Binadhan-5, Binadhan-6, BRRI dhan32 and Basmati 370 were used as plant material to study different parameters associated with in vitro regeneration of plant. The experiments were carried in 2010 at the Tissue Culture Laboratory, Department of Biotechnology, Bangladesh Agricultural University, Mymensingh and Biotech Laboratory, Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh. Culture technique: For callus induction, callus proliferation, shoot regeneration and rooting MS medium (Murashige and Skoog, 1962) was used. The culture techniques were explants culture, partial desiccation, subculture or transfer into regeneration media and rooting employed. Mature embryos attached to endosperm were the main source of explants for embryo culture. The hormones and their respective number of combinations of embryo culture were as follows treatment:
a) For callus induction MS medium + 0.50 mg L-12,4-D (T1), MS medium + with 1.00 mg L-12,4-D (T2), MS medium + with 1.50 mg L-12, 4-D (T3), MS medium + 2.00 mg L-1 2,4-D (T4), MS medium + 2.50 mg L-1 2,4-D (T5), MS medium + 3.00 mg L-1 2,4-D (T6)
b) For shoot differentiation MS medium + 2 mg L-1 Kinetin + 0.5 mg L-1 NAA (T1), MS medium + 4 mg L-1 Kinetin + 0.5 mg L-1 NAA (T2), MS medium + 6 mg L-1 Kinetin + 0.5 mg L-1 NAA (T3), MS medium + 8 mg L-1 Kinetin + 0.5 mg L-1 NAA (T4), MS medium + 10 mg L-1 Kinetin + 0.5 mg L-1 NAA (T5), MS medium +12 mg L-1 Kinetin + 0.5 mg L-1 NAA (T6)
c) For root initiation MS medium + 0.4 mg L-1 IBA (TI), MS medium + 0.5 mg L-1 IBA (T2), MS medium + 0.6 mg L-1 IBA (T3)
Explants culture: Sterilized mature seeds were cultured directly in MS medium supplemented with different concentrations of hormones and sucrose required as per treatment.The culture plates containing explants were placed under fluorescent light in a room with controlled temperature (22±2°C) using 16 hrs photoperiod. Subculture in regeneration media: Three weeks after inoculation, the calli attained convenient size. Then they placed again on freshly prepared sterilized medium containing appropriate hormonal supplements for shoot induction from the calli. The subculture media were MS medium containing different combinations and concentrations of NAA and Kinetin. The subcultured petridishes were again incubated at 22±2°C with 16 hrs photoperiod and mentioned for calli and organogenesis. Rooting: The subcultured calli continued to proliferate and differentiated into shoots. When these shoots grew about 2-3 cm in length, they were separated from each other and again cultured individually on vials or petridishes with freshly prepared root induction medium to induce root. The vials or conical flasks containing plantlets were incubated at 22±2°C with 16 hrs photoperiod. Data recorded on Callus induction (days of callus initiation, number of explants with callus, size of callus) and plantlet regeneration (days to shoot initiation, number of callus with shoot, number of shoots per callus, average number of roots per plants, percent plant establishment). Complete Randomized Design (CRD) was used in growth room Tissue Culture Laboratory. The analyses of variances for different parameters were performed and means were compared by the Duncan’s Multiple Range Test (DMRT) in MSTATC program.