Transgenic banana plants carrying ihpRNA cassette targeting viral replicase gene show resistance against Banana bract mosaic virus

摘要

RNAi approach was attempted to impart resistance against Banana bract mosaic virus (BBrMV). An intron hairpin RNA (ihpRNA) vector targeting its replicase gene was constructed in pSTARLING vector. A fragment (421 bp) of the BBrMV replicase gene isolated from the infected sucker of banana cv. Nendran (Musa AAB) was used for making ihpRNA cassette. The ihpRNA cassette (NotI fragment) was mobilised to the binary vector pART27 in Agrobacterium tumefaciens strain LBA 4404 for transformation. Embryogenic calli derived from the immature male inflorescence of cv. Nendran maintained in MS medium supplemented with BA 0.1 mg L-1 and picloram 1 mg L-1 were transformed with LBA 4404. Transformed calli were subjected to embryo induction in semisolid MS medium supplemented with BA 2 mg L-1 and IAA 0.5 mg L-1 and germinated on half-strength MS medium with BA 2 mg L-1 and IAA 0.5 mg L-1, with a maximum regeneration of 25% under the selection pressure of 100 mg L-1 kanamycin. The regenerated shoots were confirmed for the presence of ihpRNA construct by PCR with primers specific for sense-intron-antisense fragment, npt II and cre intron. The transgenics challenged with BBrMV showed no infection. Untransformed plants showed infection and developed disease symptoms after 3 months.