Development of Agrobacterium -mediated transformation of highly valued hill banana cultivar Virupakshi (AAB) for resista
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ORIGINAL PAPER
Development of Agrobacterium-mediated transformation of highly valued hill banana cultivar Virupakshi (AAB) for resistance to BBTV disease Sivalingam Elayabalan • Kalaimughilan Kalaiponmani • Sreeramanan Subramaniam Ramasamy Selvarajan • Radha Panchanathan • Ramlatha Muthuvelayoutham • Krish K. Kumar • Ponnuswami Balasubramanian
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Received: 8 August 2012 / Accepted: 15 November 2012 / Published online: 27 November 2012 Ó Springer Science+Business Media Dordrecht 2012
Abstract One of the most severe viral diseases of hill banana is caused by banana bunchy top virus (BBTV), a nanovirus transmitted by the aphid Pentalonia nigronervosa. In this study, we reported the Agrobacterium-mediated transformation on a highly valued hill banana cultivar Virupakshi (AAB) for resistance to BBTV disease. The target of the RNA interference (RNAi) is the rep gene, encoded by the BBTV-DNA1. In order to develop RNAi construct targeting the BBTV rep gene, the full-length rep gene of 870 bp was polymerase chain reaction amplified from BBTV infected hill banana sample DNA, cloned and confirmed by DNA sequencing. The partial rep gene fragment was cloned in sense and anti sense orientation in the RNAi intermediate vector, pSTARLING-A. After cloning in pSTARLING-A, the cloned RNAi gene cassette was released by NotI enzyme digestion and cloned into the NotI site of binary vector, pART27. Two different explants, embryogenic cells and embryogenic cell suspension derived microcalli were used for co-cultivation. Selection was done in presence of 100 mg/ L kanamycin. In total, 143 putative transgenic hill banana lines were generated and established in green house
S. Elayabalan (&) K. Kalaiponmani R. Panchanathan R. Muthuvelayoutham K. K. Kumar P. Balasubramanian Department of Plant Molecular Biology and Biotechnology, Centre for Plant Molecular Biology, Tamil Nadu Agricultural University (TNAU), Coimbatore 641 003, Tamil Nadu, India e-mail: [email protected] S. Subramaniam School of Biological Sciences, Universiti Sains Malaysia (USM), Minden Heights, 11800 Georgetown, Penang, Malaysia e-mail: [email protected] R. Selvarajan National Research Center for Banana (NRCB), Thayanur Post, Tiruchirapalli 620 102, India
condition. The presence of the transgenes was confirmed in the selected putative transgenic hill banana lines by PCR and reverse transcription PCR analyses. Transgenic hill banana plants expressing RNAi-BBTV rep were obtained and shown to resist infection by BBTV. The transformed plants are symptomless, and the replication of challenge BBTV almost completely suppressed. Hence, the RNAi mediating resistances were shown to be effective management of BBTV in hill banana. Keywords Banana bunchy top virus (BBTV) RNAiBBTV rep Transgenic hill banana (Virupakshi AAB) Abbreviations ECS Embryogenic cell suspension RNAi RNA Interference GI Geographical indication SH Schenk and Hildebrandt pEB Plasmid Elaya Balan MS Murashige and Skoog RT-PCR Reverse transcription PCR BBTD Banana bunchy top disease THB Transgen
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