreased significantly as the radiation dosesincreased Radiation also affects organic molecules that areessential to the cell division process and thus causing celldivision to stop [36]
54 Effect of Gamma Irradiation on Flowering DevelopmentTraits in the M1V1 All control and 10Gy irradiated plantsproduced flowers while the Chiang Mai Red and SweetPink varieties which were exposed to 20Gy did not gointo the flowering stage Lamseejan et al [4] also showedthat flowering percentage decreases as gamma ray dosesare increased In the present study gamma rays caused lateflowering in all four varieties Days to appearance of firstvisible buds were also significantly different among the fourvarieties (Table 7) Gamma rays caused a noticeable delay inflowering of irradiated plants in comparison to the untreatedones First visible buds were observed at 652 and 874 days inthe control and 10Gy treatments respectively for the ChiangMai Red variety In Doi Tung 554 the first visible buds wereappeared at 474 656 and 840 days after planting at 0 10and 20Gy doses respectively In the Sweet Pink variety thenumber of days to visible bud appearance increased signif-icantly from 678 days in controls to 978 days in the 10Gyirradiated plants In comparison to other three varieties theKimono Pink variety needed the longest time to visible budappearance and same as other varieties there was a positivecorrelation between the number of days to first visible budand the gamma irradiation dose In the control and 10Gyand 20Gy irradiated individual plants flower buds were
BioMed Research International 9
Table 7 Effect of acute gamma rays on flower development characteristics of C alismatifolia in M1V1 generation
Dose (Gy) Days tovisible bud
Inflorescencelength (cm)
Days toanthesis
Number oftrue flowers
Number ofPink bracts
Days tosenescence
CMR0 652 plusmn 47
b162 plusmn 057
a742 plusmn 54
b132 plusmn 19
a104 plusmn 15
a21 plusmn 10
a
10 874 plusmn 95a
82 plusmn 397b
1020 plusmn 71a
56 plusmn 19b
50 plusmn 07b
10 plusmn 14b
20 00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
DT0 474 plusmn 43
c134 plusmn 054
a546 plusmn 54
c162 plusmn 13
a232 plusmn 04
a23 plusmn 00
a
10 656 plusmn 37b
94 plusmn 19b
780 plusmn 37b
112 plusmn 21b
166 plusmn 08b
122 plusmn 130b
20 840 plusmn 42a
76 plusmn 082c
994 plusmn 49a
88 plusmn 13b
156 plusmn 11b
106 plusmn 18b
SP0 678 plusmn 21
b146 plusmn 089
a756 plusmn 26
b162 plusmn 13
a100 plusmn 24
a188 plusmn 083
a
10 978 plusmn 181a
79 plusmn 20b
1098 plusmn 182a
68 plusmn 25b
52 plusmn 08b
92 plusmn 24b
20 00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
KP0 852 plusmn 23
c137 plusmn 044
a942 plusmn 19
c112 plusmn 12
a96 plusmn 21
a18 plusmn 18
a
10 1044 plusmn 65b
108 plusmn 108ab
1224 plusmn 100b
76 plusmn 27b
64 plusmn 16ab
102 plusmn 17b
20 1272 plusmn 65a
790 plusmn 49b
1426 plusmn 58a
60 plusmn 38b
42 plusmn 24b
66 plusmn 37b
CV () 10 23 9 22 169 15Means with the same or common letter are not significantly different least square difference test 119875 lt 005
visible at 852 1328 and 1374 days after planting respectivelyPrevious studies also showed that onset of flowering andformation of floral parts in mutants of Arabidopsis thalianamaize barely pea and tobacco involved growth regulators(or phytohormones) such as auxins cytokinins gibberellinsabscisic acid ethylene and brassinosteroids [41] There weresignificant differences among treatments for the length ofthe inflorescence In all varieties the longest inflorescencelength was observed in the untreated plants with 162134 146 and 137 cm lengths for Chiang Mai Red DoiTung 554 Sweet Pink and Kimono Pink respectively Thecorresponding inflorescence lengths were 82 94 79 and109 cm for the 10Gy irradiated plants The days to anthesisfor C alismatifolia varieties were significantly affected byvariety gamma irradiation doses and their interaction Thenumber of days to full bloom was noticeable earliest foruntreated plants at 742 546 756 and 942 days for ChiangMai Red Doi Tung 554 Sweet Pink and Kimono Pinkvarieties respectively This was then followed by plants at10Gy at 102 78 1098 and 1224 days The number of trueflowers or the secondary inflorescence developed in the axilof the primary bracts decreased as radiation dosage increasedIn the present study the gamma rays also decreased thedays to inflorescence senescence In this study there wasa strongly significantly and positively correlation (0919lowastlowast)(data not shown) between the number of true flowers andthe number of days to senescenceThe number of pink bractsalso decreasedwith increasing radiation dosageMost gammaray effects on senescence are considered as resulting from theaction of free radicals generated from water and oxygen bythe ionizing energy on the cellular components Membrane
deterioration is a general feature of natural senescence andstress-induced aging [42]
Irradiation induced some mutation spectrum of flowercolor variation that included colors such as purple palepurple rather pale purple white purple white (marbledpattern) and two-tone purple color
Mutation spectrum of flower shape variation includeddouble inflorescence within one stalk double stalk per plantinflorescence without bracts two-flag petal true flowersand chlorophyll mutation in the leaves which are generallycaused by induced gamma rays (Figure 2) Ionizing radiationincluding gamma rays induces fragment deletions or inser-tions that eventually lead to changes in amino acids and amodification of leaf and stem pigmentation [43] A mutationin the biosynthetic pathway of structural or regulatory genesmay cause a change in flower color [44] When the blockageoccurs at the early stages of anthocyanin synthesis whiteflowers will result while a blockage at later stages leads todifferent flower colors due to the accumulation of particularanthocyanins [45] Chloroplasts were extremely sensitive togamma radiation compared to other cell organelles [46]
55 Effect of Gamma Irradiation on Rhizome Characteristicsin Selected Doses in M1V1 The number of new rhizomes andthe number of storage roots per rhizome were significantlyaffected by varieties doses and their interaction (Table 8)The number of new rhizomes only in Kimono Pink varietydid not show any differences between untreated and treatedplants The rhizome size was influenced only by doses Asdose level increased the rhizome size decreased Amonguntreated plants the Sweet Pink rhizomes had the most
10 BioMed Research International
(a) (b) (c) (d)
(e)
(f)
Figure 2 Effect of acute gamma rays on flower traits in C alismatifolia (a) Untreated inflorescence bracts color Doi Tung 554 (b) Two tonebract color Doi Tung 554 (20Gy) (c) Marbled pattern of bract color Doi Tung 554 (20Gy) (d) Double inflorescence within one stalk varChiang Mai Red (10Gy) (e) True flower in nontreated plants (f) Two-flag petals true flowers (10Gy)
Figure 3 Dendrogram representing themorphological variation among 44 irradiated and nonirradiated individuals ofC alismatifolia across14 variables CMR = Chiang Mai Red DT = Doi Tung 554 SP = Sweet Pink Kp = Kimono Pink
number of storage roots (78) and the Kimono Pink rhizomeshad the least number of storage roots (40)This was followedby 10Gy irradiated rhizomes with 4 46 48 and 28 storageroot numbers for Chiang Mai Red Doi Tung 554 SweetPink and Kimono Pink respectively Irradiation with 20Gydecreased significantly the number of storage roots in allstudied varietiesTheplantC alismatifolia is propagated froma propagule (one rhizome + 5-6 t-roots) The rhizome sizedose matters in the growth of the plant Smaller rhizome sizeusually resulted in smaller plant size with narrow grass-likeleaves The storage roots play a very important role in thegrowth and flowering ofCurcumaThe t-rootsmake up about85 of the total fresh weight and 70 of the total dry weightof a typical propaguleThe storage organs act for plant growth
during dormancy and emergence [47] More storage root perpropagule resulted in faster flowering and higher yield plant[48] In this study also there was significant and negativecorrelation between number of storage roots and number ofdays to visible bud appearance (minus0525lowastlowast)
56 Cluster Analysis of C alismatifolia for MorphologicalTraits The morphological data were used to calculate thesimilarity between the treated and non-treated C alismat-ifolia samples and UPGMA dendrogram was constructed(Figure 3) The cophenetic correlation coefficient (CCC)value between the genetic dissimilaritymatrix estimated fromthe morphological characters and the UPGMA clusteringmethod was 119903 = 093 showing a proof fit This value was
BioMed Research International 11
Table 8 Effect of acute gamma rays on rhizome characteristics ofC alismatifolia in M1V1 generation
Dose (Gray) Rhizomesize
No of newrhizomes
No of storageroot
CMR0 23 plusmn 02
a26 plusmn 055
a46 plusmn 054
a
10 18 plusmn 005a12 plusmn 044
b40 plusmn 07
a
20 08 plusmn 07b
06 plusmn 054b
14 plusmn 13b
DT0 22 plusmn 01
a30 plusmn 00
a72 plusmn 109
a
10 18 plusmn 007b14 plusmn 054
b46 plusmn 13
b
20 16 plusmn 005b
10 plusmn 00b
28 plusmn 13c
SP0 22 plusmn 02
a24 plusmn 054
a78 plusmn 21
a
10 17 plusmn 007b
10 plusmn 00b
48 plusmn 044b
20 12 plusmn 02c
10 plusmn 00b
28 plusmn 083b
KP0 21 plusmn 005
a10 plusmn 00
a40 plusmn 00
a
10 16 plusmn 01ab
10 plusmn 00a
28 plusmn 044b
20 11 plusmn 06b
10 plusmn 00a
22 plusmn 083b
CV () 18 23 26Means with the same or common letter are not significantly different leastsquare difference test 119875 lt 005
higher than of studies on olive for morphological traits (119903 =069) [49] In this dendrogram the 44C alismatifolia samplesappeared to form three main clusters and five minor clustersat coefficient level 56 cluster I included three non-irradiatedmembers and cluster II included 10Gy irradiated individualplants of all four varieties and 20Gy irradiated samples ofDoi Tung 554 and Kimono Pink varieties except KP20-4 (noflowering) Main cluster III included the 20Gy irradiatedindividuals Chiang Mai Red and Sweet Pink varieties (asmentioned in morphological part these individuals didnot go to flowering stage) and KP20-4 Cluster II can bedivided to five subclusters The first subcluster included 10members (CMR10-1 CMR10-5 SP10-1 SP10-2 SP10-3 SP10-5 CMR10-2 CMR10-4 SP10-4 and CMR10-3) the secondone included nine members (KP10-1 KP10-2 KP10-3 KP10-4 KP10-5KP20-1 KP20-2 KP20-5 and KP20-3) KP0 wasthe lone member of the third sub-cluster The fourth sub-cluster included two members only (DT10-2 and DT10-3) The last sub-cluster main cluster II had eight membersincluded (DT10-1 DT10-4 DT10-5 DT20-1 DT20-2 DT20-3 DT20-4 and DT20-5) Mean value of groups for each traitis presented in Table 9 This table clearly shows the differentmean values of the three main clusters The minimum meanvalue referred to main cluster III while the maximum meanvalue for number of shoots leaf length leaf width plantheight number of true flowers inflorescence length rhizomesize number of new rhizomes and number of storage rootsbelonged to cluster I which was included non-treated plantsThese results showed that the gamma irradiation has inducedmorphological changes in C alismatifolia individuals of
four studied varieties so that they showed phenotypicallydifferences from their controls
57 Principal Component Analysis of C alismatifolia forMorphological Traits To assess the patterns of variation PCAwas done by considering all of the 14 variables The first threecomponents of PCA explained 828 of the total variation(Table 10) Only the first component which accounted for562 of the total variation was attributed to inflorescencelength plant height days to senescence number of trueflowers and rhizome size In PCA three-dimensional graphthe grouping of individuals confirmed the clustering results(Figure 4) The PCA graph proved that all irradiated indi-vidual plants of C alismatifolia varieties are phenotypicallydifferent from their non-irradiated individuals
6 Molecular Characterization
61 SSR Polymorphism Amplifications were successful forall the 17 SSR markers assayed This reflects a high levelof homology between SSR flanking regions in C longa andC alismatifolia Out of 17 primer pairs eight primer pairsresulted in polymorphic PCR products Table 11 summarizesthe results obtained based on the analysis of individuals of thefour studied varieties using the polymorphic SSR loci for 010 and 20Gy irradiated plants The number of alleles variedwidely among these loci A total of 25 36 and 41 alleles wereobserved among 0 10 and 20Gy irradiated individual plantsrespectively In the 10Gy treatment the number of allelesranged from three (clon09 and clon14) to seven (clon08) withan average value of 45 per locus In the 20Gy treatmentthe number of alleles varied from three (clon09 and clon14)to seven (clon08 and clon12) with an average value of 51per locus In the untreated individuals the number of allelesranged from two (clon04 clon09 clon11 and clon14) to five(clon12) with an average value of 31 The difference betweenthe average number of observed alleles and effective numberof alleles was due to the uneven frequency of each allele[50] For each of the eight SSR primers PIC values (whichmeasures allele diversity and frequency among varieties)varied from 019 (clon04) to 071 (clon01) in untreated plantsand from 025 (clon04) to 075 (clon08) in 10Gy acutelyirradiated plants In 20Gy irradiated individual plants thePIC value ranged from 042 (clon14) to 075 (clon08 andclon12) The mean PIC for all loci was 047 054 and 061for 0 10 and 20Gy irradiated plants respectively
The PIC value provides an estimate of the discriminatorypower of amarker by taking into account not only the numberof alleles at a locus but also the relative frequencies of thesealleles [51] Thus except for clon09 and clon14 which weremoderately polymorphic (025 lt PIC lt 05) all other lociwere highly polymorphic (05 lt PIC) while none of theloci showed low polymorphism The average discriminatingpower ofmicrosatellitemarkers (061) observed in the presentstudy ensures the future utility of microsatellite markersfor genetic variation studies in C alismatifolia varietiesOur results reflect similar findings as reported earlier inthe study of genetic variation in other species of Curcuma
12 BioMed Research International
Table9Clusterm
eans
for14charactersestim
ated
in44
individu
also
fCalismatifolia
Clusters
Shoo
tnu
mber
Leaf
leng
thLeaf
width
Leaf
number
Visib
lebu
d(days)
Inflo
rescence
leng
thPlant
height
Anthesis
(days)
Senescence
(days)
True
flow
number
Pink
bract
number
Rhizom
esiz
eNew
rhizom
enu
mber
Storage
root
number
I220
5464
736
300
6013
1473
9233
6926
2093
152
1453
229
266
653
II1
100
2735
512
360
9260
809
5457
1059
960
620
510
177
110
440
II2
122
1922
298
344
11422
1043
3910
1312
293
3755
588
156
100
266
II3
100
3452
416
460
8520
1370
5910
9420
180
120
960
206
100
400
II4
150
2500
45
250
6500
925
4950
7700
1150
950
1700
185
200
600
II5
137
2637
417
262
7725
840
5356
9162
1137
1012
1587
170
100
312
III
054
1500
465
181
000
000
000
000
000
000
000
090
081
200
BioMed Research International 13
Table 10 Eigenvectors eigenvalues and proportions of variabilityfor three principle components among 14 characters for 44 Calismatifolia
Variable PC1 PC2 PC3Number of new shoot 0270 minus0076 minus0173Leaf length 0269 minus0305 0033Leaf width 0096 minus0455 0430Leaf number 0210 0285 0490Days to visible bud 0199 0462 0181Plant height 0321 0027 minus0071Inflorescence length 0318 0166 minus0040Days to anthesis 0205 0460 0148Days to senescence 0342 0017 minus0131Number of true flower 0323 0029 minus0238Number of pink bracts 0266 minus0028 minus0546Rhizome size 0302 minus0101 0147Number of new rhizome 0250 minus0293 0028Number of storage roots 0264 minus0247 0298Eigenvalue 7877 2694 1032Proportion 56264 19246 7371Cumulative 56264 75511 82882
using SSR markers [13] The highest Neirsquos gene diversity(ℎ) was obtained with the 20Gy treatment (061) followedby the 10Gy treatment (06) and the untreated individuals(05) The mean Shannonrsquos information index (119868) was 092114 and 130 in the 0 10 and 20Gy irradiated plantsrespectively The high value of Shannonrsquos information indexrepresents the effectiveness of microsatellite loci to revealthe variation among these varieties at the different dosesused Overall genetic variability for the varieties studiedrepresented by the Shannon-Weiner index was relatively highin comparison to other studies involving C longa accessions[10 52] Additionally the 20Gy acutely irradiated individualsshowed a higher mean percentage of polymorphic loci (63)than the 10Gy (58) and untreated (022) ones This impliesthat irradiation with a dose of 20Gy induced more geneticvariation in the M
1V1generation
62 Cluster Analysis Thedendrogramwas constructed usingPowerMarker 323The 44 studied individuals were clusteredinto seven groups (G1ndashG7) Colors were applied accordingto our model-based cluster analysis results Group I iscomprised of nine individual plants of Chiang Mai Redvariety receiving 0 10 and 20Gy treatments (blue colorin Figure 5) One individual plant DT10-1 was assignedto group II (green color in Figure 5) All irradiated andnon-irradiated individuals of Doi Tung 554 variety wereassigned to group III (yellow color in Figure 5) DT20-1 andSP10-5 were the sole members of groups IV (red color inFigure 5) and V (gray color in Figure 5) respectively GroupVI included treated and untreated individuals of Sweet Pinkvariety (pink color in Figure 5) Lastly all Kimono Pinkindividuals were assigned to cluster VII (orange color in
Figure 5) The genetic similarity coefficient (data not shown)among the 44 individuals amplified using eight SSR primersvaried from 00 to 10 The highest value (10) correspondedto [(CMR10-3 CMR10-5 CMR and CMR20-2) (DT10-2and DT10-3) (DT and DT20-2) (KP20-3 KP-20-4 KP10-3andKP10-4)] individuals that generated identical fingerprintsacross the markers studied Among the studied varieties theSweet Pink individual plants showed the lowest similarity(00) withKimono Pink individuals which indicated that theywere relatively remote in relationship (Figure 5) An overviewof the clustering pattern indicates that the grouping of thestudied individual plants was based on different varieties andlargely independent of the doses of gamma irradiation Awide range in similarity values had also been observed indifferent species of Curcuma [12 13 52 53]
63 Principle Component Analysis (PCA) Thedata generatedfrom 44 C alismatifolia individual plants were subjected toprincipal component analysis (PCA) to visualize individualsin a multivariate space In the three-dimensional graphderived from the SSR analysis all studied individuals weregrouped into seven clusters (Figure 6) In PCA the firstthree principal components (PC) extracted a cumulative of6826 of the total variation among the 44 individuals of CalismatifoliaThe first three coordinates PC1 PC2 and PC3accounted for 4136 1578 1112 of the total variationrespectively The distribution of the individual plants in athree-dimensional plot using the first three principal com-ponents showed the genetic relationship between individualplants It was evident that both methods dendrogram andthree-dimensional plots of PCA were effective in illustratinggenetic relationships and the groups found were comparable[11 13] The PCA results were similar to those obtained fromcluster analysis where all individuals from different varietieswere assigned to different groups These graphical illus-trations enable the assessment of the relationshipdistancesamong all of the individuals in the study [50]
7 Conclusion
This is the first attempt to evaluate the effect of acutegamma irradiation on C alismatifolia varieties using bothmorphological characteristics and molecular markers Inplant breeding programs mutagenic treatments with lownegative physiological effects and strong genetic effects aredesirable Hence we used more effective doses of gammairradiation (10 and 20Gy) of which particularly 20Gy dosewas effective to influence morphological and molecularcharacteristics of studiedCurcuma alismatifolia varietiesThelower dose (10Gy) of radiation probably caused little damageto the plants genetic material so that the cells could repairthemselves in next generation As a result in this study 20Gyacute gamma irradiation resulted in a higher percentage ofmutation and getting desired mutants was more possibleBased on the LD
50values determined in this study it was
apparent that the varieties Chiang Mai Red and Sweet Pinkwere more sensitive to gamma rays compared to Doi Tung554 and Kimono Pink Our results show that the variety Doi
Figure 4 Two-dimensional graph of principal component analysis (PCA) for 14 morphological variables indicating relationships amongirradiated and nonirradiated four varieties of C alismatifolia
CMR10-1CMR10-5
SP10-1SP10-2SP10-3
SP10-5
CMR10-4
SP10-4
KP10-1KP10-3
KP20-1
KP20-2
KP10-5KP20-5
KP20-3
KP10-2
DT10-2
DT10-1
DT10-5
DT20-1
DT10-4DT20-4DT20-2
DT20-5DT20-3
CMR20-1
SP20-3SP20-4
SP20-5SP20-1
SP20-2
KP
SP
CMR 10-2
SP10-5
Figure 5 Unrooted neighbor joining tree showing genetic relationship among 44 irradiated and nonirradiated C alismatifolia using SSRmarkers The colors of the branches correspond to those of the same cluster
Figure 6Three-dimensional graph of principal component analysis (PCA) indicating relationships among irradiated and nonirradiated fourvarieties of C alismatifolia
Tung 554 (Curcuma hybrid) had the most morphologicalresponses to gamma rays The use of microsatellite markersas a codominant marker will facilitate the exploration ofgenetic variability among treated and non-treated plants andwill help to distinguish the plants showing differences inmorphological characters The overall effects on the M
1V1
generation revealed that acute gamma irradiation at optimumdoses has the potential for developing new varieties of Calismatifolia with improved commercial properties suitablefor the Malaysian flower industry
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work was funded by the Fundamental Research GrantScheme (FRGS) under the Ministry of Higher Education inMalaysia
References
[1] P Apavatjrut S Anuntalabhochai P Sirirugsa and C AlisildquoMolecularmarkers in the identification of some early floweringCurcuma L (Zingiberaceae) speciesrdquo Annals of Botany vol 84no 4 pp 529ndash534 1999
[2] K Bunya-Atichart S Ketsa andW G VanDoorn ldquoPostharvestphysiology of Curcuma alismatifolia flowersrdquo Postharvest Biol-ogy and Technology vol 34 no 2 pp 219ndash226 2004
[3] M Nakano J Amano Y Watanabe et al ldquoMorphologicalvariation in Tricyrtis hirta plants regenerated from heavy ionbeam-irradiated embryogenic callusesrdquo Plant Biotechnologyvol 27 no 2 pp 155ndash160 2010
[4] S Lamseejan P Jompuk A Wongpiyasatid A Deeseepan andP Kwanthammachart ldquoGamma-rays induced morphological
[5] G-J Lee S J Chung I S Park et al ldquoVariation in thephenotypic features and transcripts of colormutants of chrysan-themum (Dendranthema grandiflorum) derived from gammaraymutagenesisrdquo Journal of Plant Biology vol 51 no 6 pp 418ndash423 2008
[6] O K Kikuchi ldquoOrchid flowers tolerance to gamma-radiationrdquoRadiation Physics and Chemistry vol 57 no 3ndash6 pp 555ndash5572000
[7] N P Arnold N N Barthakur and M Tanguay ldquoMutageniceffects of acute gamma irradiation on miniature roses targettheory approachrdquo HortScience vol 33 no 1 pp 127ndash129 1998
[8] A A Youssef M S Aly and M S Hussein ldquoResponse ofgeranium (Pelargonium graveolenus L) to gamma irradiationand foliar application of Speed Growrdquo Egyptian Journal ofHorticulture vol 27 pp 41ndash53 2000
[9] N Chuantang and L Yazhi ldquoThe radiation inducedmutation ofcanna (Canna L)rdquo Acta Agricultute Nucleatae Sinica vol 2 pp33ndash39 1998
[10] M S Sigrist J B Pinheiro J A A Filho and M I ZucchildquoGenetic diversity of turmeric germplasm (Curcuma longaZingiberaceae) identified by microsatellite markersrdquo Geneticsand Molecular Research vol 10 no 1 pp 419ndash428 2011
[11] S Taheri T L Abdullah N A P Abdullah and Z AhmadldquoGenetic relationships among five varieties of Curcuma alis-matifolia (Zingiberaceae) based on ISSRmarkersrdquoGenetics andMolecular Research vol 11 pp 3069ndash3076 2012
[12] A Das V Kesari V M Satyanarayana A Parida and L Ran-gan ldquoGenetic relationship of Curcuma species from NortheastIndia using PCR-based markersrdquo Molecular Biotechnology vol49 no 1 pp 65ndash76 2011
[13] S Siju K Dhanya S Syamkumar et al ldquoDevelopment char-acterization and utilization of genomic microsatellite markersin turmeric (Curcuma longa L)rdquo Biochemical Systematics andEcology vol 38 no 4 pp 641ndash646 2010
[14] M K Panda S Mohanty E Subudhi L Acharya and SNayak ldquoAssessment of genetic stability of micropropagated
BioMed Research International 17
plants of Curcuma L by cytophotometery and RAPD analysisrdquoInternational Journal of Integrative Biology (IJIB) vol 1 pp 189ndash195 2007
[15] S Syamkumar and B Sasikumar ldquoMolecular marker basedgenetic diversity analysis of Curcuma species from IndiardquoScientia Horticulturae vol 112 no 2 pp 235ndash241 2007
[16] D Tautz and M Renz ldquoSimple sequences are ubiquitousrepetitive components of eukaryotic genomesrdquo Nucleic AcidsResearch vol 12 no 10 pp 4127ndash4138 1984
[17] G Toth Z Gaspari and J Jurka ldquoMicrosatellites in differenteukaryotic genomes surveys and analysisrdquo Genome Researchvol 10 no 7 pp 967ndash981 2000
[18] W Powell G C Machray and J Proven ldquoPolymorphismrevealed by simple sequence repeatsrdquo Trends in Plant Sciencevol 1 no 7 pp 215ndash222 1996
[19] S-Y Lee W K Fai M Zakaria et al ldquoCharacterizationof polymorphic microsatellite markers isolated from ginger(Zingiber officinale Rosc)rdquo Molecular Ecology Notes vol 7 no6 pp 1009ndash1011 2007
[20] S Bory D Da Silva A-M Risterucci M Grisoni P Besseand M-F Duval ldquoDevelopment of microsatellite markers incultivated vanilla polymorphism and transferability to othervanilla speciesrdquo Scientia Horticulturae vol 115 no 4 pp 420ndash425 2008
[21] I C Menezes F W Cidade A P Souza and I C SampaioldquoIsolation and characterization ofmicrosatellite loci in the blackpepper Piper Nigrum L (Piperaceae)rdquo Conservation GeneticsResources vol 1 no 1 pp 209ndash212 2009
[22] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[23] V Kumar Morphological and molecular characterization ofinduced mutants in Groundnut [PhD thesis] University ofAgricultural Sciences Dharwad India 2008
[24] J R Sharma Statistical and Biometrical Techniques in PlantBreeding New Age International New Delhi India 1998
[25] J J Doyle and J L Doyle ldquoA rapid DNA isolation procedure forsmall quantities of fresh leaf tissuerdquo Phytochemical Bulletin vol19 pp 11ndash15 1987
[26] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[27] Oslash Hammer D A T Harper and P D Ryan ldquoPast paleontolog-ical statistics software package for education and data analysisrdquoPalaeontologia Electronica vol 4 no 1 pp 4ndash9 2001
[28] K Liu and S V Muse ldquoPowerMaker an integrated analysisenvironment for genetic maker analysisrdquo Bioinformatics vol 21no 9 pp 2128ndash2129 2005
[29] F J RohlfNTSYS-Pc Numerical Taxonomy System Version 2 1Exeter Publishing Setauket New York NY USA 2002
[30] M Nei ldquoAnalysis of gene diversity in subdivided populationsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 70 no 12 1973
[31] S Kadkhodaei M Shahnazari M K Nekouei et al ldquoA compar-ative study of morphological and molecular diversity analysisamong cultivated almonds (Prunus dulcis)rdquo Australian Journalof Crop Science vol 5 no 1 pp 82ndash91 2011
[32] T L Abdullah J Endan and B M Nazir ldquoChanges in flowerdevelopment chlorophyll mutation and alteration in plant
morphology of Curcuma alismatifolia by gamma irradiationrdquoAmerican Journal of Applied Sciences vol 6 no 7 pp 1436ndash14392009
[33] E Kovacs and A Keresztes ldquoEffect of gamma and UV-BCradiation on plant cellsrdquoMicron vol 33 no 2 pp 199ndash210 2002
[34] H L Ramesh Y Murthy and V N Munirajappa ldquoEffect ofgamma radiation on morphological and growth parametersof Mulberry variety M5rdquo International Journal of Science andNature vol 3 pp 447ndash452 2010
[35] K Pongchawee R Pradissan andW Pipatcharoenchai ldquoInducemutation in Anubias spp through in vitro Irradiationrdquo ThaiFisheries Gazette vol 60 pp 493ndash497 2007
[36] P Tangpong T Taychasinpitak C Jompuk and P JompukldquoEffects of acute and chronic gamma irradiations on in vitroculture ofAnubias congensisNE BrownrdquoKasetsart Journal vol43 no 3 pp 449ndash457 2009
[37] R K Hegde Studies on induced mutagenesis and in vitroregeneration in turmeric (Curcuma longa L) [PhD thesis]University of Agricultural Sciences Dharwad India 2006
[38] M P Giridharan and S Balakrishnan ldquoGamma ray inducedvariability in vegetative and floral characters of ginger IndianCocoardquo Arecanut and Spices Journal vol 15 pp 68ndash672 1992
[39] A P Cesarett ldquoEffect of radiation on higher plants and plantcommunitiesrdquo inRadiation Biology United States Atomic EnergyCommission pp 284ndash309 Washington DC USA 1968
[40] G W Seung Y C Byung J-H Kim et al ldquoUltrastructuralchanges of cell organelles in Arabidopsis stems after gammairradationrdquo Journal of Plant Biology vol 48 no 2 pp 195ndash2002005
[41] B S Ahloowalia and M Maluszynski ldquoInduced mutationsmdashanew paradigm in plant breedingrdquo Euphytica vol 118 no 2 pp167ndash173 2001
[42] R Voisine L-P Vezina and C Willemot ldquoInduction ofsenescence-like deterioration of microsomal membranes fromcauliflower by free radicals generated during gamma irradia-tionrdquo Plant Physiology vol 97 no 2 pp 545ndash550 1991
[43] N Shikazono Y Yokota S Kitamura et al ldquoMutation rate andnovel tt mutants of Arabidopsis thaliana induced by carbonionsrdquo Genetics vol 163 no 4 pp 1449ndash1455 2003
[44] T Nakatsuka M Nishihara K Mishiba and S YamamuraldquoTwo different mutations are involved in the formation ofwhite-flowered gentian plantsrdquo Plant Science vol 169 no 5 pp949ndash958 2005
[45] M Mato T Onozaki Y Ozeki et al ldquoFlavonoid biosynthesisin white-flowered Sim carnations (Dianthus caryophyllus)rdquoScientia Horticulturae vol 84 no 3-4 pp 333ndash347 2000
[46] S G Wi B Y Chung J-S Kim et al ldquoEffects of gammairradiation on morphological changes and biological responsesin plantsrdquoMicron vol 38 no 6 pp 553ndash564 2007
[47] F Lee Chin Effects of light intensity and daylength on growth andflowering of siam tulip (Curcuma alismatifolia var Chiang MaiPink) [PhD thesis] University Putra Malaysia 2007
[48] A Hagiladi N Umiel and X H Yang ldquoCurcuma alismatifoliaII Effects of temperature and daylength on the development offlowers and propagulesrdquo Acta Horticulture vol 430 pp 755ndash761 1997
[49] MHagidimitriou A Katsiotis GMenexes C Pontikis andMLoukas ldquoGenetic diversity of major greek olive cultivars usingmolecular (AFLPs and RAPDs) markers and morphologicaltraitsrdquo Journal of the American Society for Horticultural Sciencevol 130 no 2 pp 211ndash217 2005
18 BioMed Research International
[50] N Babaei N A P Abdullah G Saleh and T L AbdullahldquoIsolation and characterization of microsatellite markers andanalysis of genetic variability in Curculigo latifolia DryandrdquoMolecular Biology Reports vol 39 pp 9869ndash9877 2012
[51] B Shiran N Amirbakhtiar S Kiani S Mohammadi B ESayed-Tabatabaei and H Moradi ldquoMolecular characterizationand genetic relationship among almond cultivars assessed byRAPD and SSR markersrdquo Scientia Horticulturae vol 111 no 3pp 280ndash292 2007
[52] S Singh M K Panda and S Nayak ldquoEvaluation of geneticdiversity in turmeric (Curcuma longa L) using RAPD and ISSRmarkersrdquo Industrial Crops and Products vol 37 no 1 pp 284ndash291 2012
[53] Y Paisooksantivatana S Kako and H Seko ldquoGenetic diversityof Curcuma alismatifolia Gagnep (Zingiberaceae) in Thailandas revealed by allozyme polymorphismrdquo Genetic Resources andCrop Evolution vol 48 no 5 pp 459ndash465 2001
Table 7 Effect of acute gamma rays on flower development characteristics of C alismatifolia in M1V1 generation
Dose (Gy) Days tovisible bud
Inflorescencelength (cm)
Days toanthesis
Number oftrue flowers
Number ofPink bracts
Days tosenescence
CMR0 652 plusmn 47
b162 plusmn 057
a742 plusmn 54
b132 plusmn 19
a104 plusmn 15
a21 plusmn 10
a
10 874 plusmn 95a
82 plusmn 397b
1020 plusmn 71a
56 plusmn 19b
50 plusmn 07b
10 plusmn 14b
20 00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
DT0 474 plusmn 43
c134 plusmn 054
a546 plusmn 54
c162 plusmn 13
a232 plusmn 04
a23 plusmn 00
a
10 656 plusmn 37b
94 plusmn 19b
780 plusmn 37b
112 plusmn 21b
166 plusmn 08b
122 plusmn 130b
20 840 plusmn 42a
76 plusmn 082c
994 plusmn 49a
88 plusmn 13b
156 plusmn 11b
106 plusmn 18b
SP0 678 plusmn 21
b146 plusmn 089
a756 plusmn 26
b162 plusmn 13
a100 plusmn 24
a188 plusmn 083
a
10 978 plusmn 181a
79 plusmn 20b
1098 plusmn 182a
68 plusmn 25b
52 plusmn 08b
92 plusmn 24b
20 00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
00 plusmn 00c
KP0 852 plusmn 23
c137 plusmn 044
a942 plusmn 19
c112 plusmn 12
a96 plusmn 21
a18 plusmn 18
a
10 1044 plusmn 65b
108 plusmn 108ab
1224 plusmn 100b
76 plusmn 27b
64 plusmn 16ab
102 plusmn 17b
20 1272 plusmn 65a
790 plusmn 49b
1426 plusmn 58a
60 plusmn 38b
42 plusmn 24b
66 plusmn 37b
CV () 10 23 9 22 169 15Means with the same or common letter are not significantly different least square difference test 119875 lt 005
visible at 852 1328 and 1374 days after planting respectivelyPrevious studies also showed that onset of flowering andformation of floral parts in mutants of Arabidopsis thalianamaize barely pea and tobacco involved growth regulators(or phytohormones) such as auxins cytokinins gibberellinsabscisic acid ethylene and brassinosteroids [41] There weresignificant differences among treatments for the length ofthe inflorescence In all varieties the longest inflorescencelength was observed in the untreated plants with 162134 146 and 137 cm lengths for Chiang Mai Red DoiTung 554 Sweet Pink and Kimono Pink respectively Thecorresponding inflorescence lengths were 82 94 79 and109 cm for the 10Gy irradiated plants The days to anthesisfor C alismatifolia varieties were significantly affected byvariety gamma irradiation doses and their interaction Thenumber of days to full bloom was noticeable earliest foruntreated plants at 742 546 756 and 942 days for ChiangMai Red Doi Tung 554 Sweet Pink and Kimono Pinkvarieties respectively This was then followed by plants at10Gy at 102 78 1098 and 1224 days The number of trueflowers or the secondary inflorescence developed in the axilof the primary bracts decreased as radiation dosage increasedIn the present study the gamma rays also decreased thedays to inflorescence senescence In this study there wasa strongly significantly and positively correlation (0919lowastlowast)(data not shown) between the number of true flowers andthe number of days to senescenceThe number of pink bractsalso decreasedwith increasing radiation dosageMost gammaray effects on senescence are considered as resulting from theaction of free radicals generated from water and oxygen bythe ionizing energy on the cellular components Membrane
deterioration is a general feature of natural senescence andstress-induced aging [42]
Irradiation induced some mutation spectrum of flowercolor variation that included colors such as purple palepurple rather pale purple white purple white (marbledpattern) and two-tone purple color
Mutation spectrum of flower shape variation includeddouble inflorescence within one stalk double stalk per plantinflorescence without bracts two-flag petal true flowersand chlorophyll mutation in the leaves which are generallycaused by induced gamma rays (Figure 2) Ionizing radiationincluding gamma rays induces fragment deletions or inser-tions that eventually lead to changes in amino acids and amodification of leaf and stem pigmentation [43] A mutationin the biosynthetic pathway of structural or regulatory genesmay cause a change in flower color [44] When the blockageoccurs at the early stages of anthocyanin synthesis whiteflowers will result while a blockage at later stages leads todifferent flower colors due to the accumulation of particularanthocyanins [45] Chloroplasts were extremely sensitive togamma radiation compared to other cell organelles [46]
55 Effect of Gamma Irradiation on Rhizome Characteristicsin Selected Doses in M1V1 The number of new rhizomes andthe number of storage roots per rhizome were significantlyaffected by varieties doses and their interaction (Table 8)The number of new rhizomes only in Kimono Pink varietydid not show any differences between untreated and treatedplants The rhizome size was influenced only by doses Asdose level increased the rhizome size decreased Amonguntreated plants the Sweet Pink rhizomes had the most
10 BioMed Research International
(a) (b) (c) (d)
(e)
(f)
Figure 2 Effect of acute gamma rays on flower traits in C alismatifolia (a) Untreated inflorescence bracts color Doi Tung 554 (b) Two tonebract color Doi Tung 554 (20Gy) (c) Marbled pattern of bract color Doi Tung 554 (20Gy) (d) Double inflorescence within one stalk varChiang Mai Red (10Gy) (e) True flower in nontreated plants (f) Two-flag petals true flowers (10Gy)
Figure 3 Dendrogram representing themorphological variation among 44 irradiated and nonirradiated individuals ofC alismatifolia across14 variables CMR = Chiang Mai Red DT = Doi Tung 554 SP = Sweet Pink Kp = Kimono Pink
number of storage roots (78) and the Kimono Pink rhizomeshad the least number of storage roots (40)This was followedby 10Gy irradiated rhizomes with 4 46 48 and 28 storageroot numbers for Chiang Mai Red Doi Tung 554 SweetPink and Kimono Pink respectively Irradiation with 20Gydecreased significantly the number of storage roots in allstudied varietiesTheplantC alismatifolia is propagated froma propagule (one rhizome + 5-6 t-roots) The rhizome sizedose matters in the growth of the plant Smaller rhizome sizeusually resulted in smaller plant size with narrow grass-likeleaves The storage roots play a very important role in thegrowth and flowering ofCurcumaThe t-rootsmake up about85 of the total fresh weight and 70 of the total dry weightof a typical propaguleThe storage organs act for plant growth
during dormancy and emergence [47] More storage root perpropagule resulted in faster flowering and higher yield plant[48] In this study also there was significant and negativecorrelation between number of storage roots and number ofdays to visible bud appearance (minus0525lowastlowast)
56 Cluster Analysis of C alismatifolia for MorphologicalTraits The morphological data were used to calculate thesimilarity between the treated and non-treated C alismat-ifolia samples and UPGMA dendrogram was constructed(Figure 3) The cophenetic correlation coefficient (CCC)value between the genetic dissimilaritymatrix estimated fromthe morphological characters and the UPGMA clusteringmethod was 119903 = 093 showing a proof fit This value was
BioMed Research International 11
Table 8 Effect of acute gamma rays on rhizome characteristics ofC alismatifolia in M1V1 generation
Dose (Gray) Rhizomesize
No of newrhizomes
No of storageroot
CMR0 23 plusmn 02
a26 plusmn 055
a46 plusmn 054
a
10 18 plusmn 005a12 plusmn 044
b40 plusmn 07
a
20 08 plusmn 07b
06 plusmn 054b
14 plusmn 13b
DT0 22 plusmn 01
a30 plusmn 00
a72 plusmn 109
a
10 18 plusmn 007b14 plusmn 054
b46 plusmn 13
b
20 16 plusmn 005b
10 plusmn 00b
28 plusmn 13c
SP0 22 plusmn 02
a24 plusmn 054
a78 plusmn 21
a
10 17 plusmn 007b
10 plusmn 00b
48 plusmn 044b
20 12 plusmn 02c
10 plusmn 00b
28 plusmn 083b
KP0 21 plusmn 005
a10 plusmn 00
a40 plusmn 00
a
10 16 plusmn 01ab
10 plusmn 00a
28 plusmn 044b
20 11 plusmn 06b
10 plusmn 00a
22 plusmn 083b
CV () 18 23 26Means with the same or common letter are not significantly different leastsquare difference test 119875 lt 005
higher than of studies on olive for morphological traits (119903 =069) [49] In this dendrogram the 44C alismatifolia samplesappeared to form three main clusters and five minor clustersat coefficient level 56 cluster I included three non-irradiatedmembers and cluster II included 10Gy irradiated individualplants of all four varieties and 20Gy irradiated samples ofDoi Tung 554 and Kimono Pink varieties except KP20-4 (noflowering) Main cluster III included the 20Gy irradiatedindividuals Chiang Mai Red and Sweet Pink varieties (asmentioned in morphological part these individuals didnot go to flowering stage) and KP20-4 Cluster II can bedivided to five subclusters The first subcluster included 10members (CMR10-1 CMR10-5 SP10-1 SP10-2 SP10-3 SP10-5 CMR10-2 CMR10-4 SP10-4 and CMR10-3) the secondone included nine members (KP10-1 KP10-2 KP10-3 KP10-4 KP10-5KP20-1 KP20-2 KP20-5 and KP20-3) KP0 wasthe lone member of the third sub-cluster The fourth sub-cluster included two members only (DT10-2 and DT10-3) The last sub-cluster main cluster II had eight membersincluded (DT10-1 DT10-4 DT10-5 DT20-1 DT20-2 DT20-3 DT20-4 and DT20-5) Mean value of groups for each traitis presented in Table 9 This table clearly shows the differentmean values of the three main clusters The minimum meanvalue referred to main cluster III while the maximum meanvalue for number of shoots leaf length leaf width plantheight number of true flowers inflorescence length rhizomesize number of new rhizomes and number of storage rootsbelonged to cluster I which was included non-treated plantsThese results showed that the gamma irradiation has inducedmorphological changes in C alismatifolia individuals of
four studied varieties so that they showed phenotypicallydifferences from their controls
57 Principal Component Analysis of C alismatifolia forMorphological Traits To assess the patterns of variation PCAwas done by considering all of the 14 variables The first threecomponents of PCA explained 828 of the total variation(Table 10) Only the first component which accounted for562 of the total variation was attributed to inflorescencelength plant height days to senescence number of trueflowers and rhizome size In PCA three-dimensional graphthe grouping of individuals confirmed the clustering results(Figure 4) The PCA graph proved that all irradiated indi-vidual plants of C alismatifolia varieties are phenotypicallydifferent from their non-irradiated individuals
6 Molecular Characterization
61 SSR Polymorphism Amplifications were successful forall the 17 SSR markers assayed This reflects a high levelof homology between SSR flanking regions in C longa andC alismatifolia Out of 17 primer pairs eight primer pairsresulted in polymorphic PCR products Table 11 summarizesthe results obtained based on the analysis of individuals of thefour studied varieties using the polymorphic SSR loci for 010 and 20Gy irradiated plants The number of alleles variedwidely among these loci A total of 25 36 and 41 alleles wereobserved among 0 10 and 20Gy irradiated individual plantsrespectively In the 10Gy treatment the number of allelesranged from three (clon09 and clon14) to seven (clon08) withan average value of 45 per locus In the 20Gy treatmentthe number of alleles varied from three (clon09 and clon14)to seven (clon08 and clon12) with an average value of 51per locus In the untreated individuals the number of allelesranged from two (clon04 clon09 clon11 and clon14) to five(clon12) with an average value of 31 The difference betweenthe average number of observed alleles and effective numberof alleles was due to the uneven frequency of each allele[50] For each of the eight SSR primers PIC values (whichmeasures allele diversity and frequency among varieties)varied from 019 (clon04) to 071 (clon01) in untreated plantsand from 025 (clon04) to 075 (clon08) in 10Gy acutelyirradiated plants In 20Gy irradiated individual plants thePIC value ranged from 042 (clon14) to 075 (clon08 andclon12) The mean PIC for all loci was 047 054 and 061for 0 10 and 20Gy irradiated plants respectively
The PIC value provides an estimate of the discriminatorypower of amarker by taking into account not only the numberof alleles at a locus but also the relative frequencies of thesealleles [51] Thus except for clon09 and clon14 which weremoderately polymorphic (025 lt PIC lt 05) all other lociwere highly polymorphic (05 lt PIC) while none of theloci showed low polymorphism The average discriminatingpower ofmicrosatellitemarkers (061) observed in the presentstudy ensures the future utility of microsatellite markersfor genetic variation studies in C alismatifolia varietiesOur results reflect similar findings as reported earlier inthe study of genetic variation in other species of Curcuma
12 BioMed Research International
Table9Clusterm
eans
for14charactersestim
ated
in44
individu
also
fCalismatifolia
Clusters
Shoo
tnu
mber
Leaf
leng
thLeaf
width
Leaf
number
Visib
lebu
d(days)
Inflo
rescence
leng
thPlant
height
Anthesis
(days)
Senescence
(days)
True
flow
number
Pink
bract
number
Rhizom
esiz
eNew
rhizom
enu
mber
Storage
root
number
I220
5464
736
300
6013
1473
9233
6926
2093
152
1453
229
266
653
II1
100
2735
512
360
9260
809
5457
1059
960
620
510
177
110
440
II2
122
1922
298
344
11422
1043
3910
1312
293
3755
588
156
100
266
II3
100
3452
416
460
8520
1370
5910
9420
180
120
960
206
100
400
II4
150
2500
45
250
6500
925
4950
7700
1150
950
1700
185
200
600
II5
137
2637
417
262
7725
840
5356
9162
1137
1012
1587
170
100
312
III
054
1500
465
181
000
000
000
000
000
000
000
090
081
200
BioMed Research International 13
Table 10 Eigenvectors eigenvalues and proportions of variabilityfor three principle components among 14 characters for 44 Calismatifolia
Variable PC1 PC2 PC3Number of new shoot 0270 minus0076 minus0173Leaf length 0269 minus0305 0033Leaf width 0096 minus0455 0430Leaf number 0210 0285 0490Days to visible bud 0199 0462 0181Plant height 0321 0027 minus0071Inflorescence length 0318 0166 minus0040Days to anthesis 0205 0460 0148Days to senescence 0342 0017 minus0131Number of true flower 0323 0029 minus0238Number of pink bracts 0266 minus0028 minus0546Rhizome size 0302 minus0101 0147Number of new rhizome 0250 minus0293 0028Number of storage roots 0264 minus0247 0298Eigenvalue 7877 2694 1032Proportion 56264 19246 7371Cumulative 56264 75511 82882
using SSR markers [13] The highest Neirsquos gene diversity(ℎ) was obtained with the 20Gy treatment (061) followedby the 10Gy treatment (06) and the untreated individuals(05) The mean Shannonrsquos information index (119868) was 092114 and 130 in the 0 10 and 20Gy irradiated plantsrespectively The high value of Shannonrsquos information indexrepresents the effectiveness of microsatellite loci to revealthe variation among these varieties at the different dosesused Overall genetic variability for the varieties studiedrepresented by the Shannon-Weiner index was relatively highin comparison to other studies involving C longa accessions[10 52] Additionally the 20Gy acutely irradiated individualsshowed a higher mean percentage of polymorphic loci (63)than the 10Gy (58) and untreated (022) ones This impliesthat irradiation with a dose of 20Gy induced more geneticvariation in the M
1V1generation
62 Cluster Analysis Thedendrogramwas constructed usingPowerMarker 323The 44 studied individuals were clusteredinto seven groups (G1ndashG7) Colors were applied accordingto our model-based cluster analysis results Group I iscomprised of nine individual plants of Chiang Mai Redvariety receiving 0 10 and 20Gy treatments (blue colorin Figure 5) One individual plant DT10-1 was assignedto group II (green color in Figure 5) All irradiated andnon-irradiated individuals of Doi Tung 554 variety wereassigned to group III (yellow color in Figure 5) DT20-1 andSP10-5 were the sole members of groups IV (red color inFigure 5) and V (gray color in Figure 5) respectively GroupVI included treated and untreated individuals of Sweet Pinkvariety (pink color in Figure 5) Lastly all Kimono Pinkindividuals were assigned to cluster VII (orange color in
Figure 5) The genetic similarity coefficient (data not shown)among the 44 individuals amplified using eight SSR primersvaried from 00 to 10 The highest value (10) correspondedto [(CMR10-3 CMR10-5 CMR and CMR20-2) (DT10-2and DT10-3) (DT and DT20-2) (KP20-3 KP-20-4 KP10-3andKP10-4)] individuals that generated identical fingerprintsacross the markers studied Among the studied varieties theSweet Pink individual plants showed the lowest similarity(00) withKimono Pink individuals which indicated that theywere relatively remote in relationship (Figure 5) An overviewof the clustering pattern indicates that the grouping of thestudied individual plants was based on different varieties andlargely independent of the doses of gamma irradiation Awide range in similarity values had also been observed indifferent species of Curcuma [12 13 52 53]
63 Principle Component Analysis (PCA) Thedata generatedfrom 44 C alismatifolia individual plants were subjected toprincipal component analysis (PCA) to visualize individualsin a multivariate space In the three-dimensional graphderived from the SSR analysis all studied individuals weregrouped into seven clusters (Figure 6) In PCA the firstthree principal components (PC) extracted a cumulative of6826 of the total variation among the 44 individuals of CalismatifoliaThe first three coordinates PC1 PC2 and PC3accounted for 4136 1578 1112 of the total variationrespectively The distribution of the individual plants in athree-dimensional plot using the first three principal com-ponents showed the genetic relationship between individualplants It was evident that both methods dendrogram andthree-dimensional plots of PCA were effective in illustratinggenetic relationships and the groups found were comparable[11 13] The PCA results were similar to those obtained fromcluster analysis where all individuals from different varietieswere assigned to different groups These graphical illus-trations enable the assessment of the relationshipdistancesamong all of the individuals in the study [50]
7 Conclusion
This is the first attempt to evaluate the effect of acutegamma irradiation on C alismatifolia varieties using bothmorphological characteristics and molecular markers Inplant breeding programs mutagenic treatments with lownegative physiological effects and strong genetic effects aredesirable Hence we used more effective doses of gammairradiation (10 and 20Gy) of which particularly 20Gy dosewas effective to influence morphological and molecularcharacteristics of studiedCurcuma alismatifolia varietiesThelower dose (10Gy) of radiation probably caused little damageto the plants genetic material so that the cells could repairthemselves in next generation As a result in this study 20Gyacute gamma irradiation resulted in a higher percentage ofmutation and getting desired mutants was more possibleBased on the LD
50values determined in this study it was
apparent that the varieties Chiang Mai Red and Sweet Pinkwere more sensitive to gamma rays compared to Doi Tung554 and Kimono Pink Our results show that the variety Doi
Figure 4 Two-dimensional graph of principal component analysis (PCA) for 14 morphological variables indicating relationships amongirradiated and nonirradiated four varieties of C alismatifolia
CMR10-1CMR10-5
SP10-1SP10-2SP10-3
SP10-5
CMR10-4
SP10-4
KP10-1KP10-3
KP20-1
KP20-2
KP10-5KP20-5
KP20-3
KP10-2
DT10-2
DT10-1
DT10-5
DT20-1
DT10-4DT20-4DT20-2
DT20-5DT20-3
CMR20-1
SP20-3SP20-4
SP20-5SP20-1
SP20-2
KP
SP
CMR 10-2
SP10-5
Figure 5 Unrooted neighbor joining tree showing genetic relationship among 44 irradiated and nonirradiated C alismatifolia using SSRmarkers The colors of the branches correspond to those of the same cluster
Figure 6Three-dimensional graph of principal component analysis (PCA) indicating relationships among irradiated and nonirradiated fourvarieties of C alismatifolia
Tung 554 (Curcuma hybrid) had the most morphologicalresponses to gamma rays The use of microsatellite markersas a codominant marker will facilitate the exploration ofgenetic variability among treated and non-treated plants andwill help to distinguish the plants showing differences inmorphological characters The overall effects on the M
1V1
generation revealed that acute gamma irradiation at optimumdoses has the potential for developing new varieties of Calismatifolia with improved commercial properties suitablefor the Malaysian flower industry
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work was funded by the Fundamental Research GrantScheme (FRGS) under the Ministry of Higher Education inMalaysia
References
[1] P Apavatjrut S Anuntalabhochai P Sirirugsa and C AlisildquoMolecularmarkers in the identification of some early floweringCurcuma L (Zingiberaceae) speciesrdquo Annals of Botany vol 84no 4 pp 529ndash534 1999
[2] K Bunya-Atichart S Ketsa andW G VanDoorn ldquoPostharvestphysiology of Curcuma alismatifolia flowersrdquo Postharvest Biol-ogy and Technology vol 34 no 2 pp 219ndash226 2004
[3] M Nakano J Amano Y Watanabe et al ldquoMorphologicalvariation in Tricyrtis hirta plants regenerated from heavy ionbeam-irradiated embryogenic callusesrdquo Plant Biotechnologyvol 27 no 2 pp 155ndash160 2010
[4] S Lamseejan P Jompuk A Wongpiyasatid A Deeseepan andP Kwanthammachart ldquoGamma-rays induced morphological
[5] G-J Lee S J Chung I S Park et al ldquoVariation in thephenotypic features and transcripts of colormutants of chrysan-themum (Dendranthema grandiflorum) derived from gammaraymutagenesisrdquo Journal of Plant Biology vol 51 no 6 pp 418ndash423 2008
[6] O K Kikuchi ldquoOrchid flowers tolerance to gamma-radiationrdquoRadiation Physics and Chemistry vol 57 no 3ndash6 pp 555ndash5572000
[7] N P Arnold N N Barthakur and M Tanguay ldquoMutageniceffects of acute gamma irradiation on miniature roses targettheory approachrdquo HortScience vol 33 no 1 pp 127ndash129 1998
[8] A A Youssef M S Aly and M S Hussein ldquoResponse ofgeranium (Pelargonium graveolenus L) to gamma irradiationand foliar application of Speed Growrdquo Egyptian Journal ofHorticulture vol 27 pp 41ndash53 2000
[9] N Chuantang and L Yazhi ldquoThe radiation inducedmutation ofcanna (Canna L)rdquo Acta Agricultute Nucleatae Sinica vol 2 pp33ndash39 1998
[10] M S Sigrist J B Pinheiro J A A Filho and M I ZucchildquoGenetic diversity of turmeric germplasm (Curcuma longaZingiberaceae) identified by microsatellite markersrdquo Geneticsand Molecular Research vol 10 no 1 pp 419ndash428 2011
[11] S Taheri T L Abdullah N A P Abdullah and Z AhmadldquoGenetic relationships among five varieties of Curcuma alis-matifolia (Zingiberaceae) based on ISSRmarkersrdquoGenetics andMolecular Research vol 11 pp 3069ndash3076 2012
[12] A Das V Kesari V M Satyanarayana A Parida and L Ran-gan ldquoGenetic relationship of Curcuma species from NortheastIndia using PCR-based markersrdquo Molecular Biotechnology vol49 no 1 pp 65ndash76 2011
[13] S Siju K Dhanya S Syamkumar et al ldquoDevelopment char-acterization and utilization of genomic microsatellite markersin turmeric (Curcuma longa L)rdquo Biochemical Systematics andEcology vol 38 no 4 pp 641ndash646 2010
[14] M K Panda S Mohanty E Subudhi L Acharya and SNayak ldquoAssessment of genetic stability of micropropagated
BioMed Research International 17
plants of Curcuma L by cytophotometery and RAPD analysisrdquoInternational Journal of Integrative Biology (IJIB) vol 1 pp 189ndash195 2007
[15] S Syamkumar and B Sasikumar ldquoMolecular marker basedgenetic diversity analysis of Curcuma species from IndiardquoScientia Horticulturae vol 112 no 2 pp 235ndash241 2007
[16] D Tautz and M Renz ldquoSimple sequences are ubiquitousrepetitive components of eukaryotic genomesrdquo Nucleic AcidsResearch vol 12 no 10 pp 4127ndash4138 1984
[17] G Toth Z Gaspari and J Jurka ldquoMicrosatellites in differenteukaryotic genomes surveys and analysisrdquo Genome Researchvol 10 no 7 pp 967ndash981 2000
[18] W Powell G C Machray and J Proven ldquoPolymorphismrevealed by simple sequence repeatsrdquo Trends in Plant Sciencevol 1 no 7 pp 215ndash222 1996
[19] S-Y Lee W K Fai M Zakaria et al ldquoCharacterizationof polymorphic microsatellite markers isolated from ginger(Zingiber officinale Rosc)rdquo Molecular Ecology Notes vol 7 no6 pp 1009ndash1011 2007
[20] S Bory D Da Silva A-M Risterucci M Grisoni P Besseand M-F Duval ldquoDevelopment of microsatellite markers incultivated vanilla polymorphism and transferability to othervanilla speciesrdquo Scientia Horticulturae vol 115 no 4 pp 420ndash425 2008
[21] I C Menezes F W Cidade A P Souza and I C SampaioldquoIsolation and characterization ofmicrosatellite loci in the blackpepper Piper Nigrum L (Piperaceae)rdquo Conservation GeneticsResources vol 1 no 1 pp 209ndash212 2009
[22] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[23] V Kumar Morphological and molecular characterization ofinduced mutants in Groundnut [PhD thesis] University ofAgricultural Sciences Dharwad India 2008
[24] J R Sharma Statistical and Biometrical Techniques in PlantBreeding New Age International New Delhi India 1998
[25] J J Doyle and J L Doyle ldquoA rapid DNA isolation procedure forsmall quantities of fresh leaf tissuerdquo Phytochemical Bulletin vol19 pp 11ndash15 1987
[26] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[27] Oslash Hammer D A T Harper and P D Ryan ldquoPast paleontolog-ical statistics software package for education and data analysisrdquoPalaeontologia Electronica vol 4 no 1 pp 4ndash9 2001
[28] K Liu and S V Muse ldquoPowerMaker an integrated analysisenvironment for genetic maker analysisrdquo Bioinformatics vol 21no 9 pp 2128ndash2129 2005
[29] F J RohlfNTSYS-Pc Numerical Taxonomy System Version 2 1Exeter Publishing Setauket New York NY USA 2002
[30] M Nei ldquoAnalysis of gene diversity in subdivided populationsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 70 no 12 1973
[31] S Kadkhodaei M Shahnazari M K Nekouei et al ldquoA compar-ative study of morphological and molecular diversity analysisamong cultivated almonds (Prunus dulcis)rdquo Australian Journalof Crop Science vol 5 no 1 pp 82ndash91 2011
[32] T L Abdullah J Endan and B M Nazir ldquoChanges in flowerdevelopment chlorophyll mutation and alteration in plant
morphology of Curcuma alismatifolia by gamma irradiationrdquoAmerican Journal of Applied Sciences vol 6 no 7 pp 1436ndash14392009
[33] E Kovacs and A Keresztes ldquoEffect of gamma and UV-BCradiation on plant cellsrdquoMicron vol 33 no 2 pp 199ndash210 2002
[34] H L Ramesh Y Murthy and V N Munirajappa ldquoEffect ofgamma radiation on morphological and growth parametersof Mulberry variety M5rdquo International Journal of Science andNature vol 3 pp 447ndash452 2010
[35] K Pongchawee R Pradissan andW Pipatcharoenchai ldquoInducemutation in Anubias spp through in vitro Irradiationrdquo ThaiFisheries Gazette vol 60 pp 493ndash497 2007
[36] P Tangpong T Taychasinpitak C Jompuk and P JompukldquoEffects of acute and chronic gamma irradiations on in vitroculture ofAnubias congensisNE BrownrdquoKasetsart Journal vol43 no 3 pp 449ndash457 2009
[37] R K Hegde Studies on induced mutagenesis and in vitroregeneration in turmeric (Curcuma longa L) [PhD thesis]University of Agricultural Sciences Dharwad India 2006
[38] M P Giridharan and S Balakrishnan ldquoGamma ray inducedvariability in vegetative and floral characters of ginger IndianCocoardquo Arecanut and Spices Journal vol 15 pp 68ndash672 1992
[39] A P Cesarett ldquoEffect of radiation on higher plants and plantcommunitiesrdquo inRadiation Biology United States Atomic EnergyCommission pp 284ndash309 Washington DC USA 1968
[40] G W Seung Y C Byung J-H Kim et al ldquoUltrastructuralchanges of cell organelles in Arabidopsis stems after gammairradationrdquo Journal of Plant Biology vol 48 no 2 pp 195ndash2002005
[41] B S Ahloowalia and M Maluszynski ldquoInduced mutationsmdashanew paradigm in plant breedingrdquo Euphytica vol 118 no 2 pp167ndash173 2001
[42] R Voisine L-P Vezina and C Willemot ldquoInduction ofsenescence-like deterioration of microsomal membranes fromcauliflower by free radicals generated during gamma irradia-tionrdquo Plant Physiology vol 97 no 2 pp 545ndash550 1991
[43] N Shikazono Y Yokota S Kitamura et al ldquoMutation rate andnovel tt mutants of Arabidopsis thaliana induced by carbonionsrdquo Genetics vol 163 no 4 pp 1449ndash1455 2003
[44] T Nakatsuka M Nishihara K Mishiba and S YamamuraldquoTwo different mutations are involved in the formation ofwhite-flowered gentian plantsrdquo Plant Science vol 169 no 5 pp949ndash958 2005
[45] M Mato T Onozaki Y Ozeki et al ldquoFlavonoid biosynthesisin white-flowered Sim carnations (Dianthus caryophyllus)rdquoScientia Horticulturae vol 84 no 3-4 pp 333ndash347 2000
[46] S G Wi B Y Chung J-S Kim et al ldquoEffects of gammairradiation on morphological changes and biological responsesin plantsrdquoMicron vol 38 no 6 pp 553ndash564 2007
[47] F Lee Chin Effects of light intensity and daylength on growth andflowering of siam tulip (Curcuma alismatifolia var Chiang MaiPink) [PhD thesis] University Putra Malaysia 2007
[48] A Hagiladi N Umiel and X H Yang ldquoCurcuma alismatifoliaII Effects of temperature and daylength on the development offlowers and propagulesrdquo Acta Horticulture vol 430 pp 755ndash761 1997
[49] MHagidimitriou A Katsiotis GMenexes C Pontikis andMLoukas ldquoGenetic diversity of major greek olive cultivars usingmolecular (AFLPs and RAPDs) markers and morphologicaltraitsrdquo Journal of the American Society for Horticultural Sciencevol 130 no 2 pp 211ndash217 2005
18 BioMed Research International
[50] N Babaei N A P Abdullah G Saleh and T L AbdullahldquoIsolation and characterization of microsatellite markers andanalysis of genetic variability in Curculigo latifolia DryandrdquoMolecular Biology Reports vol 39 pp 9869ndash9877 2012
[51] B Shiran N Amirbakhtiar S Kiani S Mohammadi B ESayed-Tabatabaei and H Moradi ldquoMolecular characterizationand genetic relationship among almond cultivars assessed byRAPD and SSR markersrdquo Scientia Horticulturae vol 111 no 3pp 280ndash292 2007
[52] S Singh M K Panda and S Nayak ldquoEvaluation of geneticdiversity in turmeric (Curcuma longa L) using RAPD and ISSRmarkersrdquo Industrial Crops and Products vol 37 no 1 pp 284ndash291 2012
[53] Y Paisooksantivatana S Kako and H Seko ldquoGenetic diversityof Curcuma alismatifolia Gagnep (Zingiberaceae) in Thailandas revealed by allozyme polymorphismrdquo Genetic Resources andCrop Evolution vol 48 no 5 pp 459ndash465 2001
Figure 2 Effect of acute gamma rays on flower traits in C alismatifolia (a) Untreated inflorescence bracts color Doi Tung 554 (b) Two tonebract color Doi Tung 554 (20Gy) (c) Marbled pattern of bract color Doi Tung 554 (20Gy) (d) Double inflorescence within one stalk varChiang Mai Red (10Gy) (e) True flower in nontreated plants (f) Two-flag petals true flowers (10Gy)
Figure 3 Dendrogram representing themorphological variation among 44 irradiated and nonirradiated individuals ofC alismatifolia across14 variables CMR = Chiang Mai Red DT = Doi Tung 554 SP = Sweet Pink Kp = Kimono Pink
number of storage roots (78) and the Kimono Pink rhizomeshad the least number of storage roots (40)This was followedby 10Gy irradiated rhizomes with 4 46 48 and 28 storageroot numbers for Chiang Mai Red Doi Tung 554 SweetPink and Kimono Pink respectively Irradiation with 20Gydecreased significantly the number of storage roots in allstudied varietiesTheplantC alismatifolia is propagated froma propagule (one rhizome + 5-6 t-roots) The rhizome sizedose matters in the growth of the plant Smaller rhizome sizeusually resulted in smaller plant size with narrow grass-likeleaves The storage roots play a very important role in thegrowth and flowering ofCurcumaThe t-rootsmake up about85 of the total fresh weight and 70 of the total dry weightof a typical propaguleThe storage organs act for plant growth
during dormancy and emergence [47] More storage root perpropagule resulted in faster flowering and higher yield plant[48] In this study also there was significant and negativecorrelation between number of storage roots and number ofdays to visible bud appearance (minus0525lowastlowast)
56 Cluster Analysis of C alismatifolia for MorphologicalTraits The morphological data were used to calculate thesimilarity between the treated and non-treated C alismat-ifolia samples and UPGMA dendrogram was constructed(Figure 3) The cophenetic correlation coefficient (CCC)value between the genetic dissimilaritymatrix estimated fromthe morphological characters and the UPGMA clusteringmethod was 119903 = 093 showing a proof fit This value was
BioMed Research International 11
Table 8 Effect of acute gamma rays on rhizome characteristics ofC alismatifolia in M1V1 generation
Dose (Gray) Rhizomesize
No of newrhizomes
No of storageroot
CMR0 23 plusmn 02
a26 plusmn 055
a46 plusmn 054
a
10 18 plusmn 005a12 plusmn 044
b40 plusmn 07
a
20 08 plusmn 07b
06 plusmn 054b
14 plusmn 13b
DT0 22 plusmn 01
a30 plusmn 00
a72 plusmn 109
a
10 18 plusmn 007b14 plusmn 054
b46 plusmn 13
b
20 16 plusmn 005b
10 plusmn 00b
28 plusmn 13c
SP0 22 plusmn 02
a24 plusmn 054
a78 plusmn 21
a
10 17 plusmn 007b
10 plusmn 00b
48 plusmn 044b
20 12 plusmn 02c
10 plusmn 00b
28 plusmn 083b
KP0 21 plusmn 005
a10 plusmn 00
a40 plusmn 00
a
10 16 plusmn 01ab
10 plusmn 00a
28 plusmn 044b
20 11 plusmn 06b
10 plusmn 00a
22 plusmn 083b
CV () 18 23 26Means with the same or common letter are not significantly different leastsquare difference test 119875 lt 005
higher than of studies on olive for morphological traits (119903 =069) [49] In this dendrogram the 44C alismatifolia samplesappeared to form three main clusters and five minor clustersat coefficient level 56 cluster I included three non-irradiatedmembers and cluster II included 10Gy irradiated individualplants of all four varieties and 20Gy irradiated samples ofDoi Tung 554 and Kimono Pink varieties except KP20-4 (noflowering) Main cluster III included the 20Gy irradiatedindividuals Chiang Mai Red and Sweet Pink varieties (asmentioned in morphological part these individuals didnot go to flowering stage) and KP20-4 Cluster II can bedivided to five subclusters The first subcluster included 10members (CMR10-1 CMR10-5 SP10-1 SP10-2 SP10-3 SP10-5 CMR10-2 CMR10-4 SP10-4 and CMR10-3) the secondone included nine members (KP10-1 KP10-2 KP10-3 KP10-4 KP10-5KP20-1 KP20-2 KP20-5 and KP20-3) KP0 wasthe lone member of the third sub-cluster The fourth sub-cluster included two members only (DT10-2 and DT10-3) The last sub-cluster main cluster II had eight membersincluded (DT10-1 DT10-4 DT10-5 DT20-1 DT20-2 DT20-3 DT20-4 and DT20-5) Mean value of groups for each traitis presented in Table 9 This table clearly shows the differentmean values of the three main clusters The minimum meanvalue referred to main cluster III while the maximum meanvalue for number of shoots leaf length leaf width plantheight number of true flowers inflorescence length rhizomesize number of new rhizomes and number of storage rootsbelonged to cluster I which was included non-treated plantsThese results showed that the gamma irradiation has inducedmorphological changes in C alismatifolia individuals of
four studied varieties so that they showed phenotypicallydifferences from their controls
57 Principal Component Analysis of C alismatifolia forMorphological Traits To assess the patterns of variation PCAwas done by considering all of the 14 variables The first threecomponents of PCA explained 828 of the total variation(Table 10) Only the first component which accounted for562 of the total variation was attributed to inflorescencelength plant height days to senescence number of trueflowers and rhizome size In PCA three-dimensional graphthe grouping of individuals confirmed the clustering results(Figure 4) The PCA graph proved that all irradiated indi-vidual plants of C alismatifolia varieties are phenotypicallydifferent from their non-irradiated individuals
6 Molecular Characterization
61 SSR Polymorphism Amplifications were successful forall the 17 SSR markers assayed This reflects a high levelof homology between SSR flanking regions in C longa andC alismatifolia Out of 17 primer pairs eight primer pairsresulted in polymorphic PCR products Table 11 summarizesthe results obtained based on the analysis of individuals of thefour studied varieties using the polymorphic SSR loci for 010 and 20Gy irradiated plants The number of alleles variedwidely among these loci A total of 25 36 and 41 alleles wereobserved among 0 10 and 20Gy irradiated individual plantsrespectively In the 10Gy treatment the number of allelesranged from three (clon09 and clon14) to seven (clon08) withan average value of 45 per locus In the 20Gy treatmentthe number of alleles varied from three (clon09 and clon14)to seven (clon08 and clon12) with an average value of 51per locus In the untreated individuals the number of allelesranged from two (clon04 clon09 clon11 and clon14) to five(clon12) with an average value of 31 The difference betweenthe average number of observed alleles and effective numberof alleles was due to the uneven frequency of each allele[50] For each of the eight SSR primers PIC values (whichmeasures allele diversity and frequency among varieties)varied from 019 (clon04) to 071 (clon01) in untreated plantsand from 025 (clon04) to 075 (clon08) in 10Gy acutelyirradiated plants In 20Gy irradiated individual plants thePIC value ranged from 042 (clon14) to 075 (clon08 andclon12) The mean PIC for all loci was 047 054 and 061for 0 10 and 20Gy irradiated plants respectively
The PIC value provides an estimate of the discriminatorypower of amarker by taking into account not only the numberof alleles at a locus but also the relative frequencies of thesealleles [51] Thus except for clon09 and clon14 which weremoderately polymorphic (025 lt PIC lt 05) all other lociwere highly polymorphic (05 lt PIC) while none of theloci showed low polymorphism The average discriminatingpower ofmicrosatellitemarkers (061) observed in the presentstudy ensures the future utility of microsatellite markersfor genetic variation studies in C alismatifolia varietiesOur results reflect similar findings as reported earlier inthe study of genetic variation in other species of Curcuma
12 BioMed Research International
Table9Clusterm
eans
for14charactersestim
ated
in44
individu
also
fCalismatifolia
Clusters
Shoo
tnu
mber
Leaf
leng
thLeaf
width
Leaf
number
Visib
lebu
d(days)
Inflo
rescence
leng
thPlant
height
Anthesis
(days)
Senescence
(days)
True
flow
number
Pink
bract
number
Rhizom
esiz
eNew
rhizom
enu
mber
Storage
root
number
I220
5464
736
300
6013
1473
9233
6926
2093
152
1453
229
266
653
II1
100
2735
512
360
9260
809
5457
1059
960
620
510
177
110
440
II2
122
1922
298
344
11422
1043
3910
1312
293
3755
588
156
100
266
II3
100
3452
416
460
8520
1370
5910
9420
180
120
960
206
100
400
II4
150
2500
45
250
6500
925
4950
7700
1150
950
1700
185
200
600
II5
137
2637
417
262
7725
840
5356
9162
1137
1012
1587
170
100
312
III
054
1500
465
181
000
000
000
000
000
000
000
090
081
200
BioMed Research International 13
Table 10 Eigenvectors eigenvalues and proportions of variabilityfor three principle components among 14 characters for 44 Calismatifolia
Variable PC1 PC2 PC3Number of new shoot 0270 minus0076 minus0173Leaf length 0269 minus0305 0033Leaf width 0096 minus0455 0430Leaf number 0210 0285 0490Days to visible bud 0199 0462 0181Plant height 0321 0027 minus0071Inflorescence length 0318 0166 minus0040Days to anthesis 0205 0460 0148Days to senescence 0342 0017 minus0131Number of true flower 0323 0029 minus0238Number of pink bracts 0266 minus0028 minus0546Rhizome size 0302 minus0101 0147Number of new rhizome 0250 minus0293 0028Number of storage roots 0264 minus0247 0298Eigenvalue 7877 2694 1032Proportion 56264 19246 7371Cumulative 56264 75511 82882
using SSR markers [13] The highest Neirsquos gene diversity(ℎ) was obtained with the 20Gy treatment (061) followedby the 10Gy treatment (06) and the untreated individuals(05) The mean Shannonrsquos information index (119868) was 092114 and 130 in the 0 10 and 20Gy irradiated plantsrespectively The high value of Shannonrsquos information indexrepresents the effectiveness of microsatellite loci to revealthe variation among these varieties at the different dosesused Overall genetic variability for the varieties studiedrepresented by the Shannon-Weiner index was relatively highin comparison to other studies involving C longa accessions[10 52] Additionally the 20Gy acutely irradiated individualsshowed a higher mean percentage of polymorphic loci (63)than the 10Gy (58) and untreated (022) ones This impliesthat irradiation with a dose of 20Gy induced more geneticvariation in the M
1V1generation
62 Cluster Analysis Thedendrogramwas constructed usingPowerMarker 323The 44 studied individuals were clusteredinto seven groups (G1ndashG7) Colors were applied accordingto our model-based cluster analysis results Group I iscomprised of nine individual plants of Chiang Mai Redvariety receiving 0 10 and 20Gy treatments (blue colorin Figure 5) One individual plant DT10-1 was assignedto group II (green color in Figure 5) All irradiated andnon-irradiated individuals of Doi Tung 554 variety wereassigned to group III (yellow color in Figure 5) DT20-1 andSP10-5 were the sole members of groups IV (red color inFigure 5) and V (gray color in Figure 5) respectively GroupVI included treated and untreated individuals of Sweet Pinkvariety (pink color in Figure 5) Lastly all Kimono Pinkindividuals were assigned to cluster VII (orange color in
Figure 5) The genetic similarity coefficient (data not shown)among the 44 individuals amplified using eight SSR primersvaried from 00 to 10 The highest value (10) correspondedto [(CMR10-3 CMR10-5 CMR and CMR20-2) (DT10-2and DT10-3) (DT and DT20-2) (KP20-3 KP-20-4 KP10-3andKP10-4)] individuals that generated identical fingerprintsacross the markers studied Among the studied varieties theSweet Pink individual plants showed the lowest similarity(00) withKimono Pink individuals which indicated that theywere relatively remote in relationship (Figure 5) An overviewof the clustering pattern indicates that the grouping of thestudied individual plants was based on different varieties andlargely independent of the doses of gamma irradiation Awide range in similarity values had also been observed indifferent species of Curcuma [12 13 52 53]
63 Principle Component Analysis (PCA) Thedata generatedfrom 44 C alismatifolia individual plants were subjected toprincipal component analysis (PCA) to visualize individualsin a multivariate space In the three-dimensional graphderived from the SSR analysis all studied individuals weregrouped into seven clusters (Figure 6) In PCA the firstthree principal components (PC) extracted a cumulative of6826 of the total variation among the 44 individuals of CalismatifoliaThe first three coordinates PC1 PC2 and PC3accounted for 4136 1578 1112 of the total variationrespectively The distribution of the individual plants in athree-dimensional plot using the first three principal com-ponents showed the genetic relationship between individualplants It was evident that both methods dendrogram andthree-dimensional plots of PCA were effective in illustratinggenetic relationships and the groups found were comparable[11 13] The PCA results were similar to those obtained fromcluster analysis where all individuals from different varietieswere assigned to different groups These graphical illus-trations enable the assessment of the relationshipdistancesamong all of the individuals in the study [50]
7 Conclusion
This is the first attempt to evaluate the effect of acutegamma irradiation on C alismatifolia varieties using bothmorphological characteristics and molecular markers Inplant breeding programs mutagenic treatments with lownegative physiological effects and strong genetic effects aredesirable Hence we used more effective doses of gammairradiation (10 and 20Gy) of which particularly 20Gy dosewas effective to influence morphological and molecularcharacteristics of studiedCurcuma alismatifolia varietiesThelower dose (10Gy) of radiation probably caused little damageto the plants genetic material so that the cells could repairthemselves in next generation As a result in this study 20Gyacute gamma irradiation resulted in a higher percentage ofmutation and getting desired mutants was more possibleBased on the LD
50values determined in this study it was
apparent that the varieties Chiang Mai Red and Sweet Pinkwere more sensitive to gamma rays compared to Doi Tung554 and Kimono Pink Our results show that the variety Doi
Figure 4 Two-dimensional graph of principal component analysis (PCA) for 14 morphological variables indicating relationships amongirradiated and nonirradiated four varieties of C alismatifolia
CMR10-1CMR10-5
SP10-1SP10-2SP10-3
SP10-5
CMR10-4
SP10-4
KP10-1KP10-3
KP20-1
KP20-2
KP10-5KP20-5
KP20-3
KP10-2
DT10-2
DT10-1
DT10-5
DT20-1
DT10-4DT20-4DT20-2
DT20-5DT20-3
CMR20-1
SP20-3SP20-4
SP20-5SP20-1
SP20-2
KP
SP
CMR 10-2
SP10-5
Figure 5 Unrooted neighbor joining tree showing genetic relationship among 44 irradiated and nonirradiated C alismatifolia using SSRmarkers The colors of the branches correspond to those of the same cluster
Figure 6Three-dimensional graph of principal component analysis (PCA) indicating relationships among irradiated and nonirradiated fourvarieties of C alismatifolia
Tung 554 (Curcuma hybrid) had the most morphologicalresponses to gamma rays The use of microsatellite markersas a codominant marker will facilitate the exploration ofgenetic variability among treated and non-treated plants andwill help to distinguish the plants showing differences inmorphological characters The overall effects on the M
1V1
generation revealed that acute gamma irradiation at optimumdoses has the potential for developing new varieties of Calismatifolia with improved commercial properties suitablefor the Malaysian flower industry
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work was funded by the Fundamental Research GrantScheme (FRGS) under the Ministry of Higher Education inMalaysia
References
[1] P Apavatjrut S Anuntalabhochai P Sirirugsa and C AlisildquoMolecularmarkers in the identification of some early floweringCurcuma L (Zingiberaceae) speciesrdquo Annals of Botany vol 84no 4 pp 529ndash534 1999
[2] K Bunya-Atichart S Ketsa andW G VanDoorn ldquoPostharvestphysiology of Curcuma alismatifolia flowersrdquo Postharvest Biol-ogy and Technology vol 34 no 2 pp 219ndash226 2004
[3] M Nakano J Amano Y Watanabe et al ldquoMorphologicalvariation in Tricyrtis hirta plants regenerated from heavy ionbeam-irradiated embryogenic callusesrdquo Plant Biotechnologyvol 27 no 2 pp 155ndash160 2010
[4] S Lamseejan P Jompuk A Wongpiyasatid A Deeseepan andP Kwanthammachart ldquoGamma-rays induced morphological
[5] G-J Lee S J Chung I S Park et al ldquoVariation in thephenotypic features and transcripts of colormutants of chrysan-themum (Dendranthema grandiflorum) derived from gammaraymutagenesisrdquo Journal of Plant Biology vol 51 no 6 pp 418ndash423 2008
[6] O K Kikuchi ldquoOrchid flowers tolerance to gamma-radiationrdquoRadiation Physics and Chemistry vol 57 no 3ndash6 pp 555ndash5572000
[7] N P Arnold N N Barthakur and M Tanguay ldquoMutageniceffects of acute gamma irradiation on miniature roses targettheory approachrdquo HortScience vol 33 no 1 pp 127ndash129 1998
[8] A A Youssef M S Aly and M S Hussein ldquoResponse ofgeranium (Pelargonium graveolenus L) to gamma irradiationand foliar application of Speed Growrdquo Egyptian Journal ofHorticulture vol 27 pp 41ndash53 2000
[9] N Chuantang and L Yazhi ldquoThe radiation inducedmutation ofcanna (Canna L)rdquo Acta Agricultute Nucleatae Sinica vol 2 pp33ndash39 1998
[10] M S Sigrist J B Pinheiro J A A Filho and M I ZucchildquoGenetic diversity of turmeric germplasm (Curcuma longaZingiberaceae) identified by microsatellite markersrdquo Geneticsand Molecular Research vol 10 no 1 pp 419ndash428 2011
[11] S Taheri T L Abdullah N A P Abdullah and Z AhmadldquoGenetic relationships among five varieties of Curcuma alis-matifolia (Zingiberaceae) based on ISSRmarkersrdquoGenetics andMolecular Research vol 11 pp 3069ndash3076 2012
[12] A Das V Kesari V M Satyanarayana A Parida and L Ran-gan ldquoGenetic relationship of Curcuma species from NortheastIndia using PCR-based markersrdquo Molecular Biotechnology vol49 no 1 pp 65ndash76 2011
[13] S Siju K Dhanya S Syamkumar et al ldquoDevelopment char-acterization and utilization of genomic microsatellite markersin turmeric (Curcuma longa L)rdquo Biochemical Systematics andEcology vol 38 no 4 pp 641ndash646 2010
[14] M K Panda S Mohanty E Subudhi L Acharya and SNayak ldquoAssessment of genetic stability of micropropagated
BioMed Research International 17
plants of Curcuma L by cytophotometery and RAPD analysisrdquoInternational Journal of Integrative Biology (IJIB) vol 1 pp 189ndash195 2007
[15] S Syamkumar and B Sasikumar ldquoMolecular marker basedgenetic diversity analysis of Curcuma species from IndiardquoScientia Horticulturae vol 112 no 2 pp 235ndash241 2007
[16] D Tautz and M Renz ldquoSimple sequences are ubiquitousrepetitive components of eukaryotic genomesrdquo Nucleic AcidsResearch vol 12 no 10 pp 4127ndash4138 1984
[17] G Toth Z Gaspari and J Jurka ldquoMicrosatellites in differenteukaryotic genomes surveys and analysisrdquo Genome Researchvol 10 no 7 pp 967ndash981 2000
[18] W Powell G C Machray and J Proven ldquoPolymorphismrevealed by simple sequence repeatsrdquo Trends in Plant Sciencevol 1 no 7 pp 215ndash222 1996
[19] S-Y Lee W K Fai M Zakaria et al ldquoCharacterizationof polymorphic microsatellite markers isolated from ginger(Zingiber officinale Rosc)rdquo Molecular Ecology Notes vol 7 no6 pp 1009ndash1011 2007
[20] S Bory D Da Silva A-M Risterucci M Grisoni P Besseand M-F Duval ldquoDevelopment of microsatellite markers incultivated vanilla polymorphism and transferability to othervanilla speciesrdquo Scientia Horticulturae vol 115 no 4 pp 420ndash425 2008
[21] I C Menezes F W Cidade A P Souza and I C SampaioldquoIsolation and characterization ofmicrosatellite loci in the blackpepper Piper Nigrum L (Piperaceae)rdquo Conservation GeneticsResources vol 1 no 1 pp 209ndash212 2009
[22] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[23] V Kumar Morphological and molecular characterization ofinduced mutants in Groundnut [PhD thesis] University ofAgricultural Sciences Dharwad India 2008
[24] J R Sharma Statistical and Biometrical Techniques in PlantBreeding New Age International New Delhi India 1998
[25] J J Doyle and J L Doyle ldquoA rapid DNA isolation procedure forsmall quantities of fresh leaf tissuerdquo Phytochemical Bulletin vol19 pp 11ndash15 1987
[26] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[27] Oslash Hammer D A T Harper and P D Ryan ldquoPast paleontolog-ical statistics software package for education and data analysisrdquoPalaeontologia Electronica vol 4 no 1 pp 4ndash9 2001
[28] K Liu and S V Muse ldquoPowerMaker an integrated analysisenvironment for genetic maker analysisrdquo Bioinformatics vol 21no 9 pp 2128ndash2129 2005
[29] F J RohlfNTSYS-Pc Numerical Taxonomy System Version 2 1Exeter Publishing Setauket New York NY USA 2002
[30] M Nei ldquoAnalysis of gene diversity in subdivided populationsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 70 no 12 1973
[31] S Kadkhodaei M Shahnazari M K Nekouei et al ldquoA compar-ative study of morphological and molecular diversity analysisamong cultivated almonds (Prunus dulcis)rdquo Australian Journalof Crop Science vol 5 no 1 pp 82ndash91 2011
[32] T L Abdullah J Endan and B M Nazir ldquoChanges in flowerdevelopment chlorophyll mutation and alteration in plant
morphology of Curcuma alismatifolia by gamma irradiationrdquoAmerican Journal of Applied Sciences vol 6 no 7 pp 1436ndash14392009
[33] E Kovacs and A Keresztes ldquoEffect of gamma and UV-BCradiation on plant cellsrdquoMicron vol 33 no 2 pp 199ndash210 2002
[34] H L Ramesh Y Murthy and V N Munirajappa ldquoEffect ofgamma radiation on morphological and growth parametersof Mulberry variety M5rdquo International Journal of Science andNature vol 3 pp 447ndash452 2010
[35] K Pongchawee R Pradissan andW Pipatcharoenchai ldquoInducemutation in Anubias spp through in vitro Irradiationrdquo ThaiFisheries Gazette vol 60 pp 493ndash497 2007
[36] P Tangpong T Taychasinpitak C Jompuk and P JompukldquoEffects of acute and chronic gamma irradiations on in vitroculture ofAnubias congensisNE BrownrdquoKasetsart Journal vol43 no 3 pp 449ndash457 2009
[37] R K Hegde Studies on induced mutagenesis and in vitroregeneration in turmeric (Curcuma longa L) [PhD thesis]University of Agricultural Sciences Dharwad India 2006
[38] M P Giridharan and S Balakrishnan ldquoGamma ray inducedvariability in vegetative and floral characters of ginger IndianCocoardquo Arecanut and Spices Journal vol 15 pp 68ndash672 1992
[39] A P Cesarett ldquoEffect of radiation on higher plants and plantcommunitiesrdquo inRadiation Biology United States Atomic EnergyCommission pp 284ndash309 Washington DC USA 1968
[40] G W Seung Y C Byung J-H Kim et al ldquoUltrastructuralchanges of cell organelles in Arabidopsis stems after gammairradationrdquo Journal of Plant Biology vol 48 no 2 pp 195ndash2002005
[41] B S Ahloowalia and M Maluszynski ldquoInduced mutationsmdashanew paradigm in plant breedingrdquo Euphytica vol 118 no 2 pp167ndash173 2001
[42] R Voisine L-P Vezina and C Willemot ldquoInduction ofsenescence-like deterioration of microsomal membranes fromcauliflower by free radicals generated during gamma irradia-tionrdquo Plant Physiology vol 97 no 2 pp 545ndash550 1991
[43] N Shikazono Y Yokota S Kitamura et al ldquoMutation rate andnovel tt mutants of Arabidopsis thaliana induced by carbonionsrdquo Genetics vol 163 no 4 pp 1449ndash1455 2003
[44] T Nakatsuka M Nishihara K Mishiba and S YamamuraldquoTwo different mutations are involved in the formation ofwhite-flowered gentian plantsrdquo Plant Science vol 169 no 5 pp949ndash958 2005
[45] M Mato T Onozaki Y Ozeki et al ldquoFlavonoid biosynthesisin white-flowered Sim carnations (Dianthus caryophyllus)rdquoScientia Horticulturae vol 84 no 3-4 pp 333ndash347 2000
[46] S G Wi B Y Chung J-S Kim et al ldquoEffects of gammairradiation on morphological changes and biological responsesin plantsrdquoMicron vol 38 no 6 pp 553ndash564 2007
[47] F Lee Chin Effects of light intensity and daylength on growth andflowering of siam tulip (Curcuma alismatifolia var Chiang MaiPink) [PhD thesis] University Putra Malaysia 2007
[48] A Hagiladi N Umiel and X H Yang ldquoCurcuma alismatifoliaII Effects of temperature and daylength on the development offlowers and propagulesrdquo Acta Horticulture vol 430 pp 755ndash761 1997
[49] MHagidimitriou A Katsiotis GMenexes C Pontikis andMLoukas ldquoGenetic diversity of major greek olive cultivars usingmolecular (AFLPs and RAPDs) markers and morphologicaltraitsrdquo Journal of the American Society for Horticultural Sciencevol 130 no 2 pp 211ndash217 2005
18 BioMed Research International
[50] N Babaei N A P Abdullah G Saleh and T L AbdullahldquoIsolation and characterization of microsatellite markers andanalysis of genetic variability in Curculigo latifolia DryandrdquoMolecular Biology Reports vol 39 pp 9869ndash9877 2012
[51] B Shiran N Amirbakhtiar S Kiani S Mohammadi B ESayed-Tabatabaei and H Moradi ldquoMolecular characterizationand genetic relationship among almond cultivars assessed byRAPD and SSR markersrdquo Scientia Horticulturae vol 111 no 3pp 280ndash292 2007
[52] S Singh M K Panda and S Nayak ldquoEvaluation of geneticdiversity in turmeric (Curcuma longa L) using RAPD and ISSRmarkersrdquo Industrial Crops and Products vol 37 no 1 pp 284ndash291 2012
[53] Y Paisooksantivatana S Kako and H Seko ldquoGenetic diversityof Curcuma alismatifolia Gagnep (Zingiberaceae) in Thailandas revealed by allozyme polymorphismrdquo Genetic Resources andCrop Evolution vol 48 no 5 pp 459ndash465 2001
Table 8 Effect of acute gamma rays on rhizome characteristics ofC alismatifolia in M1V1 generation
Dose (Gray) Rhizomesize
No of newrhizomes
No of storageroot
CMR0 23 plusmn 02
a26 plusmn 055
a46 plusmn 054
a
10 18 plusmn 005a12 plusmn 044
b40 plusmn 07
a
20 08 plusmn 07b
06 plusmn 054b
14 plusmn 13b
DT0 22 plusmn 01
a30 plusmn 00
a72 plusmn 109
a
10 18 plusmn 007b14 plusmn 054
b46 plusmn 13
b
20 16 plusmn 005b
10 plusmn 00b
28 plusmn 13c
SP0 22 plusmn 02
a24 plusmn 054
a78 plusmn 21
a
10 17 plusmn 007b
10 plusmn 00b
48 plusmn 044b
20 12 plusmn 02c
10 plusmn 00b
28 plusmn 083b
KP0 21 plusmn 005
a10 plusmn 00
a40 plusmn 00
a
10 16 plusmn 01ab
10 plusmn 00a
28 plusmn 044b
20 11 plusmn 06b
10 plusmn 00a
22 plusmn 083b
CV () 18 23 26Means with the same or common letter are not significantly different leastsquare difference test 119875 lt 005
higher than of studies on olive for morphological traits (119903 =069) [49] In this dendrogram the 44C alismatifolia samplesappeared to form three main clusters and five minor clustersat coefficient level 56 cluster I included three non-irradiatedmembers and cluster II included 10Gy irradiated individualplants of all four varieties and 20Gy irradiated samples ofDoi Tung 554 and Kimono Pink varieties except KP20-4 (noflowering) Main cluster III included the 20Gy irradiatedindividuals Chiang Mai Red and Sweet Pink varieties (asmentioned in morphological part these individuals didnot go to flowering stage) and KP20-4 Cluster II can bedivided to five subclusters The first subcluster included 10members (CMR10-1 CMR10-5 SP10-1 SP10-2 SP10-3 SP10-5 CMR10-2 CMR10-4 SP10-4 and CMR10-3) the secondone included nine members (KP10-1 KP10-2 KP10-3 KP10-4 KP10-5KP20-1 KP20-2 KP20-5 and KP20-3) KP0 wasthe lone member of the third sub-cluster The fourth sub-cluster included two members only (DT10-2 and DT10-3) The last sub-cluster main cluster II had eight membersincluded (DT10-1 DT10-4 DT10-5 DT20-1 DT20-2 DT20-3 DT20-4 and DT20-5) Mean value of groups for each traitis presented in Table 9 This table clearly shows the differentmean values of the three main clusters The minimum meanvalue referred to main cluster III while the maximum meanvalue for number of shoots leaf length leaf width plantheight number of true flowers inflorescence length rhizomesize number of new rhizomes and number of storage rootsbelonged to cluster I which was included non-treated plantsThese results showed that the gamma irradiation has inducedmorphological changes in C alismatifolia individuals of
four studied varieties so that they showed phenotypicallydifferences from their controls
57 Principal Component Analysis of C alismatifolia forMorphological Traits To assess the patterns of variation PCAwas done by considering all of the 14 variables The first threecomponents of PCA explained 828 of the total variation(Table 10) Only the first component which accounted for562 of the total variation was attributed to inflorescencelength plant height days to senescence number of trueflowers and rhizome size In PCA three-dimensional graphthe grouping of individuals confirmed the clustering results(Figure 4) The PCA graph proved that all irradiated indi-vidual plants of C alismatifolia varieties are phenotypicallydifferent from their non-irradiated individuals
6 Molecular Characterization
61 SSR Polymorphism Amplifications were successful forall the 17 SSR markers assayed This reflects a high levelof homology between SSR flanking regions in C longa andC alismatifolia Out of 17 primer pairs eight primer pairsresulted in polymorphic PCR products Table 11 summarizesthe results obtained based on the analysis of individuals of thefour studied varieties using the polymorphic SSR loci for 010 and 20Gy irradiated plants The number of alleles variedwidely among these loci A total of 25 36 and 41 alleles wereobserved among 0 10 and 20Gy irradiated individual plantsrespectively In the 10Gy treatment the number of allelesranged from three (clon09 and clon14) to seven (clon08) withan average value of 45 per locus In the 20Gy treatmentthe number of alleles varied from three (clon09 and clon14)to seven (clon08 and clon12) with an average value of 51per locus In the untreated individuals the number of allelesranged from two (clon04 clon09 clon11 and clon14) to five(clon12) with an average value of 31 The difference betweenthe average number of observed alleles and effective numberof alleles was due to the uneven frequency of each allele[50] For each of the eight SSR primers PIC values (whichmeasures allele diversity and frequency among varieties)varied from 019 (clon04) to 071 (clon01) in untreated plantsand from 025 (clon04) to 075 (clon08) in 10Gy acutelyirradiated plants In 20Gy irradiated individual plants thePIC value ranged from 042 (clon14) to 075 (clon08 andclon12) The mean PIC for all loci was 047 054 and 061for 0 10 and 20Gy irradiated plants respectively
The PIC value provides an estimate of the discriminatorypower of amarker by taking into account not only the numberof alleles at a locus but also the relative frequencies of thesealleles [51] Thus except for clon09 and clon14 which weremoderately polymorphic (025 lt PIC lt 05) all other lociwere highly polymorphic (05 lt PIC) while none of theloci showed low polymorphism The average discriminatingpower ofmicrosatellitemarkers (061) observed in the presentstudy ensures the future utility of microsatellite markersfor genetic variation studies in C alismatifolia varietiesOur results reflect similar findings as reported earlier inthe study of genetic variation in other species of Curcuma
12 BioMed Research International
Table9Clusterm
eans
for14charactersestim
ated
in44
individu
also
fCalismatifolia
Clusters
Shoo
tnu
mber
Leaf
leng
thLeaf
width
Leaf
number
Visib
lebu
d(days)
Inflo
rescence
leng
thPlant
height
Anthesis
(days)
Senescence
(days)
True
flow
number
Pink
bract
number
Rhizom
esiz
eNew
rhizom
enu
mber
Storage
root
number
I220
5464
736
300
6013
1473
9233
6926
2093
152
1453
229
266
653
II1
100
2735
512
360
9260
809
5457
1059
960
620
510
177
110
440
II2
122
1922
298
344
11422
1043
3910
1312
293
3755
588
156
100
266
II3
100
3452
416
460
8520
1370
5910
9420
180
120
960
206
100
400
II4
150
2500
45
250
6500
925
4950
7700
1150
950
1700
185
200
600
II5
137
2637
417
262
7725
840
5356
9162
1137
1012
1587
170
100
312
III
054
1500
465
181
000
000
000
000
000
000
000
090
081
200
BioMed Research International 13
Table 10 Eigenvectors eigenvalues and proportions of variabilityfor three principle components among 14 characters for 44 Calismatifolia
Variable PC1 PC2 PC3Number of new shoot 0270 minus0076 minus0173Leaf length 0269 minus0305 0033Leaf width 0096 minus0455 0430Leaf number 0210 0285 0490Days to visible bud 0199 0462 0181Plant height 0321 0027 minus0071Inflorescence length 0318 0166 minus0040Days to anthesis 0205 0460 0148Days to senescence 0342 0017 minus0131Number of true flower 0323 0029 minus0238Number of pink bracts 0266 minus0028 minus0546Rhizome size 0302 minus0101 0147Number of new rhizome 0250 minus0293 0028Number of storage roots 0264 minus0247 0298Eigenvalue 7877 2694 1032Proportion 56264 19246 7371Cumulative 56264 75511 82882
using SSR markers [13] The highest Neirsquos gene diversity(ℎ) was obtained with the 20Gy treatment (061) followedby the 10Gy treatment (06) and the untreated individuals(05) The mean Shannonrsquos information index (119868) was 092114 and 130 in the 0 10 and 20Gy irradiated plantsrespectively The high value of Shannonrsquos information indexrepresents the effectiveness of microsatellite loci to revealthe variation among these varieties at the different dosesused Overall genetic variability for the varieties studiedrepresented by the Shannon-Weiner index was relatively highin comparison to other studies involving C longa accessions[10 52] Additionally the 20Gy acutely irradiated individualsshowed a higher mean percentage of polymorphic loci (63)than the 10Gy (58) and untreated (022) ones This impliesthat irradiation with a dose of 20Gy induced more geneticvariation in the M
1V1generation
62 Cluster Analysis Thedendrogramwas constructed usingPowerMarker 323The 44 studied individuals were clusteredinto seven groups (G1ndashG7) Colors were applied accordingto our model-based cluster analysis results Group I iscomprised of nine individual plants of Chiang Mai Redvariety receiving 0 10 and 20Gy treatments (blue colorin Figure 5) One individual plant DT10-1 was assignedto group II (green color in Figure 5) All irradiated andnon-irradiated individuals of Doi Tung 554 variety wereassigned to group III (yellow color in Figure 5) DT20-1 andSP10-5 were the sole members of groups IV (red color inFigure 5) and V (gray color in Figure 5) respectively GroupVI included treated and untreated individuals of Sweet Pinkvariety (pink color in Figure 5) Lastly all Kimono Pinkindividuals were assigned to cluster VII (orange color in
Figure 5) The genetic similarity coefficient (data not shown)among the 44 individuals amplified using eight SSR primersvaried from 00 to 10 The highest value (10) correspondedto [(CMR10-3 CMR10-5 CMR and CMR20-2) (DT10-2and DT10-3) (DT and DT20-2) (KP20-3 KP-20-4 KP10-3andKP10-4)] individuals that generated identical fingerprintsacross the markers studied Among the studied varieties theSweet Pink individual plants showed the lowest similarity(00) withKimono Pink individuals which indicated that theywere relatively remote in relationship (Figure 5) An overviewof the clustering pattern indicates that the grouping of thestudied individual plants was based on different varieties andlargely independent of the doses of gamma irradiation Awide range in similarity values had also been observed indifferent species of Curcuma [12 13 52 53]
63 Principle Component Analysis (PCA) Thedata generatedfrom 44 C alismatifolia individual plants were subjected toprincipal component analysis (PCA) to visualize individualsin a multivariate space In the three-dimensional graphderived from the SSR analysis all studied individuals weregrouped into seven clusters (Figure 6) In PCA the firstthree principal components (PC) extracted a cumulative of6826 of the total variation among the 44 individuals of CalismatifoliaThe first three coordinates PC1 PC2 and PC3accounted for 4136 1578 1112 of the total variationrespectively The distribution of the individual plants in athree-dimensional plot using the first three principal com-ponents showed the genetic relationship between individualplants It was evident that both methods dendrogram andthree-dimensional plots of PCA were effective in illustratinggenetic relationships and the groups found were comparable[11 13] The PCA results were similar to those obtained fromcluster analysis where all individuals from different varietieswere assigned to different groups These graphical illus-trations enable the assessment of the relationshipdistancesamong all of the individuals in the study [50]
7 Conclusion
This is the first attempt to evaluate the effect of acutegamma irradiation on C alismatifolia varieties using bothmorphological characteristics and molecular markers Inplant breeding programs mutagenic treatments with lownegative physiological effects and strong genetic effects aredesirable Hence we used more effective doses of gammairradiation (10 and 20Gy) of which particularly 20Gy dosewas effective to influence morphological and molecularcharacteristics of studiedCurcuma alismatifolia varietiesThelower dose (10Gy) of radiation probably caused little damageto the plants genetic material so that the cells could repairthemselves in next generation As a result in this study 20Gyacute gamma irradiation resulted in a higher percentage ofmutation and getting desired mutants was more possibleBased on the LD
50values determined in this study it was
apparent that the varieties Chiang Mai Red and Sweet Pinkwere more sensitive to gamma rays compared to Doi Tung554 and Kimono Pink Our results show that the variety Doi
Figure 4 Two-dimensional graph of principal component analysis (PCA) for 14 morphological variables indicating relationships amongirradiated and nonirradiated four varieties of C alismatifolia
CMR10-1CMR10-5
SP10-1SP10-2SP10-3
SP10-5
CMR10-4
SP10-4
KP10-1KP10-3
KP20-1
KP20-2
KP10-5KP20-5
KP20-3
KP10-2
DT10-2
DT10-1
DT10-5
DT20-1
DT10-4DT20-4DT20-2
DT20-5DT20-3
CMR20-1
SP20-3SP20-4
SP20-5SP20-1
SP20-2
KP
SP
CMR 10-2
SP10-5
Figure 5 Unrooted neighbor joining tree showing genetic relationship among 44 irradiated and nonirradiated C alismatifolia using SSRmarkers The colors of the branches correspond to those of the same cluster
Figure 6Three-dimensional graph of principal component analysis (PCA) indicating relationships among irradiated and nonirradiated fourvarieties of C alismatifolia
Tung 554 (Curcuma hybrid) had the most morphologicalresponses to gamma rays The use of microsatellite markersas a codominant marker will facilitate the exploration ofgenetic variability among treated and non-treated plants andwill help to distinguish the plants showing differences inmorphological characters The overall effects on the M
1V1
generation revealed that acute gamma irradiation at optimumdoses has the potential for developing new varieties of Calismatifolia with improved commercial properties suitablefor the Malaysian flower industry
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work was funded by the Fundamental Research GrantScheme (FRGS) under the Ministry of Higher Education inMalaysia
References
[1] P Apavatjrut S Anuntalabhochai P Sirirugsa and C AlisildquoMolecularmarkers in the identification of some early floweringCurcuma L (Zingiberaceae) speciesrdquo Annals of Botany vol 84no 4 pp 529ndash534 1999
[2] K Bunya-Atichart S Ketsa andW G VanDoorn ldquoPostharvestphysiology of Curcuma alismatifolia flowersrdquo Postharvest Biol-ogy and Technology vol 34 no 2 pp 219ndash226 2004
[3] M Nakano J Amano Y Watanabe et al ldquoMorphologicalvariation in Tricyrtis hirta plants regenerated from heavy ionbeam-irradiated embryogenic callusesrdquo Plant Biotechnologyvol 27 no 2 pp 155ndash160 2010
[4] S Lamseejan P Jompuk A Wongpiyasatid A Deeseepan andP Kwanthammachart ldquoGamma-rays induced morphological
[5] G-J Lee S J Chung I S Park et al ldquoVariation in thephenotypic features and transcripts of colormutants of chrysan-themum (Dendranthema grandiflorum) derived from gammaraymutagenesisrdquo Journal of Plant Biology vol 51 no 6 pp 418ndash423 2008
[6] O K Kikuchi ldquoOrchid flowers tolerance to gamma-radiationrdquoRadiation Physics and Chemistry vol 57 no 3ndash6 pp 555ndash5572000
[7] N P Arnold N N Barthakur and M Tanguay ldquoMutageniceffects of acute gamma irradiation on miniature roses targettheory approachrdquo HortScience vol 33 no 1 pp 127ndash129 1998
[8] A A Youssef M S Aly and M S Hussein ldquoResponse ofgeranium (Pelargonium graveolenus L) to gamma irradiationand foliar application of Speed Growrdquo Egyptian Journal ofHorticulture vol 27 pp 41ndash53 2000
[9] N Chuantang and L Yazhi ldquoThe radiation inducedmutation ofcanna (Canna L)rdquo Acta Agricultute Nucleatae Sinica vol 2 pp33ndash39 1998
[10] M S Sigrist J B Pinheiro J A A Filho and M I ZucchildquoGenetic diversity of turmeric germplasm (Curcuma longaZingiberaceae) identified by microsatellite markersrdquo Geneticsand Molecular Research vol 10 no 1 pp 419ndash428 2011
[11] S Taheri T L Abdullah N A P Abdullah and Z AhmadldquoGenetic relationships among five varieties of Curcuma alis-matifolia (Zingiberaceae) based on ISSRmarkersrdquoGenetics andMolecular Research vol 11 pp 3069ndash3076 2012
[12] A Das V Kesari V M Satyanarayana A Parida and L Ran-gan ldquoGenetic relationship of Curcuma species from NortheastIndia using PCR-based markersrdquo Molecular Biotechnology vol49 no 1 pp 65ndash76 2011
[13] S Siju K Dhanya S Syamkumar et al ldquoDevelopment char-acterization and utilization of genomic microsatellite markersin turmeric (Curcuma longa L)rdquo Biochemical Systematics andEcology vol 38 no 4 pp 641ndash646 2010
[14] M K Panda S Mohanty E Subudhi L Acharya and SNayak ldquoAssessment of genetic stability of micropropagated
BioMed Research International 17
plants of Curcuma L by cytophotometery and RAPD analysisrdquoInternational Journal of Integrative Biology (IJIB) vol 1 pp 189ndash195 2007
[15] S Syamkumar and B Sasikumar ldquoMolecular marker basedgenetic diversity analysis of Curcuma species from IndiardquoScientia Horticulturae vol 112 no 2 pp 235ndash241 2007
[16] D Tautz and M Renz ldquoSimple sequences are ubiquitousrepetitive components of eukaryotic genomesrdquo Nucleic AcidsResearch vol 12 no 10 pp 4127ndash4138 1984
[17] G Toth Z Gaspari and J Jurka ldquoMicrosatellites in differenteukaryotic genomes surveys and analysisrdquo Genome Researchvol 10 no 7 pp 967ndash981 2000
[18] W Powell G C Machray and J Proven ldquoPolymorphismrevealed by simple sequence repeatsrdquo Trends in Plant Sciencevol 1 no 7 pp 215ndash222 1996
[19] S-Y Lee W K Fai M Zakaria et al ldquoCharacterizationof polymorphic microsatellite markers isolated from ginger(Zingiber officinale Rosc)rdquo Molecular Ecology Notes vol 7 no6 pp 1009ndash1011 2007
[20] S Bory D Da Silva A-M Risterucci M Grisoni P Besseand M-F Duval ldquoDevelopment of microsatellite markers incultivated vanilla polymorphism and transferability to othervanilla speciesrdquo Scientia Horticulturae vol 115 no 4 pp 420ndash425 2008
[21] I C Menezes F W Cidade A P Souza and I C SampaioldquoIsolation and characterization ofmicrosatellite loci in the blackpepper Piper Nigrum L (Piperaceae)rdquo Conservation GeneticsResources vol 1 no 1 pp 209ndash212 2009
[22] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[23] V Kumar Morphological and molecular characterization ofinduced mutants in Groundnut [PhD thesis] University ofAgricultural Sciences Dharwad India 2008
[24] J R Sharma Statistical and Biometrical Techniques in PlantBreeding New Age International New Delhi India 1998
[25] J J Doyle and J L Doyle ldquoA rapid DNA isolation procedure forsmall quantities of fresh leaf tissuerdquo Phytochemical Bulletin vol19 pp 11ndash15 1987
[26] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[27] Oslash Hammer D A T Harper and P D Ryan ldquoPast paleontolog-ical statistics software package for education and data analysisrdquoPalaeontologia Electronica vol 4 no 1 pp 4ndash9 2001
[28] K Liu and S V Muse ldquoPowerMaker an integrated analysisenvironment for genetic maker analysisrdquo Bioinformatics vol 21no 9 pp 2128ndash2129 2005
[29] F J RohlfNTSYS-Pc Numerical Taxonomy System Version 2 1Exeter Publishing Setauket New York NY USA 2002
[30] M Nei ldquoAnalysis of gene diversity in subdivided populationsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 70 no 12 1973
[31] S Kadkhodaei M Shahnazari M K Nekouei et al ldquoA compar-ative study of morphological and molecular diversity analysisamong cultivated almonds (Prunus dulcis)rdquo Australian Journalof Crop Science vol 5 no 1 pp 82ndash91 2011
[32] T L Abdullah J Endan and B M Nazir ldquoChanges in flowerdevelopment chlorophyll mutation and alteration in plant
morphology of Curcuma alismatifolia by gamma irradiationrdquoAmerican Journal of Applied Sciences vol 6 no 7 pp 1436ndash14392009
[33] E Kovacs and A Keresztes ldquoEffect of gamma and UV-BCradiation on plant cellsrdquoMicron vol 33 no 2 pp 199ndash210 2002
[34] H L Ramesh Y Murthy and V N Munirajappa ldquoEffect ofgamma radiation on morphological and growth parametersof Mulberry variety M5rdquo International Journal of Science andNature vol 3 pp 447ndash452 2010
[35] K Pongchawee R Pradissan andW Pipatcharoenchai ldquoInducemutation in Anubias spp through in vitro Irradiationrdquo ThaiFisheries Gazette vol 60 pp 493ndash497 2007
[36] P Tangpong T Taychasinpitak C Jompuk and P JompukldquoEffects of acute and chronic gamma irradiations on in vitroculture ofAnubias congensisNE BrownrdquoKasetsart Journal vol43 no 3 pp 449ndash457 2009
[37] R K Hegde Studies on induced mutagenesis and in vitroregeneration in turmeric (Curcuma longa L) [PhD thesis]University of Agricultural Sciences Dharwad India 2006
[38] M P Giridharan and S Balakrishnan ldquoGamma ray inducedvariability in vegetative and floral characters of ginger IndianCocoardquo Arecanut and Spices Journal vol 15 pp 68ndash672 1992
[39] A P Cesarett ldquoEffect of radiation on higher plants and plantcommunitiesrdquo inRadiation Biology United States Atomic EnergyCommission pp 284ndash309 Washington DC USA 1968
[40] G W Seung Y C Byung J-H Kim et al ldquoUltrastructuralchanges of cell organelles in Arabidopsis stems after gammairradationrdquo Journal of Plant Biology vol 48 no 2 pp 195ndash2002005
[41] B S Ahloowalia and M Maluszynski ldquoInduced mutationsmdashanew paradigm in plant breedingrdquo Euphytica vol 118 no 2 pp167ndash173 2001
[42] R Voisine L-P Vezina and C Willemot ldquoInduction ofsenescence-like deterioration of microsomal membranes fromcauliflower by free radicals generated during gamma irradia-tionrdquo Plant Physiology vol 97 no 2 pp 545ndash550 1991
[43] N Shikazono Y Yokota S Kitamura et al ldquoMutation rate andnovel tt mutants of Arabidopsis thaliana induced by carbonionsrdquo Genetics vol 163 no 4 pp 1449ndash1455 2003
[44] T Nakatsuka M Nishihara K Mishiba and S YamamuraldquoTwo different mutations are involved in the formation ofwhite-flowered gentian plantsrdquo Plant Science vol 169 no 5 pp949ndash958 2005
[45] M Mato T Onozaki Y Ozeki et al ldquoFlavonoid biosynthesisin white-flowered Sim carnations (Dianthus caryophyllus)rdquoScientia Horticulturae vol 84 no 3-4 pp 333ndash347 2000
[46] S G Wi B Y Chung J-S Kim et al ldquoEffects of gammairradiation on morphological changes and biological responsesin plantsrdquoMicron vol 38 no 6 pp 553ndash564 2007
[47] F Lee Chin Effects of light intensity and daylength on growth andflowering of siam tulip (Curcuma alismatifolia var Chiang MaiPink) [PhD thesis] University Putra Malaysia 2007
[48] A Hagiladi N Umiel and X H Yang ldquoCurcuma alismatifoliaII Effects of temperature and daylength on the development offlowers and propagulesrdquo Acta Horticulture vol 430 pp 755ndash761 1997
[49] MHagidimitriou A Katsiotis GMenexes C Pontikis andMLoukas ldquoGenetic diversity of major greek olive cultivars usingmolecular (AFLPs and RAPDs) markers and morphologicaltraitsrdquo Journal of the American Society for Horticultural Sciencevol 130 no 2 pp 211ndash217 2005
18 BioMed Research International
[50] N Babaei N A P Abdullah G Saleh and T L AbdullahldquoIsolation and characterization of microsatellite markers andanalysis of genetic variability in Curculigo latifolia DryandrdquoMolecular Biology Reports vol 39 pp 9869ndash9877 2012
[51] B Shiran N Amirbakhtiar S Kiani S Mohammadi B ESayed-Tabatabaei and H Moradi ldquoMolecular characterizationand genetic relationship among almond cultivars assessed byRAPD and SSR markersrdquo Scientia Horticulturae vol 111 no 3pp 280ndash292 2007
[52] S Singh M K Panda and S Nayak ldquoEvaluation of geneticdiversity in turmeric (Curcuma longa L) using RAPD and ISSRmarkersrdquo Industrial Crops and Products vol 37 no 1 pp 284ndash291 2012
[53] Y Paisooksantivatana S Kako and H Seko ldquoGenetic diversityof Curcuma alismatifolia Gagnep (Zingiberaceae) in Thailandas revealed by allozyme polymorphismrdquo Genetic Resources andCrop Evolution vol 48 no 5 pp 459ndash465 2001
Table 10 Eigenvectors eigenvalues and proportions of variabilityfor three principle components among 14 characters for 44 Calismatifolia
Variable PC1 PC2 PC3Number of new shoot 0270 minus0076 minus0173Leaf length 0269 minus0305 0033Leaf width 0096 minus0455 0430Leaf number 0210 0285 0490Days to visible bud 0199 0462 0181Plant height 0321 0027 minus0071Inflorescence length 0318 0166 minus0040Days to anthesis 0205 0460 0148Days to senescence 0342 0017 minus0131Number of true flower 0323 0029 minus0238Number of pink bracts 0266 minus0028 minus0546Rhizome size 0302 minus0101 0147Number of new rhizome 0250 minus0293 0028Number of storage roots 0264 minus0247 0298Eigenvalue 7877 2694 1032Proportion 56264 19246 7371Cumulative 56264 75511 82882
using SSR markers [13] The highest Neirsquos gene diversity(ℎ) was obtained with the 20Gy treatment (061) followedby the 10Gy treatment (06) and the untreated individuals(05) The mean Shannonrsquos information index (119868) was 092114 and 130 in the 0 10 and 20Gy irradiated plantsrespectively The high value of Shannonrsquos information indexrepresents the effectiveness of microsatellite loci to revealthe variation among these varieties at the different dosesused Overall genetic variability for the varieties studiedrepresented by the Shannon-Weiner index was relatively highin comparison to other studies involving C longa accessions[10 52] Additionally the 20Gy acutely irradiated individualsshowed a higher mean percentage of polymorphic loci (63)than the 10Gy (58) and untreated (022) ones This impliesthat irradiation with a dose of 20Gy induced more geneticvariation in the M
1V1generation
62 Cluster Analysis Thedendrogramwas constructed usingPowerMarker 323The 44 studied individuals were clusteredinto seven groups (G1ndashG7) Colors were applied accordingto our model-based cluster analysis results Group I iscomprised of nine individual plants of Chiang Mai Redvariety receiving 0 10 and 20Gy treatments (blue colorin Figure 5) One individual plant DT10-1 was assignedto group II (green color in Figure 5) All irradiated andnon-irradiated individuals of Doi Tung 554 variety wereassigned to group III (yellow color in Figure 5) DT20-1 andSP10-5 were the sole members of groups IV (red color inFigure 5) and V (gray color in Figure 5) respectively GroupVI included treated and untreated individuals of Sweet Pinkvariety (pink color in Figure 5) Lastly all Kimono Pinkindividuals were assigned to cluster VII (orange color in
Figure 5) The genetic similarity coefficient (data not shown)among the 44 individuals amplified using eight SSR primersvaried from 00 to 10 The highest value (10) correspondedto [(CMR10-3 CMR10-5 CMR and CMR20-2) (DT10-2and DT10-3) (DT and DT20-2) (KP20-3 KP-20-4 KP10-3andKP10-4)] individuals that generated identical fingerprintsacross the markers studied Among the studied varieties theSweet Pink individual plants showed the lowest similarity(00) withKimono Pink individuals which indicated that theywere relatively remote in relationship (Figure 5) An overviewof the clustering pattern indicates that the grouping of thestudied individual plants was based on different varieties andlargely independent of the doses of gamma irradiation Awide range in similarity values had also been observed indifferent species of Curcuma [12 13 52 53]
63 Principle Component Analysis (PCA) Thedata generatedfrom 44 C alismatifolia individual plants were subjected toprincipal component analysis (PCA) to visualize individualsin a multivariate space In the three-dimensional graphderived from the SSR analysis all studied individuals weregrouped into seven clusters (Figure 6) In PCA the firstthree principal components (PC) extracted a cumulative of6826 of the total variation among the 44 individuals of CalismatifoliaThe first three coordinates PC1 PC2 and PC3accounted for 4136 1578 1112 of the total variationrespectively The distribution of the individual plants in athree-dimensional plot using the first three principal com-ponents showed the genetic relationship between individualplants It was evident that both methods dendrogram andthree-dimensional plots of PCA were effective in illustratinggenetic relationships and the groups found were comparable[11 13] The PCA results were similar to those obtained fromcluster analysis where all individuals from different varietieswere assigned to different groups These graphical illus-trations enable the assessment of the relationshipdistancesamong all of the individuals in the study [50]
7 Conclusion
This is the first attempt to evaluate the effect of acutegamma irradiation on C alismatifolia varieties using bothmorphological characteristics and molecular markers Inplant breeding programs mutagenic treatments with lownegative physiological effects and strong genetic effects aredesirable Hence we used more effective doses of gammairradiation (10 and 20Gy) of which particularly 20Gy dosewas effective to influence morphological and molecularcharacteristics of studiedCurcuma alismatifolia varietiesThelower dose (10Gy) of radiation probably caused little damageto the plants genetic material so that the cells could repairthemselves in next generation As a result in this study 20Gyacute gamma irradiation resulted in a higher percentage ofmutation and getting desired mutants was more possibleBased on the LD
50values determined in this study it was
apparent that the varieties Chiang Mai Red and Sweet Pinkwere more sensitive to gamma rays compared to Doi Tung554 and Kimono Pink Our results show that the variety Doi
Figure 4 Two-dimensional graph of principal component analysis (PCA) for 14 morphological variables indicating relationships amongirradiated and nonirradiated four varieties of C alismatifolia
CMR10-1CMR10-5
SP10-1SP10-2SP10-3
SP10-5
CMR10-4
SP10-4
KP10-1KP10-3
KP20-1
KP20-2
KP10-5KP20-5
KP20-3
KP10-2
DT10-2
DT10-1
DT10-5
DT20-1
DT10-4DT20-4DT20-2
DT20-5DT20-3
CMR20-1
SP20-3SP20-4
SP20-5SP20-1
SP20-2
KP
SP
CMR 10-2
SP10-5
Figure 5 Unrooted neighbor joining tree showing genetic relationship among 44 irradiated and nonirradiated C alismatifolia using SSRmarkers The colors of the branches correspond to those of the same cluster
Figure 6Three-dimensional graph of principal component analysis (PCA) indicating relationships among irradiated and nonirradiated fourvarieties of C alismatifolia
Tung 554 (Curcuma hybrid) had the most morphologicalresponses to gamma rays The use of microsatellite markersas a codominant marker will facilitate the exploration ofgenetic variability among treated and non-treated plants andwill help to distinguish the plants showing differences inmorphological characters The overall effects on the M
1V1
generation revealed that acute gamma irradiation at optimumdoses has the potential for developing new varieties of Calismatifolia with improved commercial properties suitablefor the Malaysian flower industry
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work was funded by the Fundamental Research GrantScheme (FRGS) under the Ministry of Higher Education inMalaysia
References
[1] P Apavatjrut S Anuntalabhochai P Sirirugsa and C AlisildquoMolecularmarkers in the identification of some early floweringCurcuma L (Zingiberaceae) speciesrdquo Annals of Botany vol 84no 4 pp 529ndash534 1999
[2] K Bunya-Atichart S Ketsa andW G VanDoorn ldquoPostharvestphysiology of Curcuma alismatifolia flowersrdquo Postharvest Biol-ogy and Technology vol 34 no 2 pp 219ndash226 2004
[3] M Nakano J Amano Y Watanabe et al ldquoMorphologicalvariation in Tricyrtis hirta plants regenerated from heavy ionbeam-irradiated embryogenic callusesrdquo Plant Biotechnologyvol 27 no 2 pp 155ndash160 2010
[4] S Lamseejan P Jompuk A Wongpiyasatid A Deeseepan andP Kwanthammachart ldquoGamma-rays induced morphological
[5] G-J Lee S J Chung I S Park et al ldquoVariation in thephenotypic features and transcripts of colormutants of chrysan-themum (Dendranthema grandiflorum) derived from gammaraymutagenesisrdquo Journal of Plant Biology vol 51 no 6 pp 418ndash423 2008
[6] O K Kikuchi ldquoOrchid flowers tolerance to gamma-radiationrdquoRadiation Physics and Chemistry vol 57 no 3ndash6 pp 555ndash5572000
[7] N P Arnold N N Barthakur and M Tanguay ldquoMutageniceffects of acute gamma irradiation on miniature roses targettheory approachrdquo HortScience vol 33 no 1 pp 127ndash129 1998
[8] A A Youssef M S Aly and M S Hussein ldquoResponse ofgeranium (Pelargonium graveolenus L) to gamma irradiationand foliar application of Speed Growrdquo Egyptian Journal ofHorticulture vol 27 pp 41ndash53 2000
[9] N Chuantang and L Yazhi ldquoThe radiation inducedmutation ofcanna (Canna L)rdquo Acta Agricultute Nucleatae Sinica vol 2 pp33ndash39 1998
[10] M S Sigrist J B Pinheiro J A A Filho and M I ZucchildquoGenetic diversity of turmeric germplasm (Curcuma longaZingiberaceae) identified by microsatellite markersrdquo Geneticsand Molecular Research vol 10 no 1 pp 419ndash428 2011
[11] S Taheri T L Abdullah N A P Abdullah and Z AhmadldquoGenetic relationships among five varieties of Curcuma alis-matifolia (Zingiberaceae) based on ISSRmarkersrdquoGenetics andMolecular Research vol 11 pp 3069ndash3076 2012
[12] A Das V Kesari V M Satyanarayana A Parida and L Ran-gan ldquoGenetic relationship of Curcuma species from NortheastIndia using PCR-based markersrdquo Molecular Biotechnology vol49 no 1 pp 65ndash76 2011
[13] S Siju K Dhanya S Syamkumar et al ldquoDevelopment char-acterization and utilization of genomic microsatellite markersin turmeric (Curcuma longa L)rdquo Biochemical Systematics andEcology vol 38 no 4 pp 641ndash646 2010
[14] M K Panda S Mohanty E Subudhi L Acharya and SNayak ldquoAssessment of genetic stability of micropropagated
BioMed Research International 17
plants of Curcuma L by cytophotometery and RAPD analysisrdquoInternational Journal of Integrative Biology (IJIB) vol 1 pp 189ndash195 2007
[15] S Syamkumar and B Sasikumar ldquoMolecular marker basedgenetic diversity analysis of Curcuma species from IndiardquoScientia Horticulturae vol 112 no 2 pp 235ndash241 2007
[16] D Tautz and M Renz ldquoSimple sequences are ubiquitousrepetitive components of eukaryotic genomesrdquo Nucleic AcidsResearch vol 12 no 10 pp 4127ndash4138 1984
[17] G Toth Z Gaspari and J Jurka ldquoMicrosatellites in differenteukaryotic genomes surveys and analysisrdquo Genome Researchvol 10 no 7 pp 967ndash981 2000
[18] W Powell G C Machray and J Proven ldquoPolymorphismrevealed by simple sequence repeatsrdquo Trends in Plant Sciencevol 1 no 7 pp 215ndash222 1996
[19] S-Y Lee W K Fai M Zakaria et al ldquoCharacterizationof polymorphic microsatellite markers isolated from ginger(Zingiber officinale Rosc)rdquo Molecular Ecology Notes vol 7 no6 pp 1009ndash1011 2007
[20] S Bory D Da Silva A-M Risterucci M Grisoni P Besseand M-F Duval ldquoDevelopment of microsatellite markers incultivated vanilla polymorphism and transferability to othervanilla speciesrdquo Scientia Horticulturae vol 115 no 4 pp 420ndash425 2008
[21] I C Menezes F W Cidade A P Souza and I C SampaioldquoIsolation and characterization ofmicrosatellite loci in the blackpepper Piper Nigrum L (Piperaceae)rdquo Conservation GeneticsResources vol 1 no 1 pp 209ndash212 2009
[22] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[23] V Kumar Morphological and molecular characterization ofinduced mutants in Groundnut [PhD thesis] University ofAgricultural Sciences Dharwad India 2008
[24] J R Sharma Statistical and Biometrical Techniques in PlantBreeding New Age International New Delhi India 1998
[25] J J Doyle and J L Doyle ldquoA rapid DNA isolation procedure forsmall quantities of fresh leaf tissuerdquo Phytochemical Bulletin vol19 pp 11ndash15 1987
[26] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[27] Oslash Hammer D A T Harper and P D Ryan ldquoPast paleontolog-ical statistics software package for education and data analysisrdquoPalaeontologia Electronica vol 4 no 1 pp 4ndash9 2001
[28] K Liu and S V Muse ldquoPowerMaker an integrated analysisenvironment for genetic maker analysisrdquo Bioinformatics vol 21no 9 pp 2128ndash2129 2005
[29] F J RohlfNTSYS-Pc Numerical Taxonomy System Version 2 1Exeter Publishing Setauket New York NY USA 2002
[30] M Nei ldquoAnalysis of gene diversity in subdivided populationsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 70 no 12 1973
[31] S Kadkhodaei M Shahnazari M K Nekouei et al ldquoA compar-ative study of morphological and molecular diversity analysisamong cultivated almonds (Prunus dulcis)rdquo Australian Journalof Crop Science vol 5 no 1 pp 82ndash91 2011
[32] T L Abdullah J Endan and B M Nazir ldquoChanges in flowerdevelopment chlorophyll mutation and alteration in plant
morphology of Curcuma alismatifolia by gamma irradiationrdquoAmerican Journal of Applied Sciences vol 6 no 7 pp 1436ndash14392009
[33] E Kovacs and A Keresztes ldquoEffect of gamma and UV-BCradiation on plant cellsrdquoMicron vol 33 no 2 pp 199ndash210 2002
[34] H L Ramesh Y Murthy and V N Munirajappa ldquoEffect ofgamma radiation on morphological and growth parametersof Mulberry variety M5rdquo International Journal of Science andNature vol 3 pp 447ndash452 2010
[35] K Pongchawee R Pradissan andW Pipatcharoenchai ldquoInducemutation in Anubias spp through in vitro Irradiationrdquo ThaiFisheries Gazette vol 60 pp 493ndash497 2007
[36] P Tangpong T Taychasinpitak C Jompuk and P JompukldquoEffects of acute and chronic gamma irradiations on in vitroculture ofAnubias congensisNE BrownrdquoKasetsart Journal vol43 no 3 pp 449ndash457 2009
[37] R K Hegde Studies on induced mutagenesis and in vitroregeneration in turmeric (Curcuma longa L) [PhD thesis]University of Agricultural Sciences Dharwad India 2006
[38] M P Giridharan and S Balakrishnan ldquoGamma ray inducedvariability in vegetative and floral characters of ginger IndianCocoardquo Arecanut and Spices Journal vol 15 pp 68ndash672 1992
[39] A P Cesarett ldquoEffect of radiation on higher plants and plantcommunitiesrdquo inRadiation Biology United States Atomic EnergyCommission pp 284ndash309 Washington DC USA 1968
[40] G W Seung Y C Byung J-H Kim et al ldquoUltrastructuralchanges of cell organelles in Arabidopsis stems after gammairradationrdquo Journal of Plant Biology vol 48 no 2 pp 195ndash2002005
[41] B S Ahloowalia and M Maluszynski ldquoInduced mutationsmdashanew paradigm in plant breedingrdquo Euphytica vol 118 no 2 pp167ndash173 2001
[42] R Voisine L-P Vezina and C Willemot ldquoInduction ofsenescence-like deterioration of microsomal membranes fromcauliflower by free radicals generated during gamma irradia-tionrdquo Plant Physiology vol 97 no 2 pp 545ndash550 1991
[43] N Shikazono Y Yokota S Kitamura et al ldquoMutation rate andnovel tt mutants of Arabidopsis thaliana induced by carbonionsrdquo Genetics vol 163 no 4 pp 1449ndash1455 2003
[44] T Nakatsuka M Nishihara K Mishiba and S YamamuraldquoTwo different mutations are involved in the formation ofwhite-flowered gentian plantsrdquo Plant Science vol 169 no 5 pp949ndash958 2005
[45] M Mato T Onozaki Y Ozeki et al ldquoFlavonoid biosynthesisin white-flowered Sim carnations (Dianthus caryophyllus)rdquoScientia Horticulturae vol 84 no 3-4 pp 333ndash347 2000
[46] S G Wi B Y Chung J-S Kim et al ldquoEffects of gammairradiation on morphological changes and biological responsesin plantsrdquoMicron vol 38 no 6 pp 553ndash564 2007
[47] F Lee Chin Effects of light intensity and daylength on growth andflowering of siam tulip (Curcuma alismatifolia var Chiang MaiPink) [PhD thesis] University Putra Malaysia 2007
[48] A Hagiladi N Umiel and X H Yang ldquoCurcuma alismatifoliaII Effects of temperature and daylength on the development offlowers and propagulesrdquo Acta Horticulture vol 430 pp 755ndash761 1997
[49] MHagidimitriou A Katsiotis GMenexes C Pontikis andMLoukas ldquoGenetic diversity of major greek olive cultivars usingmolecular (AFLPs and RAPDs) markers and morphologicaltraitsrdquo Journal of the American Society for Horticultural Sciencevol 130 no 2 pp 211ndash217 2005
18 BioMed Research International
[50] N Babaei N A P Abdullah G Saleh and T L AbdullahldquoIsolation and characterization of microsatellite markers andanalysis of genetic variability in Curculigo latifolia DryandrdquoMolecular Biology Reports vol 39 pp 9869ndash9877 2012
[51] B Shiran N Amirbakhtiar S Kiani S Mohammadi B ESayed-Tabatabaei and H Moradi ldquoMolecular characterizationand genetic relationship among almond cultivars assessed byRAPD and SSR markersrdquo Scientia Horticulturae vol 111 no 3pp 280ndash292 2007
[52] S Singh M K Panda and S Nayak ldquoEvaluation of geneticdiversity in turmeric (Curcuma longa L) using RAPD and ISSRmarkersrdquo Industrial Crops and Products vol 37 no 1 pp 284ndash291 2012
[53] Y Paisooksantivatana S Kako and H Seko ldquoGenetic diversityof Curcuma alismatifolia Gagnep (Zingiberaceae) in Thailandas revealed by allozyme polymorphismrdquo Genetic Resources andCrop Evolution vol 48 no 5 pp 459ndash465 2001
Table 10 Eigenvectors eigenvalues and proportions of variabilityfor three principle components among 14 characters for 44 Calismatifolia
Variable PC1 PC2 PC3Number of new shoot 0270 minus0076 minus0173Leaf length 0269 minus0305 0033Leaf width 0096 minus0455 0430Leaf number 0210 0285 0490Days to visible bud 0199 0462 0181Plant height 0321 0027 minus0071Inflorescence length 0318 0166 minus0040Days to anthesis 0205 0460 0148Days to senescence 0342 0017 minus0131Number of true flower 0323 0029 minus0238Number of pink bracts 0266 minus0028 minus0546Rhizome size 0302 minus0101 0147Number of new rhizome 0250 minus0293 0028Number of storage roots 0264 minus0247 0298Eigenvalue 7877 2694 1032Proportion 56264 19246 7371Cumulative 56264 75511 82882
using SSR markers [13] The highest Neirsquos gene diversity(ℎ) was obtained with the 20Gy treatment (061) followedby the 10Gy treatment (06) and the untreated individuals(05) The mean Shannonrsquos information index (119868) was 092114 and 130 in the 0 10 and 20Gy irradiated plantsrespectively The high value of Shannonrsquos information indexrepresents the effectiveness of microsatellite loci to revealthe variation among these varieties at the different dosesused Overall genetic variability for the varieties studiedrepresented by the Shannon-Weiner index was relatively highin comparison to other studies involving C longa accessions[10 52] Additionally the 20Gy acutely irradiated individualsshowed a higher mean percentage of polymorphic loci (63)than the 10Gy (58) and untreated (022) ones This impliesthat irradiation with a dose of 20Gy induced more geneticvariation in the M
1V1generation
62 Cluster Analysis Thedendrogramwas constructed usingPowerMarker 323The 44 studied individuals were clusteredinto seven groups (G1ndashG7) Colors were applied accordingto our model-based cluster analysis results Group I iscomprised of nine individual plants of Chiang Mai Redvariety receiving 0 10 and 20Gy treatments (blue colorin Figure 5) One individual plant DT10-1 was assignedto group II (green color in Figure 5) All irradiated andnon-irradiated individuals of Doi Tung 554 variety wereassigned to group III (yellow color in Figure 5) DT20-1 andSP10-5 were the sole members of groups IV (red color inFigure 5) and V (gray color in Figure 5) respectively GroupVI included treated and untreated individuals of Sweet Pinkvariety (pink color in Figure 5) Lastly all Kimono Pinkindividuals were assigned to cluster VII (orange color in
Figure 5) The genetic similarity coefficient (data not shown)among the 44 individuals amplified using eight SSR primersvaried from 00 to 10 The highest value (10) correspondedto [(CMR10-3 CMR10-5 CMR and CMR20-2) (DT10-2and DT10-3) (DT and DT20-2) (KP20-3 KP-20-4 KP10-3andKP10-4)] individuals that generated identical fingerprintsacross the markers studied Among the studied varieties theSweet Pink individual plants showed the lowest similarity(00) withKimono Pink individuals which indicated that theywere relatively remote in relationship (Figure 5) An overviewof the clustering pattern indicates that the grouping of thestudied individual plants was based on different varieties andlargely independent of the doses of gamma irradiation Awide range in similarity values had also been observed indifferent species of Curcuma [12 13 52 53]
63 Principle Component Analysis (PCA) Thedata generatedfrom 44 C alismatifolia individual plants were subjected toprincipal component analysis (PCA) to visualize individualsin a multivariate space In the three-dimensional graphderived from the SSR analysis all studied individuals weregrouped into seven clusters (Figure 6) In PCA the firstthree principal components (PC) extracted a cumulative of6826 of the total variation among the 44 individuals of CalismatifoliaThe first three coordinates PC1 PC2 and PC3accounted for 4136 1578 1112 of the total variationrespectively The distribution of the individual plants in athree-dimensional plot using the first three principal com-ponents showed the genetic relationship between individualplants It was evident that both methods dendrogram andthree-dimensional plots of PCA were effective in illustratinggenetic relationships and the groups found were comparable[11 13] The PCA results were similar to those obtained fromcluster analysis where all individuals from different varietieswere assigned to different groups These graphical illus-trations enable the assessment of the relationshipdistancesamong all of the individuals in the study [50]
7 Conclusion
This is the first attempt to evaluate the effect of acutegamma irradiation on C alismatifolia varieties using bothmorphological characteristics and molecular markers Inplant breeding programs mutagenic treatments with lownegative physiological effects and strong genetic effects aredesirable Hence we used more effective doses of gammairradiation (10 and 20Gy) of which particularly 20Gy dosewas effective to influence morphological and molecularcharacteristics of studiedCurcuma alismatifolia varietiesThelower dose (10Gy) of radiation probably caused little damageto the plants genetic material so that the cells could repairthemselves in next generation As a result in this study 20Gyacute gamma irradiation resulted in a higher percentage ofmutation and getting desired mutants was more possibleBased on the LD
50values determined in this study it was
apparent that the varieties Chiang Mai Red and Sweet Pinkwere more sensitive to gamma rays compared to Doi Tung554 and Kimono Pink Our results show that the variety Doi
Figure 4 Two-dimensional graph of principal component analysis (PCA) for 14 morphological variables indicating relationships amongirradiated and nonirradiated four varieties of C alismatifolia
CMR10-1CMR10-5
SP10-1SP10-2SP10-3
SP10-5
CMR10-4
SP10-4
KP10-1KP10-3
KP20-1
KP20-2
KP10-5KP20-5
KP20-3
KP10-2
DT10-2
DT10-1
DT10-5
DT20-1
DT10-4DT20-4DT20-2
DT20-5DT20-3
CMR20-1
SP20-3SP20-4
SP20-5SP20-1
SP20-2
KP
SP
CMR 10-2
SP10-5
Figure 5 Unrooted neighbor joining tree showing genetic relationship among 44 irradiated and nonirradiated C alismatifolia using SSRmarkers The colors of the branches correspond to those of the same cluster
Figure 6Three-dimensional graph of principal component analysis (PCA) indicating relationships among irradiated and nonirradiated fourvarieties of C alismatifolia
Tung 554 (Curcuma hybrid) had the most morphologicalresponses to gamma rays The use of microsatellite markersas a codominant marker will facilitate the exploration ofgenetic variability among treated and non-treated plants andwill help to distinguish the plants showing differences inmorphological characters The overall effects on the M
1V1
generation revealed that acute gamma irradiation at optimumdoses has the potential for developing new varieties of Calismatifolia with improved commercial properties suitablefor the Malaysian flower industry
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work was funded by the Fundamental Research GrantScheme (FRGS) under the Ministry of Higher Education inMalaysia
References
[1] P Apavatjrut S Anuntalabhochai P Sirirugsa and C AlisildquoMolecularmarkers in the identification of some early floweringCurcuma L (Zingiberaceae) speciesrdquo Annals of Botany vol 84no 4 pp 529ndash534 1999
[2] K Bunya-Atichart S Ketsa andW G VanDoorn ldquoPostharvestphysiology of Curcuma alismatifolia flowersrdquo Postharvest Biol-ogy and Technology vol 34 no 2 pp 219ndash226 2004
[3] M Nakano J Amano Y Watanabe et al ldquoMorphologicalvariation in Tricyrtis hirta plants regenerated from heavy ionbeam-irradiated embryogenic callusesrdquo Plant Biotechnologyvol 27 no 2 pp 155ndash160 2010
[4] S Lamseejan P Jompuk A Wongpiyasatid A Deeseepan andP Kwanthammachart ldquoGamma-rays induced morphological
[5] G-J Lee S J Chung I S Park et al ldquoVariation in thephenotypic features and transcripts of colormutants of chrysan-themum (Dendranthema grandiflorum) derived from gammaraymutagenesisrdquo Journal of Plant Biology vol 51 no 6 pp 418ndash423 2008
[6] O K Kikuchi ldquoOrchid flowers tolerance to gamma-radiationrdquoRadiation Physics and Chemistry vol 57 no 3ndash6 pp 555ndash5572000
[7] N P Arnold N N Barthakur and M Tanguay ldquoMutageniceffects of acute gamma irradiation on miniature roses targettheory approachrdquo HortScience vol 33 no 1 pp 127ndash129 1998
[8] A A Youssef M S Aly and M S Hussein ldquoResponse ofgeranium (Pelargonium graveolenus L) to gamma irradiationand foliar application of Speed Growrdquo Egyptian Journal ofHorticulture vol 27 pp 41ndash53 2000
[9] N Chuantang and L Yazhi ldquoThe radiation inducedmutation ofcanna (Canna L)rdquo Acta Agricultute Nucleatae Sinica vol 2 pp33ndash39 1998
[10] M S Sigrist J B Pinheiro J A A Filho and M I ZucchildquoGenetic diversity of turmeric germplasm (Curcuma longaZingiberaceae) identified by microsatellite markersrdquo Geneticsand Molecular Research vol 10 no 1 pp 419ndash428 2011
[11] S Taheri T L Abdullah N A P Abdullah and Z AhmadldquoGenetic relationships among five varieties of Curcuma alis-matifolia (Zingiberaceae) based on ISSRmarkersrdquoGenetics andMolecular Research vol 11 pp 3069ndash3076 2012
[12] A Das V Kesari V M Satyanarayana A Parida and L Ran-gan ldquoGenetic relationship of Curcuma species from NortheastIndia using PCR-based markersrdquo Molecular Biotechnology vol49 no 1 pp 65ndash76 2011
[13] S Siju K Dhanya S Syamkumar et al ldquoDevelopment char-acterization and utilization of genomic microsatellite markersin turmeric (Curcuma longa L)rdquo Biochemical Systematics andEcology vol 38 no 4 pp 641ndash646 2010
[14] M K Panda S Mohanty E Subudhi L Acharya and SNayak ldquoAssessment of genetic stability of micropropagated
BioMed Research International 17
plants of Curcuma L by cytophotometery and RAPD analysisrdquoInternational Journal of Integrative Biology (IJIB) vol 1 pp 189ndash195 2007
[15] S Syamkumar and B Sasikumar ldquoMolecular marker basedgenetic diversity analysis of Curcuma species from IndiardquoScientia Horticulturae vol 112 no 2 pp 235ndash241 2007
[16] D Tautz and M Renz ldquoSimple sequences are ubiquitousrepetitive components of eukaryotic genomesrdquo Nucleic AcidsResearch vol 12 no 10 pp 4127ndash4138 1984
[17] G Toth Z Gaspari and J Jurka ldquoMicrosatellites in differenteukaryotic genomes surveys and analysisrdquo Genome Researchvol 10 no 7 pp 967ndash981 2000
[18] W Powell G C Machray and J Proven ldquoPolymorphismrevealed by simple sequence repeatsrdquo Trends in Plant Sciencevol 1 no 7 pp 215ndash222 1996
[19] S-Y Lee W K Fai M Zakaria et al ldquoCharacterizationof polymorphic microsatellite markers isolated from ginger(Zingiber officinale Rosc)rdquo Molecular Ecology Notes vol 7 no6 pp 1009ndash1011 2007
[20] S Bory D Da Silva A-M Risterucci M Grisoni P Besseand M-F Duval ldquoDevelopment of microsatellite markers incultivated vanilla polymorphism and transferability to othervanilla speciesrdquo Scientia Horticulturae vol 115 no 4 pp 420ndash425 2008
[21] I C Menezes F W Cidade A P Souza and I C SampaioldquoIsolation and characterization ofmicrosatellite loci in the blackpepper Piper Nigrum L (Piperaceae)rdquo Conservation GeneticsResources vol 1 no 1 pp 209ndash212 2009
[22] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[23] V Kumar Morphological and molecular characterization ofinduced mutants in Groundnut [PhD thesis] University ofAgricultural Sciences Dharwad India 2008
[24] J R Sharma Statistical and Biometrical Techniques in PlantBreeding New Age International New Delhi India 1998
[25] J J Doyle and J L Doyle ldquoA rapid DNA isolation procedure forsmall quantities of fresh leaf tissuerdquo Phytochemical Bulletin vol19 pp 11ndash15 1987
[26] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[27] Oslash Hammer D A T Harper and P D Ryan ldquoPast paleontolog-ical statistics software package for education and data analysisrdquoPalaeontologia Electronica vol 4 no 1 pp 4ndash9 2001
[28] K Liu and S V Muse ldquoPowerMaker an integrated analysisenvironment for genetic maker analysisrdquo Bioinformatics vol 21no 9 pp 2128ndash2129 2005
[29] F J RohlfNTSYS-Pc Numerical Taxonomy System Version 2 1Exeter Publishing Setauket New York NY USA 2002
[30] M Nei ldquoAnalysis of gene diversity in subdivided populationsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 70 no 12 1973
[31] S Kadkhodaei M Shahnazari M K Nekouei et al ldquoA compar-ative study of morphological and molecular diversity analysisamong cultivated almonds (Prunus dulcis)rdquo Australian Journalof Crop Science vol 5 no 1 pp 82ndash91 2011
[32] T L Abdullah J Endan and B M Nazir ldquoChanges in flowerdevelopment chlorophyll mutation and alteration in plant
morphology of Curcuma alismatifolia by gamma irradiationrdquoAmerican Journal of Applied Sciences vol 6 no 7 pp 1436ndash14392009
[33] E Kovacs and A Keresztes ldquoEffect of gamma and UV-BCradiation on plant cellsrdquoMicron vol 33 no 2 pp 199ndash210 2002
[34] H L Ramesh Y Murthy and V N Munirajappa ldquoEffect ofgamma radiation on morphological and growth parametersof Mulberry variety M5rdquo International Journal of Science andNature vol 3 pp 447ndash452 2010
[35] K Pongchawee R Pradissan andW Pipatcharoenchai ldquoInducemutation in Anubias spp through in vitro Irradiationrdquo ThaiFisheries Gazette vol 60 pp 493ndash497 2007
[36] P Tangpong T Taychasinpitak C Jompuk and P JompukldquoEffects of acute and chronic gamma irradiations on in vitroculture ofAnubias congensisNE BrownrdquoKasetsart Journal vol43 no 3 pp 449ndash457 2009
[37] R K Hegde Studies on induced mutagenesis and in vitroregeneration in turmeric (Curcuma longa L) [PhD thesis]University of Agricultural Sciences Dharwad India 2006
[38] M P Giridharan and S Balakrishnan ldquoGamma ray inducedvariability in vegetative and floral characters of ginger IndianCocoardquo Arecanut and Spices Journal vol 15 pp 68ndash672 1992
[39] A P Cesarett ldquoEffect of radiation on higher plants and plantcommunitiesrdquo inRadiation Biology United States Atomic EnergyCommission pp 284ndash309 Washington DC USA 1968
[40] G W Seung Y C Byung J-H Kim et al ldquoUltrastructuralchanges of cell organelles in Arabidopsis stems after gammairradationrdquo Journal of Plant Biology vol 48 no 2 pp 195ndash2002005
[41] B S Ahloowalia and M Maluszynski ldquoInduced mutationsmdashanew paradigm in plant breedingrdquo Euphytica vol 118 no 2 pp167ndash173 2001
[42] R Voisine L-P Vezina and C Willemot ldquoInduction ofsenescence-like deterioration of microsomal membranes fromcauliflower by free radicals generated during gamma irradia-tionrdquo Plant Physiology vol 97 no 2 pp 545ndash550 1991
[43] N Shikazono Y Yokota S Kitamura et al ldquoMutation rate andnovel tt mutants of Arabidopsis thaliana induced by carbonionsrdquo Genetics vol 163 no 4 pp 1449ndash1455 2003
[44] T Nakatsuka M Nishihara K Mishiba and S YamamuraldquoTwo different mutations are involved in the formation ofwhite-flowered gentian plantsrdquo Plant Science vol 169 no 5 pp949ndash958 2005
[45] M Mato T Onozaki Y Ozeki et al ldquoFlavonoid biosynthesisin white-flowered Sim carnations (Dianthus caryophyllus)rdquoScientia Horticulturae vol 84 no 3-4 pp 333ndash347 2000
[46] S G Wi B Y Chung J-S Kim et al ldquoEffects of gammairradiation on morphological changes and biological responsesin plantsrdquoMicron vol 38 no 6 pp 553ndash564 2007
[47] F Lee Chin Effects of light intensity and daylength on growth andflowering of siam tulip (Curcuma alismatifolia var Chiang MaiPink) [PhD thesis] University Putra Malaysia 2007
[48] A Hagiladi N Umiel and X H Yang ldquoCurcuma alismatifoliaII Effects of temperature and daylength on the development offlowers and propagulesrdquo Acta Horticulture vol 430 pp 755ndash761 1997
[49] MHagidimitriou A Katsiotis GMenexes C Pontikis andMLoukas ldquoGenetic diversity of major greek olive cultivars usingmolecular (AFLPs and RAPDs) markers and morphologicaltraitsrdquo Journal of the American Society for Horticultural Sciencevol 130 no 2 pp 211ndash217 2005
18 BioMed Research International
[50] N Babaei N A P Abdullah G Saleh and T L AbdullahldquoIsolation and characterization of microsatellite markers andanalysis of genetic variability in Curculigo latifolia DryandrdquoMolecular Biology Reports vol 39 pp 9869ndash9877 2012
[51] B Shiran N Amirbakhtiar S Kiani S Mohammadi B ESayed-Tabatabaei and H Moradi ldquoMolecular characterizationand genetic relationship among almond cultivars assessed byRAPD and SSR markersrdquo Scientia Horticulturae vol 111 no 3pp 280ndash292 2007
[52] S Singh M K Panda and S Nayak ldquoEvaluation of geneticdiversity in turmeric (Curcuma longa L) using RAPD and ISSRmarkersrdquo Industrial Crops and Products vol 37 no 1 pp 284ndash291 2012
[53] Y Paisooksantivatana S Kako and H Seko ldquoGenetic diversityof Curcuma alismatifolia Gagnep (Zingiberaceae) in Thailandas revealed by allozyme polymorphismrdquo Genetic Resources andCrop Evolution vol 48 no 5 pp 459ndash465 2001
Figure 4 Two-dimensional graph of principal component analysis (PCA) for 14 morphological variables indicating relationships amongirradiated and nonirradiated four varieties of C alismatifolia
CMR10-1CMR10-5
SP10-1SP10-2SP10-3
SP10-5
CMR10-4
SP10-4
KP10-1KP10-3
KP20-1
KP20-2
KP10-5KP20-5
KP20-3
KP10-2
DT10-2
DT10-1
DT10-5
DT20-1
DT10-4DT20-4DT20-2
DT20-5DT20-3
CMR20-1
SP20-3SP20-4
SP20-5SP20-1
SP20-2
KP
SP
CMR 10-2
SP10-5
Figure 5 Unrooted neighbor joining tree showing genetic relationship among 44 irradiated and nonirradiated C alismatifolia using SSRmarkers The colors of the branches correspond to those of the same cluster
Figure 6Three-dimensional graph of principal component analysis (PCA) indicating relationships among irradiated and nonirradiated fourvarieties of C alismatifolia
Tung 554 (Curcuma hybrid) had the most morphologicalresponses to gamma rays The use of microsatellite markersas a codominant marker will facilitate the exploration ofgenetic variability among treated and non-treated plants andwill help to distinguish the plants showing differences inmorphological characters The overall effects on the M
1V1
generation revealed that acute gamma irradiation at optimumdoses has the potential for developing new varieties of Calismatifolia with improved commercial properties suitablefor the Malaysian flower industry
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work was funded by the Fundamental Research GrantScheme (FRGS) under the Ministry of Higher Education inMalaysia
References
[1] P Apavatjrut S Anuntalabhochai P Sirirugsa and C AlisildquoMolecularmarkers in the identification of some early floweringCurcuma L (Zingiberaceae) speciesrdquo Annals of Botany vol 84no 4 pp 529ndash534 1999
[2] K Bunya-Atichart S Ketsa andW G VanDoorn ldquoPostharvestphysiology of Curcuma alismatifolia flowersrdquo Postharvest Biol-ogy and Technology vol 34 no 2 pp 219ndash226 2004
[3] M Nakano J Amano Y Watanabe et al ldquoMorphologicalvariation in Tricyrtis hirta plants regenerated from heavy ionbeam-irradiated embryogenic callusesrdquo Plant Biotechnologyvol 27 no 2 pp 155ndash160 2010
[4] S Lamseejan P Jompuk A Wongpiyasatid A Deeseepan andP Kwanthammachart ldquoGamma-rays induced morphological
[5] G-J Lee S J Chung I S Park et al ldquoVariation in thephenotypic features and transcripts of colormutants of chrysan-themum (Dendranthema grandiflorum) derived from gammaraymutagenesisrdquo Journal of Plant Biology vol 51 no 6 pp 418ndash423 2008
[6] O K Kikuchi ldquoOrchid flowers tolerance to gamma-radiationrdquoRadiation Physics and Chemistry vol 57 no 3ndash6 pp 555ndash5572000
[7] N P Arnold N N Barthakur and M Tanguay ldquoMutageniceffects of acute gamma irradiation on miniature roses targettheory approachrdquo HortScience vol 33 no 1 pp 127ndash129 1998
[8] A A Youssef M S Aly and M S Hussein ldquoResponse ofgeranium (Pelargonium graveolenus L) to gamma irradiationand foliar application of Speed Growrdquo Egyptian Journal ofHorticulture vol 27 pp 41ndash53 2000
[9] N Chuantang and L Yazhi ldquoThe radiation inducedmutation ofcanna (Canna L)rdquo Acta Agricultute Nucleatae Sinica vol 2 pp33ndash39 1998
[10] M S Sigrist J B Pinheiro J A A Filho and M I ZucchildquoGenetic diversity of turmeric germplasm (Curcuma longaZingiberaceae) identified by microsatellite markersrdquo Geneticsand Molecular Research vol 10 no 1 pp 419ndash428 2011
[11] S Taheri T L Abdullah N A P Abdullah and Z AhmadldquoGenetic relationships among five varieties of Curcuma alis-matifolia (Zingiberaceae) based on ISSRmarkersrdquoGenetics andMolecular Research vol 11 pp 3069ndash3076 2012
[12] A Das V Kesari V M Satyanarayana A Parida and L Ran-gan ldquoGenetic relationship of Curcuma species from NortheastIndia using PCR-based markersrdquo Molecular Biotechnology vol49 no 1 pp 65ndash76 2011
[13] S Siju K Dhanya S Syamkumar et al ldquoDevelopment char-acterization and utilization of genomic microsatellite markersin turmeric (Curcuma longa L)rdquo Biochemical Systematics andEcology vol 38 no 4 pp 641ndash646 2010
[14] M K Panda S Mohanty E Subudhi L Acharya and SNayak ldquoAssessment of genetic stability of micropropagated
BioMed Research International 17
plants of Curcuma L by cytophotometery and RAPD analysisrdquoInternational Journal of Integrative Biology (IJIB) vol 1 pp 189ndash195 2007
[15] S Syamkumar and B Sasikumar ldquoMolecular marker basedgenetic diversity analysis of Curcuma species from IndiardquoScientia Horticulturae vol 112 no 2 pp 235ndash241 2007
[16] D Tautz and M Renz ldquoSimple sequences are ubiquitousrepetitive components of eukaryotic genomesrdquo Nucleic AcidsResearch vol 12 no 10 pp 4127ndash4138 1984
[17] G Toth Z Gaspari and J Jurka ldquoMicrosatellites in differenteukaryotic genomes surveys and analysisrdquo Genome Researchvol 10 no 7 pp 967ndash981 2000
[18] W Powell G C Machray and J Proven ldquoPolymorphismrevealed by simple sequence repeatsrdquo Trends in Plant Sciencevol 1 no 7 pp 215ndash222 1996
[19] S-Y Lee W K Fai M Zakaria et al ldquoCharacterizationof polymorphic microsatellite markers isolated from ginger(Zingiber officinale Rosc)rdquo Molecular Ecology Notes vol 7 no6 pp 1009ndash1011 2007
[20] S Bory D Da Silva A-M Risterucci M Grisoni P Besseand M-F Duval ldquoDevelopment of microsatellite markers incultivated vanilla polymorphism and transferability to othervanilla speciesrdquo Scientia Horticulturae vol 115 no 4 pp 420ndash425 2008
[21] I C Menezes F W Cidade A P Souza and I C SampaioldquoIsolation and characterization ofmicrosatellite loci in the blackpepper Piper Nigrum L (Piperaceae)rdquo Conservation GeneticsResources vol 1 no 1 pp 209ndash212 2009
[22] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[23] V Kumar Morphological and molecular characterization ofinduced mutants in Groundnut [PhD thesis] University ofAgricultural Sciences Dharwad India 2008
[24] J R Sharma Statistical and Biometrical Techniques in PlantBreeding New Age International New Delhi India 1998
[25] J J Doyle and J L Doyle ldquoA rapid DNA isolation procedure forsmall quantities of fresh leaf tissuerdquo Phytochemical Bulletin vol19 pp 11ndash15 1987
[26] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[27] Oslash Hammer D A T Harper and P D Ryan ldquoPast paleontolog-ical statistics software package for education and data analysisrdquoPalaeontologia Electronica vol 4 no 1 pp 4ndash9 2001
[28] K Liu and S V Muse ldquoPowerMaker an integrated analysisenvironment for genetic maker analysisrdquo Bioinformatics vol 21no 9 pp 2128ndash2129 2005
[29] F J RohlfNTSYS-Pc Numerical Taxonomy System Version 2 1Exeter Publishing Setauket New York NY USA 2002
[30] M Nei ldquoAnalysis of gene diversity in subdivided populationsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 70 no 12 1973
[31] S Kadkhodaei M Shahnazari M K Nekouei et al ldquoA compar-ative study of morphological and molecular diversity analysisamong cultivated almonds (Prunus dulcis)rdquo Australian Journalof Crop Science vol 5 no 1 pp 82ndash91 2011
[32] T L Abdullah J Endan and B M Nazir ldquoChanges in flowerdevelopment chlorophyll mutation and alteration in plant
morphology of Curcuma alismatifolia by gamma irradiationrdquoAmerican Journal of Applied Sciences vol 6 no 7 pp 1436ndash14392009
[33] E Kovacs and A Keresztes ldquoEffect of gamma and UV-BCradiation on plant cellsrdquoMicron vol 33 no 2 pp 199ndash210 2002
[34] H L Ramesh Y Murthy and V N Munirajappa ldquoEffect ofgamma radiation on morphological and growth parametersof Mulberry variety M5rdquo International Journal of Science andNature vol 3 pp 447ndash452 2010
[35] K Pongchawee R Pradissan andW Pipatcharoenchai ldquoInducemutation in Anubias spp through in vitro Irradiationrdquo ThaiFisheries Gazette vol 60 pp 493ndash497 2007
[36] P Tangpong T Taychasinpitak C Jompuk and P JompukldquoEffects of acute and chronic gamma irradiations on in vitroculture ofAnubias congensisNE BrownrdquoKasetsart Journal vol43 no 3 pp 449ndash457 2009
[37] R K Hegde Studies on induced mutagenesis and in vitroregeneration in turmeric (Curcuma longa L) [PhD thesis]University of Agricultural Sciences Dharwad India 2006
[38] M P Giridharan and S Balakrishnan ldquoGamma ray inducedvariability in vegetative and floral characters of ginger IndianCocoardquo Arecanut and Spices Journal vol 15 pp 68ndash672 1992
[39] A P Cesarett ldquoEffect of radiation on higher plants and plantcommunitiesrdquo inRadiation Biology United States Atomic EnergyCommission pp 284ndash309 Washington DC USA 1968
[40] G W Seung Y C Byung J-H Kim et al ldquoUltrastructuralchanges of cell organelles in Arabidopsis stems after gammairradationrdquo Journal of Plant Biology vol 48 no 2 pp 195ndash2002005
[41] B S Ahloowalia and M Maluszynski ldquoInduced mutationsmdashanew paradigm in plant breedingrdquo Euphytica vol 118 no 2 pp167ndash173 2001
[42] R Voisine L-P Vezina and C Willemot ldquoInduction ofsenescence-like deterioration of microsomal membranes fromcauliflower by free radicals generated during gamma irradia-tionrdquo Plant Physiology vol 97 no 2 pp 545ndash550 1991
[43] N Shikazono Y Yokota S Kitamura et al ldquoMutation rate andnovel tt mutants of Arabidopsis thaliana induced by carbonionsrdquo Genetics vol 163 no 4 pp 1449ndash1455 2003
[44] T Nakatsuka M Nishihara K Mishiba and S YamamuraldquoTwo different mutations are involved in the formation ofwhite-flowered gentian plantsrdquo Plant Science vol 169 no 5 pp949ndash958 2005
[45] M Mato T Onozaki Y Ozeki et al ldquoFlavonoid biosynthesisin white-flowered Sim carnations (Dianthus caryophyllus)rdquoScientia Horticulturae vol 84 no 3-4 pp 333ndash347 2000
[46] S G Wi B Y Chung J-S Kim et al ldquoEffects of gammairradiation on morphological changes and biological responsesin plantsrdquoMicron vol 38 no 6 pp 553ndash564 2007
[47] F Lee Chin Effects of light intensity and daylength on growth andflowering of siam tulip (Curcuma alismatifolia var Chiang MaiPink) [PhD thesis] University Putra Malaysia 2007
[48] A Hagiladi N Umiel and X H Yang ldquoCurcuma alismatifoliaII Effects of temperature and daylength on the development offlowers and propagulesrdquo Acta Horticulture vol 430 pp 755ndash761 1997
[49] MHagidimitriou A Katsiotis GMenexes C Pontikis andMLoukas ldquoGenetic diversity of major greek olive cultivars usingmolecular (AFLPs and RAPDs) markers and morphologicaltraitsrdquo Journal of the American Society for Horticultural Sciencevol 130 no 2 pp 211ndash217 2005
18 BioMed Research International
[50] N Babaei N A P Abdullah G Saleh and T L AbdullahldquoIsolation and characterization of microsatellite markers andanalysis of genetic variability in Curculigo latifolia DryandrdquoMolecular Biology Reports vol 39 pp 9869ndash9877 2012
[51] B Shiran N Amirbakhtiar S Kiani S Mohammadi B ESayed-Tabatabaei and H Moradi ldquoMolecular characterizationand genetic relationship among almond cultivars assessed byRAPD and SSR markersrdquo Scientia Horticulturae vol 111 no 3pp 280ndash292 2007
[52] S Singh M K Panda and S Nayak ldquoEvaluation of geneticdiversity in turmeric (Curcuma longa L) using RAPD and ISSRmarkersrdquo Industrial Crops and Products vol 37 no 1 pp 284ndash291 2012
[53] Y Paisooksantivatana S Kako and H Seko ldquoGenetic diversityof Curcuma alismatifolia Gagnep (Zingiberaceae) in Thailandas revealed by allozyme polymorphismrdquo Genetic Resources andCrop Evolution vol 48 no 5 pp 459ndash465 2001
Figure 4 Two-dimensional graph of principal component analysis (PCA) for 14 morphological variables indicating relationships amongirradiated and nonirradiated four varieties of C alismatifolia
CMR10-1CMR10-5
SP10-1SP10-2SP10-3
SP10-5
CMR10-4
SP10-4
KP10-1KP10-3
KP20-1
KP20-2
KP10-5KP20-5
KP20-3
KP10-2
DT10-2
DT10-1
DT10-5
DT20-1
DT10-4DT20-4DT20-2
DT20-5DT20-3
CMR20-1
SP20-3SP20-4
SP20-5SP20-1
SP20-2
KP
SP
CMR 10-2
SP10-5
Figure 5 Unrooted neighbor joining tree showing genetic relationship among 44 irradiated and nonirradiated C alismatifolia using SSRmarkers The colors of the branches correspond to those of the same cluster
Figure 6Three-dimensional graph of principal component analysis (PCA) indicating relationships among irradiated and nonirradiated fourvarieties of C alismatifolia
Tung 554 (Curcuma hybrid) had the most morphologicalresponses to gamma rays The use of microsatellite markersas a codominant marker will facilitate the exploration ofgenetic variability among treated and non-treated plants andwill help to distinguish the plants showing differences inmorphological characters The overall effects on the M
1V1
generation revealed that acute gamma irradiation at optimumdoses has the potential for developing new varieties of Calismatifolia with improved commercial properties suitablefor the Malaysian flower industry
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work was funded by the Fundamental Research GrantScheme (FRGS) under the Ministry of Higher Education inMalaysia
References
[1] P Apavatjrut S Anuntalabhochai P Sirirugsa and C AlisildquoMolecularmarkers in the identification of some early floweringCurcuma L (Zingiberaceae) speciesrdquo Annals of Botany vol 84no 4 pp 529ndash534 1999
[2] K Bunya-Atichart S Ketsa andW G VanDoorn ldquoPostharvestphysiology of Curcuma alismatifolia flowersrdquo Postharvest Biol-ogy and Technology vol 34 no 2 pp 219ndash226 2004
[3] M Nakano J Amano Y Watanabe et al ldquoMorphologicalvariation in Tricyrtis hirta plants regenerated from heavy ionbeam-irradiated embryogenic callusesrdquo Plant Biotechnologyvol 27 no 2 pp 155ndash160 2010
[4] S Lamseejan P Jompuk A Wongpiyasatid A Deeseepan andP Kwanthammachart ldquoGamma-rays induced morphological
[5] G-J Lee S J Chung I S Park et al ldquoVariation in thephenotypic features and transcripts of colormutants of chrysan-themum (Dendranthema grandiflorum) derived from gammaraymutagenesisrdquo Journal of Plant Biology vol 51 no 6 pp 418ndash423 2008
[6] O K Kikuchi ldquoOrchid flowers tolerance to gamma-radiationrdquoRadiation Physics and Chemistry vol 57 no 3ndash6 pp 555ndash5572000
[7] N P Arnold N N Barthakur and M Tanguay ldquoMutageniceffects of acute gamma irradiation on miniature roses targettheory approachrdquo HortScience vol 33 no 1 pp 127ndash129 1998
[8] A A Youssef M S Aly and M S Hussein ldquoResponse ofgeranium (Pelargonium graveolenus L) to gamma irradiationand foliar application of Speed Growrdquo Egyptian Journal ofHorticulture vol 27 pp 41ndash53 2000
[9] N Chuantang and L Yazhi ldquoThe radiation inducedmutation ofcanna (Canna L)rdquo Acta Agricultute Nucleatae Sinica vol 2 pp33ndash39 1998
[10] M S Sigrist J B Pinheiro J A A Filho and M I ZucchildquoGenetic diversity of turmeric germplasm (Curcuma longaZingiberaceae) identified by microsatellite markersrdquo Geneticsand Molecular Research vol 10 no 1 pp 419ndash428 2011
[11] S Taheri T L Abdullah N A P Abdullah and Z AhmadldquoGenetic relationships among five varieties of Curcuma alis-matifolia (Zingiberaceae) based on ISSRmarkersrdquoGenetics andMolecular Research vol 11 pp 3069ndash3076 2012
[12] A Das V Kesari V M Satyanarayana A Parida and L Ran-gan ldquoGenetic relationship of Curcuma species from NortheastIndia using PCR-based markersrdquo Molecular Biotechnology vol49 no 1 pp 65ndash76 2011
[13] S Siju K Dhanya S Syamkumar et al ldquoDevelopment char-acterization and utilization of genomic microsatellite markersin turmeric (Curcuma longa L)rdquo Biochemical Systematics andEcology vol 38 no 4 pp 641ndash646 2010
[14] M K Panda S Mohanty E Subudhi L Acharya and SNayak ldquoAssessment of genetic stability of micropropagated
BioMed Research International 17
plants of Curcuma L by cytophotometery and RAPD analysisrdquoInternational Journal of Integrative Biology (IJIB) vol 1 pp 189ndash195 2007
[15] S Syamkumar and B Sasikumar ldquoMolecular marker basedgenetic diversity analysis of Curcuma species from IndiardquoScientia Horticulturae vol 112 no 2 pp 235ndash241 2007
[16] D Tautz and M Renz ldquoSimple sequences are ubiquitousrepetitive components of eukaryotic genomesrdquo Nucleic AcidsResearch vol 12 no 10 pp 4127ndash4138 1984
[17] G Toth Z Gaspari and J Jurka ldquoMicrosatellites in differenteukaryotic genomes surveys and analysisrdquo Genome Researchvol 10 no 7 pp 967ndash981 2000
[18] W Powell G C Machray and J Proven ldquoPolymorphismrevealed by simple sequence repeatsrdquo Trends in Plant Sciencevol 1 no 7 pp 215ndash222 1996
[19] S-Y Lee W K Fai M Zakaria et al ldquoCharacterizationof polymorphic microsatellite markers isolated from ginger(Zingiber officinale Rosc)rdquo Molecular Ecology Notes vol 7 no6 pp 1009ndash1011 2007
[20] S Bory D Da Silva A-M Risterucci M Grisoni P Besseand M-F Duval ldquoDevelopment of microsatellite markers incultivated vanilla polymorphism and transferability to othervanilla speciesrdquo Scientia Horticulturae vol 115 no 4 pp 420ndash425 2008
[21] I C Menezes F W Cidade A P Souza and I C SampaioldquoIsolation and characterization ofmicrosatellite loci in the blackpepper Piper Nigrum L (Piperaceae)rdquo Conservation GeneticsResources vol 1 no 1 pp 209ndash212 2009
[22] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[23] V Kumar Morphological and molecular characterization ofinduced mutants in Groundnut [PhD thesis] University ofAgricultural Sciences Dharwad India 2008
[24] J R Sharma Statistical and Biometrical Techniques in PlantBreeding New Age International New Delhi India 1998
[25] J J Doyle and J L Doyle ldquoA rapid DNA isolation procedure forsmall quantities of fresh leaf tissuerdquo Phytochemical Bulletin vol19 pp 11ndash15 1987
[26] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[27] Oslash Hammer D A T Harper and P D Ryan ldquoPast paleontolog-ical statistics software package for education and data analysisrdquoPalaeontologia Electronica vol 4 no 1 pp 4ndash9 2001
[28] K Liu and S V Muse ldquoPowerMaker an integrated analysisenvironment for genetic maker analysisrdquo Bioinformatics vol 21no 9 pp 2128ndash2129 2005
[29] F J RohlfNTSYS-Pc Numerical Taxonomy System Version 2 1Exeter Publishing Setauket New York NY USA 2002
[30] M Nei ldquoAnalysis of gene diversity in subdivided populationsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 70 no 12 1973
[31] S Kadkhodaei M Shahnazari M K Nekouei et al ldquoA compar-ative study of morphological and molecular diversity analysisamong cultivated almonds (Prunus dulcis)rdquo Australian Journalof Crop Science vol 5 no 1 pp 82ndash91 2011
[32] T L Abdullah J Endan and B M Nazir ldquoChanges in flowerdevelopment chlorophyll mutation and alteration in plant
morphology of Curcuma alismatifolia by gamma irradiationrdquoAmerican Journal of Applied Sciences vol 6 no 7 pp 1436ndash14392009
[33] E Kovacs and A Keresztes ldquoEffect of gamma and UV-BCradiation on plant cellsrdquoMicron vol 33 no 2 pp 199ndash210 2002
[34] H L Ramesh Y Murthy and V N Munirajappa ldquoEffect ofgamma radiation on morphological and growth parametersof Mulberry variety M5rdquo International Journal of Science andNature vol 3 pp 447ndash452 2010
[35] K Pongchawee R Pradissan andW Pipatcharoenchai ldquoInducemutation in Anubias spp through in vitro Irradiationrdquo ThaiFisheries Gazette vol 60 pp 493ndash497 2007
[36] P Tangpong T Taychasinpitak C Jompuk and P JompukldquoEffects of acute and chronic gamma irradiations on in vitroculture ofAnubias congensisNE BrownrdquoKasetsart Journal vol43 no 3 pp 449ndash457 2009
[37] R K Hegde Studies on induced mutagenesis and in vitroregeneration in turmeric (Curcuma longa L) [PhD thesis]University of Agricultural Sciences Dharwad India 2006
[38] M P Giridharan and S Balakrishnan ldquoGamma ray inducedvariability in vegetative and floral characters of ginger IndianCocoardquo Arecanut and Spices Journal vol 15 pp 68ndash672 1992
[39] A P Cesarett ldquoEffect of radiation on higher plants and plantcommunitiesrdquo inRadiation Biology United States Atomic EnergyCommission pp 284ndash309 Washington DC USA 1968
[40] G W Seung Y C Byung J-H Kim et al ldquoUltrastructuralchanges of cell organelles in Arabidopsis stems after gammairradationrdquo Journal of Plant Biology vol 48 no 2 pp 195ndash2002005
[41] B S Ahloowalia and M Maluszynski ldquoInduced mutationsmdashanew paradigm in plant breedingrdquo Euphytica vol 118 no 2 pp167ndash173 2001
[42] R Voisine L-P Vezina and C Willemot ldquoInduction ofsenescence-like deterioration of microsomal membranes fromcauliflower by free radicals generated during gamma irradia-tionrdquo Plant Physiology vol 97 no 2 pp 545ndash550 1991
[43] N Shikazono Y Yokota S Kitamura et al ldquoMutation rate andnovel tt mutants of Arabidopsis thaliana induced by carbonionsrdquo Genetics vol 163 no 4 pp 1449ndash1455 2003
[44] T Nakatsuka M Nishihara K Mishiba and S YamamuraldquoTwo different mutations are involved in the formation ofwhite-flowered gentian plantsrdquo Plant Science vol 169 no 5 pp949ndash958 2005
[45] M Mato T Onozaki Y Ozeki et al ldquoFlavonoid biosynthesisin white-flowered Sim carnations (Dianthus caryophyllus)rdquoScientia Horticulturae vol 84 no 3-4 pp 333ndash347 2000
[46] S G Wi B Y Chung J-S Kim et al ldquoEffects of gammairradiation on morphological changes and biological responsesin plantsrdquoMicron vol 38 no 6 pp 553ndash564 2007
[47] F Lee Chin Effects of light intensity and daylength on growth andflowering of siam tulip (Curcuma alismatifolia var Chiang MaiPink) [PhD thesis] University Putra Malaysia 2007
[48] A Hagiladi N Umiel and X H Yang ldquoCurcuma alismatifoliaII Effects of temperature and daylength on the development offlowers and propagulesrdquo Acta Horticulture vol 430 pp 755ndash761 1997
[49] MHagidimitriou A Katsiotis GMenexes C Pontikis andMLoukas ldquoGenetic diversity of major greek olive cultivars usingmolecular (AFLPs and RAPDs) markers and morphologicaltraitsrdquo Journal of the American Society for Horticultural Sciencevol 130 no 2 pp 211ndash217 2005
18 BioMed Research International
[50] N Babaei N A P Abdullah G Saleh and T L AbdullahldquoIsolation and characterization of microsatellite markers andanalysis of genetic variability in Curculigo latifolia DryandrdquoMolecular Biology Reports vol 39 pp 9869ndash9877 2012
[51] B Shiran N Amirbakhtiar S Kiani S Mohammadi B ESayed-Tabatabaei and H Moradi ldquoMolecular characterizationand genetic relationship among almond cultivars assessed byRAPD and SSR markersrdquo Scientia Horticulturae vol 111 no 3pp 280ndash292 2007
[52] S Singh M K Panda and S Nayak ldquoEvaluation of geneticdiversity in turmeric (Curcuma longa L) using RAPD and ISSRmarkersrdquo Industrial Crops and Products vol 37 no 1 pp 284ndash291 2012
[53] Y Paisooksantivatana S Kako and H Seko ldquoGenetic diversityof Curcuma alismatifolia Gagnep (Zingiberaceae) in Thailandas revealed by allozyme polymorphismrdquo Genetic Resources andCrop Evolution vol 48 no 5 pp 459ndash465 2001
Figure 6Three-dimensional graph of principal component analysis (PCA) indicating relationships among irradiated and nonirradiated fourvarieties of C alismatifolia
Tung 554 (Curcuma hybrid) had the most morphologicalresponses to gamma rays The use of microsatellite markersas a codominant marker will facilitate the exploration ofgenetic variability among treated and non-treated plants andwill help to distinguish the plants showing differences inmorphological characters The overall effects on the M
1V1
generation revealed that acute gamma irradiation at optimumdoses has the potential for developing new varieties of Calismatifolia with improved commercial properties suitablefor the Malaysian flower industry
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work was funded by the Fundamental Research GrantScheme (FRGS) under the Ministry of Higher Education inMalaysia
References
[1] P Apavatjrut S Anuntalabhochai P Sirirugsa and C AlisildquoMolecularmarkers in the identification of some early floweringCurcuma L (Zingiberaceae) speciesrdquo Annals of Botany vol 84no 4 pp 529ndash534 1999
[2] K Bunya-Atichart S Ketsa andW G VanDoorn ldquoPostharvestphysiology of Curcuma alismatifolia flowersrdquo Postharvest Biol-ogy and Technology vol 34 no 2 pp 219ndash226 2004
[3] M Nakano J Amano Y Watanabe et al ldquoMorphologicalvariation in Tricyrtis hirta plants regenerated from heavy ionbeam-irradiated embryogenic callusesrdquo Plant Biotechnologyvol 27 no 2 pp 155ndash160 2010
[4] S Lamseejan P Jompuk A Wongpiyasatid A Deeseepan andP Kwanthammachart ldquoGamma-rays induced morphological
[5] G-J Lee S J Chung I S Park et al ldquoVariation in thephenotypic features and transcripts of colormutants of chrysan-themum (Dendranthema grandiflorum) derived from gammaraymutagenesisrdquo Journal of Plant Biology vol 51 no 6 pp 418ndash423 2008
[6] O K Kikuchi ldquoOrchid flowers tolerance to gamma-radiationrdquoRadiation Physics and Chemistry vol 57 no 3ndash6 pp 555ndash5572000
[7] N P Arnold N N Barthakur and M Tanguay ldquoMutageniceffects of acute gamma irradiation on miniature roses targettheory approachrdquo HortScience vol 33 no 1 pp 127ndash129 1998
[8] A A Youssef M S Aly and M S Hussein ldquoResponse ofgeranium (Pelargonium graveolenus L) to gamma irradiationand foliar application of Speed Growrdquo Egyptian Journal ofHorticulture vol 27 pp 41ndash53 2000
[9] N Chuantang and L Yazhi ldquoThe radiation inducedmutation ofcanna (Canna L)rdquo Acta Agricultute Nucleatae Sinica vol 2 pp33ndash39 1998
[10] M S Sigrist J B Pinheiro J A A Filho and M I ZucchildquoGenetic diversity of turmeric germplasm (Curcuma longaZingiberaceae) identified by microsatellite markersrdquo Geneticsand Molecular Research vol 10 no 1 pp 419ndash428 2011
[11] S Taheri T L Abdullah N A P Abdullah and Z AhmadldquoGenetic relationships among five varieties of Curcuma alis-matifolia (Zingiberaceae) based on ISSRmarkersrdquoGenetics andMolecular Research vol 11 pp 3069ndash3076 2012
[12] A Das V Kesari V M Satyanarayana A Parida and L Ran-gan ldquoGenetic relationship of Curcuma species from NortheastIndia using PCR-based markersrdquo Molecular Biotechnology vol49 no 1 pp 65ndash76 2011
[13] S Siju K Dhanya S Syamkumar et al ldquoDevelopment char-acterization and utilization of genomic microsatellite markersin turmeric (Curcuma longa L)rdquo Biochemical Systematics andEcology vol 38 no 4 pp 641ndash646 2010
[14] M K Panda S Mohanty E Subudhi L Acharya and SNayak ldquoAssessment of genetic stability of micropropagated
BioMed Research International 17
plants of Curcuma L by cytophotometery and RAPD analysisrdquoInternational Journal of Integrative Biology (IJIB) vol 1 pp 189ndash195 2007
[15] S Syamkumar and B Sasikumar ldquoMolecular marker basedgenetic diversity analysis of Curcuma species from IndiardquoScientia Horticulturae vol 112 no 2 pp 235ndash241 2007
[16] D Tautz and M Renz ldquoSimple sequences are ubiquitousrepetitive components of eukaryotic genomesrdquo Nucleic AcidsResearch vol 12 no 10 pp 4127ndash4138 1984
[17] G Toth Z Gaspari and J Jurka ldquoMicrosatellites in differenteukaryotic genomes surveys and analysisrdquo Genome Researchvol 10 no 7 pp 967ndash981 2000
[18] W Powell G C Machray and J Proven ldquoPolymorphismrevealed by simple sequence repeatsrdquo Trends in Plant Sciencevol 1 no 7 pp 215ndash222 1996
[19] S-Y Lee W K Fai M Zakaria et al ldquoCharacterizationof polymorphic microsatellite markers isolated from ginger(Zingiber officinale Rosc)rdquo Molecular Ecology Notes vol 7 no6 pp 1009ndash1011 2007
[20] S Bory D Da Silva A-M Risterucci M Grisoni P Besseand M-F Duval ldquoDevelopment of microsatellite markers incultivated vanilla polymorphism and transferability to othervanilla speciesrdquo Scientia Horticulturae vol 115 no 4 pp 420ndash425 2008
[21] I C Menezes F W Cidade A P Souza and I C SampaioldquoIsolation and characterization ofmicrosatellite loci in the blackpepper Piper Nigrum L (Piperaceae)rdquo Conservation GeneticsResources vol 1 no 1 pp 209ndash212 2009
[22] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[23] V Kumar Morphological and molecular characterization ofinduced mutants in Groundnut [PhD thesis] University ofAgricultural Sciences Dharwad India 2008
[24] J R Sharma Statistical and Biometrical Techniques in PlantBreeding New Age International New Delhi India 1998
[25] J J Doyle and J L Doyle ldquoA rapid DNA isolation procedure forsmall quantities of fresh leaf tissuerdquo Phytochemical Bulletin vol19 pp 11ndash15 1987
[26] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[27] Oslash Hammer D A T Harper and P D Ryan ldquoPast paleontolog-ical statistics software package for education and data analysisrdquoPalaeontologia Electronica vol 4 no 1 pp 4ndash9 2001
[28] K Liu and S V Muse ldquoPowerMaker an integrated analysisenvironment for genetic maker analysisrdquo Bioinformatics vol 21no 9 pp 2128ndash2129 2005
[29] F J RohlfNTSYS-Pc Numerical Taxonomy System Version 2 1Exeter Publishing Setauket New York NY USA 2002
[30] M Nei ldquoAnalysis of gene diversity in subdivided populationsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 70 no 12 1973
[31] S Kadkhodaei M Shahnazari M K Nekouei et al ldquoA compar-ative study of morphological and molecular diversity analysisamong cultivated almonds (Prunus dulcis)rdquo Australian Journalof Crop Science vol 5 no 1 pp 82ndash91 2011
[32] T L Abdullah J Endan and B M Nazir ldquoChanges in flowerdevelopment chlorophyll mutation and alteration in plant
morphology of Curcuma alismatifolia by gamma irradiationrdquoAmerican Journal of Applied Sciences vol 6 no 7 pp 1436ndash14392009
[33] E Kovacs and A Keresztes ldquoEffect of gamma and UV-BCradiation on plant cellsrdquoMicron vol 33 no 2 pp 199ndash210 2002
[34] H L Ramesh Y Murthy and V N Munirajappa ldquoEffect ofgamma radiation on morphological and growth parametersof Mulberry variety M5rdquo International Journal of Science andNature vol 3 pp 447ndash452 2010
[35] K Pongchawee R Pradissan andW Pipatcharoenchai ldquoInducemutation in Anubias spp through in vitro Irradiationrdquo ThaiFisheries Gazette vol 60 pp 493ndash497 2007
[36] P Tangpong T Taychasinpitak C Jompuk and P JompukldquoEffects of acute and chronic gamma irradiations on in vitroculture ofAnubias congensisNE BrownrdquoKasetsart Journal vol43 no 3 pp 449ndash457 2009
[37] R K Hegde Studies on induced mutagenesis and in vitroregeneration in turmeric (Curcuma longa L) [PhD thesis]University of Agricultural Sciences Dharwad India 2006
[38] M P Giridharan and S Balakrishnan ldquoGamma ray inducedvariability in vegetative and floral characters of ginger IndianCocoardquo Arecanut and Spices Journal vol 15 pp 68ndash672 1992
[39] A P Cesarett ldquoEffect of radiation on higher plants and plantcommunitiesrdquo inRadiation Biology United States Atomic EnergyCommission pp 284ndash309 Washington DC USA 1968
[40] G W Seung Y C Byung J-H Kim et al ldquoUltrastructuralchanges of cell organelles in Arabidopsis stems after gammairradationrdquo Journal of Plant Biology vol 48 no 2 pp 195ndash2002005
[41] B S Ahloowalia and M Maluszynski ldquoInduced mutationsmdashanew paradigm in plant breedingrdquo Euphytica vol 118 no 2 pp167ndash173 2001
[42] R Voisine L-P Vezina and C Willemot ldquoInduction ofsenescence-like deterioration of microsomal membranes fromcauliflower by free radicals generated during gamma irradia-tionrdquo Plant Physiology vol 97 no 2 pp 545ndash550 1991
[43] N Shikazono Y Yokota S Kitamura et al ldquoMutation rate andnovel tt mutants of Arabidopsis thaliana induced by carbonionsrdquo Genetics vol 163 no 4 pp 1449ndash1455 2003
[44] T Nakatsuka M Nishihara K Mishiba and S YamamuraldquoTwo different mutations are involved in the formation ofwhite-flowered gentian plantsrdquo Plant Science vol 169 no 5 pp949ndash958 2005
[45] M Mato T Onozaki Y Ozeki et al ldquoFlavonoid biosynthesisin white-flowered Sim carnations (Dianthus caryophyllus)rdquoScientia Horticulturae vol 84 no 3-4 pp 333ndash347 2000
[46] S G Wi B Y Chung J-S Kim et al ldquoEffects of gammairradiation on morphological changes and biological responsesin plantsrdquoMicron vol 38 no 6 pp 553ndash564 2007
[47] F Lee Chin Effects of light intensity and daylength on growth andflowering of siam tulip (Curcuma alismatifolia var Chiang MaiPink) [PhD thesis] University Putra Malaysia 2007
[48] A Hagiladi N Umiel and X H Yang ldquoCurcuma alismatifoliaII Effects of temperature and daylength on the development offlowers and propagulesrdquo Acta Horticulture vol 430 pp 755ndash761 1997
[49] MHagidimitriou A Katsiotis GMenexes C Pontikis andMLoukas ldquoGenetic diversity of major greek olive cultivars usingmolecular (AFLPs and RAPDs) markers and morphologicaltraitsrdquo Journal of the American Society for Horticultural Sciencevol 130 no 2 pp 211ndash217 2005
18 BioMed Research International
[50] N Babaei N A P Abdullah G Saleh and T L AbdullahldquoIsolation and characterization of microsatellite markers andanalysis of genetic variability in Curculigo latifolia DryandrdquoMolecular Biology Reports vol 39 pp 9869ndash9877 2012
[51] B Shiran N Amirbakhtiar S Kiani S Mohammadi B ESayed-Tabatabaei and H Moradi ldquoMolecular characterizationand genetic relationship among almond cultivars assessed byRAPD and SSR markersrdquo Scientia Horticulturae vol 111 no 3pp 280ndash292 2007
[52] S Singh M K Panda and S Nayak ldquoEvaluation of geneticdiversity in turmeric (Curcuma longa L) using RAPD and ISSRmarkersrdquo Industrial Crops and Products vol 37 no 1 pp 284ndash291 2012
[53] Y Paisooksantivatana S Kako and H Seko ldquoGenetic diversityof Curcuma alismatifolia Gagnep (Zingiberaceae) in Thailandas revealed by allozyme polymorphismrdquo Genetic Resources andCrop Evolution vol 48 no 5 pp 459ndash465 2001
plants of Curcuma L by cytophotometery and RAPD analysisrdquoInternational Journal of Integrative Biology (IJIB) vol 1 pp 189ndash195 2007
[15] S Syamkumar and B Sasikumar ldquoMolecular marker basedgenetic diversity analysis of Curcuma species from IndiardquoScientia Horticulturae vol 112 no 2 pp 235ndash241 2007
[16] D Tautz and M Renz ldquoSimple sequences are ubiquitousrepetitive components of eukaryotic genomesrdquo Nucleic AcidsResearch vol 12 no 10 pp 4127ndash4138 1984
[17] G Toth Z Gaspari and J Jurka ldquoMicrosatellites in differenteukaryotic genomes surveys and analysisrdquo Genome Researchvol 10 no 7 pp 967ndash981 2000
[18] W Powell G C Machray and J Proven ldquoPolymorphismrevealed by simple sequence repeatsrdquo Trends in Plant Sciencevol 1 no 7 pp 215ndash222 1996
[19] S-Y Lee W K Fai M Zakaria et al ldquoCharacterizationof polymorphic microsatellite markers isolated from ginger(Zingiber officinale Rosc)rdquo Molecular Ecology Notes vol 7 no6 pp 1009ndash1011 2007
[20] S Bory D Da Silva A-M Risterucci M Grisoni P Besseand M-F Duval ldquoDevelopment of microsatellite markers incultivated vanilla polymorphism and transferability to othervanilla speciesrdquo Scientia Horticulturae vol 115 no 4 pp 420ndash425 2008
[21] I C Menezes F W Cidade A P Souza and I C SampaioldquoIsolation and characterization ofmicrosatellite loci in the blackpepper Piper Nigrum L (Piperaceae)rdquo Conservation GeneticsResources vol 1 no 1 pp 209ndash212 2009
[22] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[23] V Kumar Morphological and molecular characterization ofinduced mutants in Groundnut [PhD thesis] University ofAgricultural Sciences Dharwad India 2008
[24] J R Sharma Statistical and Biometrical Techniques in PlantBreeding New Age International New Delhi India 1998
[25] J J Doyle and J L Doyle ldquoA rapid DNA isolation procedure forsmall quantities of fresh leaf tissuerdquo Phytochemical Bulletin vol19 pp 11ndash15 1987
[26] M S Sigrist J B Pinheiro J A Azevedo-Filho et al ldquoDevel-opment and characterization of microsatellite markers forturmeric (Curcuma longa)rdquo Plant Breeding vol 129 no 5 pp570ndash573 2010
[27] Oslash Hammer D A T Harper and P D Ryan ldquoPast paleontolog-ical statistics software package for education and data analysisrdquoPalaeontologia Electronica vol 4 no 1 pp 4ndash9 2001
[28] K Liu and S V Muse ldquoPowerMaker an integrated analysisenvironment for genetic maker analysisrdquo Bioinformatics vol 21no 9 pp 2128ndash2129 2005
[29] F J RohlfNTSYS-Pc Numerical Taxonomy System Version 2 1Exeter Publishing Setauket New York NY USA 2002
[30] M Nei ldquoAnalysis of gene diversity in subdivided populationsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 70 no 12 1973
[31] S Kadkhodaei M Shahnazari M K Nekouei et al ldquoA compar-ative study of morphological and molecular diversity analysisamong cultivated almonds (Prunus dulcis)rdquo Australian Journalof Crop Science vol 5 no 1 pp 82ndash91 2011
[32] T L Abdullah J Endan and B M Nazir ldquoChanges in flowerdevelopment chlorophyll mutation and alteration in plant
morphology of Curcuma alismatifolia by gamma irradiationrdquoAmerican Journal of Applied Sciences vol 6 no 7 pp 1436ndash14392009
[33] E Kovacs and A Keresztes ldquoEffect of gamma and UV-BCradiation on plant cellsrdquoMicron vol 33 no 2 pp 199ndash210 2002
[34] H L Ramesh Y Murthy and V N Munirajappa ldquoEffect ofgamma radiation on morphological and growth parametersof Mulberry variety M5rdquo International Journal of Science andNature vol 3 pp 447ndash452 2010
[35] K Pongchawee R Pradissan andW Pipatcharoenchai ldquoInducemutation in Anubias spp through in vitro Irradiationrdquo ThaiFisheries Gazette vol 60 pp 493ndash497 2007
[36] P Tangpong T Taychasinpitak C Jompuk and P JompukldquoEffects of acute and chronic gamma irradiations on in vitroculture ofAnubias congensisNE BrownrdquoKasetsart Journal vol43 no 3 pp 449ndash457 2009
[37] R K Hegde Studies on induced mutagenesis and in vitroregeneration in turmeric (Curcuma longa L) [PhD thesis]University of Agricultural Sciences Dharwad India 2006
[38] M P Giridharan and S Balakrishnan ldquoGamma ray inducedvariability in vegetative and floral characters of ginger IndianCocoardquo Arecanut and Spices Journal vol 15 pp 68ndash672 1992
[39] A P Cesarett ldquoEffect of radiation on higher plants and plantcommunitiesrdquo inRadiation Biology United States Atomic EnergyCommission pp 284ndash309 Washington DC USA 1968
[40] G W Seung Y C Byung J-H Kim et al ldquoUltrastructuralchanges of cell organelles in Arabidopsis stems after gammairradationrdquo Journal of Plant Biology vol 48 no 2 pp 195ndash2002005
[41] B S Ahloowalia and M Maluszynski ldquoInduced mutationsmdashanew paradigm in plant breedingrdquo Euphytica vol 118 no 2 pp167ndash173 2001
[42] R Voisine L-P Vezina and C Willemot ldquoInduction ofsenescence-like deterioration of microsomal membranes fromcauliflower by free radicals generated during gamma irradia-tionrdquo Plant Physiology vol 97 no 2 pp 545ndash550 1991
[43] N Shikazono Y Yokota S Kitamura et al ldquoMutation rate andnovel tt mutants of Arabidopsis thaliana induced by carbonionsrdquo Genetics vol 163 no 4 pp 1449ndash1455 2003
[44] T Nakatsuka M Nishihara K Mishiba and S YamamuraldquoTwo different mutations are involved in the formation ofwhite-flowered gentian plantsrdquo Plant Science vol 169 no 5 pp949ndash958 2005
[45] M Mato T Onozaki Y Ozeki et al ldquoFlavonoid biosynthesisin white-flowered Sim carnations (Dianthus caryophyllus)rdquoScientia Horticulturae vol 84 no 3-4 pp 333ndash347 2000
[46] S G Wi B Y Chung J-S Kim et al ldquoEffects of gammairradiation on morphological changes and biological responsesin plantsrdquoMicron vol 38 no 6 pp 553ndash564 2007
[47] F Lee Chin Effects of light intensity and daylength on growth andflowering of siam tulip (Curcuma alismatifolia var Chiang MaiPink) [PhD thesis] University Putra Malaysia 2007
[48] A Hagiladi N Umiel and X H Yang ldquoCurcuma alismatifoliaII Effects of temperature and daylength on the development offlowers and propagulesrdquo Acta Horticulture vol 430 pp 755ndash761 1997
[49] MHagidimitriou A Katsiotis GMenexes C Pontikis andMLoukas ldquoGenetic diversity of major greek olive cultivars usingmolecular (AFLPs and RAPDs) markers and morphologicaltraitsrdquo Journal of the American Society for Horticultural Sciencevol 130 no 2 pp 211ndash217 2005
18 BioMed Research International
[50] N Babaei N A P Abdullah G Saleh and T L AbdullahldquoIsolation and characterization of microsatellite markers andanalysis of genetic variability in Curculigo latifolia DryandrdquoMolecular Biology Reports vol 39 pp 9869ndash9877 2012
[51] B Shiran N Amirbakhtiar S Kiani S Mohammadi B ESayed-Tabatabaei and H Moradi ldquoMolecular characterizationand genetic relationship among almond cultivars assessed byRAPD and SSR markersrdquo Scientia Horticulturae vol 111 no 3pp 280ndash292 2007
[52] S Singh M K Panda and S Nayak ldquoEvaluation of geneticdiversity in turmeric (Curcuma longa L) using RAPD and ISSRmarkersrdquo Industrial Crops and Products vol 37 no 1 pp 284ndash291 2012
[53] Y Paisooksantivatana S Kako and H Seko ldquoGenetic diversityof Curcuma alismatifolia Gagnep (Zingiberaceae) in Thailandas revealed by allozyme polymorphismrdquo Genetic Resources andCrop Evolution vol 48 no 5 pp 459ndash465 2001
[50] N Babaei N A P Abdullah G Saleh and T L AbdullahldquoIsolation and characterization of microsatellite markers andanalysis of genetic variability in Curculigo latifolia DryandrdquoMolecular Biology Reports vol 39 pp 9869ndash9877 2012
[51] B Shiran N Amirbakhtiar S Kiani S Mohammadi B ESayed-Tabatabaei and H Moradi ldquoMolecular characterizationand genetic relationship among almond cultivars assessed byRAPD and SSR markersrdquo Scientia Horticulturae vol 111 no 3pp 280ndash292 2007
[52] S Singh M K Panda and S Nayak ldquoEvaluation of geneticdiversity in turmeric (Curcuma longa L) using RAPD and ISSRmarkersrdquo Industrial Crops and Products vol 37 no 1 pp 284ndash291 2012
[53] Y Paisooksantivatana S Kako and H Seko ldquoGenetic diversityof Curcuma alismatifolia Gagnep (Zingiberaceae) in Thailandas revealed by allozyme polymorphismrdquo Genetic Resources andCrop Evolution vol 48 no 5 pp 459ndash465 2001
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