2.6. Shoot Proliferation from Callus
Well-established organogenic callus (4-week-old) grown on MS medium supplemented with combination of BAP (1.0 mg/L) and 2,4-D (1.0 mg/L) for all accessions were used. About 0.5gram of calli were transferred to shoot regeneration media, consisting of MS basal medium containing various combinations of cytokinins viz. BAP (0, 0.5, 1.0 mg/L) and TDZ (0, 0.25, 0.5, 1.0 mg/L) individually and in combination with different concentrations of IBA (0, 0.1, 0.2 mg/L). There were three callus clumps per jar and five replicates per treatment. Then the culture was maintained in a growth room at a temperature of 25±20C and 16 h photoperiod provided by white florescent lamps. The cultures were subcultured once transferring into fresh media after three weeks for further initiation of adventitious shoots. During subculture removal of dead, dark brown cells was done. The percentage of shoot organogenesis and number of adventitious shoot initiated from callus was recorded after six weeks of transferring the callus on shoot regeneration media.