Indian Journal of Fundamental and Applied Life Sciences ISSN: 2231-6345 (Online)
An Online International Journal Available at http://www.cibtech.org/jls.htm
2013 Vol. 3 (4) October-December, pp.17-26/Tiwari et al.
Research Article
ENHANCED IN VITRO REGENERATION OF TWO SUGARCANE
VARIETIES COS 8820 AND COS 767 THROUGH ORGANOGENESIS
Satish Tiwari1, Arvind Arya2 Poonam Yadav1, Purnima Kumari1 and *Sandeep Kumar1
1
Department of Biotechnology, NIET, NIMS University Rajasthan, Jaipur, India
2
Department of Biotechnology, Meerut Institute of Engineering and Technology, Meerut, India
*Author for Correspondence
ABSTRACT
Enhanced in vitro regeneration protocols for two sugarcane varieties CoS 8820 and CoS 767 were
developed. Effective and efficient callogenesis from different explants were obtained. Young leaf explant
showed maximum callus induction when cultured on MS Medium supplemented with 2,4-D (3.0 mg/l). In
case of CoS 8820, 100% callus formation was observed in leaf, 90% in shoot apical meristem and 60% in
pith. Similarly, in case of CoS 767 the best results were obtained in the leaf explant followed by shoot
apical meristem and pith where percentage of callus formation was 100%, 90% and 70% respectively.
Various auxins and cytokinins combinations were also tested and the best results were obtained at lower
concentrations. Highest proliferation rate and the morphogenic response of callus were observed in 3rd
subculture while subsequent culturing resulted in poor response. Further organogenesis was maximized
by increasing the time of incubation for more than 13 weeks. Variety CoS 767 showed better organogenic
response compared to CoS 8820. Rooting response (80%) was obtained in both the varieties using
different types and concentrations auxins along with different strength MS medium.
Key Words: Sugarcane, Callus, Organogenesis, in vitro, Subculture
INTRODUCTION
In vitro regeneration is a routinely used method for producing large clonal population for wide range of
plants. In present investigation, two important sugarcane varieties were investigated for their large-scale
propagation through callogenesis and organogenesis from different explants. Callus formation in
sugarcane has been obtained mostly from young leaves (Liao and Chen, 1981; Kasten, 1990; Aftab and
Iqbal, 1999; Parmessur et al., 2002; Van Der Vyver et al., 2013; Cho et al., 2013), shoot tips (Lee, 1987;
Baksha et al., 2002a) and immature inflorescences (Gallo-Meagher and Irvine, 1996).
In a comparison of callus formation from leaf explants and apical meristem, Most (1971) concluded that
the leaf explants took less line to initiate callus. Authors observed a significant interaction between
genotypes and media regarding number of days to callus initiation. Callus volume was found to be larger
for the leaf rather than the apical meristem explants. Kumari and Shahi (2002) also demonstrated the
interaction of media and genotype as regard to callus initiation and growth by using young scaly leaves of
four genotypes of sugarcane. Mamun et al., (2004) also observed the response on callus differed with
various genotypes, 2.4-D levels and explants sources.
In most of the studies made so far, callus was induced in the presence of auxin, either 2, 4-D or piclorain.
Varying concentration of 2,4-D is explored but most frequently used concentration is 3.0 mg/l alone
(Homhual et al., 2003; Ali et al., 2008) or in combination with CM (Virupakshi et al., 2002; Alam et al.,
2003) or BAP (Gupta et al., 1995). However, some cultivars of sugarcane respond to even high amount
(5.0 mg/l) (Lal, 2003) and some proliferate at low concentration (0.7mg/l) (Pawar et al., 2002).
For maintenance of callus, subculturing to new medium and lower concentration of 2, 4-D is normally
used. Lal (2003) maintained calluses of cv B.O. 91 at low 2, 4-D concentration (3.0 mg/l).
Lal and Singh (1994) also reported organogenesis in hybrid varieties CoS 91269 and CoS 687 by
completely eliminating auxin from the medium and using only BA (0.5—2.0 mg/l) developed at the base
of 85% of these shoots with NAA (1.0 mg/l) and formed plantlets.
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Indian Journal of Fundamental and Applied Life Sciences ISSN: 2231-6345 (Online)
An Online International Journal Available at http://www.cibtech.org/jls.htm
2013 Vol. 3 (4) October-December, pp.17-26/Tiwari et al.
Research Article
Baksha et al., (2002b) obtained best shoot regeneration on MS medium supplemented with high cytokinin
and low auxin (1.0 mg BA/l and 0.5 mg NAA/l). Regenerated shoots were rooted on both full and half
strength MS medium supplemented with NAA. Derived plantlets, showed satisfactory establishment in
soil. In addition to that, Karim et al., (2002) observed genotype specific requirement of auxin for
regeneration of shoots in three sugarcane cultivars. They also observed that calluses induced in MS
medium, containing 2, 4-D showed better regenerating capacity than the MS medium containing NAA.
MATERIALS AND METHODS
Callus Induction
For callus induction MS medium supplemented with different combinations of different hormones
(auxins and cytokinins) were tested. Sixteen different combinations of auxins and cytokinins were used in
MS medium. Shoot apical meristem, spindle leaves and pith parenchyma in different sizes were used for
the induction of callus. Calluses were grown for 28-30 days. After 30 days data was recorded on the
frequency of callus formation. Callus color, type of callus i.e. embryogenic and non-embryogenic callus.
Organogenesis
Different experiments were conducted to investigate the regeneration potential of callus. To standardize
the medium for regeneration frequency of callus, different concentrations of growth hormones were used.
To find the regeneration ability of undifferentiated somatic embryos, they were first transferred to MS
basal medium and then into media supplemented with different concentrations of auxins and cytokinins
and the regeneration potential of embryos were observed.
RESULTS AND DISCUSSION
1.1 Callus Formation
Callus could be obtained from explants of almost any part of the original plant. However, callus formation
was greatly influenced by type of explants, culture medium, photoperiod and temperature.
1.1.1 Relative Potential of Different Explants for Callus Formation
Among various types of leaves, young and newly formed leaves resulted in better callus formation. When
mature leaves were used mostly rooting callus was formed, which did not show further proliferation or
generation response. On the other hand, explant from pith proved less efficient for callus formation and
growth (Hoque and Mansfield, 2004) also confirmed that younger explants were more efficient in callus
induction. However, the inner leaf explants provided larger callus volume than explants from apical
meristem. (Sorory and Hosien-Zadeh, 2000; Rahman et al., 2008) and (Shahnewaz et al., 2004) also
reported the better performance for callus formation from leaf explants. (Cui et al., 2002) also used leaf
explants of wheat for callus induction.
1.1.2 Effect of Medium Composition on Callus Growth and Development
MS medium (
Graph 1 and Graph 2) containing sixteen different concentrations of auxins, cytokinins and auxincytokinin combinations were formulated to find the most suitable medium for optimum callus induction
and proliferation.
1.1.2.1 Auxins
Among the various concentrations of auxins used for callus induction, the concentration of 2, 4-D at
3.0 mg/l proved best for maximum callus induction and proliferation in all kinds of explants of both the
varieties. At this concentration the rate of callus formation in CoS 8820 was 100% in leaf, 90% in
meristem and 60% in pith while in CoS 767 it was 100%, 90% and 70% in leaf, meristem and pith
respectively. Increase or decrease in the concentration of 2, 4-D adversely affected the rate of callus
formation and growth. At low concentration of 2, 4-D (i.e. 2 mg/l) the rate of callus formation was
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Indian Journal of Fundamental and Applied Life Sciences ISSN: 2231-6345 (Online)
An Online International Journal Available at http://www.cibtech.org/jls.htm
2013 Vol. 3 (4) October-December, pp.17-26/Tiwari et al.
Research Article
decreased. It was 80% in leaf, 70% in meristem and 40% in pith in CoS 8820 and 80%, 70% and 50% in
leaf, shoot apical meristem and pith in case of CoS 767 (
Graph 1). Almost similar results were obtained when the concentration of 2, 4-D was increased to 4.0
mg/l. It was also observed that explants of variety CoS 767 were more amenable for callus formation as
compared to CoS 8820 (Graph 2) (Fitch and Moore 1990) reported the better performance of 2,4-D for
callus induction and proliferation in sugarcane. Present study also reaffirms the unnecessary role of
cytokinins in callus forming medium. However, high concentration of 2, 4-D for maximum callus
induction is also reported by (Lal and Singh, 1994; Somashekhar et al., 2000).
Four different combinations of BAP and NAA were also used (
Graph 1) but these two auxins together in different combinations did not show good results for callus
induction or proliferation in both the varieties, therefore, combinations of these two auxins were not used
in further experiments.
1.1.2.2 Auxin and Cytokinin
Eight different auxin–cytokinin combinations were tested for callus formation and proliferation (
Graph 1, Graph 2) The combination of 2, 4-D with BAP did not show good results in variety CoS
767 while in CoS 8820 this combination proved effective (
Graph 1). In variety CoS 8820, combination of 2.0 mg/l 2, 4-D along with 0.5 mg/l of BAP did not show
good results for callus formation. By increasing the concentration of 2, 4-D to 3.0 mg/l with same amount
of BAP (0.5 mg/l) rate of callus formation was enhanced. However, when the concentration of BAP was
decreased to 0.25 mg/l, good results were obtained not only for callus formation but also for callus
proliferation. The percentage of callus induction was 90%, 80% and 60% in leaf, shoot apical meristem
and pith respectively.
A combination of 2, 4-D and kinetin was promotive for CoS 767, but the results were not good for
CoS 8820 (
Graph 1). In case of CoS 767, in medium containing 3.0 mg/.l of 2,4-D in combination with 0.5 mg/l of
kinetin showed good results for callus formation. It was 90%, 60% and 50% in leaf, shoot apical
meristem and pith, respectively but proliferation of callus was not satisfactory. Further increase in the
concentration of 2, 4-D or decrease in the concentration of kinetin, rate of callus formation was decrease
(Graph 2). From the data it is concluded that 2, 4-D alone provided best for callus growth and
development as compared with any other combination in both the varieties.
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Indian Journal of Fundamental and Applied Life Sciences ISSN: 2231-6345 (Online)
An Online International Journal Available at http://www.cibtech.org/jls.htm
2013 Vol. 3 (4) October-December, pp.17-26/Tiwari et al.
Research Article
Graph 1: Effect of different hormones on callus growth and development in variety CoS 8820
Graph 2: Effect of different hormones on callus growth and development in variety CoS 767
1.1.3 The Effect of Subculturing on Callus Growth
The proliferation response of callus varied respect to sub culturing. Callus induced from different explants
was less prolific as compare to subsequent cell cultures. One gm of callus was used for inoculation in
each cell culture. Proliferation response increased weeks of culturing and highest proliferation response
was noticed at 3rd subculture. At this stage callus induced from the explant gained maximum weight (1.98
gm in CoS 8820 and 1.84 gm in CoS 767), followed by short apical Meristem explant (1.94 gm in CoS
8820 and 1.45 gm in CoS 767). From fourth subculture onward there was gradual decrease in callus
growth and proliferation (Graph 3).
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Indian Journal of Fundamental and Applied Life Sciences ISSN: 2231-6345 (Online)
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2013 Vol. 3 (4) October-December, pp.17-26/Tiwari et al.
Research Article
Graph 3: Effect of subculture on callus growth
1.1.4 Morphogenic Response of Callus
Morphogenic the response of callus (induction of short and root primordia) was dependent on incubation
period and type of explant.
1.1.4.1 Effect of Subculturing on Morphogenic Response
It was noticed that when callus derived from different explants was transferred to first subculture, no
morphogenic response was observed. However, in second subculture both from leaf and shoot apical
Meristem 20 % and form pith 5% morphogenic response was noticed. Maximum morphogenic response
was noted in third subculture where 50% morphogenic and response was observed in leaves followed by
shoot apical Meristem (45%) and pith (25%). In fourth subculture no further increase in morphogenic
response was observed and necrosis initiated which increased in subsequent subculture. In sixth and
seventh sub culturing the morphogenic response of all the explants was zero and in seventh subculture
almost all the calli became dead (Graph 4).
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Indian Journal of Fundamental and Applied Life Sciences ISSN: 2231-6345 (Online)
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2013 Vol. 3 (4) October-December, pp.17-26/Tiwari et al.
Research Article
Graph 4: Proliferation and morphogenic response of callus on sub-culturing from different
explants
1.2 Organogenesis
Two types of callus i.e. morphogenic and non-morphogenic, obtained from third subculture after eight
weeks of inoculation was what the subculture and either on growth regulators free or supplemented MS
medium for efficient organ induction.
1.2.1 Organogenesis from Morphogenic Callus
When morphogenic callus of both the varieties was transferred to growth-regulator-free MS
medium, the rate of regeneration was almost same for both the varieties, which were 60%, 40% and 20%
in CoS 8820 and 60%, 60% and 20% in CoS 767 in leaf, shoot apical meristem and pith, respectively (
Graph 5). Meristematic cells of callus retain relatively higher totipotency and swiftly induce the organ
formation even on the basal medium (Siddiqui, 1993).
To further accelerate the rate of regeneration, MS medium was supplemented with different
concentrations and combinations of the BAP and kinetin.
In case of variety CoS 8820, maximum shoot differentiation was obtained in fifth subculture in MS
medium supplemented with 1.0 mg/l BAP (
Graph 5). At this concentration 70% shoot induction was observed in callus obtained both from leaf and
shoot apical meristem and 40% in callus from pith explants.
In case of CoS 767 the best organogenic response was obtained in MS medium supplemented with 0.
25mg/l BAP and 0.25mg/l Kn. At this concentration 70%, 60% and 40% shoot induction was observed
from callus of leaf, shoot apical Meristem and pith respectively.
Among the callus obtained from different explants, the callus derived from leaf showed better results
for organ (shoot and root) induction, followed by shoot apical Meristem and pith explants respectively (
Graph 5).
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Indian Journal of Fundamental and Applied Life Sciences ISSN: 2231-6345 (Online)
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2013 Vol. 3 (4) October-December, pp.17-26/Tiwari et al.
Research Article
Graph 5: Organogenesis from morphogenic callus in variety CoS 8820
Graph 6: Organogenesis from morphogenic callus in variety CoS 767
1.2.1.1 The Effect of Incubation Time and Subculturing of Callus on Organogenesis
Maximum organogenic response from morphogenic callus was noticed after 10 weeks of inoculation
in fifth subculture. By increasing the time of incubation for more than 13 weeks that is 6 th subculture, rate
of organogenesis was decreased and rate of necrosis was increased (
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Indian Journal of Fundamental and Applied Life Sciences ISSN: 2231-6345 (Online)
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2013 Vol. 3 (4) October-December, pp.17-26/Tiwari et al.
Research Article
Graph 7). After 15 weeks i.e. in 7th subculture 60 % callus became dead with very poor organogenic
response.
Graph 7: Effect of incubation time and sub-culturing on organogenesis of morphogenic callus.
1.2.1.2 Organogenesis from Callus
The maximum organogenic response obtain was 40% from leaf explant in both the varieties, while
explants from parenchymatous pith did not show more than 10% of organogenic response (
Graph 8, Graph 9). Of the two varieties CoS 767 showed better organogenic response from leaf and shoot
apical Meristem as compared to CoS 8820 for the same explants.
24
Indian Journal of Fundamental and Applied Life Sciences ISSN: 2231-6345 (Online)
An Online International Journal Available at http://www.cibtech.org/jls.htm
2013 Vol. 3 (4) October-December, pp.17-26/Tiwari et al.
Research Article
Graph 8: Organogenesis from non-morphogenic callus in variety CoS 8820
Graph 9: Organogenesis from non-morphogenic callus in variety CoS 767
1.2.1.3 Effect of Subculturing and Incubation time on Organogenic Potential of Callus
For organogenesis from non-morphogenic callus the maximum organogenic response of callogenic
tissues was obtained after 10 weeks in 5th subculture. In variety CoS 8820 regeneration was 40%, 30%
and 10% in leaf, shoot apical meristem and pith respectively while in CoS 767 shoot regeneration was
obtained in leaf and shoot apical Meristem and 10% in pith (Yeoman and Forche, 1980) also reported that
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Indian Journal of Fundamental and Applied Life Sciences ISSN: 2231-6345 (Online)
An Online International Journal Available at http://www.cibtech.org/jls.htm
2013 Vol. 3 (4) October-December, pp.17-26/Tiwari et al.
Research Article
the loss of morphogenic competence is directly correlated with age of culture (Moreno et al., 1985) also
confirmed that morphogenic potential of embryogenic callus decreased with an increasing number of
subcultures. By increasing the incubation time a number of subculture the rate of organogenesis was
decreased, while rate of necrosis was increased in both the varieties. At 4th subculture in organogenic
medium 70% necrosis of callus was obtained in all both the varieties.
1.2.2 Rooting of Induced Shoots
The shoot produced both from morphogenic and non-morphogenic calluses were subjected to half as well
as fully as strengths of MS medium both in basal form as well as in combination with different auxins for
rooting. Shoots grown on MS basal medium either in half or fully strength did not show good rooting
response. Only 20% rooting was obtained both at half as well as full strength MS basal medium within 14
days of inoculation. However, when full strength MS medium was supplemented with 2.0 mg/l NAA,
90% rooting was obtained, within seven days of incubation in CoS 8820 medium and six days of
inoculation in CoS 767. Numbers of roots were also higher at this concentration. It was 4 roots per
plantlets in CoS 8820 and 3.4 roots per plantlet in CoS 767. An increase or decrease in the concentration
of NAA, not only rate of root induction a number of roots per plant was decreased but days for root
formation were also increased. MS medium supplemented with 1.0 mg/l NAA and 2.0 mg/l IBA also
gave good rooting response under this treatment, 80% rooting was obtained in both the varieties, within
seven days of inoculation. By increasing the concentration of either IBA, or NAA, rate of root induction
was decreased. Among both the varieties CoS 767 showed better rooting as compared to CoS 8820.
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