The commercial cultivar Co261 of sugarcane grown in Madhya Pradesh was employed as the source of explants. Healthy and disease-free sugarcane tops obtained from KVK Narsinghpur and from other sugarcane cultivating farmers of Narsinghpur, Madhya Pradesh, India.were used as explants.

In order to study the morphogenic response of sugarcane explants the most widely accepted MS medium (Murashige and Skoog 1962) was used as basal medium. The stock solutions were prepared fresh after every 4 to 6 weeks and that of the growth regulators were prepared fresh after every week. The supplement to be incorporated such as sucrose 30gl-1 into the basal medium were added before final adjustment of the volume prepared by double distilled water and further the plant growth regulators were added and then pH of the medium was adjusted at 5.8 ± 0.5 using either 0.1N NaOH or 0.1N HCl. The medium was solidified with bacteriological grade agar added before autoclaving at 120 ºC at 15 psi. Surface sterilization of explant leaf roll (sugarcane tops of about 10 cm) was done by 4-5 time washing under running tap water and then initially treated with treated with Tween-20 (5-7 drops) for 20 min proceeded by seventy percent of ethanol and different concentration of mercuric chloride for 3, 4 and 5 min respectively. Sterilized leaf roll explants were washed thoroughly with sterile distilled water for 5-7 times to eliminate the mercuric chloride in a laminar air- flow cabinet.

The segments of leaf roll were excised from contamination-free shoot apices of sugarcane variety Co261and cultured on MS medium supplemented with different levels of 2,4-D to know the response on callus induction and maintained by subculturing at every 21 days with 2,4-D (3.0 mg/l). After 60 days, the callus were transferred to callus induction medium containing 8 ìM Ethyl methane sulfonate (EMS). After induction of somaclonal variation, callus were transferred to regeneration medium containing different concentrations of BAP. After shoot differentiation from established callus cultures DNA was isolated, purified, quantified and diluted PCR conditions were standardized considering different parameters viz. initial denaturation, denaturation, annealing, extension and final extension. PCR thermal and reaction profile was optimized for amplification purpose by using primers of unique sequence. PCR products mixed with gel loading dye was loaded on 1.5% agarose gel along with appropriate size DNA ladder (1kb) and run under constant voltage of 70 volt. The gels were visualized under UV light in gel documentation system and photographed through the same. DNA fingerprints on ISSR and RAPD gels were scored for the presence (1) or absence (0) of bands of molecular weight size in the form of binary matrix for all the accessions studied using DARwin6 Software. A dendogram was constructed using UPGMA (un weighted pair-group method with arithmetic averages) with the SAHN (sequential, agglomerative, hierarchical, and nested clustering) routine.

MS medium fortified with different concentrations of 2,4-D were used for callus induction were incubated at 25±2°C with photosynthetic photon of 50 μmol-2s^-1 under 16h/8h photo period. Callus induction was observed only on MS medium supplemented with 2,4-D alone. Callus of loose and fragile nature was observed on plumule region after 10-12 days of incubation which further spread throughout the seedling. On Maximum callus induction (52.22%) was obtained on MS medium fortified with 3 mg/l 2,4-D (Plate 2 B and D) followed by 47% callus induction on MS medium fortified with 4mg/l 2,4-D (Plate 2 A and C ). With the increase in concentration from 1 mg/l to 4 mg/l of 2,4-D into MS medium, callus induction gradually enhanced in mature leaf roll explants. It is evident from the results that concentrations of auxin significantly affected both the traits. These results are in consonance with Kale (2004). According to Lal et al. (1992), in fast growing callus DNA, RNA and protein content increase rapidly and the condition becomes different during callus growth.

Calli were treated with Ethyl methane sulfonate (EMS) in order to increase the mutation frequency and enhance somaclonal variation after 60 days, the callus were transferred to callus induction medium containing 8 ìM EMS. Studies have shown that EMS induces mutations (Hoffmann et al. 2004), thus generating variation amongst plants produced from the treated calli. Further, several studies have reported on EMS treatments to calli and subsequent selection of plants with desired traits using appropriate selection protocols. These traits include herbicide resistance, salt tolerance and disease resistance (Matsumoto et al. 2010).

After callus induction, calli were sub-cultured on shoot induction medium. Callus culture in shoot induction medium turned in embryogenic callus after 7 days and leaf primordia were induced within 15 days of culturing. These leaf primordia were elongated in the form of shoots within one month of culturing. MS medium supplemented with different concentrations of BAP, maximum shoot induction (55.75%) and shoots per callus (4.36) were obtained on MS medium fortified with 3 mg/l BAP followed by (48.85%) callus exhibiting shoot induction with (3.62) shoots on MS medium fortified with 2 mg/l BAP.

Shoot induction response and numbers of shoots were significantly reduced with increasing concentration of BAP. MS medium fortified with BAP was found to be significantly superior in terms of both shoot induction percent and number of shoot per callus. Study done by Kumar et al. (2012)showed a large number of shoots were regenerated from such calli by subculturing on Murashige and Skoog’s medium containing BAP and Kinetin (0.5 mg/l each). These results clearly indicate that a lower concentration of cytokinin is required for shoot induction in sugarcane.

In vitro culture and induced mutagenesis are potentially-useful complementary approaches to conventional breeding for developing somaclonal variant plants exhibiting disease tolerance. The findings from the present study indicate that the treatment of calli (variety Co261) with EMS in combination with selection of tolerant calli. Indirect organogenesis clearly indicated its potential in inducing variability. Actually, genetic variation may have occured as a result of dedifferentiation of mature cells into callus stage, in the presence of plant growth regulators such as 2,4-D. Leaf roll proved as an better explants. Effective utilization of regeneration from callus may prove to be another way for creating some desirable variations with optimize conditions for increasing regeneration frequency. Plant tissue culture could be used in rejuvenating aging varieties like Co261 (Indian variety).

I would like to take this opportunity to thank all the people who have helped me, in one way or another to gain this experience. Words cannot express my deepest core of my soul to my family whose selfless love constant and encouragement, sincere prayers to God, expectation and blessing have always been the most vital source of inspiration in my life.

Words are inadequate in the available lexicon to avouch the excellent guidance given by my major advisor Dr. Sushma Nema, Professor, Biotechnology Centre, JNKVV, Jabalpur and Dr. Amitava Rakshit Assistant Professor, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, U.P. Their dedication to research, meticulous planning, consecutive consul and reserved help serve as a beacon light throughout the research work.