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

ABSTRACT

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.

KEYWORDS:

• Okra
• RNAi
• Agrobacterium
• tissue culture
• AC1/AC2 or AC1/AC4 and qRT-PCR

Acknowledgements

The authors are grateful to Punjab Agricultural University, Ludhiana (Punjab), India for providing a laboratory facility to carry out the present work. We are also thankful to Department of Vegetable Sciences for maintaining and providing the seeds of okra variety Punjab 8 for performing transformation experiments.

Disclosure statement

No potential conflict of interest was reported by the authors.

Author contributions

Nity Sharma performed the entire research and wrote the manuscript. Navraj Kaur Sarao, Prashant Mohanpuria, and Abhishek Sharma designed the research and analysed the data.

Data availability statement

No data related to this research are deposited in any repositories.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/14620316.2023.2251978

Additional information

Funding

This work was financially supported by Punjab Agricultural University.