Integration of antifungal β-1,3-glucanase gene in japonica rice variety Kitaake
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Date
2017
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Punjab Agricultural University, Ludhiana
Abstract
Agrobacterium mediated genetic transformation was carried out in japonica rice cultivar ‘Kitaake’ for integration of β-1,3-glucanase gene. The callus obtained from mature seeds on culture media supplemented with 3 mg/L 2,4-D, 0.25 mg/L BAP, 600 mg/L proline, 300 mg/L casein hydrolysate, 40 g/L maltose and 3 g/L clarigel was co-cultivated with Agrobacterium tumefaciens strain LBA4404 carrying H1 plasmid with pBI121 backbone harbouring β-1,3-glucanase gene. The Agrobacterium broth at O.D-0.25-0.3 was used for infection and the co-cultivation was performed for two days in the presence of 100 µM acetosyringone. The 38% regeneration was obtained after infection on media containing 4 mg/L BAP, 0.2 mg/L NAA, 30 g/L sucrose, 8 g/L agar, 2 g/L clarigel supplemented with 250 ppm cefotaxime. Out of 1215 calli subjected to infection, 368 calli regenerated and produced 620 plantlets after rooting. When transferred to glasshouse, the surviving 500 plants were analysed by PCR which revealed 16 PCR positive plants. Their T1 generation was grown and the 10 lines were found PCR positive. The reverse-transcriptase PCR analysis showed the expression of gene in 5 plants. RT-PCR positive plants were further analysed by qRT-PCR using actin as the reference gene. The results revealed the 1.5 fold increase in expression of β-1,3-glucanase gene in two lines.
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