Effective Agrobacterium-mediated genetic transformation of okra (Abelmoschus esculentus L.) and generation of RNAi plants resistant to Begomovirus infecting okra

Okra is an important vegetable crop of the Malvaceae family and is infected by varying numbers of viruses of the genus Begomovirus. Regardless of the importance of the crop, very little consideration has been given to its genetic improvement. RNA interference (RNAi), a potent biotechnological tool, is known to control Begomovirus in many crops. For the implementation of successful RNAi, there is a need for an efficient genetic transformation system in okra. In the present study, we developed a procedure for Agrobacterium-mediated tissue culture-dependent regeneration of okra plants for the application of RNAi. Eleven transgenic okra RNAi plants were regenerated by utilising hypocotyls as explants. Transformed plants were screened with hygromycin at the regeneration stage and the presence of transgenes (AC1, AC2 & AC4 codes for replication-associated protein, transcriptional activator protein and suppressor of PTGS) in putative transformed plants was confirmed by polymerase chain reaction (PCR). Wild and transgenic lines were challenged with a dimeric Begomovirus clone or viruliferous whiteflies and the level of resistance was estimated with quantitative real-time reverse transcriptase PCR (qRT-PCR) by utilising viral gene-specific primers. The resistant transgenic lines accumulated very low titres of viral gene products according to the qRT-PCR assays compared to the control plants. This is the first report of tissue culture-mediated RNAi-derived resistance in okra against Begomovirus infection.