Explant: Mature dehusked seeds from two local Aus rice cultivars viz. Chamak and Shama were collected during October 2012 to July 2013 as explants to conduct the experiment. Prior to culture establishment, seed germination test was done by petridish method. The germination percentage of Chamak was 90% and that of Shama was 95%.
Design of experiment: The experiment was laid out in the laboratory following Completely Randomized Design with two factors and three replications. Each culture bottle containing 25 ml of nutrient medium with 10 seeds was considered as one replication for callus induction.
Factor A: i) Chamak and ii) Shama
Factor B: Hormonal concentrations (2,4-D @ of 1.0, 1.5, 2.0 mgL-1 and NAA at 0.0, 0.5, 1.0 mgL-1 )
Transfer of calli onto the regeneration media: After 4 weeks of inoculation of seeds, the calli were plated in test tube containing 5ml regeneration medium (MS medium supplemented with 0.5 mgL-1 NAA and 3.0 mgL-1 BAP). After 3 weeks of transference of calli, a single subculture was done on the same regeneration medium. The test tubes were placed under fluorescent light in a growth room with controlled temperature (25±1 0C) and under 16 hours photoperiod with light intensity of 3000 lux.
Transfer of rootless plantlets onto the liquid media: The plantlets which had only shoot were transferred to the liquid media supplemented with 1.5 mgL-1 IBA and 0.5 mgL-1 NAA. Two subcultures were done at 3 weeks interval.
Hardening: Regenerated plants with profuse roots were transferred in sand supplemented with MS liquid medium and were kept in hardening room for pot culture. Before transferring the plants were treated with Bavistin solution @ 0.1% (w/v).
Established plants from small pots were transferred to larger pots containing puddle field soil and were kept in net house. They were frequently watered and kept under observation for 3 weeks. Data on survival were then recorded by using the following formula:
Survival(%) = No of plants survived after 3 weeks / Total no. of plants transferred to larger pots x 100
Collection, calculation and analysis of data: Data were collected by counting the calli induced and plants regenerated in the test tubes. After four weeks of inoculation of rice seeds, callus induction frequency was calculated. Data on plant regeneration were collected after 3 weeks of transference of calli onto regeneration medium. All the calli originated from a single seed and all the plants regenerated from single plated callus was considered as one. The weight and diameter of calli were measured by digital balance and slide calipers, respectively. Callus induction frequency (%), weight of callus (g), diameter of callus (mm), response of calli to caulogenesis (%), response of calli to rhizogenesis (%), response of calli to caulo-rhizogenesis (%), total regeneration (%), frequency of root regeneration in root inducing media (%) and ex vitro survival (%) were considered as parameters. The parameters were recorded by using the following formula: Callusinduction frequency(%) = No of seeds produced callus / No of seeds inoculated x 100