Article
Induction and Evaluation of Somaclonal Variation in
Sugarcane (Saccharum officinarum L.) var. Isd-16
Mohashweta Roy1, Monzur Hossain2, Animesh Biswas1, Rafiul Islam2, Shipra Rani Sarker2
and Sharoni Akhter3
1Department of Botany, Govt. B. L. College, Khulna, Bangladesh.
2Plant Breeding and Gene Engineering Laboratory, Department of Botany, University of
Rajshahi, Rajshahi-6205, Bangladesh.
3Division of Genome and Biodiversity Research, National Institute of Agrobiological
Sciences (NIAS), Tsukuba Ibaraki, Japan.
Received July 23, 2010 Accepted August 23, 2010
Abstract
Plant regeneration was established through somatic embryogenesis for sugarcane
variety Isd-16 using leaf sheath explants. The explants were cultured onto MS medium
supplemented with different concentrations of 2,4-D for callus induction. The calli were
underwent embryogenesis and produced huge number of somatic embryos when they
were transferred onto MS medium fortified with L-proline. The embryos were
germinated and developed to plantlets on half-strength MS0 and successfully
transplanted into the experimental field and grown to maturity. A large number of
somatic embryos derived plants (SEDPs) were found morphologically different with
some distinct characters such as stool habit, tillering habit, tillering density, auricle,
legule, stalk colour and bud shape as compared to setts derived plants (SETDPs).
Significant differences were also noted between SEDPs and SETDPs in respect of stalk
height, tillers/plant, five internodes length/stem, single stalk weight, individual clump
weight, millable cane/clump, and stalk density. Biochemical properties of juice for
SEDPs and SETDPs were almost similar in SETDPs and SEDPs. Significant difference
between SEDPs and SETDPs was noted only in HR brix% of juice. Somaclonal
variations occurred among the plants regenerated through somatic embryogenesis
(SEDPs) could be used in breeding programme for the improvement of sugarcane
cultivars.
Key words: Embryogenesis, Somaclonal variation, Sugarcane.
INTRODUCTION
Sugarcane (Saccharum officinarum L.) is one of the economically important crops
widely cultivated in the tropics to subtropics and annually provides around 60 to 70 % of
the world’s sugar (Shah et al., 2009). It is an important food-cum cash crops and the
only source of sugar production in Bangladesh. It has long been observed that the
sugarcane varieties tend to run-out or decline in yield after a few years in a particular
area (Khan et al., 2009).The use of tissue culture for creation of somaclonal variation
can be used to increase the speed or efficiency of the breeding process to improve the
accessibility of existing germplasm of sugarcane and create new variation for crop
improvement. It has been recognized that all plants regenerated from tissue culture are
not exact replicas of a parental form and exhibit great variability in agronomic traits
(Heinz et al., 1977; Larkin and Scowcroft, 1983; Ramos Leal and Maribona, 1991). This
genetic alternation termed somaclonal variation (Larkin and Scowcroft, 1981), which is
being exploited to shorten the time needed to produce new breeding lines with
desirable traits. The somaclonal variation may be attributed to either (i) pre-existing
variation in the somatic cells of the explant (genetic) or (ii) variation generation during
tissue culture (epigenetic). Often both factors may contribute. Factors such as explants
source, explant age, duration of culture, number of sub-cultures, culture environment,
chemical additives or growth stimulants or regulators, media composition, the level of
ploidy and genetic mosaicism are capable of inducing in vitro variability (Silvarolla,
1992). Plant regeneration from calli, cells, protoplast have been shown to exhibit great
variability in agronomic traits reported in potato (Shepard et al., 1980), rice (Oono,
1978), maize (Green, 1977) and even in sugarcane (Heinz et al., 1977; Larkin and
Scowcroft, 1993 and Ramos Leal and Maribona, 1991). In sugarcane somaclones have
been identified with increased resistance to both Fiji and downy mildew disease
(Krishnamurthi, 1974; Krishnamurthi and Tlaskal, 1974) and eyespot disease (Ramos
Leal et al., 1996). Salt tolerance somaclones have also been generated by a tissue
culture cycle (Samad and Begum, 2000 and Khan et al., 2004). The present study was
undertaken with a view to evaluate the somaclonal variation in somatic embryo derived
plants of sugarcane var. Isd-16 which can be used in local agronomic field for mass
production with efficient output.
Methods and Materials
Plant material
Young shoot apics of 3, 6 and 12 months field grown clones of sugarcane var Isd-16
were used as the source of explants. The explants were provided by the Bangladesh
Sugarcane Research Institute (BSRI), Ishurdi, Pubna and maintained at the
experimental field of Botanical Garden, Department of Botany, University of Rajshahi,
Bangladesh.
Culture conditions
The pH of all media adjusted to 5.7 and autoclaved at 15 psi for 20 min at 1210C. Initial
cultures i.e. serial slices were cultured on agar (8mg/L) solidified medium in test tubes.
After shoot induction the explants were transferred onto MS medium in jars containing
agent. All cultures were incubated in a culture room at 25±20C under cool white
fluorescent light (60 µE/m2/s) with a 16 h photoperiod. Sub-culturing was carried out
every fortnightly.
Callus induction and multiplication
Apical portion of healthy shoots were stripped to the terminal bud and attached
immature leaf rolls were surface sterilized with 0.1% HgCl2 for 9 min. Approximately 3
slices (5 x 3 mm) were taken from each cylindrical leaf roll and cultured on MS medium
(Murashige and Skoog, 1962) supplemented with 1-5 mg/L 2,4-D alone and incubated
in dark for callus induction. When the callus was abundant, it was transferred to the
regeneration medium.
Protocol for somatic embryogenesis and shoot proliferation
Plantlets were regenerated through somatic embryogenesis from the embryogenic
callus derived from leaf sheath explants of sugarcane var. Isd-16 using the protocol
developed by Roy (2006). The embryogenic calli with induced shoot were transferred to
MS liquid medium supplemented with various concentrations of BAP (0-2mg/L) for
shoot proliferation and multiplication.
Field evaluation of somaclones
The plantlets after proper acclimatization were transplanted in the experimental field of
Botanical Garden, Department of Botany, University of Rajshahi, during 2004.
Conventional setts derived plants (SETDPs) also planted side by side as control. The
experiment was laid out in randomized block design with three replications. Each
replication consisted 5 x 3 m plots. Row to row and plant to plant distance were 1 m
and 0.3 m respectively. To evaluate the incidence of somaclonal variation among
somatic embryo derived field grown plants (SEDPs) and their control (SETDPs), data
on different qualitative and quantitative parameters were recorded after 8-12 months of
planting.
Biometric parameters
The biometric parameters viz. shoot habit, tillering habit, tillering density, auricle, legule,
stalk colour, bud shape, stalk height, tillers/plant, internodes length/stem, single stalk
weight, individual clump weight, millable cane/clump, and stalk density of both the
somatic embryos derived plants (SEDPs) and setts derived plants (SETDPs) were
taken at the age of 12 months.
Biochemical parameters
The biochemical parameters viz. brix per cent juice, pol per cent juice, purity per cent
juice, pol per cent cane, recovery percent and RS (Reducing Sugar) percent were
estimated in the laboratory using CSR method. The analysis was performed on six stalk
samples which were collected at the age of 12 months. The stalks were shredded using
a cutter grinder and juice squeezed with a hydraulic press at 60 lbs/sq.inch.
Statistical analysis
Student ‘t’ test was performed to find out the difference between the individual sett
derived plants (SETDPs) compared to somatic embryo derived plants (SEDPs) for
biometric and biochemical parameters.
Results
Callus induction
Explants showed variation for callus development in MS medium with different
concentrations of 2,4-D. 3 month old immature leaf sheath explants produced highest
calli (51%) in MS medium supplemented with 4 mg/L 2,4-D. Explants of 6 and 12 month
old leaf sheath developed optimum calli (91 and 62%, respectively) in MS medium
fortified with 3mg/L 2,4-D.
Differentiation and somatic embryogenesis
In the present study, Optimum (42%) somatic embryo induction from immature calli was
obtained in MS medium supplemented with 0.5 mg/L 2,4-D. Callus underwent
embryogenesis producing huge number of somatic embryos when sub-cultured on MS
medium supplemented with 25 mg/L L-proline. Plant regeneration was achieved (98%)
by transferring them onto half-strength MS0. The highest shoot multiplication was
achieved when BAP was at a concentration of 1.5mg/L in MS medium. Plantlets with
well-developed roots were transferred into poly bags contained compost for primary
acclimatization and then transplanted to the field.
Field evaluation of somaclones
In the current investigation, the researchers found that a large number of somatic
embryo derived plants (SEDPs) were morphologically different with some distinct
characters as compared to setts derived plants (SETDPs) (Table1). Variations in almost
all qualitative characters were noticed between SEDPs and SETDPs. The highest 60%
of SEDPs was exhibited variation in stalk colour. Among them 33% had brownish green
stalk, 20% had yellowish green and 7% had reddish green. Profuse tillering density was
another exceptional character found in SEDPs and 53% plants possessed this
character. Similar percentage (33%) of SEDPs showed variations in terms of outer
auricle and bud shape. Plant habit, tillering and internodes habits of some SEDPs were
also different from SETDPs. Results also reveals that occurrence of different types of
somaclonal variants were more frequent among SEDPs populations. Occurrence of
dwarf off-types plant was very common. Other morphological characteristics such as
short leaves, deformed internodes and semi-erect stem were found to be linked with
dwarfism.
Present findings showed that SEDPs of sugarcane var. Isd-16 possessed significant
differences from setts derived plants (SETDPs) populations as control in the
characteristics of stalk height, tillers/plant, five internodes length/stem, individual clump
weight, millable cane/clump, and stalk density.
Biochemical properties of juice for SEDPs and SETDPs were almost similar in SETDPs
and SEDPs (Table 2 and 3). Significant difference between SEDPs and SETDPs was
noted only in HR brix% of juice. These results of occurrence of somaclonal variations in
SEDPs populations in sugarcane var. Isd-16 due to tissue culture are more or less
concomitant with the previous studies. Moreover, these results elucidated those genetic
variations of sugarcane var. Isd-16 could open an opportunity for selection of elite
clones.
Discussion
Callus induction
Immature sugarcane leaves are established good explant source for callus production
(Brisbe et al., 1994; Chengalrayan & Gallo-Meagher, 2001, Shah et al., 2009) and has
been used to induce callus from various sugarcane explants (Oropeza and Garcia,
1996; Gallo-Meagher et al., 2000, Shah et al., 2009). Callus induction in Ms medium
fortified with 2,4-D was reported by Chen et al (1988). Behera and Sahoo (2009)
reported the highest percentage of callus induction in MS medium supplemented with
2.5 mg/L 2,4-D. High amount of calli (100%) were produced using 2,4-D in the
concentration of 3 mg/L with MS medium by Ather et al (2009).
Differentiation and somatic embryogenesis
Induction of somatic embryogenesis was reported by many workers in MS medium
supplemented with 2,4-D and coconut water (Ahloowalia and Maretzki, 1983; Ho and
Vasil, 1983) and in MS medium fortified with proline, ABA and 2,4-D (Gosal et al,
2003). Higher shoot regeneration was achieved on a medium containing only BAP at
concentration of 0.5 mg/L (Gosal et al, 2003).
Field evaluation of somaclones
Heinz and Mee (1971) found variations among callus-derived H 50-7209-14-24 plants
when compare to their control with respect to tops, dewlap, auricle shape, leaf sheath
colour and hair group. Sood et al. (2006) demonstrated that tissue culture derived
sugarcane var. CoJ 64 plants attained better height, millable cane height, a greater
number of live buds, increased cane yield and sugar recovery % as compared to
conventionally propagated sugarcane plants under parallel agronomic practices in the
field. They also reported that high tillering is resulted in thinner canes because
thickness of the canes is directly proportional to the number of tillers per clump and is
also related to the cytokinin effect. The tissue-cultured plants in the first year were
thinner but the thickness was increased in first ratoon and subsequent generations. Lat
and Lantin (1976) reported that some of the somaclones of sugarcane cultivars CAC
57-13 showed significant differences from the parental variety in cane diameter stalk
length and weight. Liu and Chen (1976, 1978 a, b) found significant variation amongst
sugarcane somaclones from eight varieties in characters such as cane yield, sugar
yield, stalk number, length, diameter, volume, density and weight, per cent fiber, auricle
length, dewlap shape, hair group and attitude of top leaf. Some of this somaclones
showed significant improvements over the parental performance. Siddiqui et al. (1994)
compared the brix % of canes of somaclones with those of their parents and found the
somaclones were better than their parents in this character. On the other hand, Khan et
al. (2004) reported that brix % of canes of somaclones was less than those of their
parents. They also reported that the somaclones were found better in the characters of
tillers/plant, stalk height, number of nodes/stem and root band width but they found no
difference in the length of internodes of somaclones and source plants.
The original ploidy level of the plant or the plant organ from which the explants were
taken may play an important role in such somaclonal variations. Non-meristematic
explants generally produce genetic variability as compared to meristamatic explants.
However the cells in non-meristematic explants are the derivatives of meristematic part
of the plant and during their subsequent dedifferentiation, gross changes in their
genome may be arise including endopolyploidy, polyteny and amplification or
diminuition of DNA sequences (D’ Amato, 1989). Moreover, when the cells were
various genomic constitution of the initial explants are induced to divide in culture; the
cells may exhibit changes in chromosome number such as aneuploids and polyploids
but very often from these mixoploid callus cultures.
The culture environment specially the choice and particularly by the concentration of
growth regulators in the medium is influenced the somaclonal variation (Karp, 1992). It
is possible that growth regulators act as mutagens. The synthetic auxin (2,4-D) has
been shown to increase the frequency of blue to pink mutation in the Tradescantia
stamen hair system (Dolezel and Novak, 1984) and to induce significant increases in
the frequency of sister chromatid exchanges in root tip cells of Allium sativum (Dolezel
et al., 1987). However, there is a paucity of examples of this kind and most evidence
points to growth regulators influencing somaclonal variations during the culture phase
through their effects on cell division (Gould, 1984), the degree of disorganized growth
(Karp, 1992), and selective proliferation of specific cell types (Ghosh and Gadgil, 1979).
Conclusion
The cause of the morphological, agronomical and biochemical variations observed in
the present study, though not well defined but it may be linked with the use of synthetic
auxin (2,4-D) and inherent chromosomal instability in callus culture. Larkin et al. (1985)
evidenced that transposable elements play a vital role on somaclonal variation.
Whereas, Siddiqui et al. (1994) mentioned that one did not know the phase at which the
variation arise. According to them the variations are caused by a combination of
physical and chemical phenomenon. In the present study, somaclonal variations
occurred among the plants regenerated through somatic embryogenesis (SEDPs) also
could be used in breeding programme for the improvement of sugarcane cultivars.
Acknowledgements
We would like to extend thanks to the BSRI, Ishurdi, Pubna for providing researc
materials, laboratory facilities for conducting biochemical analysis. Special thanks to Dr.
Md. Amzad Hossain, Associate Cane Nutritionist, BSRI, Ishurdi, Pubna for giving
necessary information during the research work.
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Table 1. Morphological characters of conventional setts derived (SETDPs) and somatic
embryo derived plants (SEDPs) of sugarcane var. Isd-16. Data were recorded 7-8
months after planting.
- = No variation
Table 2. Mean performance for stalk characters of sett derived (SETDPs) and somatic
embryo derived plants (SEDPs) of sugarcane var. Isd-16 after 12 months of planting.
Characters
Sett derived plants
(SETDPs)
Somatic embryo
derived plants
(SEDPs)
Somaclonal variants
Exceptional char. in
SEDPs
%
Plant habit Erect Erect and semi-erect Semi -erect 13
Tillering habit Compact Compact and
spreading
Spreading 20
Tillering
density
Intermediate Intermediate and
profuse
Profuse 53
Leaf blade Green, curved near
tip
do
-
-
Outer auricle Straight transitional Straight transitional
and deltoid
Deltoid 33
Legule Crescent with broad
lozenge
Crescent with broad
lozenge and Broad
crescent
Broad crescent 13
Stalk colour
Green, greenish
yellow
Green, greenish
yellow, yellowish
green, brownish green,
reddish green
Brownish green,
yellowish green,
reddish green
60
Internode habit Cylindrical Cylindrical, bobbin and
conoidial
Bobbin and
conoidial
20
Bud Simple ovate Simple or narrow ovate Narrow ovate 33
Table 3. Mean performance for Juice characters of sett derived (SETDPs) and somatic
embryo derived plants (SEDPs) of sugarcane var. Isd-16. Data were recorded from 12-
months-old plants.
Characters
Setts derived plants
(SETDPs)
Somatic embryo derived
plants (SEDPs)
t value Range Mean ± SE Range Mean ± SE
Stalk
height
(cm)
198-250 221.05 ± 4.94 160-280 247.25 ± 6.50 **
Stalk girth
(cm)
2.0-2.5 2.15 ± 0.04 1.7-3.0 2.08 ± 0.74 ns
Nos. of
nodes/stem
20-28 24.75 ± 0.37 18-28 23.05 ± 0.63 ns
Five
internodes
length
(cm)/stem
40-60 51.49 ± 1.20 46.5-70 55.25 ± 1.37 *
Tillers/plant
(nos.)
5-8 6.25 ± 0.20 4-14 8.5 ± 0.55 **
Single
stalk
weight
(kg)
0.52-0.80 0.63 ± 0.02 0.53-0.80 0.61 ± 0.02 ns
Individual
clump
weight
(kg)
1.2-3.12 2.45 ± 0.12 1.74-4.0 2.80 ± 0.10 *
Millable
cane/clump
(nos.)
2-5 3.90 ± 0.19 3-5 4.55 ± 0.13 *
Stalk
density
(c.c)
0.55-1.12 0.73 ± 0.38 0.52-1.15 0.87 ± 0.40 *
Characters
Sett derived plants (SETDPs) Somatic embryo derived plants
(SEDPs)
t value Range Mean ± SE Range Mean ± SE
ns = non significant
* = significant at 5% level
** = significant at 1% level
ns = non significant
* = significant at 5% level
Figure legends
Figure 1. In vitro regenerated sugercane plant var. Isd-16 developed through somatic
embryogenesis.
Figure 2. Field grown micropropagated sugarcane var. Isd-16 after 2 months of
planting.
Figure3. Field grown micropropagated sugarcane var. Isd-16 after 12 months of
planting.
Figure 4. Internode, girth and colour differences among somatic embryo derived plants.
HR brix % juice 14.48-23.0 19.20 ± 0.49 19.0-23.0 20.55 ± 0.29 *
Pol % juice 11.88-17.87 14.97 ± 0.40 12.07-17.88 14.75 ± 0.42 ns
Purity % 57.41-89.30 74.92 ± 0.48 58.99-89.38 71.05 ± 2.58 ns
Recovery % 4.74-10.91 7.96 ± 0.47 4.74-10.91 7.25 ± 0.49 ns
Pole % cane 7.24-13.94 10.33 ± 0.56 7.24-13.94 9.91 ± 0.55 ns
R.S. % 0.86-3.0 1.83 ± 0.20 0.86-3.0 2.24 ± 0.12 ns
Figures 1-4. Roy et. al.,2010