Abstract
Five experiments were conducted with ten indica rice genotypes to study gene cloning following INVITROGEN technology, in vitro regeneration, genetic transformation and variability among parents and regenerants using embryogenic callus as explants following standard protocols. Two 1694 bp DNA bands each obtained after electrophoresis confirmed the cloning of the gene AtNHXI into Agrobacterium tumefaciens. Among the varieties BRRI dhan40 took the least number of days (14.50) for callusing, exhibited the highest callus induction frequency (39.60 %), the highest embryogenic callus induction frequency (35.20 %), the best size of calli (3.80 mm) and also the best callus weight (1.11 mg). The least number of days required for green bud formation (13.76), the highest callus induction frequency (62.20 %), the highest embryogenic callus induction frequency (59.80 %), the best callus size (3.43 mm) and the best callus weight (1.89 mg) were observed in MS medium supplemented with 2.0 mg/L 2, 4-D, 0.5 mg/L BAP and 500 mg/L L-Proline. BRRI dhan40 showed rapid callusing (10.60 days), the highest callus induction frequency (96.0%), the highest embryogenic callus induction frequency (94.0 %), the best callus size (4.96 mm) and the best callus weight (2.44 mg) when cultured in MS medium supplemented with 2.0 mg/L 2, 4-D, 0.5 mg/L BAP and 500 mg/L L-Proline. The least number of days (11.46) for green bud formation, the highest callus regeneration (72.66 %), the highest number of shoots per callus (5.26) was reported in BRRI dhan29. The least number of days for green bud formation (12.50), the highest callus regeneration (68.26%) and the highest number of shoots per callus (6.24) was observed in MS medium supplemented with 6.0 mg/L Kinetin and 0.2 mg/L NAA. BRRI dhan29 in MS medium supplemented with 6.0 mg/L Kinetin and 0.2 mg/L NAA took the least number of days (7.60) for green bud formation, the highest callus regeneration (97.80%) as well as the highest number of shoots per callus (9.80). The highest number (25.20) of roots per shoot and the highest plant establishments (68.05%) were observed in BRRI dhan29. However, the highest numbers of roots per shoot (32.18) and the highest plant establishment (66.18%) were obtained in MS medium supplemented with 0.5 mg/L IBA. BRRI dhan29 in combination with MS medium supplemented with 0.5 mg/L IBA showed the best performance in producing maximum roots per shoot (43.60) and also the highest plant establishment (43.60%). The highest level of gene diversity value and Shannon's Information index were found respectively in loci OPA 20-3, OPA 20-11 and G 7-9, whereas the highest genetic distance (0.9904) was observed between BRRI dhan36 control and Bina dhan-6 control. The highest percentage of callus regeneration as well as number of shoots per callus was observed when no antibiotic was used. The regeneration gradually decreased when antibiotic hygromycin at 10, 20, 30, 40 and 50 mg/L, cefotaxime and carbenicillin respectively at 100, 200, 300, 400 and 500 mg/L were used in regeneration medium. In case of genetic transformation, the least number of days (12.60) was required to green budding in BRRI dhan29 when the calli were infected for 30 minutes and co-cultivated for 3 days with Agrobacterium strain EHA105 containing plasmid pH2GW7-pUbq-AtNHXI. The highest transformation (12.33%), the highest number of transgenic shoots per callus (5.20) and also the highest establishment of transgenic plants (63.21) were also found in BRRI dhan29 for 30 minutes infection time and for 3 days co- cultivation period. The transformed plantlets of ten genotypes produced expected bands 1694 bp by the primer AtNHX1, whereas non transformed control plantlets did not show any band in PCR reaction by the same primer. The presence of 1694 bp bands in the putative transgenic plantlets proved the integration of the transgene in the tested genotypes. This protocol will help develop salt tolerant transgenic rice variety and thus will solve the crucial salinity problem of our coastal region for rice cultivation.