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Experiments were conducted to establish an efficient and simple protocol for regeneration and Agrobacterium mediated genetic transformation of an agronomically important indica rice variety Bg 250. Callus induction was achieved on modified N6B5 medium supplemented with 2,4 –D (2.0 mg/L), BAP (1.0 mg/L) and NAA (1.0 mg/L). The maximum callusing frequency of 90% was observed after 21 days followed by 4 days of incubation on callus induction medium under dark. The highly prolific, nodular, compact yellowish large calli produced after 25 days were first checked for regeneration ability. Ninety five percent of regeneration frequency was observed with N 6 B 5 medium supplemented with 3.0 mg/L BAP and 1.5 mg/L NAA. Therefore, embryonic calli induced after 25 days were used for genetic transformation in subsequent experiments. Agrobacterium tumefaciens strain GV 3101 was transformed with pCAMBIA 1303 binary vector which contains hygromycin marker and GUS reporter gene. The transformed colonies were selected on 50 mg/L kanamycin and 25 mg/L rifampicin and confirmed by colony PCR. The PCR positive colonies were used to transform Bg 250 rice calli. The maximum transformation efficiency of 20% was obtained using 500 mg/L cefotaxime as a bacteriostatic agent to inhibit growth of Agrobacterium . 100 μM acetosyringone in co-cultivation medium and cocultivation for 3 days were the optimum conditions for maximum transformation. The expression of GUS gene revealed that the calli were successfully transformed. Key words: Agrobacterium ; Bg 250; Callus induction; Rice transformation. DOI: 10.4038/tar.v22i1.2669 Tropical Agricultural Research Vol. 22 (1): 45-53 (2010) |
