results and discussion 4.1 tissue culture and sugarcane...
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Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 55
Results and Discussion
4.1 Tissue culture and sugarcane improvement
Tissue culture technique works as a bridge between the conventional breeding
and the high-tech molecular breeding. The developments in sugarcane tissue culture
have already boosted sugarcane improvement to a great extent. Recent advances in
sugarcane biotechnology, especially marker assisted selections and genetic
transformation has speeded up the progress being achieved in sugarcane improvement
in a precise and an efficient way. Tissue culture initiates with the successful
establishment of cultures and regeneration protocol. Even though various protocols
are available, improvements in variety specific protocols are needed to optimize their
use in embryogenic callus development and its efficient regeneration.
4.2 Callus initiation and regeneration
Embryogenic callus was established by using young inflorescence panicle
from 9-10 month old plants and nodal buds from vigorously growing sugarcane stalk
of variety CoC 671. PEG at (100 mg/l) improved the callusing, embryogenesis and
regeneration in sugarcane (Dalvi et al., 2012), (Table 13), (Fig.7a, b).
Table 13: Effect of PEG on callus development from inflorescence of variety
CoC 671.
Sr.No.
PEG mg/l
Test tubes Inoculated
(No)
Test tubes with callus
(No)
Callus development type
Remarks
1 0 20 18 Slow No greening
2 50 20 16 Yellowish compact No greening
3 75 20 17 Yellow compact No greening
4 100 20 15Yellow compact, globular, friable, some green calli
Fast development with direct shoot regeneration
5 125 20 13Yellow compact, globular, dry
Slow development
6 150 20 11Yellow compact, globular, dry, slight browning
Slow development Slightly reddish
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For the optimization of embryogenic callus/somatic embryos development
from different explants various variety specific protocols have been reported.
Different compounds viz. 2-4.D., benzyl adenine, kinetin, naphthalene acetic acid,
proline, abscisic acid, zeatin, thidiazuron, paclobutrazol have been tried and reviewed
by many workers from time to time (Basnayake et al., 2011, Gopitha et al., 2010;
Joyce et al., 2010; Lakshmanan et al.,2005, 2006a,b; Snyman et al., 2011).
Optimization in protocol for embryogenic callus development in sugarcane variety
CoC 671 has been reported (Dalvi et al., 2012; Desai et al., 2004, 2006; Patade et al.,
2006, 2008 a,b; Suprasanna et al., 2009). It is observed that callus development with
inflorescence tissue was faster and there were higher number of variants from the
inflorescence tissue callus than callus from the young leaf role discs.
Various reports have indicated that MS medium supplemented with 2-4.D (3
mg/l) is most efficient for embryogenic callus development and other amendments
viz. malt extract, casein acid hydrolyset and glutamine have boosted the development
further. Even though proline, zeatin, thiadiazuron reported for embryogenic callusing
and regeneration in sugarcane their further reports are scanty (Gallo-Meagher et al,
2000; Suprasanna et al., 2005). Munir and Aftab (2009) has reported that stress
related enzyme activity and soluble proteins were increased in 1% PEG treated callus
enhancing salt tolerance in sugarcane. In present study enhanced regeneration may be
because of PEG resulting direct somatic embryogenesis from inflorescence tissue.
It has been reported that abiotic stress is essential for somatic embryo
formation and plant regeneration (Chen and Dribenenki, 2004; Oleszczuk, 2006).
Incorporation of PEG in tissue culture medium for development of somatic embryos
in different plants has demonstrated that it has improved the process in terms of
number, maturity and regeneration. Use of PEG in medium for the production of
somatic embryos resembling the zygotic embryos has been reported in flax (Chen and
Dribnenki, 2004), conifer embryonic tissue (Winkle and Pullman, 2003;) enhancing
the quality and quantity of microspore-derived embryos of cruciferous species (Ferrie
et al., 2007) and microspore embryogenesis in barley (Oleszczuk et al., 2006). The
main advantage in using PEG was that it produced embryos that were
morphologically more similar to zygotic embryos with enhanced germination
capabilities. Additionally the beneficial effect of priming with PEG and NaCl has
been also reported for better regeneration of Brassica seeds (Srivastava et al., 2010),
Chapter 4 [Results and discussion]
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sugarcane callus (Munir and Aftab, 2009) and in shoot buds (Patade et al., 2009), in
vitro propagated shoots of different plants (Nowak and Shulaev, 2003).
4.3 Cotton as support matrix in sugarcane tissue culture
In present study a pilot experiment with four different cotton samples
without any analysis were used as support matrix with liquid medium for callus
initiation /regeneration showed varied performance (Fig.8). It was observed that with
cotton sample ‘D’, there was better shoot development (shoot number and shoot
height) compared to other cotton samples.
In tissue culture, use of liquid medium is advantageous in terms of easier
dispensing, uniform nutrients and temperature dispersion and cost as compared to
semisolid medium prepared with agar. However, liquid medium causes
hyperhydricity of explants and requires illuminated shakers which are not cost
effective. Agar is most widely used gelling agent in plant tissue culture, considered as
non-toxic and biologically inert. However, it has a lot of ionic contaminants and
accounts to 80% of medium cost (Scholten and Pierik, 1998). Therefore efforts were
made by research workers to develop an alternative support matrix to agar. An ideal
support matrix would be the one in which there is miniumum ionic contamination
after autoclaving the medium and which does not interfere with availability of
nutrients through medium to growing tissue. Agar has many disadvantages such as (1)
a chemically undefined substance, (2) costly, (3) contains impurities that may affect
the growth of the cultured plant cells and organs, (4) hinders dissolved oxygen to the
explants, (5) its low solubility and viscosity creates problems in dispensing it in
molten condition (Bhattacharya et al., 1994; Debergh, 1983; Jain et al., 2009;
Scholten and Pierik, 1998).
Different alternatives to gelling agents tried by different authors have been
reviewed by George et al., (2008). Various gelling agents / inert support matrices
viz. filter paper (Goodwin, 1966, Jaime and D.Silva, 2003), sago, isabgol, nylon cloth,
polystyrene foam and glass wool (Babbar and Jain, 1998;Bhattacharya et al., 1994),
wheat flour, laundry starch, semolina, potato powder, rice powder (Prakash et al.,
1993, 2003), vermiculite and paper pulp (Afreen -Zobayed et al., 2000), Sago (Naik
and Sarkar, 2001), xanthan gum (Jain and Babbar, 2006), glass beads (Goel, et al.,
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2007), silica sand (Prknova, 2007), Cassava floor (Kuria et al., 2008), corn and potato
starch (Mohamed et al., 2009), polyurethane foam, membrane rafts (Prasad and
Gupta, 2010), blends of alternative gelling agents with agar viz. guar gum, xanthan
gum, isabgol (Jain-Raina and Babbar, 2011) had been tried but none was found
suitable for the use. Various demerits have been attributed to their use in culture.
Although starch from different sources has been used as cheap alternative, it
gets metabolized, resulting in decreased medium consistency and reduced growth rate
of cultures. Which is because of significant ionic variation in elemental and organic
impurities affecting morphological and molecular responses of seedlings (Jain et al.,
2009; Jain and Babbar, 2002; Kuria et al., 2008; Naik and Sarkar, 2001). Starch
upon autoclaving, yields sugars which cause the enhancement of osmotic potential of
medium that could result in growth reduction. Nevertheless, these alternatives are also
uncertain in quality and they may be chemically unstable in hot acidic solutions.
Cellulose base support matrices viz. filter paper (Goodwin,1966; Jaime and
D.Silva, 2003), paper plugs- sorbarods (Roberts and Smith, 1990), paper pulp and
vermiculite mixture-Florialite (Afreen-Zobayed et al., 2000), coir (Gangopadhyay et
al., 2002), Luffa fiber (Gangopadhyay et al., 2004), sugarcane bagasse (Mohan et al.,
2005), absorbent cotton (Dalvi et al., 2011; Khan et al., 2001; Moraes-Cerdeira et al.,
1995; Shah et al., 2009) has been reported as good alternatives to agar. It has been
reported that the cellulose base support matrices buffers antibiotic’s phytotoxic effect
and influence growth of callus/plants positively. Among all these cellulose based
alternative matrices, absorbent cotton was uniform in quality, free from non-cellulose
compounds (hemicelluloses, waxes, pectin, proteins), carried no contaminating ions
with it and available in ready to use form. Further, absorbent cotton is easily available
in local market by different manufacturers and extremely cheap in comparison with
filter paper/Sorboards/agar price.
Cotton does not absorb water to a greater extent, is insoluble in organic
solvents and does not undergo hydrolysis below 3000C. For making cotton absorbent,
scouring and maceration of the fiber is done. Scouring is an alkali treatment process
helps in removal of non cellulosic impurities, waxes and increases fiber
hydrophilicity. This process also alters super molecular structure and fiber
morphology that in turn alters the mechanical properties, as well as increased affinity
to aqueous medium (Sauperl, 2009). Traces of chemicals used in scouring process
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were found contaminating the absorbent cotton during its use in tissue culture.
Therefore attempts were made to evaluate absorbent cotton quality before its use as an
alternative support matrix in sugarcane tissue culture
4.3.1 Variations in pH, EC and sodium content of leachet of cotton samples
In order to determine its primary suitability as support matrix, pH , EC and
sodium content of leachets of absorbent cotton samples under study were analyzed
(Fig. 9). There was little difference in pH of absorbent cotton samples tested (Fig. 9a).
Sample ‘D’ indicated the lowest pH value (6.47 ± 0.05), sample ‘A’ indicated highest
pH vale (7.71 ± 0.08), cotton sample ‘B’ indicated pH 7.51 ± 0.08 and sample ‘C’
indicated pH 7.37 ± 0.08. Among these pH of agar was 5.3 ± 0.09 which was lowest.
The electrical conductivity (EC) of sample ‘A’ was highest (0.12 ± 0.001
S/cm2) and sample ‘D’ was lowest (0.038 ± 0.002 S/cm2) among four cotton
samples. It indicated low amount of ionic contaminants in sample ‘D’ (Fig. 9b). Low
ionic contaminants indicated the proper processing of cotton while making it
absorbent. The EC of leachet from agar sample was 0.057 ± 0.002 S/cm2 (Fig.9 b).
Cotton sample ‘D’ showed lowest amount of sodium (0.63 ± 0.49 ppm) and cotton
sample ‘A’ showed highest sodium content (12.07 ± 0.55 ppm). Sodium content in the
agar sample was 21.2 ± 0.56 ppm. Variation in pH and EC in cotton samples was
attributed to insufficient washing during the scouring and maceration process. The
cotton sample ‘D’ showed the minimum EC level (Fig. 9b). Chloride contamination
was observed visually and lowest browning in sample ‘D’ was noticed. These
observations indicated that cotton sample ‘D’ was best support matrix for tissue
culture use.
4.3.2 Drift in pH of tissue culture medium
A drift in pH after autoclacing of the medium towards alkaline or acidic range
was observed for different cotton samples tested (Fig. 10). To determine the ability of
support matrix on pH stability after autoclacing, pH of the medium was adjusted to
different pH between 4.0 to 6.0 before the addition of agar or cotton. Determination of
pH of the sterilized medium revealed that in agar infused medium drift in pH of the
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medium was observed at various pH ranges. Present study indicated no pH drift in
sample ‘D’ within the pH range 5.6 to 6.0 (Fig. 10). Similar to sample ‘D’ sample ‘C’
also stabilized pH of the medium at 5.6. This is attributed to the individual buffering
capacity of samples ‘C’ and ‘D’.
While preparing any plant tissue culture medium, pH is usually adjusted to 5.6
or 5.8 before its autoclacing and changes in pH after medium autoclacing and during
the explant’s in vitro growth are normally not monitored. The drift in pH of the liquid
or semisolid medium after autoclacing is a common phenomenon (Owen and
Wozniak 1991; Sarma et al., 1990; Williams, et al., 1990). The post autoclacing drift
in pH of the medium is almost unavoidable due to many chemical reactions during
medium autoclacing. Skirvin et al., (1986) noted that decrease in pH was significantly
correlated with the original pH of the medium (i.e. pH before autoclaving).
Observations in present study has significance, as drift in pH of the medium was
prevented by buffering action of cotton samples.
pH of the medium is one of the important factors of the physico-chemical
environment during plant tissues development under in vitro conditions; which gets
modified during growth and development of explants. Optimum pH is essential for
better plant growth as the suboptimal pH levels leading to abnormalities in
development of explants (Anderson and Ievinsh et al, 2008; Lal et al., 1995;
Ostrolucka et al., 2010; Patil et al., 2010; Piza et al., 2003; Shibili et al., 1999)
George et al., (2008) have stated that explant expends a certain amount of energy to
maintain pH of the culture medium surrounding the explants to ensure its optimal
growth. It is revealed from the present studies that absorbent cotton’s role as support
matrix in prevention of the drift in pH of the medium and stabilizing it at 5.8 ± 0.01 is
very significant. Similar results were also observed in commercial potato
micropropgation, where absorbant cotton was used as low cost support matrix (Dalvi
et al., 2011). It was clear from the present investigation that cotton sample ‘D’ was
good as support matrix for the use in tissue culture studies because of prescence of
low ionic contaminants and ability to prevent drift in pH of medium after autoclacing.
An experiment was carried out with different cotton samples and agar to find
out the buffering capacity of different absorbent cotton samples. Amount of 0.2N
NaOH required to raise the pH of cotton fiber incorporated and agar incorporated
medium to 8.0 were determined. It was observed that sample ‘D’ required highest
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amount of NaOH (84.0 ± 4.0 l of 0.2N NaOH) to raise the medium pH to 8.0 while
agar required the least (43.33 ± 3.3 l of 0.2N NaOH). This indicated that sample ‘D’
has higher buffering capacity than agar which is almost double than agar.
Organic acids or synthetic buffers have been used in plant tissue culture to
stabilize pH of the medium. MES is widely used buffer in plant tissue culture medium
having buffering capacity in the pH range 5-6 but in some cases it has also shown
toxic effects, inhibition of root primordia development and precipitation of
manganese (George et al., 2008; Klerk et al., 2008; Stahl et al., 1999).
In plant transformation optimum pH of the medium is very important. The
selection system and stability of pH in co-cultivation medium was the most important
factor in determining the success of transformation and transgenic plant regeneration.
Cotton support matrix may be beneficial for overcoming these lacunae.
The induction of vir gene expression in different types of Agrobacterium
strains showed different pH sensitivity profiles (Turk et al., 1991). Although A.
tumefaciens was reported to show the best growth at neutral pH (Li et al., 2002),
various reports on the Agrobacterium-mediated transformation showed that an acidic
pH favored for the optimal expression of the vir genes. The acetosyringone mediated
vir gene induction increases with the decreasing of pH from 6.2 to 5.1 and the optimal
induction of vir gene is attained when pH is lower (Stachel et al., 1986) than those
commonly used in plant tissue culture medium (pH 5.8). Godwin et al. (1991) have
reported that acetosyringone assisted gene transfer frequency was higher at pH 5.5 to
5.8 than at pH 5.2. For effective vir induction requires a medium with pH< 5.7
(Gelvin, 2000; Godwin et al., 1991; Ogaki et al., 2008). Therefore, buffers have been
utilized for maintaining the pH of the medium for better transformation efficiency.
Further, Gelvin, (2006) has stated that acetosyringone does not work well to induce
vir genes at neutral pH or in rich bacterial growth medium AB minimal medium with
MES buffer for maintaining the pH (5.6) for induction of Agrobacterium is used. In
another reports co-cultivation medium was buffered with 10 mM MES to retain pH
5.8 and improved transformation efficiency has been demonstrated (Joyce et al.,
2010; Ogaki et al., 2008). Possible toxicities of buffers used and high potassium,
phosphorus ions in medium remains cofounding problems as these elements increase
the induced defense resistance in plants (Dordas, 2009; Pasqualetto et al., 1988;
Williams, 1993). Thus in order to get better transformation and regeneration
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efficiency there should be inert support matrix which may enhance the process by
buffering of the medium, minimizing nutrient precipitation, phytotoxic effect of
antibiotics and preventing stressful atmosphere due to drift in pH. It has been
reported that Whatman No.1 filter paper improved the morphogenesis and buffers
phytotoxic effects of antibiotics in tobacco and Chrysanthemum in agar incorporated
medium (Jaime and da Silva, 2003). All these factors can be monitored in better way
with cotton support matrix than the agar.
4.3.3 Osmotic potential of culture medium
It was observed that agar incorporated medium was more negative for the
water potential (-0.002) than that of a cotton incorporated medium (-0.001). This
indicated that there was higher diffusion of medium components in cotton
incorporated medium as compared to semisolid agar medium. This may be due to the
micro-capillary action of cotton fibers. Bhattacharya et al., (1994) have shown that
higher the water content, greater the hydraulic conductivity, thermal conductivity and
diffusion coefficients of solute in the gel. Thus diffusibility of cotton incorporated
medium was an important attribute which avoids accumulation of toxic metabolites
(Fig. 11a). In agar incorporated medium there was blackening of medium and the base
of explants. This was due to accumulation of phenolics. While in case of cotton there
was no such phenomenon observed (Fig.11b).
Solutions of inorganic salts and sugars which compose tissue culture medium
besides having a purely nutritive effect, influence plant cell growth through their
osmotic properties (George et al., 2008). Concentration gradient of medium
constituents due to uptake by the explants is responsible for water movement across
the cell membranes resulting in differences of osmotic potentials. Thus availability
and uptake of nutrients was better when cotton was used as support matrix.
4.3.4 Support matrix interaction with medium ingredients and mineral uptake
Interaction of support matrix/gelling agent on medium with respect to addition
of some elements carried with them and their role has been reported by many authors
as discussed in 4.3. It has been indicated that agar is responsible for carrying some
toxic elements with it and making them less available due to fixing (George et al.
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2008; Jain et al., 2009; Naik and Sarkar, 2001; Prakash et al., 2003). The toxic/heavy
metals contaminants carried with agar were not carried with cotton (Lewin, 2007).
Further it has been reported that all the cellulosic support matrices viz. filter paper,
paper pulp, cotton were responsible for some growth promoting substance which had
influenced the better development of callus and explants (Ichimura and Oda, 1998).
Stability of pH in tissue culture medium with absorbent cotton as support
matrix indicated no significant change in pH of medium which was set at 5.6 in
sample ’C’ and 5.8 in sample ‘D’. Earlier reports with agar as support matrix in
sugarcane tissue culture (Lal and Singh, 1995; Piza et al., 2003), and in other crops
(Anderson and Ievinsh, 2008; Ramage et al., 2002; Scholten and Pierik, 1998; Thorpe
et al., 2008) have shown drift in pH of the medium after autoclaving, due to storage,
culture growth and precipitation of nutrients in the medium. pH plays an important
role by solublizing minerals and dissociation of mineral salts and perhaps preventing
fixation/ precipitation of soluble salts resulting their higher availability. pH role in
vitamins stability and availability has been also reported by Shibili et al., (1999). At
low pH, phosphate forms sparingly soluble precipitates with Al3+ and Fe3+ and as the
pH increases and reaches to 7.0, phosphorus forms complexes with calcium and
magnesium ions (Marschner, 1996) becoming less available to explants.
4.3.5 Embryogenic callus induction
The callus with explants on cotton incorporated medium was friable,
embryogenic and uniform than the callus with explants on agar incorporated medium
(Fig.11). The callus development was noticed within 12-15 days on cotton
incorporated medium where as it took 20-22 days on the agar incorporated medium.
Callus fresh weight and dry weight (704.00 ± 194 mg, 70.16 ± 18 mg) in cotton
incorporated medium was significantly higher than the callus on agar incorporated
medium (555.86 ± 106 mg, 59.58 ± 15 mg) amounting 28% and 18% increase
respectively (Table 14). Higher accumulation of biomass indicated that callus/shoot
growth on cotton incorporated medium was better than that of on the agar
incorporated medium. Similar reports has been reported in potato, banana, sugarcane ,
Artemisia, Agrostis, and Taxus (Dalvi et al., 2011; Khan et al., 2001; Moraes-Cerdeira
et al., 1995; Shah et al., 2009)
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4.3.6 Shoot and root induction
Greening of callus on agar incorporated medium was better than that on cotton
incorporated medium but shoot primordial initiation and development was earlier and
more (Fig. 11c and d) in cotton incorporated medium than on the agar incorporated
medium. The shoot primordial initiation and development was recorded visually in
growing cultures. It was observed that the percent shoot initiation was 46% more as
well as and shoots height was 19% more on cotton incorporated medium than on the
agar incorporated medium (Fig. 11e, f, 12a,b). The shoot growth was significantly
superior for shoot number (20.00 ± 4.0 cm) and shoot height (5.37± 0.4 cm) on cotton
incorporated medium than on the agar incorporated medium (13.75 ± 2 cm and 4.53 ±
0.5 cm) respectively (Table 14). The shoots were having higher chlorophyll a,
chlorophyll b and total chlorophyll content for the callus on cotton incorporated
medium (0.682 ± 0.79 mg/gFw, 0.309 ± 0.54 mg/gFw, 0.986 ± 0.52 mg/gFw
respectively) than the callus on agar incorporated medium (0.659 ± 0.93, 0.287 ±
0.69, 0.960± 0.05 mg/gFw) respectively. The higher regeneration capacity of callus
cells and biomass accumulation with cotton as support matrix indicated that the callus
and shoot growth on cotton incorporated medium was better than on agar incorporated
medium.
The agar incorporated medium has shown profuse root hairs on the roots
(Fig.12c). The roots regenerated from shoots on cotton incorporated medium were
with hardly any root hairs (Fig.12d). In this regard it has been reported that agar
incorporated medium showed drop in medium pH after autoclaving, making
phosphorus non-available to explants and thus causing phosphorus deficiency. Under
phosphorus deficiency the plants produce lots of root hairs to absorb more phosphorus
(Jain et al., 2009). In Arabidopsis due to low-phosphorus, root hairs have greater tip
growth rate, and roots of those plants have increased total root surface area which
indicated low-phosphorus plants may expend more energy or invest plant resources
for the purpose of acquiring limiting phosphorus (Bates et al., 2000). However, in
case of cotton incorporated medium, cotton matrix maintained the pH of the medium
to 5.8 (± 0.01) which facilitated the availability of phosphorus and different elements
for callusing as well as in tissue regeneration and growth.
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4.3.7 Mineral uptake by callus tissue
In present studies there was no difference in nitrogen uptake in callus grown
on both type of support matrices. Significantly higher phosphorus accumulation in
callus on cotton incorporated medium (1.23 ± 0.16 %) than the callus on agar
incorporated medium (1.06 ± 0.22%) was observed (Table 14). Uptake of potassium
by the callus on cotton incorporated medium was also higher (1.536 ± 0.30 %) than
callus on the agar incorporated medium (1.414 ± 0.22%) however it was not
statistically significant.
Similarly magnesium accumulation was higher (1.56 ± 0.29) in callus on
cotton supported medium than callus on agar incorporated medium (1.46 ± 0.90 mg).
Significantly higher accumulation of calcium (8.988± 4.5 mg) was observed in callus
grown on the cotton incorporated medium as compared to callus grown on agar
incorporated medium (5.651 ± 2.2 mg). Further it has been observed that manganese
accumulated significantly higher (0.55 ± 0.06 mg) on agar incorporated medium than
cotton incorporated medium (0.48 ± 0.05 mg). Zinc accumulation was significantly
higher in callus on cotton supported medium (0.83 ± 0.21mg) than callus on agar
incorporated medium (0.68 ± 0.21mg). While sodium content of callus on agar
incorporated medium was higher (9.81 ± 1.81 %) than callus on cotton supported
medium (9.16 ± 1.34%). Other elements as ferrous (2.04± 0.23mg) and copper (0.13 ±
0.09 mg) accumulated higher in callus on cotton supported medium than callus on
agar incorporated medium.
The higher amount of total soluble sugar (0.75 ± 0.08 mg) observed in callus
on cotton supported medium than callus on agar incorporated medium (0.60 ±
0.24mg).
Potassium is an important macronutrient having role in stress bearing capacity
of plants. In vitro plants are poor in abiotic and biotic stress bearing capacity due to
poorly developed stomata, root system and other morphological and physiological
characters (Ziv, 1987). It has been reported that potassium level adversely affected
vitrification and shoot quality preventing incidence of various diseases by increasing
defense related enzymes viz. peroxidase and 1-3-glucanase (Dordas, 2009;
Pasqualetto et al., 1988).
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Table 14: Comparison of different parameters and element uptake in
sugarcane callus grown with agar and absorbent cotton as support
matrix.
*significant at P< 0.05
Due to significant role of potassium and phosphorus in defense related activities in
many crops, KH2PO4 (0.1%) is sprayed for better acclimatization of in vitro plants and
reduced disease incidence (Mucharromah and Kuc,1991). Thus the better uptake of
Sr.
NoPARAMETER
AGAR
MATRIX
COTTON
MATRIXP VALUE
1 Fresh weight (mg) 555.86+106.92 704.42*+ 194.46 0.001
2 Dry weight (mg) 59.58 +14.85 70.16*+ 17.86 0.019
3 Water content (%) 89.49+0.47 90.20+ 0.141 0.26
4 No. of shoot buds (No) 13.75 +2.22 20.00* + 3.74 0.01
5 Shoot height (cm) 04.53 + 0.57 05.37* + 0.44 0.01
6 Total Chlorophyll (mg,) 0.986 + 0.02 0.960 + 0.05 0.01
7 Nitrogen (%) 05.10+ 0.27 05.20 + 0.52 0.39
8 Potassium (%) 01.41+ 0.21 1.56+ 0.29 0.12
9 Phosphorus (%) 01.06 + 0.22 1.23* + 0.16 0.03
10 Magnesium (mg) 01.46 + 0.90 1.64 + 0.69 0.58
11 Calcium (mg) 05.65 + 2.20 8.98+ 4.48* 0.02
12 Sodium (%) 09.81+1.81 9.16 + 1.34 0.04
13 Manganese (mg) 0.55* + 0.06 0.48 + 0.05 0.02
14 Zinc(mg) 0.68 + 0.32 0.83* + 0.21 0.11
16 Copper (mg) 0.07 + 0.09 0.13 + 0.09 0.03
17 Ferrous(mg) 01.96 + 0.35 2.04 + 0.23 0.53
18 Cadmium (mg) 01.04 + 0.11 0.98 +0.06 0.11
19 Soluble sugar (mg) 0.60 + 0.24 0.75 + 0.08 0.10
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phosphorus and potassium during in-vitro growth will definitely help tissue culture
plants to bear stress in acclimatization process as observed in present studies.
It has been reported that (Amiri 2008; Dalton et al., 1983; Scholten and Pierik,
1998) there was non-availability of mineral nutrients especially ferrous 45%, zinc
20% and phosphates 13% due to sequestration of phosphorus with ferrous and zinc
present in the medium. As the pH exceeded 5.8 precipitations became pronounced
resulting reductions in availability of manganese and ferrous (50%), smaller
reductions in calcium (20%) and phosphorus (15%) (Winkle and Pullman, 2003).
Phosphorus deficiency-induced reduction in the total phosphorus content in leaves
and roots affects the concentrations of macro elements and microelements such as
potassium, sulfur, ferrous and zinc (Misson et al., 2005). This results in altered ion
uptake and toxicity to the cells (Thorpe et al., 2008).
Further, it has been reported that increasing the concentration of phosphorus in
MS medium to ameliorate phosphorus deficiency retards the culture growth. This was
attributed to the tittering out of calcium by the phosphorus and increased precipitation
of cations as phosphates, reducing their availability in stages of cell development
starting from callusing to plant regeneration (Jain et al., 2009; Sheng et al., 2008;
Shekafandeh, 2010). However, even though phosphorus is a major nutrient in plant
growth, tissue culture medium contains relatively low concentrations of phosphorus to
prevent accumulation of calcium phosphate. There are limitations for increasing
concentration of phosphorus in MS medium (Shekafandeh, 2010). Jain et al., (2009)
and Sheng et al.,(2008) have shown that gelling agents significantly affect morpho-
physiological and molecular responses of the seedlings to deficiencies of nutrients;
which decreased the photosynthesis due to prolonged phosphorus starvation in agar
gelled medium. Mucharromah and Kuc (1991) have reported that deficiency of
phosphorus and potassium was responsible for developmental abnormalities in the in-
vitro plants causing poor acclimatization. Kavanova et al., (2006) have reported that
phosphorus deficiency reduced cell division and elongation of cells in grasses.
From our observations it seems that stabilization of pH of the medium
minimizes the phosphorus precipitation/fixation in cotton incorporated medium
resulting sufficient uptake of phosphorus for growing callus.
Magnesium is a cation in plant to balance negative ions which is required by a
large number of enzymes involved in energy transfer, particularly those utilizing
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 68
ATP. It is a constituent of the chlorophyll molecule and is required for the normal
structural development of the chloroplast as well as other organelles such as the
mitochondrion. Thus, it is expected that magnesium deficiency would have damaging
effects on photosynthesis and respiration (George et al., 2008). The calcium ion is
involved in many of the responses induced by plant growth substances particularly
auxins and cytokinins in in vitro morphogenesis (George et al., 2008). Calcium
deficiency in plants results in poor root growth, blackening and curling of the margins
of apical leaves, often followed by a cessation of growth and death of the shoot tip in
Amelanchier, Betula, Populous, Sequoia, Ulmus, Cydonia (Singha et al., 1990; Sha et
al, 1985). Bairu et al., (2009) had reviewed calcium deficiency and its physiological
interactions in different plants. Since calcium is usually used as CaCl2, increasing
calcium has limitations as chlorine ion toxicity increases. Higher accumulation of
sodium may be due to higher sodium content and the nutrient availability stress in
agar. Zinc is important component of enzymes responsible for plant growth (George
et al., 2008). Thus higher accumulation of calcium, magnesium, zinc, copper and
ferrous is the cross talk between the accumulated macronutrients.
For understanding the special role of absorbent cotton as a support matrix in
plant tissue culture, attention is to be focused on the dynamics of the interaction
between explants, growth medium and cotton matrix during growth. Cotton fibers
have β-1, 4-D glucopyranose, the principle building blocks of cotton cellulose chain
linked by l, 4-glucodic bonds. Cotton fiber has several –OH groups present at
different positions on backbone of cotton fiber in its amorphous and crystalline
regions. Oxidation of –OH groups results into formation of carbonyl (=C=O) and
carboxyl (-COOH) groups which are weak acids (Hsieh, 2007). Carboxylic groups of
cellulose are prone to hydrogen bonds and thus cotton has weak acidic property
exchanges cations which maintain the pH and EC of the medium by working as
buffer. This is further improved due to the presence of salts in MS medium. This
ultimately increases the charged ions to transport the electrons. Processing natural
cotton fiber by scouring and mercerization making it absorbent has effect on number
of carboxyl groups of the cotton fiber.
From the foregoing account it is clear that the absorbent cotton as a support
matrix is very much beneficial for in-vitro nutritional studies as it prevents drift in pH
of medium and increases the availability and uptake of nutrients for better growth of
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 69
the explant. It is evident from present study that quality of absorbent cotton needs to
be checked for the tissue culture use.
4.3.8 Economics of cotton and agar as support matrix in tissue culture
To calculate the economics, current market price for agar is .2329/500g (Hi
Media Laboratories Ltd. Mumbai) and Rs.100/500g (for different cotton samples with
different manufactures) has been used. The approximate cost of matrices per bottle
works out to be . 0.24 , ( . 6.72/l) in case of cotton and . 0.85 (i.e. .35.60/l) for
agar. This results in direct saving of . 0.61/- per bottle a significant saving (i.e. ~ .
29.00/l) on medium cost. Since in case of cotton pre-heating of medium to melt agar
is not required, which makes medium dispensing easy and saves energy. Aseptic
conditions needs to be maintained with high vigil to dispense the molten agar
medium, a step that can result in microbial contamination. Use of cotton for replacing
agar has no such limitation as it is easy to incorporate in bottles and pouring medium.
It has been observed that there is increase in production efficiency in terms of higher
number of shoots/bottle with same amount of medium with cotton as a support matrix
against agar (Dalvi et al., 2011). The work presented here has detailed the benefits for
not only lowering the cost of medium but also improving quality of product and
production process.
For commercial micropropagation laboratories used agar disposal is a problem
as agar does not get degraded in soil easily and therefore invites rapid microbial
development over it. However, cotton matrix has no such problems further it also
avoids over-exploitation of natural resources. Hence, cotton as support matrix has
tremendous potential in commercial micropropagation.
4.4 Embryogenic callus development for induced mutations
The embryogenic callus is of prime importance in tissue culture development
activity in any crop. The embryogenic callus development with alternative support
matrix has been reported and discussed in above part. The callus development
protocol was further modified by incorporation of Poly Ethylene Glycol (PEG) in
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 70
callus initiation medium. Different concentrations of PEG (8000) were incorporated
in the callus induction medium. It was observed that PEG (100mg/l) in the callus
initiation medium produced better embryogenic, friable compact callus than the
medium without PEG (i.e. control). The modified protocol was utilized for further
experimental work in induction of mutations and Agrobacterium transformation in
sugarcane.
4.5 Ethyl methyl sulphonate (EMS) induced mutagenesis in in vitro grown calli of
sugarcane variety CoC 671
Embryogenic calli (Fig.7b) were treated with EMS in two batches and
progressed in further stages of development.
4.5.1 Shoot regeneration, rooting and acclimatization of mutant plantlets
The vigorously growing calli were isolated and shifted to the shoot
regeneration medium containing 10% PEG ((Fig. 13 (i)) and well grown shoots were
shifted to rooting medium and later to green house for acclimatization.
Well acclimatized plantlets (300) of first batch and similarly (300) of second
batch were transplanted in the field for seed multiplication and screening the morpho-
physiological variations (Fig. 13ii).
4.6 Screening of sugarcane mutants
At the 12th month maturity, eighteen mutants were selected from the first batch
and eleven mutants from second batch on the basis of Brix%, morphological
distinctness, height of cane and cane girth. The canes from the stool were harvested
and a rod row trial was performed as described in Chapter Materials and methods
3.14. Observations of the rod row trial were recorded at 11th and 12th months crop age
for Brix%, CCS%, Sucrose%, Purity%, number of millable canes, millable height
(cm), cane diameter (cm), number of internodes/cane, weight of single cane (kg), for
all sugarcane mutants (Table 15 and 16).
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 71
4.6.1 Performance analysis of sugarcane mutants in rod row trial I (Batch I)
It was observed that mutant TC 928 has shown highest cane yield (143.50
t/ha) followed by TC 906 (137.02 t/ha) as compared to donor CoC 671(141.1 t/ha) at
12th month crop age (Table 15 and 16).
For CCS t/ha TC 917 has shown highest CCS t/ha (20.79) followed by TC 906
(20.01 t/ha), TC 929 (18.80 t/ha), TC 925 (18.28 t/ha) and TC 922 (18.26 t/ha) as
compared to CoC 671(17.92 t/ha) at 12th month crop age (Table 15).
It has been observed that TC 929 was significantly superior for millable
canes/ha (109.67) as compared to CoC 671 (97.53). TC 925 (107.33), TC 930
(104.07), TC 928 (102.67) have shown higher millable cane number than CoC 671
(97.53) however, statistically non significant (Table 15).
For cane height TC 926 (300.44 cm), TC 924 (297.78 cm), TC 923 (291.22
cm), TC 922 (289.00 cm) TC 929 (284.56 cm), TC 925 (282.45 cm), TC 909 (279.56
cm), TC 906 (275.00 cm) TC 928 (274.89 cm), TC 932 (274.70 cm) have shown
significantly superior cane height than CoC 671 (263.53 cm) at 12th month crop age
(Table 15).
As compared to CoC 671 (2.76 cm), TC 909 (2.97 cm) has shown higher cane
diameter followed by TC 927 (2.90 cm) and TC 906 (2.88 cm) however, not statically
significant at 12th month crop age (Table 15).
For the single cane weight parameter none of the mutant has shown
significantly higher single cane weight over CoC 671 at 12th month crop age (Table
15).
TC 926 has shown significantly superior number of internodes (24.55) as
compared to CoC 671 (21.31). Sugarcane mutant TC 922 has shown higher number of
internodes (22.78) followed by TC 926 at 12th month crop age (Table 15).
For brix% TC 917 (21.48%), TC 906 (21.00%), TC 924 (21.01%), TC 933
(20.96%) have shown higher brix% than CoC 671 (20.2%) at 11th month while TC
933 (22.79%), TC 924 (22.15%), TC 921 (22.14%) and TC 906 (21.59%) have shown
higher brix% than CoC 671(20.71) at 12th month crop age (Table 16).
For brix% TC 906 (21.0 %), TC 917 (21.46 %), TC 921 (20.77 %), TC
924(21.01 %), TC 932 (20.97 %) and TC 933 (20.96 %) have shown significantly
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 72
higher brix % over standard check Co 86032 (18.65 %) at 11th month crop age (Table
16).
Table 15: Yield and quality parameters of sugarcane mutants developed from
sugarcane variety CoC 671 in Rod row trial I (Batch I)
* Significant at p<0.05
Sugarcanemutants
& Checks
Yield
(T/ha)
CCS
(T/ha)
No. of millable canes /
ha (‘000)
Cane
height
(cm)
Single
cane
weight
( Kg)
No. of inter
nodes
Cane diameter
(cm)
TC-906 137.02 20.01 95.67 275.00* 1.47 21.78 2.88
TC-909 114.52 15.16 89.43 279.56* 1.37 21.22 2.97
TC-910 130.76 17.46 99.87 268.11 1.22 21.33 2.85
TC-914 114.29 15.30 86.33 246.66 1.32 19.33 2.76
TC-917 133.00 20.79 98.00 260.34 1.41 20.56 2.53
TC 921 119.42 16.96 104.53 273.67 1.23 21.44 2.72
TC-922 129.67 18.26 86.33 289.00* 1.53 22.78 2.80
TC-923 127.38 18.05 84.00 291.22* 1.41 23.33 2.65
TC-924 129.04 15.33 89.60 297.78* 1.37 22.44 2.79
TC-925 129.13 18.28 107.33 282.45* 1.24 21.78 2.81
TC-926 133.16 18.16 107.8 300.44* 1.30 24.55 2.71
TC-927 120.66 16.37 86.33 269.33 1.38 19.89 2.90
TC-928 143.50 17.96 102.67 274.89* 1.29 21.67 2.52
TC-929 120.71 18.80 109.67 284.56* 1.26 21.11 2.64
TC-930 103.72 13.96 104.07 272.89 1.08 21.11 2.55
TC-931 121.24 13.28 80.27 269.33 1.36 20.89 2.88
TC-932 117.98 16.40 93.33 274.70* 1.38 21.22 2.69
TC933 117.69 15.69 84.93 264.53 1.25 22.00 2.88
CoC 671 141.10 17.92 97.53 263.42 1.36 21.31 2.76
Co 86032 141.66 16.78 110.47 250.05 1.29 18.8 2.57
S.E 7.39 - 4.44 3.54 - 0.844 -
C.D.5% 21.23 NS 12.76 10.19 NS 2.42 NS
CV% 10.14 - 8.03 2.23 - 6.81 -
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 73
Table 16: Juice quality parameters of the selected sugarcane mutants from
sugarcane variety CoC 671 in rod row trial-I (Batch I)
*Significant at p < 0.05
TC 933 (20.71%), TC 923(19.96%) and TC 906(19.81%) have shown higher
sucrose % than CoC 671 (18.59%) at 11th month of crop age while TC 923 (21.03%),
TC 917 (21.01%) and TC 933(20.72%) have shown higher sucrose% than CoC
671(19.00 %) at 12th month crop age (Table 16).
It has been observed that TC 933 (14.80%), TC 923 ( 14.47%), TC 906
(14.66%), TC 917 (14.36%), have shown higher CCS% than CoC 671 (13.73%), at
11th month crop age (Table 16). While TC 923 (15.23%), TC 933 (14.71%), TC 910
Sugarcane mutants
& Checks
Brix % Sucrose % CCS% Purity %
11M 12M 11M 12M 11M 12M 11M 12M
TC-906 21.00 21.59 19.81 19.92 14.66 14.24 94.67 92.15
TC-909 19.05 20.86 18.17 18.19 13.44 12.67 94.67 83.03
TC-910 18.54 21.45 18.13 19.94 13.42 14.30 97.67 92.97
TC-917 21.46 21.21 19.41 21.01 14.36 15.21 93.33 94.86
TC-921 20.77 22.14 18.22 18.95 13.48 13.52 90.67 91.60
TC-922 20.04 20.62 17.08 19.91 12.64 14.21 89.67 91.48
TC-923 19.08 21.67 19.96 21.03 14.77 15.23 95.00 95.05
TC-924 21.01 22.15 18.94 17.91 14.02 12.73 93.67 90.30
TC-925 20.23 19.15 17.98 19.01 13.31 13.64 91.0 95.52
TC-926 19.77 19.95 17.31 17.98 12.81 13.02 95.67 93.93
TC-927 19.4 20.57 17.32 19.06 12.82 13.66 91.0 92.81
TC-928 18.52 21.07 17.25 19.14 12.76 13.59 93.00 90.66
TC-929 19.00 19.91 17.04 18.79 12.61 13.58 89.67 94.33
TC-930 20.83 19.78 19.13 17.89 12.16 12.67 91.67 90.25
TC-931 18.33 19.18 17.16 17.04 12.70 11.97 93.33 88.85
TC-932 20.97 20.07 18.96 18.21 14.04 12.93 90.67 90.56
TC933 20.96 22.79 20.07 20.72 14.80 14.71 93.00 91.7
CoC 671 20.02 20.71 18.59 19.00 13.73 13.55 92.67 90.20
Co 86032 18.65 17.61 16.80 15.88 12.43 11.23 90.00 90.20
S.E 0.64 - - - - - - -
C.D.5% 1.82 NS NS NS NS NS NS NS
CV% 5.57 - - - - -
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 74
(14.30%) and TC 906 (14.24%) have shown higher CCS% than CoC 671(13.55% ) at
12th month.
It has been observed that TC 910 (97.67%) has shown highest purity %
followed by TC 926 (95.67%), TC 925 (95.52 %), TC 906 and TC 909 ( 94%) over
CoC 671 (92.67%) at 11th month crop age. While all other mutants have shown higher
purity % than CoC 671, except TC 909 and TC 931 at 12th month crop age (Table
16).
4.6.2 Analysis of performance of sugarcane mutant in rod row trial I (Batch II)
Eleven mutants showing variations in brix%, morphological differences in
cane color, internodes arrangement, leaf canopy, cane height and cane girth were
selected for the rod row trial I (Batch II).
Rod row trial for batch II was planted with standard checks for assessing their
qualitative and quantitative performance of mutants in replicated field trial as per rod
row trial for batch I. Observations of various agronomic parameters recorded were as
follows
The results of rod row trial I (Batch II) have been presented in Table 17 and
18. It has been observed that mutants TC 2826 (97.91 t/ha) and TC 2907 (99.69 t/ha)
has shown significantly superior cane yield as compared to its parent CoC 671 (81.53
t/ha) while TC 2925 (95.39 t/ha) and TC 2924 (93.65 t/ha) and TC 2813 (94.63 t/ha)
were higher than CoC 671(81.53 t/ha) for yield level but not statistically significant
(Table 17).
It has been observed that none of the mutant showed statistically significant
CCS t/ha over the parent CoC 671, however, TC 2813 (12.71 t/ha), TC 2819 (11.13
t/ha), TC 2881 (14.36 t/ha), TC 2907 (13.59 t/ha), TC 2924 (12.52 t/ha), TC 2925
(12.53 t/ha) have shown higher CCS t/ha than CoC 671 (11.32 t/ha) but not
statistically significant (Table 17)
For number of millable canes/ha none of the mutant was statistically superior
to CoC 671 or Co 86032 but TC 2813 (80.73), TC 2819 (89.60), TC 2881(77.93)
were having higher number of thousand millable canes/ha than CoC 671 (67.20) and
Co 86032 (72.33) (Table 17).
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 75
For cane height, cane weight, number of internodes none of the sugarcane
mutant has shown statistically superior to CoC 671(Table 17).
However, for cane diameter TC 2924 (9.67cm) has shown statistically
significant cane diameter than CoC 671 (8.74cm) at 12th month crop age (Table 17).
It has been observed that TC 2813 (21.01%), TC 2881 (19.74%), TC 2931 (
19.38%) have shown higher brix% than CoC 671 ( 19.15%) at 10th month crop age
and TC 2813 (22.46%) has shown higher brix % than CoC 671 (20.49%) at 11th
month crop age (Table 18). While TC 2813 (22.52%), TC 2907 (21.78%), TC 2875
(21.15%) have shown higher brix % than CoC 671 (20.98%) at 12th month crop age
(Table 18).
Table 17: Yield and quality parameters of selected sugarcane mutants developed
from sugarcane variety CoC 671 under rod row trial I (Batch II)
Significant at p < 0.05
Sugarcane mutants &
Checks
Yield
(T/ha)
CCS
(T/ha)
No. of
millable Canes/ha
( ‘000)
Cane
height
(cm)
Single
cane
weight
(Kg)
No. of
inter-
nodes
Cane
diameter
(cm)
TC 2813 94.63 12.71 80.73 219.44 1.04 20.44 8.22
TC 2819 74.18 11.13 89.60 236.11 0.84 19.67 7.65
TC 2826 97.91* 10.75 62.73 252.00 1.58 22.22 9.11
TC 2875 73.21 10.17 63.00 225.11 1.17 19.89 8.50
TC 2881 88.63 14.36 77.93 238.32 1.37 21.11 8.98
TC 2907 99.69* 13.59 68.60 260.00 1.53 21.89 8.61
TC 2912 91.19 11.26 56.00 244.11 1.63 20.56 9.44
TC 2924 93.65 12.52 55.53 267.56 1.73 22.22 9.67*
TC 2925 95.39 12.53 70.00 243.33 1.45 21.89 9.17
TC 2929 87.51 11.84 66.27 255.11 1.33 21.67 8.83
TC 2931 91.65 11.94 71.40 230.11 1.31 20.55 9.34
CoC 671 81.53 11.32 67.20 230.00 1.20 21.00 8.74
Co 86032 112.3 16.99 72.33 246.22 1.56 22.04 9.28
SE 5.6 - - - 0.16 - 0.3
CD 5% 16.21 NS NS NS 0.46 NS 0.86
CV % 9.95 - - 5.39
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 76
Table 18: Juice quality parameters for the selected sugarcane mutants developed from sugarcane variety CoC 671 in
rod row trial I (Batch II)
*Significant at p < 0.05
Significant at p < 0.05
Sugarcane mutants
& Checks
Brix% Sucrose% CCS% Purity%
10 M 11 M 12 M 10 M 11 M 12 M 10 M 11 M 12 M 10 M 11 M 12 M
TC 2813 21.01 22.46 22.52 17.80 20.71 19.52 12.23 14.81 13.56 85.00 85.00 92.00
TC 2819 16.08 18.94 19.75 14.68 16.92 16.71 10.21 11.92 14.46 87.00 87.00 89.33
TC 2826 16.99 19.97 19.78 12.85 18.23 16.51 8.27 12.97 11.24 89.00 84.00 91.67
TC 2875 18.54 20.04 21.11 16.06 17.82 19.50 11.15 12.54 13.95 86.67 86.67 89.00
TC 2881 19.74 20.32 21.08 17.20 18.68 19.11 12.06 13.34 13.56 88.33 88.33 92.00
TC 2907 16.60 18.48 21.78 14.05 15.73 16.61 9.64 10.83 14.53 84.33 84.33 85.00
TC 2912 17.73 19.45 19.44 15.75 18.07 17.47 11.07 12.97 12.34 89.00 89.00 93.33
TC 2924 16.21 20.01 20.33 13.67 18.41 18.83 9.37 13.15 13.49 84.33 84.33 91.67
TC 2925 18.02 19.54 20.06 15.57 17.55 18.49 10.89 12.40 13.22 86.00 86.00 89.67
TC 2929 16.53 19.07 20.45 13.93 16.73 18.84 9.54 11.69 13.47 84.33 84.33 87.67
TC 2931 19.38 20.11 20.40 17.32 18.22 18.39 12.21 12.93 13.02 89.33 89.33 90.67
CoC 671 19.15 20.49 20.98 16.97 18.12 19.64 11.91 12.71 14.13 88.67 88.67 83.33
Co 86032 17.89 19.57 19.98 16.42 17.60 18.08 11.72 12.44 12.82 91.97 91.97 90.00
SE 0.94 - - 0.98 - - 0.8 - - - - -
CD 5% 2.72 NS - 2.83 NS - NS 2.33 NS - - NS - NS NS - NS -
CV % 8.33 - - 10.06 - - 11.93 - - 85.00 85.00 92.00
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 77
For sucrose% it was observed that there was none of the mutant statistically superior
over CoC 671 at 10th, 11th and 12th month but mutants TC 2813 (17.80%), TC 2931
(17.32%), TC 2881 (17.20%) have shown higher sucrose % than CoC 671 (16.97%) at 10th
month crop age. While TC 2813 (20.71%), TC 2881 (18.68%), TC 2924 (18.41%), TC 2826
(18.23%) have shown higher brix % than CoC 671 (18.12%) at 11th month crop age. At 12th
month CoC 671 has shown higher CCS % than all the mutants(Table 18).
It has been observed that TC 2813 (12.23%), TC 2831 (12.21%) and TC 2881
(12.06%) have shown higher CCS % than CoC 671 (11.91%) at 10th month crop age while
TC 2813( 14.81%), TC 2881 ( 13.34%), TC 2924 ( 13.15%), TC 2826 (12.97%) have shown
higher CCS% than CoC 671 ( 12.71%) at 11th month crop age and TC 2907(14.53) and TC
2819 (14.46%) have shown higher CCS% than CoC 671( 14.13%) at 12th month crop age
(Table 18).
CoC 671 has shown higher purity % values than all the mutants in 10th and 12th
month. The purity % values of all the mutants in 1oth and 11th month were more than 85%
where as in 12th month crop age TC 2813 (92.00%), TC 2819 (91.67%), TC 1912 9
93.33%), TC 2924 ( 91.67%), and TC 2931 ( 90.67%) have shown higher purity % than Co
671 (83.33%)
From the CCS t/ha, yield, quality parameters i.e. brix%, sucrose%, CCS% and
morphological distinctness of mutants TC 2813, TC 2819, TC 2826 and TC 2875 were
preliminarily selected for further screening in clonal trial to further evaluate their
performance and screening for smut disease resistance..
4.7 Sugarcane mutants in clonal trial
4.7.1 Sugarcane mutants in clonal trial I (Batch I)
Selected mutants TC 906 and TC 922 were further screened in field (clonal trial I) for
recording their performance following the guidelines by All India Coordinated Research
Program for Sugarcane. Quantitative and qualitative parameters were recorded at 10th, 11th
and 12th month (Table 19 and 20).
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 78
From the data it was revealed that the mutant TC 922 was significantly superior in
cane yield (165 t/ha) and the mutant TC 906 was higher in cane yield (144.11 t/ha) over CoC
671 (128.44 t/ha) at 12th month crop age (Table 19)
TC 922 (22.27 t/ha) and TC 906 (21.61 t/ha) has shown higher CCS t/ha as compared
to CoC 671 (19.27 for CCS t/ha) respectively at 12th month crop age (Table 19).
In TC 906 and TC 922 there was no significant difference for number of millable
canes as compared to CoC 671 and Co 86032.
TC 922 (317.11 cm) and TC 906 (316.65 cm) have shown higher cane height than
CoC 671(307.11cm) and Co 86032(307.00) at 12th month crop age.
Table 19: Yield and quality parameters of selected sugarcane mutants developed from
sugarcane variety CoC 671 in Clonal Trial I (Batch I)
*Significant at p < 0.05
Significantly superior single cane weight (2.07 kg) for TC 906 and (2.30 kg) for TC 922 as
compared to CoC 671 (1.72 kg) at 12th month crop age was observed.
For brix% TC 906 (19.45% at 10th month, 21.67% at 11th month, 22.95 % at 12th
month) and TC 922 (18.94 10th month, 21.80% at 11th month, 23.11% at 12th month) have
shown higher values as compared to CoC 671 (18.05% at 10th month, 20.78% at 11th month
and 22.34% at 12th month) respectively.
Sugarcane Mutants &
Checks
Yield
(t/ha)
CCS
(t/ha)
No. of millable
canes
Cane height
(cm)
Single cane
Weight
(cm)
No. of Inter-
nodes
Cane
Diameter (cm)
TC- 906 144.11 21.61 70.03 316.65 2.07* 24.22 3.25
TC-922 165.33* 22.27 71.87 317.11 2.30* 26.11 3.34*
Co 86032 133.56 17.86 88.13 307.00 1.52 22.33 3.07
CoC 671 128.44 19.27 74.53 307.11 1.72 26.00 2.74
S.E. 7.37 - 3.47 - 0.08 0.63 0.19
C.D. 5% 21.89 NS 10.3 NS 0.22 1.86 0.57
C.V. % 9.51 - 8.07 - 7.15 4.42 3.48
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 79
Table 20: Juice quality data of selected sugarcane mutant from sugarcane variety CoC 671 in clonal trial-I (Batch I)
*Significant at p < 0.05
.
Sugarcane
mutants &
Checks
Brix % Sucrose % CCS% Purity%
10M 11M 12M 10M 11M 12M 10M 11M 12M 10M 11M 12M
TC- 906 19.45 21.67 22.95 16.67 19.80 21.04 11.51 14.10 15.00 85.70 91.39 91.63
TC-922 18.94 21.80 23.11 15.84 19.79 21.33 10.81 14.05 15.26 83.61 90.78 92.26
Co 86032 16.55 19.10 20.50 13.50 16.98 18.76 9.08 11.93 13.33 81.47 88.88 91.51
CoC 671 18.05 20.78 22.34 15.48 19.23 20.87 10.70 13.77 15.01 85.66 92.49 93.21
S.E. 0.57 0.59 0.44 0.72 0.73 0.54 0.59 0.60 0.43 1.48 1.6 0.69
C.D. 5% NS 1.67 1.30 NS NS 1.59 NS NS NS NS NS NS
C.V. % 5.48 4.72 3.33 8.3 6.73 4.41 9.89 11.04 6.89 3.08 3.06 1.29
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 80
Similarly for Sucrose% TC 906 has shown higher values (16.67% at 10th month,
19.80 % at 11th month, 21.04 at 12th month) and TC 922 has shown higher values (( 15.84
% at 10th month, 19.79 % at 11th month and 21.33 at 12th month) over CoC671 (15.48% at
10th month, 19.23 % at 11th month and 20.87 at 12th month) respectively.
For CCS%, TC 906 (11.51% at 10th month, 14.10% at 11th month and 15.00% at 12th
month), and TC 922 (10.81% at 10th month, 14.05% at 11th month and 15.26 % at 12th
month) were higher than CoC 671 (10.70 at 10th month, 13.77 at 11th month, and 15.01% at
12th month) respectively.
For Purity% there was no significant difference between the mutants TC 906 and
TC 922 over to CoC 671 at 10th, 11th and 12th month respectively.
The CCS%, Sucrose% and Brix% values of both mutants have shown higher in 10th and 11th
month than CoC 671. Clonal trial I results indicated that the both mutants were early in
maturity than its CoC 671 with statistically superior yield.
4.7.2 Analysis of sugarcane mutants in clonal trial I (Batch II)
Analysis of performance of selected mutants from batch II in their clonal trial I has
presented in the Tables 21 and 22, it has been seen that TC 2813 (129.22 t/ha) TC 2819
(128.65 t/ha) have higher cane yield over CoC 671 (94.42 t/ha) followed by TC 2826 (107.16
t/ha) as compared to donor CoC 671 (94.42 t/ha) at 14th month crop age.
TC 2813(21.94 t/ha) and TC 2819 (21.56 t/ha) have shown significantly higher CCS
t/ha than CoC 671(14.55t/ha) at 14th month crop age. The CCS t/ha of TC 2826 (16.99 t/ha)
and TC 2875 (15.68 t/ha) was higher than CoC 671(14.55 t/ha) but it was statistically non-
significant (Table 21).
It has been observed that TC 2813 (86.00), TC 2819 (87.00), TC 2826 (92.4) have
higher number of millable canes than CoC 671 (82.66) but not statistically significant (Table
21). Cane height of mutants TC 2813 (285.33cm), TC 2819 (244.89cm) and TC 2875
(250.56cm) was higher than CoC 671 (241.66cm) but it was statistically non-significant
(Table 19).
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 81
Table 21: Yield and quality parameters of selected sugarcane mutants developed from
sugarcane variety CoC 671 in clonal trial I (Batch II)
*Significant at p < 0.05
TC 2813 (1.48kg) has significantly superior and TC 2819 (1.41kg), TC 2826 (1.16kg)
and TC 2875 (1.23kg) have higher single cane weight than CoC 671 (1.13kg) (Table 21).
The cane girth, number of internodes of all the mutants was statically non significant
than CoC 671 (Table21).
From the data for brix% it has been observed that the TC 2813 (24.27%), TC 2819
(24.07%) and TC 2826 (23.97%) have shown significantly higher brix% values respectively
than CoC 671 (22.63) at 12th month cop age. All the mutants have shown higher brix % than
CoC671 at 10th and 14th month crop age except TC 2875 (19.23%) (Table 22).
Similarly it has been observed that TC 2813 has shown significantly higher sucrose %
(23.71%) than CoC 671 (21.39%) at 12th month crop age and higher sucrose % (18.53% and
23.32%) as compared to CoC 671 (17.67% and 21.35%) at 10th and 14th month crop age
respectively. TC 2819 has shown significantly higher sucrose % (20.33 and 23.98%) than
CoC 671 (17.67 and 21.35%) at 10th and 14th month crop age (Table 22).
Sugarcane mutants & Checks
Yield
(t/ha)
CCS
(t/ha)
No of millable
canes
(‘000/ha)
Cane
height
(cm)
Single
cane
Weight
(kg)
No. of
Inter
Nodes
Cane
diameter
(cm)
TC 2813 129.22 21.94* 86.000 285.33 1.48* 23.00 2.90
TC 2819 128.65 22.56* 87.000 244.89 1.41 23.67 3.05
TC 2826 107.16 16.99 92.400 227.22 1.16 18.33 2.93
TC 2875 97.56 15.68 78.667 250.67 1.23 21.78 2.93
CoC671 94.42 14.55 82.667 241.66 1.13 21.89 2.68
Co86032 125.14 18.23 90.133 254.44 1.37 21.33 2.90
SE 26.27 2.38 5.6399 17.21 0.11 1.06 0.17
CD at 5% 76.07 6.89 16.334 49.84 0.33 3.06 0.5
CV % 10.38 19.7 11.15 11.39 13.64 8.05 10.1
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 82
Table 22: Juice quality data of selected sugarcane mutants from sugarcane variety CoC 671 in clonal trial I (Batch II)
*Significant at p < 0.05
Sugarcane mutants &
Checks
Brix% Sucrose % CCS% Purity%
10M 12M 14M 10M 12M 14M 10M 12M 14M 10M 12M 14M
TC 2813 20.30 24.27* 23.90 18.53 23.71* 23.32 13.13 17.42* 17.02 90.29 98.21 96.66
TC 2819 22.10 24.07* 24.30 20.33* 22.49 23.98* 14.47* 16.18 17.56* 91.37 93.49 97.55
TC 2826 19.47 23.97* 23.37 18.11 23.13* 22.08 12.97 16.90 15.90 92.24 96.96 93.64
TC 2875 19.23 23.37 23.70 17.04 22.11 22.22 11.91 16.02 15.94 87.74 94.96 92.88
CoC671 19.53 22.63 22.93 17.67 21.39 21.35 12.47 15.45 15.26 89.46 94.40 92.21
Co86032 18.80 21.90 21.87 17.53 20.91 20.46 12.54 15.18 14.66 92.19 95.37 92.65
SE 1.13 0.41 0.5 0.83 0.54 0.72 0.65 0.49 0.66 1.65 1.7 2.1
CD at 5% 3.28 1.18 1.44 2.41 1.57 2.07 1.87 1.42 1.9 4.77 4.92 6.09
CV% 10 3.04 3.66 8.05 4.28 5.6 7.02 5.38 8.96 3.15 3.11 3.89
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 83
TC 2826 has shown significantly higher sucrose % (23.13%) over CoC 671
(21.39%) at 12th month crop age and higher sucrose % (18.11% and 22.08%) than
CoC671 (17.67% and 21.35%) in 10th and 14th month crop age respectively. The mutant
TC 2875 has shown higher values of sucrose % in 12th and 14th month crop age but those
were not significant statistically (Table 22).
It has been revealed that for CCS% TC 2813 (17.42%) was significantly superior
in 12th month and TC 2819 (14.47% and 17.56%) was significantly superior in CCS%
than CoC 671 (12.54% and 14.66 % respectively) at 10th and 14th month (Table 22).TC
2813 (13.13, 17.42, 17.02), TC 2819 (14.47, 16.18, 17.56), TC 2826 (12.97, 16.90,
15.90) was higher than CoC 671 (12.47, 15.45, 15.26) respectively at 10th 12th and 14th
month crop age.
For the juice Purity% record data it has been observed that all the mutants showed
higher purity% than CoC 671 at 10th, 12th and 14th month crop age except TC 2875 in
10th moth where it was less than CoC 671 (Table 22). In all the mutants and CoC 671
purity % was above 90 % (Table 22).
Thus from the present study it has been observed that tissue culture technique
with induced mutations has resulted in better mutants viz. TC 906, TC 922, TC 2813,
TC 2819, TC 2826 and TC 2875 showing considerable improvement in agronomical
traits over CoC 671.
Various reports are available for induced mutations in sugarcane for the
development of varieties for change in morphological, biochemical traits governing the
agronomical characters which have been reviewed from time to time (Sreenivasan and
Jalaja, 1998; Jain 2001; Suprasanna et al., 2009). Mutations in agronomical traits such
as tolerance to drought and salt tolerance in sugarcane have been reported (Balasundaran,
1981; Douel, 2006, Naik and Manjunatha, 2001; Patade et al., 2006, Radhakrishnan,
1990; Sreenivasan and Jalaja, 1998; Suprasanna et al., 2009). In CoC 671 mutants
generated through physical mutagenesis has shown that out of 64, 63 resistant to smut, 8
moderately resistant and 18 moderately susceptible and 5 were susceptible to smut
(Sreenivasan and Jalaja, 1998).
Wide ranges of chemical mutagenic agents have been utilized to induce gene
mutations in sugarcane (Micke and Donini, 1993; Sreenivasan and Jalaja, 1998; Wagih et
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 84
al., 2004). It has been also noticed that higher rate of gene mutations in sugarcane is
obtained by chemical mutagenesis than physical mutagenesis.
Present work is in agreement with earlier reports of induced mutations in
sugarcane. Further improvements in qualitative as well as quantitative traits are possible
through induced mutations. Thus using traditional breeding methods when genetic
variability is narrowed for a long period, induced mutations are one of the most important
approaches for broadening the genetic variation to circumvent the bottleneck conditions
in sugarcane. The induced mutation work with in vitro selection for the mutants
exhibiting an early maturity with enhanced sugar accumulation and tolerance to smut
disease may prove beneficial to improve the competitiveness of the popular sugarcane
cultivars and their commercial cultivation
4.8 Screening for smut resistance in laboratory using PCR
Samples from the field grown sugarcane mutants were collected every month up
to 12th month and genomic DNA was prepared for PCR analysis. Amplification of smut
specific primers (18S rRNA- 5.8S rRNA- 28S rRNA Intergenic spacer region of S.
scitamineum, results in 460bp amplicon was carried out which revealed that among the
mutants tested, mutant TC 906 and TC 2826 did not show the presence of smut specific
band amplicon of 460 bp size (Fig. 14a,b) till the harvest. However in the susceptible
varieties CoC 671, Co 740 smut specific band amplicon of 460bp size was detected from
4th month growth. The PCR amplified band was eluted and sequenced. The sequence for
Sporisorium scitamineum partial 18S rRNA gene, ITS1, 5.8S rRNA gene and partial
ITS2, isolate SG 671" has been submitted to European Nucleotide Archive, EMBL
Nucleotide Sequence Database with the accession number HE800528.
PCR analysis data showed high correlation with field screening results for smut
resistance. PCR screening tests are faster than conventional methods for screening
sugarcane for smut resistance (takes ~12-18 months). By PCR analysis, it was made
possible to detect smut at an early stage of infection before the symptoms are visualized.
PCR detection using sugarcane smut specific primers is sensitive that it detected smut
DNA in spite of microscopic observations failed to detect even one week after
inoculation (Schenck, 1998; Singh et al., 2004). Further it was suggested that resistance
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 85
expressed in fully–grown and well developed seedlings should be considered as an
indicator, as the young plantlets register high mortality rates (Olweny et al., 2008). PCR
screening provides sensitive assay to detect the presence of smut pathogen (Schenck,
1998). Although, PCR analysis cannot be used to declare the sugarcane variety as
resistant, it can be used to screen the population for presence of smut infection. We used
PCR analysis to screen sugarcane somaclones and mutants to detect smut and also
screened morphologically for presence of whip formation. This technique will be simple
and reliable when there is development of smut hyphae but no whip formation due to
environmental factors. However, the infested plants show reduced tillering and stunted
growth (Moosawi-Jorf and Izadi, 2007; Singh et al., 2004; Singh and Somai, 2005). The
results from this study put together suggest that the sugarcane TC 906 and TC 2826 are
relatively resistant to smut with good agronomic traits as against their parent CoC 671.
4.9 Screening for smut resistance in field by artificial smut inoculation method
Two years field screening revealed that TC906 and TC 2826 show resistance to
smut. After two years (with field screening and artificial smut inoculation screening
methods) TC 906 and TC 2826 showed 0.0% smut incidence, while TC 922 has shown
2.77 % (Table 23), TC 2813 (4 %) and TC 2875 (12%) smut disease incidence (Table
24).
Table 23: Screening of smut resistance in field by artificial smut inoculation method
for sugarcane mutant (Batch I)
[*DI % Rating: 1) 0.0% - Resistant 2) 0.1 to 10 % - Moderately Resistant
3) 10.1 to 20.0 – Moderately Susceptible 4) 20.1 to 30 % - Susceptible 5) Above 30% highly susceptible]
Sr.
No.
Sugarcane
mutant and
Check
Disease Incidence %
(DI %)
Year I Year II
Average
(%)
Resistance *
Level
1 TC 906 0 0 0 R
2 TC 922 0 5.55 2.77 MR
3 CoC 671 9.10 7.49 8.29 MR
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 86
Table 24: Screening of smut resistance in field by artificial smut inoculation method
for sugarcane mutant (Batch II)
Artificial inoculation of smut spores to sugarcane sets is routine and established
method for screening resistance to smut. Whip production is the most reliable symptom
of smut disease in sugarcane (Fig.14c) but when there is no whip formation due to
environmental factors, the infested sugarcane plants, often tiller profusely with the shoots
being more spindly and the leaves being more upright and narrow (‘‘grass-like’’ in
appearance) emerging from the shoots following infection (Que et al. 2011). Less
common symptoms are leaf and stem galls, and proliferating buds (Moosawi-Jorf and
Izadi 2007; Singh et al. 2004; Singh and Somani, 2005).
4.10 Analysis of parameters for screening drought tolerance
Various parameters related to drought tolerance capability of sugarcane mutants
obtained through chemical mutagenesis were analyzed over the variety CoC671 and
results are recorded in Table 25.
It has been revealed that TC2813 (1356.40g/gFw), TC 2819 (1388.10 g/gFw),
TC 2826 (1207g/g Fw) and TC2875 (1267 g/gFw) accumulated significantly higher
proline compared to CoC671 (1190.40 ggFw TC906 (2.35g/gFw) reported higher
MDA while TC2813 (1.97 g/gFw) and TC2819 (1.96g/gFw) were similar to CoC 671
(1.98g/gFw).
Sr.
No.
Sugarcane
mutant and
Check
Disease Incidence %
(DI %)
Year I Year II
Average
(%)
Resistance *
Level
1 TC 2813 7.6 9.09 8 MR
2 TC 2819 3.5 15.3 9.4 MR
3 TC 2826 0.0 0.0 0.0 R
4 TC 2875 9.5 16.26 12 MR
5 CoC 671 12.4 15.23 13 MR
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 87
Table 25: Biophysical parameters of drought tolerance capacity in selected mutants
in sugarcane variety CoC 671
WRA for TC 906 (90.62), TC 2813 (92.00%), TC 2819 (93.20%) and TC 2875 (90.36%)
were on par with CoC 671 (89.50%). Sugarcane mutants showed no significant
difference with CoC 671 in relative water retention capacity (RWC) and REC. The
results indicated that the drought tolerance capacity of mutants is on par with CoC 671.
Hemaprabha et al., (2006) reported that CoC 671 has better drought tolerance capacity
compared to Co 740, and performed well under drought conditions. Patade et al., (2006)
reported that sugarcane somaclones developed on PEG medium did show variations in
drought and salt tolerance and were not genetically distinct yet phylogeneticaly close to
their parent CoC 671.
4.11 Scoring of morphological variations in sugarcane mutants
Distinct morphological differences in the variants are essential for phasing out the
original parents from the commercial cultivation. Otherwise it would be difficult to
Sr.
No
Sugarcanemutant &
Check
Proline
g/g Fw
MDA
/gFw
WRA
%
RWC
%
REC
S/cm2
1 TC 906 1014.00 2.35 90.62 86.13 0.01
2 TC 922 1009.00 1.81 88.42 84.52 0.011
3 TC 2813 1356.40* 1.97 92.00 85.5 0.010
4 TC 2819 1388.10* 1.96 93.20 89.31 0.009
5 TC 2826 1207.00 1.40 87.88 85.93 0.011
6 TC 2875 1267.6* 1.74 90.36 85.12 0.013
7 Co 86032 919.30 1.81 87.57 80.33 0.012
8 CoC 671 1190.40 1.98 89.50 85.55 0.007
10 SE 19.90 0.13 1.97 1.461 0.001
11 CD at 5 % 60.36 0.39 5.96 4.43 0.003
12 CV 2.95 11.98 2.78 2.96 18.31
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 88
identify the mixture of parental material from the newly introduced genotype. The
morphological changes therefore were critically observed in this study and were
documented. Morphological variations in TC 906 and TC 922 (Table 26a and Fig. 15 a,
16a), TC 2813, TC 2819, TC 2826 and TC 2875 (Table. 26b, Fig. 15 b and c, 16 b) were
recorded for three years. Distinct variations in morphological characters viz. stem color
(exposed and un-exposed), internodes alignment, internodes waxiness, leaf width, and
leaf sheath color, internodal length, stool habit, bulging of leaf sheath, presence or
absence of spines on leaf sheath, root band number and color, cane girth, bud shape etc.
were recorded observed (Table.26b).
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 89
Table 26a: Morphological variations in mutants developed from variety CoC 671
Sr.NoMorphological
parameterCoC 671 TC 906 TC 922
1Stem color -exposed
PurpleYellowish
purpleYellowish purple
2Stem color -Un exposed
Light greenishyellow
Greenish yellow
Green
3Internodes alignment
Zig Zag Straight Slightly zigzag
4Internodes diameter
2.74cm 3.25cm 3.34cm
5 Pithiness Absent Absent Absent
6Internodes waxiness Absent Heavy Heavy (Black)
7 Bud shape Ovate Ovate Ovate
8 Growth ring color Light green Light green Light green
9 Leaf width Medium to broad
Broad Broad
10 Lamina color Dark green Dark Green Dark green
11 Leaf carriage Open drooping Open drooping Open drooping
12 Leaf sheath Color Light green Light green Slight purple ting
13Leaf sheath waxiness
Absent Absent Absent
15 Leaf sheath Spines Hard profuse Hard profuse Hard profuse
16Leaf sheath clasping
Self detrashing Medium Self detrashing
17 Dewlap color Light green Light green Light green
18Ligular Process (Auricle)
Present Present Present
19Shape of the auricle
Crescent, thin Crescent, thin Crescent, thin
20 FloweringFlowers in
South Maharashtra
Flowers in South
Maharashtra
Flowers in South Maharashtra
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 90
Table 26b: Morphological variations in mutants developed from variety CoC 671
Sr.No
Morphological parameter CoC 671 TC 2813 TC 2819 TC 2826 TC 2875
1 Stem color -exposed Purple
Pinkish purple
Pinkish purple
Pinkish purple
Whitish purple
2 Stem color –Un exposed
Light greenish yellow
greenish yellow
greenish yellow
greenish yellow
greenish yellow
3 Internodes alignment Zig Zag Straight Straight Straight Straight
4 Internodes diameter
2.74cm 2.90 cm 3.05 2.93 2.93
5 Pithiness Absent Absent Absent Absent Absent
6 Internodes Waxiness Absent Heavy
Heavy white
Medium white
Heavy (Black)
7 Bud shape Ovate Round Round Round Round8 Growth ring
colorLight green Light green Light green Light green Light green
9 Leaf width Medium to broad
Medium medium medium Medium
10 Lamina color Dark green Dark Green Green Green Dark green
11 Leaf carriage Open drooping
Open drooping
Open drooping
Open drooping
Open drooping
12 Leaf sheath Color
Light Green
Light green Light greenLight Green
Slight purple
13 Leaf sheath wax Absent Absent Absent Absent Absent
15 Leaf sheath Spines
Hard profuse
Hard profuse
Hard profuse
Hard profuse
Hard profuse
16 Leaf carriage Open drooping
Open drooping
Open drooping
Open drooping
Open drooping
16 Leaf sheath clasping
Self detrashing
Hard Hard HardSelf
detrashing
17 Dewlap colorLight green Light green Light green Light green Light green
18 Ligular Process Present Present Present Present Present
19 Shape of auricle Crescent, thin
Crescent, thin
Crescent, thin
Crescent, thin
Crescent, thin
20 Flowering Flowers in South
Maharashtra
Flowers in South
Maharashtra
Flowers in South
Flowers in South
Flowers in South
Maharashtra
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 91
4.12 RAPD analysis to evaluate genetic variation of sugarcane mutants
The molecular marker analysis was carried out to detect variations at genetic level
using RAPD amplification profile.
4.12.1Optimization of PCR conditions for RAPD analysis
MgCl2 at 2.5 mM concentration produced scorable banding pattern where as the
concentrations below 2.5 mM produced faint bands or no bands. Of the different
concentrations of genomic DNA tried (50, 100, 150 and 200 ng per 20l reaction mix),
reaction mixture containing DNA 50 ng/ml found optimum. The lower DNA quantity
yielded less intense bands, whereas the higher concentrations added background effect.
Taq DNA polymerase (1U) has resulted good amplification profile compared to 0.5U.
Among the primer annealing temperatures (35, 36, 37, 38, 39 and 400C), tested, 37 0C
was found to be optimum. Band number decreased above the annealing temperature of 37 0C and no bands were observed above 39 0C.
The molecular characterization of sugarcane varieties CoC 671, Co 86032 and
mutants (TC 906, TC 922, TC 2813, TC 2819, TC 2826 and TC 2875) of CoC 671 was
assessed by using RAPD primers (Fig. 17 and Table 27). The genetic similarity between
mutants was assessed on the basis of the Jaccard’s similarity coefficient and
complemented with a UPGMA-based cluster analysis. Scoring of morphological
characters and molecular marker data support the distinctness of mutants from the parent
CoC 671. RAPD analysis of DNA isolated from sugarcane embryogenic cultures and
mutants was carried out using 60 decamer oligonucleotide primers (OPA, OPB and OPC)
from Operon Technology Inc., USA. Among the primers screened, five primers, OPA-01,
OPA-05, OPB-02, OPC-01 and OPC-19 showed distinct and good banding pattern (Fig.
17 and Table 27).
4.12.2 Primer selection
From the 20 different primers screened, five primers produced clear polymorphic
bands in all the mutants on preliminary analysis and were selected for further analysis.
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 92
Only clear and un-ambiguous bands were taken for scoring. It has been observed that the
RAPD profile showed totally 34 bands from which 13 were polymorphic and there was
38.23 % polymorphism (Fig. 17 and Table 27). The number of bands for each primer
varied from 2 to 10 with average 6.8 bands per primer. The size of amplicons generated
by five primers ranged from 300 to 1300 bp.
Table 27 : Details of RAPD primer sequences and the band polymorphism
Genetic similarity between genotypes was assessed on the basis of the Jaccard’s
similarity coefficient and complemented with a UPGMA-based cluster analysis. The
scoring of morphological characters and molecular marker data supported the distinctness
of the genotypes from the parent CoC671.
A cophenetic correlation coefficient r = 0.82476 was obtained from two way
Mantel test (Mantel, 1967) which indicates a good fit between the original similarity
matrix and the resulting clustering analysis. Pair wise comparisons of RAPD profiles
resulted in a similarity matrix used to develop a consensus tree and estimate their
similarity indices for these mutants (Fig.18)
Genotypes CoC 671, TC 2813, TC 2819, TC 2826, TC 2875 formed a separate
cluster and TC 906, TC 922 and TC 906 B* formed separate cluster distinct from the
earlier one with parent (CoC 671) and standard Check (Co 86023).
Sr.
No
Primer Code
SequenceTotal No.
of Bands
No. of Polymorphic
Bands
%
Polymorphism
1 OPA-01 CAGGCCCTTC 8 2 25
2 OPA-05 AGGGGTCTTG 10 6 60
3 OPB-02 TGATCCCTGG 3 2 66.66
4 OPC-01 GTTGCCAGCC 8 1 12.5
5 OPC-19 GTCCCGACGA 5 2 40.00
Total 34 13 38.23
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 93
(* TC 906B sugarcane mutant data has not has been not included as it was highly
susceptible for smut. It was included in RAPD analysis as smut susceptible variant for
looking variability of susceptible and resistant genes)
CoC 671 and TC 2813 formed a tertiary sub cluster making them genetically
separate from remaining genotypes. Among the remaining genotypes TC 2819 was
distinct from the parent CoC 671, TC 2826 and TC 2875. Sugarcane mutants TC 2826
and TC 2875 were closely associated forming a separate cluster group.
0.79 0.84 0.89 0.95 1.00
Coefficient
Figure 18. Dendrogram showing the genetic relationships among sugarcane mutants
with source variety CoC 671 and standard check Co 86032.
Mean distance of individual varieties with the rest was computed from the
distance matrix for comparison. Mean genetic distance among the varieties within a
particular cluster, between standard check, CoC 671, Co 86032 and between mutants
developed is calculated. The genetic distance between the groups was found to be only
TC 906
TC 2875
TC 2826
TC 2819
TC 2813
CoC 671
Co 86032
TC 922
TC 906B*
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 94
marginally higher than the respective within group distances and the overall mean genetic
distance. The similarity indices indicated that there was 15 % genetic dissimilarity
between CoC 671 and Co 86032. This may be because CoC 671 was one of the parents
for Co 86032.
Table 28: Partial similarity matrix, showing the similarity indices between
sugarcane mutants developed from variety CoC 671
TC 906 (22%), TC 922 (12%), TC 2819 (10%), TC 2826 (10%) and TC 2875
(13%) showed genetic dissimilarities with the parent CoC671. TC 2813 showed 100%
genetic similarity with CoC 671 while TC 922 and TC 906B showed 100% genetic
similarity between themselves even though they are morphologically distinct (Table 28).
Nair et al., (2002) reported that despite of the sexual reproduction, the mean genetic
distance among 28 sugarcane varieties was only 29.31%, implying that a large part of the
sugarcane genome is similar among the varieties. This probably arises from little parental
diversity among the clones used in hybridization. RAPD analysis indicates that EMS
induces point mutations resulting in specific rectifications without much change in
genetic backbone of genotype CoC 671. However studies on radiation induced mutants
reveal 37% genetic dissimilarity (Patade et al., 2006). In present investigation genetic
similarity value ranged from 0.97 to 0.78 among the mutants (Table.27). Since this
analysis was carried out on mutants after three successive generations, we presume that
these mutants/genotypes are stable and express the variations minimizing the possibility
GenotypeCo
86032CoC 671
TC 906
TC 922
TC 2813
TC 2819
TC 2826
TC 2875
Co 86032 1.00
CoC 671 0.85 1.00
TC 906 0.76 0.78 1.00
TC 922 0.79 0.88 0.90 1.00
TC 2813 0.85 1.00 0.78 0.88 1.00
TC 2819 0.76 0.90 0.80 0.90 0.90 1.00
TC 2826 0.76 0.90 0.80 0.90 0.90 0.93 1.00
TC 2875 0.74 0.87 0.77 0.87 0.87 0.90 0.96 1.00
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 95
of epigenecity. Thus application of molecular marker technique will be of great help to
establish efficient system for selection of clones through in vitro mutagenesis. The results
obtained in present studies confirm the efficiency of the RAPD technique for
determination and estimation of genetic distances and relatedness among different
sugarcane mutants.
4.13 Qualitative traits of sugarcane mutants
Sugarcane breeders are targeting enhanced sucrose content for genetic
improvement in sugarcane. The gains in sugar yield reported in last two decades are
attributed to the increase in biomass and/or cane yield. Inspite of this breakthrough in
sugarcane improvement a plateau has been reached with respect to sugar content.
Selection for high sugar varieties is difficult due to lack of high sugared varieties in the
germplasm (Ming et al. 2002), as sugar content and yield are negatively correlated
(Hunsingi, 1993; Jackson, 2005, Wagih et al., 2004).
Building of genetic stocks for high sugar content has been attempted using
parents with high sucrose content in conventional hybridization program. Its polyploidy
nature is bottle neck for improvement. Sugarcane breeding process is lengthy and tedious,
requires consistent and strenuous effort for a period spanning over 15-17 years. Over and
above, environmental adaptability of these varieties for a specific location also
determines the yield and sugar recovery.
However, it is possible to develop varieties with high sugar, resistance to biotic
and abiotic stress and short duration cultivar by inducing mutations and through genetic
engineering. The mutants developed in this study, TC 906, TC 922, TC 2813 and TC
2819 hold very good promise to achieve this goal. These mutants are agronomically
superior, with good quality and yield traits over their parent CoC 671. TC906 shows 7%
improvement in Sucrose %, CCS % and 12% in cane yield over CoC 671 (Table 29a, b,c
Fig. 19a).
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 96
Table 29a: Sucrose% in selected sugarcane mutants (Batch I)
Table 29b: CCS% of selected sugarcane mutants (Batch I)
Table 29c: Cane yield of sugarcane mutants over CoC671 clones at 12th month
(Batch I)
Sugarcane mutant &
Check
Sucrose %
Mean
% improvement over CoC 671
10M 11M 12M 10M 11M 12M
TC- 906 16.67 19.80 21.04 7.68 2.96 0.77
TC-922 15.84 19.79 21.33 2.27 2.91 2.20
CoC 671 15.48 19.23 20.87
Sugarcane
mutant &
Check
CCS%
Mean
% improvement over CoC 671
10M 11M 12M 10M 11M 12M
TC- 906 11.51 14.10 15.00 7.57 2.39 -
TC- 922 10.81 14.05 15.26 1.03 2.03 1.66
CoC 671 10.70 13.77 15.01
Sugarcane mutant & Check
Cane yield
t/ha% improvement over
CoC 671 t/ha
TC- 906 144.11 12.00
TC-922 165.33 28.46
CoC 671 128.44
Chapter 4 [Results and discussion]
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For another set of mutants the analysis has been given in the Tables 29a, 29b and
29c. It has been seen that TC 2813 and TC 2819 higher sucrose % and CCS % from 10th
month onwards till the crop harvest at 14th month. Mutants TC 2813 and TC 2819
showed an early maturity with higher level of CCS t/ha, CCS%, Sucrose % and Purity %
and higher sugarcane yield over the source variety CoC 671, not only at 10th month but it
has prolonged over 14th months of crop age (Table 22, Fig. 19b).
Table 30a: Comparative analysis of sugarcane mutants for sucrose% (Batch II).
Table 30b: Comparative analysis of sugarcane mutants for CCS% (Batch II).
Sr.No.
Sugarcane mutant &
CheckSucrose%
% improvement over
CoC 671
10 M 12 M 14 M 10 M 12 M 14 M
1 TC2813 18.53 23.71 23.32 4.86 10.84 9.22
2 TC2819 20.33 22.49 23.98 15.05 5.14 12.31
3 TC2826 18.11 23.13 22.08 2.49 8.13 3.50
4 TC2875 17.04 22.11 22.22 - 3.37 4.07
5 CoC 671 17.67 21.39 21.35
Sr.No.
Sugarcane mutant &
CheckCCS %
% Improvement over
CoC 671
10 M 12 M 14 M 10 M 12 M 14 M
1 TC2813 13.13 17.42 17.02 5.42 12.77 11.53
2 TC2819 14.47 16.18 17.56 16.04 4.72 15.07
3 TC2826 12.97 16.90 15.90 4.00 9.38 4.5
4 TC2875 11.91 16.02 15.94 - 3.69 4.46
5 CoC 671 12.47 15.45 15.26
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 98
Table 30c: Comparative analysis of sugarcane mutants for cane yield (Batch II).
Sugarcane harvesting is usually determined by parameters such as sucrose and
brix %. Ssugarcane mutant developed in this study show high sucrose accumulation at
early stage of its growth beginning at 10th month and are promising to replace CoC671
which was released for commercial cultivation two decades ago. High sucrose
accumulation also correlates significantly with earliness, high CCS, high purity and
improved cane yield.
Wagih et al., (2004) stated that an early maturing variant with prolonged
production and/or overlapping production period would be desirable in extending the
harvesting season. This will make the cropping season more efficient, aiming at
producing adequate sugar yield. Therefore, TC 906, TC 922, TC 2813 and TC 2819,
have longer harvesting period with early maturity and high sugar content are more
suitable for mid and late harvesting of the crop, holds sustainability in sugarcane
production.
Many research workers have reported negative correlation between sugar content
and yield using exiting genetic base but not absolute. From the mutants (Table 28 and
Table 29) identified in this study, we have observed positive improvement in cane yield
and sucrose content. These mutants can be used as parental clones in breeding program
for development of sugarcane hybrids.
It has been reported that CoC 671 is a better female parent for development of
high sugared drought tolerant variety through hybridization program (Hemaprabha et al.,
Sr.NoSugarcane mutant
& CheckCane
Yield t/ha
% improvement over
CoC 671 t/ha
1 TC 2813 129.22 36.85
2 TC 2819 128.65 36.25
3 TC 2826 107.16 13.49
4 TC 2875 97.56 3.32
5 CoC 671 94.42
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 99
2006). Shanti et al.,( 2005), and Ming et al., (2001, 2002 a,b) reported that all the high
sugared varieties developed, have cytoplasm from female parent S. officinarum and have
been traced to seventeen generations. In similar line the high sugar mutants developed
through this study will be useful as better female parents for sugarcane improvement.
Mutants TC 906, TC 922, TC 2813 and TC 2819 developed in present work show
considerable improvements over CoC 671 with respect to smut resistance, early maturity,
sugar content and yield. Hence they can serve as better germplasm than CoC 671 for the
sugarcane improvement. Identification of markers/quantitative trait loci linked to both
yield and sucrose traits could help adapting good breeding strategy and to minimize the
time frame for selecting the better genotypes. Casu et al. (2005) suggested that there is a
need to identify the genes and mechanisms that contribute to high CCS phenotypes which
can be combined to optimize sugar accumulation. This can be achieved by analyzing:- (1)
analysis of Specific gene expression patterns associated with high sucrose accumulation
and pyramiding genes and (2) use of these genes as DNA markers to test for linkage to
QTLs for high sugar content.
The maturity profiles of these variants showed that juice and cane parameters
(earliness high sucrose content, high purity and high CCS and improved yield) are
significantly correlated. The sugar recovery percentage reported in countries like Brazil,
Australia and USA is around 14 percent. The sugar recovery in India has remained
stagnant at around 10 percent for the last few years (Table 4). There is a need to take
steps towards evolving improved varieties with improved sugar recovery to meet
demands of sugar and sugarcane based industries. TC 2813, TC 2819, TC 906 and TC
922 are useful due to their better agronomic and quality parameters and have the potential
to serve as mid-maturing genotypes. Early maturing TC 2819 will have numerous
benefits to both the growers and sugar industries providing an efficient and reliable
means of achieving increased sugar yields at the beginning and end of the season. (Table,
Fig 19 a, b). It will allow earlier commencement of the harvesting and the processing, and
delivering benefits to the farmers. The present research studies has resulted in selection of
mutants TC 906, TC 922, TC 2813, TC 2819 and TC 2826 with improvement in smut
disease resistance, earliness in maturity and yield potential over CoC 671 and there by
fulfilling the objectives of genetic improvement of sugarcane.
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 100
4.14. Agrobacterium mediated AtNDPK2 transformation in sugarcane
4.14.1 Plasmid DNA isolation and confirmation
Plasmid pCsV1300 harboring NDPK2 gene (Fig. 20) was isolated from E. coli
strain DH5. The plasmid DNA was subjected to agarose gel electrophoresis to confirm
(Fig.21a). The gel purified plasmid pCsV1300 was subjected to restriction digestion
using Eco RV and Hind III restriction enzymes (Fig 21b). After restriction digestion
fragment of 10500 bp of non-digested DNA, 2600 bp and 5100 bp fragment containing
the digested backbone by the restriction enzymes. The presence of AtNDPK2 gene was
also confirmed by PCR amplification using AtNDPK2 gene specific primers (Fig.21c).
4.14.2. Agrobacterium transformation with plasmid DNA
Plasmid pCsV1300, harboring AtNDPK2 gene was transformed in to the
Agrobacterium strains EHA 105, LBA 4404 and GV3101. The positive colonies of
Agrobacterium were cultured on YEM plates/medium containing rifampicin as selection
marker. The confirmation of Agrobacterium transformation was carried out with colony
PCR amplified 560 bp fragments corresponding to AtNDPK2 gene (Fig.22). The pCsV
1300 transformed Agrobacterium colonies then were utilized for transformation of
sugarcane calli and nodal buds as mentioned in Materials and methods 3.25.
4.14.3 Optimization of Agrobacterium transformation protocol
The optimization of callus culture/ nodal buds with respect to support matrix has
been discussed in 4.2. The calli/ nodal buds (Fig.7 and Fig. 23 ) grown on the cotton
support matrix were vigorous and fast growing with lower phenolics as compared to that
of on agar medium. Other benefits of buffering pH of the medium, better nutrient
availability have been discussed in 4.3.7. The prospects discussed above were useful for
facilitating and improving Agrobacterium mediated transformation.
Chapter 4 [Results and discussion]
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4.15 Effect of biocides on Agrobacterium growth
Sodium benzoate (10mg/l) and Chitosan (500mg/l) successfully restricted the
Agrobacterium growth on YEM medium (Fig 24 a,b,c) and did not show any toxic effect
on callus development and shoot regeneration (Fig.24 d,e). It was observed that further
growth of calli was found optimum in tissues treated with sodium benzoate and chitosan
indicating their biocidal effect against Agrobacterium.
Agrobacterium contamination in the transformed calli was a major constraint
during the study. Therefore attempts were made to restrict the bacterial growth and boost
the plant cells vigor by using biocides. Sodium benzoate is a food grade antibacterial
compound (Stanojevic et al., 2010) and Chitosan is a systemic acquired resistance
inducer and antimicrobial compound (Badawy and Rabea, 2011) are non toxic to plant
cells. Inoculation of a plant tissue with Agrobacterium itself is a disruptive process and
triggers a hypersensitive response. As a result, there will be poor survival rate of the
target tissue. Therefore, minimizing the damage due to interaction of Agrobacterium with
plant tissue is critical for the success of genetic transformation experiments.
4.15.1 Agrobacterium infection to callus and nodal buds
Out of 500 transformed calli, 50% became brown/black which were discarded.
The survived calli were regenerated on shooting and rooting medium containing
hygromycin (25mg/l) (Fig 25). After 25 days, the rooted plants were transplanted in
polybags containing soil mixture and kept in humidity and temperature controlled green
house for hardening (Fig 26). Similarly, a batch of 100 nodal buds was infected with
Agrobacterium suspension and the regenerating buds were passed to hygromycin
containing medium and the regenerated buds transferred to polybags containing soil
mixture (Table 31 and Fig. 26). The leaf samples were collected and used for genomic
DNA extraction (Fig. 27) and presence of AtNDPK2 gene using PCR was analyzed (Fig
26).
Chapter 4 [Results and discussion]
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Table 31: Agrobacterium infection and selection of putative transgenic plants
4.16. Screening of putative transgenic plants by PCR
T1 generation of PCR positives have been raised by cutting the sets and planting them in
polybags (Table 31).
4.16.1 Confirmation of transgene expression in T1 generation
Among plants from regenerated calli and nodal buds, six plants were PCR
positive for NDPK2 gene. The hardened plants were grown for a period of three months
in polybags (Fig.28). Later on the putative transgenic plants (PCR positive) were
transplanted in to big earthen pots containing 5kg of soil and grown for nine months. For
further multiplication, sets generated (second generation - T1 plants) from theses six
plants were further raised in larger earthen pots.
The leaf samples were subjected to PCR analysis using gene specific primers
(Fig. 29). No band corresponding to AtNDPK2 gene of 756 bp was observed in any of
the re-germinated buds. These studies indicated that only small portion of transgenic
events show stable expression irrespective of the transformation system. Similar
observations have been made in sugarcane (Birch 1997; Efendi and Matsuoka, 2011).
Explant TypeVariety Number Number
PCR Positive
Plants
Embryonic callus
Inoculated Regenerated
CoC 671 500 50 2
CoC 671 500 74 2
CoC 671 500 64 2
Nodal buds
CoC 671 100 22 Bulk Sample B I
CoC 671 100 34 Bulk Sample II
CoC 671 100 45 Bulk Sample III
Chapter 4 [Results and discussion]
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Table 32: Bud multiplication in T1 generation of PCR positive plants
In other crops, it is common for thousands of transformed lines to be screened to
identify a few chance events that escape both transgene silencing and any undesired
incidental effects of the transformation process. The problem is exacerbated because the
onset of silencing is unpredictable, creating a risk of instability in the introduced trait
(Birch, 1997). Romano et al. (2005) have reported similar observations in soybean where
more than 300 plants of progeny were obtained, demonstrating that the phenomenon of
elimination was consistently repeated and offering an opportunity for detailed study of
transgene elimination, including the characterization of the integration sites. Yin et al.,
(2004) have reviewed that non Mendelian inheritance of transgene has been with
frequency between 10% and 50% in transgenic plants produced either by Agrobacterium
mediated transformation or through particle bombardment. Various effects such as
deletion, duplication, rearrangement, repeated sequence recombination as well as gene
interaction has been observed for transgenic loci.
Transgene elimination of the bar gene has been reported in wheat. Of six
transgenic wheat lines, five were stably transformed. However, one line with five copies
of the bar gene has lost gene expression in the R1 generation, and the transgenes were
physically eliminated in the R3 generation (Srivastava et al., 1996). Complete physical
loss of transgenes has also been reported in Cyamopsis tetragonoloba (Joersbo et al.,
1999), Nicotiana tabacum (Risseeuw et al., 1997), Nicotiana plumbaginifolia
(Cherdshewasart et al., 1993) and Arabidopsis thaliana (Feldmann et al., 1997; Howden
et al., 1998). The mechanisms involved in transgene elimination are poorly understood,
and it has been attributed to intrachromosomal recombination (Fladung, 1999), genetic
Sr. No. Plant No. No. of Buds Planted No. of Buds Regenerated
1 TGS1 3 17 10
2 TGS 4 16 9
3 TGS 13 22 13
4 TGS 14 19 9
5 TGS 70 20 12
6 TGS 71 25 14
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 104
instability resulting from the tissue culture conditions (Risseeuw et al., 1997; Joersbo et
al., 1999) or a genomic defense process (Srivastava et al., 1996).
Kumpatla et al. (1998) further suggested that transgene elimination might have a
role in a genomic defense system that acts against natural intrusive DNA, as already well
described for gene silencing (Matzke et al., 2000; Waterhouse et al., 2001; Voinnet,
2001; Plasterk et al., 2002;). Corroborating with this hypothesis, recently it was
demonstrated that elimination of transgenes acts as a genome defense system in
Tetrahymena thermophila (Yao et al., 2003).
Sugarcane has exceptional genetic complexity with 100-120 chromosomes in
cultivar that are highly heterozygous. Studies to-date have reported that sugarcane, is
more prone to genomic instability, show highly efficient and impose rapid silencing of
diverse transgene constructs. Silencing is 5′-sequence-specific; copy number
independent, developmentally regulated and post-transcriptional in the plants first
regenerated from transgenic callus may be associated with increasing endopolyploidy
during maturation of differentiated tissues (Birch et al., 2000 ; Birch et al., 2010, Samac
et al., 2004; Su-Hyun et al.,2010).
High copy number and exceptional complex integration pattern are common
causes that contribute to instability of transgene expression. Chromosome number in
sugarcane varies between cell to cell and organ to organ. However effects are over
ridden by genetic redundancy having more than two copies of the same genome. Birch et
al.(2010) have shown that transgene silencing in sugarcane is exceptionally efficient in
primary transformants (T0 Plants). They are developmentally regulated, independent of
the copy number, sequence specificity and post transcriptional modifications (Heinz and
Mee 1969; Karp 1995; Prasanna et al., 2009; Su-Hyun et al., 2010; Waguespack et al.,
2009). This could be the reason for the variability in the genetic traits not being expressed
including the transgenes as observed in present study.
Promoters are key regulatory element, direct the pattern of gene expression
whether they are constitutive, tissue specific, developmentally regulated. They play a
crucial role in successful transformation and expression of the gene. Use of strong and
tissue specific promoters are critical to success of transformation especially in crop like
sugarcane which has polyploidy genome. Earlier studies indicated that CaMV 35 like
Chapter 4 [Results and discussion]
In Vitro Approaches for Improvement of Sugarcane Cultivar Page 105
promoters show reduction in expression of the genes in mature stems of regenerated
plants (Birch et al., 2010; Mudge et al., 2009). Maize Ubi-1 promoter has been used in
sugarcane to successfully express genes of interest (Efendi and Matsuoka, 2011; Hansom
et. al., 1999; Joyce et al., 2010; Mudge et al., 2009; Wei et. al., 2003; Yang et. al., 2003).
In sugarcane transformation studies, Rice actin1 and EmuI elements have shown better
expression than CaMV35S (Gallo-Meagher and Irvine, 1993) but not sugarcane ubiquitin
promoters ub1 and ub9 (Wei et al., 2001). High level of polyploidy in sugarcane further
complicates the development of transgenes. Mendelian heritability rules hardly can be
applied to plants that are polyploidy in nature. In sugarcane each allele has 5-14 copies in
genome, replaces poor alleles with desired characters. If a recessive allele is introduced
into sugarcane, the trait it encodes does not express itself in the plant until every single
original allele has been replaced with the introduced one. The probability of finding such
a fortunate genetic recombination among transformed progeny may be practically zero
(Tammisola, 2010). It is clear from the present study that the Cassava Mosaic Virus
promoter (pCsV 1300) seems to get silenced in the regenerated tissue although we could
observe good transient expression in shoots regenerated from the callus. Our data also
corroborate with all above mentioned possibilities. Therefore, it would be expected that a
foreign sequence integrated into an rDNA unit would be identified in T0 and eliminated
in subsequent generations.