Development of transient assay for analyzing the efficacy of antifungal genes against different fungal pathogens of rice
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Date
2020
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Punjab Agricultural University, Ludhiana
Abstract
Two gene cassettes containing β-1,3 glucanase and chitinase genes under the control of CaMV 35S promoter and nos terminator mobilized in Agrobacterium strain LBA4404 were used to agroinfect rice calli for testing their antifungal activity against rice pathogens. The callus cultures were induced by culturing mature rice seeds (Oryza sativa L. cv. PR114) on MS media supplemented with 1mg/l 2,4-D and 0.15mg/l BAP. The Agrobacterium broth at O.D-0.6 was used for agroinfection of rice callus in the presence of 100 μM of acetosyringone. The agroinfected calli were transferred for first selection cycle of 14 days on MS media supplemented with 2,4-D (1mg/l) + BAP (0.5mg/l) + cefotaxime (250mg/l) and kanamycin (50 mg/l). The calli were transferred for second and third selection cycle for 10 days and 7 days on MS media supplemented with 2,4-D (1mg/l) + BAP (0.5mg/l) and kanamycin (50 mg/l). The presence of β-1,3 glucanase and chitinase transgene was verified on all surviving calli through PCR using transgene-specific primers. The amplification of 1764 bp and 1275 bp sequence confirmed the presence of β-1,3 glucanase and chitinase gene, respectively with the transformation efficiency of 26.78% and 26.73%. The calli showing amplification of β-1,3 glucanase and chitinase gene were analyzed for the antagonism against Rhizactonia solani, Magnoporthe oryzae and Fusarium moniliforme through dual culture technique. The transformed calli did not inhibit mycelium growth on R. solani and F. moniliforme probably due to the ineffectiveness of β-1,3 glucanase and chitinase transgenes against the pathogens. Whereas, inhibition of M. oryzae growth was observed in vitro. So, β-1,3-glucanase and chitinase gene integration serve as an effective control measure for the control of rice blast disease caused by M. oryzae.