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Belarmino and GonzalesAnnals of Tropical Research 30[2]:22-33(2008)
VSU, Leyte, Philippines
Somatic embryogenesis and plant regeneration in
purple food yam (Dioscorea alata L.)
Marilyn M. Belarmino1 and Jocelyn R. Gonzales2
1Department of Horticulture, College of Agriculture, Visayas State University, Visca,
Baybay, Leyte 6521-A, Philippines;2Laboratory of Vegetable Science, Faculty of Horticulture, Chiba University, 648
Matsudo, Matsudo City, Chiba 271-8510, Japan
AbstrAct
A udy wa ondued o ealih a eliale poedue fo omai emyogenei
and plan egeneaion fom allu ulue of puple food yam (Dioscorea alata L.). the
poedue involved hee ep; (1) ulue of nodal em egmen fom geenhoue-
gown plan o geneae in vitro planle; (2) induion of allu fom he leaf, peiole
and nodal em iue; and (3) iniiaion of omai emyo fom allu. reul howed
that the agar-solidied Murashige ad Skoog (MS) medium cotaiig 30 gl-1 uga,0.1 gl-1-cysteie , 10 mgl-1 calcium patotheic acid, 2.0 mgl-1 asparagie, 2.0 mgl-1
argiie, 80.0 mgl-1 adenine ulfae (AdsO4) ad 0.1 mgl-1 naphhalene aei aid (NAA)
effectively broke dormacy of lateral buds of odal stem cultures from both VU-2
and Kinampayvaieie. Poduion of muliple adveniiou hoo oued afe
trasfer of i vitro odal pieces to the same medium added with 1.0 mgl-1 enzylamino
purie (BAP) or, MSA medium. Callus was effectively iduced from the vegetative
tissues i MS medium added with 1.0 mgl-1 2,4-Dihloophenoxy aei aid (2,4-D)
o, wih piloam. Among he hee ype of explan, he nodal em wa he mo
uiale whih podued puplih nodula emyogeni allu. A highe peenage of
odal stem-derived calli produced globular embryos i MS medium cotaiig 1.0 mgl-12,4-D ad 0.5 mgl-1 BAP, or i 1.0 mgl-1 picloram ad 0.5 mgl-1 bAP han, in he plan
gowh egulao-fee medium (onol). the mauaion of emyo wa failiaed y
oe-moth culture i MS medium cotaiig 0.1 mgl-1 ABA ad 100 mgl-1 gluamine.
thi ep impoved he geminaion of omai emyo in one-half engh PGr-
free MS medium cotaiig 100 mgl-1 gluamine (egeneaion medium). All omai
emyo-deived planle wee mophologially nomal and ealihed well in oil.
Keywod: allu, muliple adveniiou hoo, nodal em explan, planle, omai
emyo
Correspondence: M. M. Belarmio Address: AVRDC-The World Vegetable Ceter, Regioal
Ceter for Africa, P.O.Box 10, Duluti, Arusha, Tazaia.E-mail:mailyn.elamino@woldveg.
og Tel No. +255 27 2553093/2553102Fax No. +255 27 2553125
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Somatic embryogenesis in purple food yam (Dioscorea alata L.) 23
InTRODUCTIOnAomai puple vaieie of geae yam (Dioscorea alata L.) ae
among he impoan food yam ha ae highly ough eaue of hei
ulinay value. Depie hei eonomi impoane, howeve, poduion
is still low due to limited supply of platig materials ad lack of
eiane o vial and fungal dieae (saleil et al. 1990). Although
in vio mulipliaion ofDioscorea peie ha een pefomed uing
explats sice 1980s (cited by Shu et al. 2005), this techique has ot bee
ouinely applied in puple yam due o iue oxidaion and neoi.so fa, epo of de novo egeneaion inD. alata wee limied o
allu deived fom peiole explan (Faue et al. 1985) ad somatic
embryos derived from root cells (Twyford ad Matell 1996) of white
yams. Likewise, somatic embryogeesis has bee reported i several
Dioscorea species (Osifo, 1988; Twyford ad Matell 1996; Shu et al.
2005) but these did ot iclude the purple yams. To date, the techical
kow how o the productio of ulimited umber of somatic embryos,
whih ae unifom in ize and hape and eain he apaiy o give ieto ormal plats, is ot available for ay species (Petrivica, 2004).
In hi pape, we epo he poduion of plan fom puple vaieie
ofD. alata hough he induion of allu and omai emyo uing
exied nodal em of in vio plan. the effe of plan gowh
egulao on adveniiou hoo fomaion, allu induion, omai
emyo fomaion and plan egeneaion ae deied.
MATERIALS AnD METHODS
Preparation of explants
Geenhoue-gown plan (2 monh-old) fom wo puple vaieie
ofD. alata, `VU-2` ad `Kinampay(Type D, LA-609) were used as
oue of nodal em explan fo iniial in vio ulue. Nodal explan
wee pepaed fom vine ha wee hooughly wahed wih ap waead the soaked i 0.1% (v/v) of fugicide solutio (Belate; Dupot,
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Belarmino and Gonzales24
Wilmigto, Delaware, USA) for 10 mi followed by risig i sterile
diilled wae fou ime. Finally, he explan wee ufae eilized
i a commercial chlorie bleach solutio (cotaiig 4.5% chlorie) for
20 miutes ad the, rised four times i a sterile distilled water prior to
cuttig ito sigle ode stem pieces about 10-15 mm log.
Culture medium and incubation condition
The MS basal medium (Murashige ad Skoog 1960) cotaiig 30
gl-1 sugar (commercial white table sugar), 0.1 gl-1-cysteie , 10 mgl-1
calcium patotheic acid, 2.0 mgl-1 asparagie, 2.0 mgl-1 argiie ad 6gl-1 agar (Proadisa, Madrid, Spai) was used throughout this study. The
pH of the medium was adjusted to 5.8 prior to autoclavig at 1.1 kg cm-2
(121 C) for 20 miutes. newly-ioculated explats were icubated i
dark for oe week followed by exposure to 16 hours of daylight (supplied
by two pieces of 40-W uorescet tubes) at 26 1 C.
Nodal stem culture of purple D. alata and adventitious shoot
formation
To break dormacy of lateral bud, odal stems were ioculated
idividually i glass vial (25 mm 95 mm) cotaiig 10 ml of medium.
The medium was supplemeted with 1.0 mgl-1 BAP ad 0.1 mgl-1 NAA
(t1); 2.0 mgl-1 BAP ad 0.1 mgl-1 NAA (t
2); 80 mgl-1 adenine ulfae
(AdsO4) ad 0.1 mgl-1 NAA (t
3); 100 mgl-1 AdsO
4ad 0.1 mgl-1 NAA
(t4). The medium lackig plat growth regulators (PGR) served as
onol (t0
). Twety-ve explats were ioculated for each treatmet
ad replicated four times. All odal cultures were kept i dark for oe
week ad the exposed to 16 h photoperiod for further eight weeks. The
explats were trasferred to fresh medium at oe week iterval util
lateral bud break ad shoot emergece.
Induction of multiple adventitious shoots
Nodal em-deived hoo onaining a lea fou node wee u
ino ingle node em piee and inoulaed veially in one-half engh
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Somatic embryogenesis in purple food yam (Dioscorea alata L.) 25
MS medium to iduce multiple advetitious shoot formatio. The rst
medium, MSA was supplemeted with 1.0 mgl-1 BAP, 80 mgl-1 AdSO4ad 0.1 mgl-1 nAA ad the other, MSB was added with 1.0 mgl-1 bAP
ad 0.2 mgl-1 nAA. The MS medium without plat growth regulator
served as cotrol (MSC). Five sigle ode stem pieces were ioculated
per culture bottle (60 mm 120 mm) cotaiig 30 ml of medium. Fifty
nodal em piee wee inoulaed fo eah eamen and epliaed fou
times. All odal cultures were kept uder 16 h photoperiod at 251 C. The
peenage of nodal explan poduing muliple hoo and he aveage
nume of hoo podued pe nodal explan wee deemined afe fouweeks of culture. The odal stem-derived shoots were maitaied by
anfeing hem o feh medium a one monh ineval.
Induction of callus from vegetative tissues
Stem (5 mm log), leaf (5 mm 5 mm), ad petiole (5 mm log)
piee oained fom one monh old in vio planle ofKinampayand
VU-2 were evaluated for callus formatio usig 1.0 mgl-1 of 2,4-D,
nAA, ad picloram that were added idividually ito the MS medium.
the medium wihou PGr eved a onol. the mo uiale ype
of explan (i.e., nodal em) and PGr (i.e., 2,4-D and piloam) wa
deemined aed on he highe peenage of allu fomaion. to
improve the quality of callus, the experimet was repeated usig 0.2,
0.5, 1.0 ad 2.0 mgl-1 of 2,4-D ad picloram. The quality of the callus
wa evaluaed afe wo monh. Fo oh expeimen, he explan wee
ioculated i glass vial (25 mm 95 mm) cotaiig 15 ml of treatmet
medium. Twety-ve explats were cultured per treatmet medium adreplicated four times. The explats were icubated i dark at 251 C for
one monh and uulued o feh medium wo ime.
Induction of somatic embryogenesis and regeneration of plants
to iniiae omai emyo fomaion, he emyogeni alli
derived from odal stem explats were trasferred ito agar-solidied
MS medium composed of macroelemets with reduced ammoiumitrate (800 mgl-1), mioelemen and viamin a full onenaion,
30 gl-1 sugar, ad 0.10 gl-1-yeine (iniiaion medium). the iniiaion
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Belarmino and Gonzales26
medium was added with combiatios of 1.0 mgl-1 2,4-D ad 0.5 mg/L
bAP (s1), or 1.0 mgl-1 picloram ad 0.5 mg/L BAP (S2). the mediumwihou PGr eved a onol (s
0). Twety calli (0.5 g fresh weight per
callus) was ioculated for each treatmet medium ad icubated uder 16
h photoperiod at 251 c. Afe wo monh, he alli onaining gloula
somatic embryos were divided ito small pieces (0.2 g fresh weight per
piece) ad the, subcultured idividually to agar-solidied MS medium
cotaiig 0.1 mgl-1 of lter sterilized abscisic acid (ABA) ad 100 mgl-1
gluamine (mauaion medium). Afe one monh, he peenage of alli
piee onaining geminaing emyo wa evaluaed. the geminaingembryos were trasferred to oe-half stregth PGR-free MS medium
cotaiig 100 mgl-1 gluamine (egeneaion medium) o ineae he
geminaion ae. the omai emyo-deived planle wee allowed
o gow in hi medium unil he poduion of a lea hee leave and
ve roots. The platlets were acclimatized at room temperature for oe
wk ad the, trasplated i a mixture of garde soil ad river sad
(1:1, v/v) ad maitaied i the greehouse. Percet survival of potted
planle wa deemined one monh afe anplaning. All expeimenwee ondued following a ompleely andomized deign. the analyi
of ample fom eah eamen wa evaluaed uing analyi of vaiane
(AnOVA,p
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Somatic embryogenesis in purple food yam (Dioscorea alata L.)
media and he onol (tale 1). t3
alo indued he highe peenage
of nodal explan wih emeging laeal ud. Ineaing he onenaionof AdsO
4to 100 mgl-1 (t
4) howeve, did no haen ud emegene.
Moreover, the medium cotaiig 1.0 mgl-1 BAP + 0.1 mgl-1 NAA (t1)
iduced bud emergece but this took loger compared to the medium
added wih ominaion of AdsO4
and NAA o, he onol. Ineaing
the cocetratio of BAP to 2.0 mgl-1 (t2) euled o he welling of he
laeal ud u did no haen hoo emegene. Adenine ulfae elong
to the cytokii group ad kow to iduce shoot formatio from excised
plat tissues (George ad Sherigto, 1984). Likewise, nAA is a auxiha i epoed o effeively indue hoo emegene when omined
with a adeie derivative (Torres, 1989).
The three media: MSA, MSB ad MSC (cotrol) iduced advetitious
hoo fomaion fom in vitro-deived nodal em explan ofKinampay
ad VU-2. MSA medium was the most effective for both varieties sice
it iitiated rapid lateral bud emergece (14.0 days i Kinampayand 12.4
days i VU-2), produced the highest percetage of shoot formatio (60%
i Kiampay ad 75% i VU-2) ad the highest average umber of
hoo pe nodal em explan (4.3 hoo in Kinampayad 5.0 shoots
i VU-2) (Table 2). The multiple advetitious shoot formatio was
meaued afe wo monh of ulue.
tale 1. Fomaion of hoo fom exied ingle nodal em explan ofD. alata vaieie
VU-1 ad Kinampay(KI) i agar-solidied Murashige ad Skoog mediumonaining ominaion of NAA and bAP, and adenine ulfae (AdsO
4) and
NAA.
teamen Ave. no. of Peen hoo Ave. no. of
(mgl-1) days to bud emergece shoots/odal
emegene explan
VU-2 KI VU-2 KI VU-2 KI
t0; 0.0 52.0a 60.0b 30.0b 0.0 1.0 0.0t
1; 1.0 BAP+0.1 nAA 57.0a 65.0a 15.0c 0.0 1.0 0.0
t2; 2.0 BAP +0.1 nAA d* d 0.0 0.0 0.0 0.0
t3; 80.0 AdSO
4+0.1 nAA 30.0c 51.0cd 50.0a 30.0 2.0 1.0
t4;100.0AdSO
4+0.1 nAA 37.0b 55.0c 45.0a 30.0 1.0 1.0
*o data
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Belarmino and Gonzales28
Table2.Multipleadvetitiou
sshootformatioofodalstemexplatsderivedfrominv
itroplanlesofD.alatavar.
KinampayadVU
-2after2mothsofculture.
treamens
Ave.no.ofd
ays
Percenadv.
Ave.no.ofshoos
(mgl-1)
toadv.shoot
shootformatio
/odalexplat
formaion
D.a
latavar.Kinampay
MSA;1.0BAP+80AdSO4+0
.1nAA
14.01.2
60.41.7
4.30.2
MSB;1.0BAP+0.2nAA
21.21.5
30.70.8
3.20.1
MSC;cotrol
35.00.6
20.11.2
1.00.0
D.a
latavar.VU-2
MSA;1.0BAP+80AdSO4+0
.1nAA
12.41.3
75.51.1
5.00.2
MSB;1.0BAP+0.2nAA
20.41.0
60.51.0
4.10.2
MSC;cotrol
30.00.8
40.91.3
2.00.1
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Somatic embryogenesis in purple food yam (Dioscorea alata L.)
Figue 1. Peen allu fomaion fom leaf, peiole and nodal em explan ofD.
alata L. vaiey Kinampayafter two moths of culture i agar-solidied
MS medium cotaiig 1.0 mgl-1 of 2,4-D, NAA and piloam
Figue 2. Peen allu fomaion fom leaf, peiole and nodal em explan ofD.
alata L. variety VU-2 after two moths of culture i agar-solidied MS
medium cotaiig 1.0 mgl-1 of 2,4-D, NAA and piloam.
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Belarmino and Gonzales30
Induction of callus and somatic embryogenesis
2,4-D, nAA ad picloram at 1.0 mgl-1 iduced callus formatio from
leaf, peiole and nodal em iue ofKinampay(Fig. 1) ad VU-2
(Fig. 2) after six weeks of culture i the dark. However, these explats
did no fom allu in he medium wihou PGr (onol). the medium
added with 1.0 mgl-1 2,4-D o piloam indued highe peenage of
explats that produced callus compared to 1.0 mgl-1NAA. Geneally,
VU-2 exhibited higher percetage of explats that produced callus
ompaed o Kinampay. callu aed foming aound he edge of he
u aea of explan iue. Among he hee ype of explan, he nodal
em podued he highe peenage of puplih nodula emyogeni
allu (Fig. 3A). In ona, he leaf and peiole explan podued of
e o-embryogeic calli that were highly vacuolated. More tha 50%
of he leaf and peiole explan eame oally own and neoi due o
phenoli uane ha wee exuded fom he u poion (thoma and
Ravidra 1997). Tissue browig ihibits the productio of callus ad
i severe cases led to the death of tissue (Waez, 1987; Taji ad Williams
1996).
Induction of somatic embryogenesis and regeneration of plants
the emyogeni nodal-deived alli iniiaed omai emyo
(Fig. 3b) ha appeaed a puplih whie gloula uue afe eigh
weeks of culture i the treatmet media (S1
and s2) and onol (s
0). A
highe peenage of alli podued gloula emyo in he medium
cotaiig 1.0 mgl-1
2,4-D ad 0.5 mgl-1
bAP (s1), or 1.0 mgl-1
piloamad 0.5 mgl-1 bAP (s
2) han in he onol (s
0). the gloula emyo
uderwet sequetial stages of developmet ad the germiated oe
moth after trasfer i PGR-free MS medium (Fig. 3C). Embryo
germiatio, however, was low (12-20%) due to slow embryo maturatio.
Hece, the embryos were cultured i maturatio medium cotaiig 0.1
mgl-1 ABA ad 100 mgl-1 gluamine pio o anfe in he egeneaion
medium. thi ep impoved he geminaion of omai emyo. AbA
effeively nomalized he developmen of emyo and foeed nomalmaturatio (Ammirato, 1983). Forty-five ad sixty-two percet of
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Somatic embryogenesis in purple food yam (Dioscorea alata L.)
Figue 3. Induion of allu and omai emyogenei in puple D. alata L. (A)
nodal em-deived alli, (b) omai emyo, (c) geminaion of planle,
(D) rootig of platlets, (E) pottig of platlets, ad (F) establishmet of
plan in oil. (bar size; A=5mm; B=1mm; C, D & E=1 cm; F= 0.5 m)
31
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Belarmino and Gonzales32
AbA-eaed omai emyo ofKinampayad VU-2, respectively,
were coverted to platlets after three weeks of culture i regeeratiomedium cotaiig 100 mgl-1 glutamie (Fig. 3D). The beecial effects
of gluamine fo plan egeneaion wee alo epoed in he explan of
Dioscoreacomposita Hemsl. adD. cayenensis Lam.,D. composita and
D. cayensis (Viaa ad Matell 1988). The regeerated platlets were
trasplated i soil (Fig. 3E), established well i the greehouse (Fig.
3F)) ad produced oe to two tubers setts at 20-40 g per tuber sett.
the peen udy, heefoe, peen a mehod fo plan egeneaion
via he induion of omai emyo fom exied nodal em. theailiy o onol omai emyogenei in puple yam i a neeay
requisite for the developmet of a efciet biotechological tool with
appliaion in apid ma lonal popagaion, genei anfomaion,
articial seed productio ad cryopreservatio.
LITERATURE CITED
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632).Proc. 7th Symp. Intl. Soc. Trop. Root Crops, INRA, Guadaloupe.
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Somatic embryogenesis in purple food yam (Dioscorea alata L.) 33
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26
VIAnA, A. M. ad S. H. MAnTELL. 1989. Callus iductio ad plat regeeratio
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