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.