Transient assay for validating the efficacy of antifungal genes in plant cells rapidly against phytopathogens

Abstract

The antifungal efcacy of 1, 3-glucanase and chitinase genes isolated from Trichoderma spp. was gauged in agroinfected rice callus cells following in vitro confrontation with the pathogen. The binary vector pBI121 containing 1, 3-glucanase or chitinase under the control of CaMV 35S promoter and nos terminator mobilized in Agrobacterium strain LBA 4404 was used to transform indica rice callus. The transformed calli were passed through three antibiotic selection sub-culture passages of 15 days each on MS medium supplemented with kanamycin (50 mg/l) and cefotaxime (250mg/l). The calli surviving after third selection passage were PCR analyzed for the presence of 3–1, 3-glucanase and chitinase genes using gene specific primers. The results revealed detection of-1, 3-glucanase gene in 28.97% transformed calli and amplifcation of chitinase in 32.67% calli. PCR verifed calli were confronted in vitro with Magnapor the oryzae the casual organism of rice blast, an important disease of the crop. The mycelial growth of M. Oryzae was inhibited (27.62%) by callus transformed with 1, 3 glucanase gene construct and the callus transformed with chitinase gene construct also exhibited the pathogen restraint (28.84%) as compared to the control. The results confrmed effectiveness of 1, 3 glucanase and chitinase against M. Oryzae, suggesting that rapid transient assay can be used to test antifungal effect of the transgenes in plant cells against plant pathogens before attempting to develop transgenic plants.