Abstract
A study was carried out to find out somaclonal variation along with induction in sugarcane through biotechnological approaches such as in vitro regeneration, acclimatization of the regenerates, mutation as well as somaclones development, screening for red rot resistant somaclones, characterization of induced somaclones using morphological tools, RAPD and SSR Markers. The collected leaf sheath explants (0.5 cm) of five BSRI released sugarcane varieties viz. Isd 36, Isd 37, Isd 38, Isd 39 and Isd 40 were inoculated on only MS medium, MS medium supplemented with different concentrations (10%, 20%, 30%, 40% and 50%) of Green Coconut Water (GCW) and MS medium supplemented with different concentrations (1, 2, 3, 4 and 5 mg/l) of 2,4-D containing GCW (10%). Only MS medium and MS medium supplemented with different concentrations (10%, 20%, 30%, 40% and 50%) of GCW did not response regarding callusing, shooting and rooting while the responses to callus induction were obtained from MS medium supplemented with all different concentrations (1, 2, 3, 4 and 5 mg/l) of 2,4-D containing GCW (10%) and the best response was found in MS medium fortified with 3 mg/l of 2,4-D containing GCW (10%) for all varieties. Best performance to shoot initiation was found in MS medium supplemented with 2 mg/l (BAP) and 1 mg/l (Kn). Moreover, MS medium containing 5 mg/l of NAA showed the best performance for root initiation. The developed somaclones, on field evaluation, exhibited huge variability for leaf, tiller, plant height, internode, bud, millable cane, brix% and flowering characters etc. The plantlets regeneration as well as mutants were developed by sodium azide (1 and 2 mg/l) and EMS (0.01M) treated calli derived from MS medium supplemented with 2,4-D. Mutants were produced having changes in different traits such as number of tillers, stalk colour, number of internodes per plant etc. Somaclones showed variation from their sources varieties in case of reaction against red rot pathogen. Out of 24 genotypes including 4 varieties, 14 were resistant, 8 were moderately resistant and 2 showed variable degree of susceptibility. Somaclones were analyzed by RAPD and SSR markers to check the variation between parents and their somaclones at molecular level. Six RAPD primers generated 237 bands with average 39.5 varied from 15 to 63 with size ranging 145 bp-1000 bp among the 4 varieties and their 12 somaclones. Genetic diversity or PIC value ranged from 0.39 to 0.50 for all loci across the 4 varieties and their 12 somaclones based on RAPD markers. Dendrogram based on linkage distance using Unweighted Pair Group Method of Arithmetic Means based on 9 SSR primers indicated segregation of the 4 varieties and their somaclones into two main clusters at linkage distance 16.5. Variety Isd 40 was found to retain in main cluster C, while other varieties (Isd 37, Isd 38 and Isd 39) and their somaclones were found in main cluster C2 having different sub-clusters. The sizes of the amplified bands in the 4 varieties and their 8 somaclones ranged from 115 bp to 434 bp based on 5 SSR primers using Polyacrylamide Gel Electrophoresis. Therefore, it may be concluded that RAPD and SSR markers can be used for identification of somaclonal variation and the relationship between parents and their somaclones. These somaclones and mutants with desirable characters could be selected for a future breeding program.