Results and Discussion 4.1 Tissue culture and sugarcane ...shodhganga.inflibnet.ac.in/bitstream/10603/23749/12/12_chapter_04.pdf · 4.1 Tissue culture and sugarcane improvement Tissue

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



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),



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.,



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



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



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



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



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.



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)



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.



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).



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



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



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



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



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).



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



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 -



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 - - - - -



(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).



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



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

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



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).



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)


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



Table 20: Juice quality data of selected sugarcane mutant from sugarcane variety CoC 671 in clonal trial-I (Batch I)


.

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



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).



Table 21: Yield and quality parameters of selected sugarcane mutants developed from

sugarcane variety CoC 671 in clonal trial I (Batch II)


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



Table 22: Juice quality data of selected sugarcane mutants from sugarcane variety CoC 671 in clonal trial I (Batch II)


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



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



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



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



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



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



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).



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



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



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.



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



(* 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*



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



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).



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



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



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



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.



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.



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).



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



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



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



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.

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