Development of Agrobacterium-mediated transformation of highly valued hill banana cultivar Virupakshi (AAB) for resistance to BBTV disease.

Authors:
Sivalingam Elayabalan
Sivalingam Elayabalan
Tamil Nadu Agricultural University (TNAU)
India
Sreeramanan Subramaniam
Sreeramanan Subramaniam
School of Biological Sciences
Malaysia
Ramasamy Selvarajan
Ramasamy Selvarajan
Tamil Nadu Agricultural University (TNAU)
India
Radha Panchanathan
Radha Panchanathan
Tamil Nadu Agricultural University (TNAU)
India
Ramlatha Muthuvelayoutham
Ramlatha Muthuvelayoutham
Tamil Nadu Agricultural University (TNAU)
India
Krish K Kumar
Krish K Kumar
Tamil Nadu Agricultural University (TNAU)
India
Ponnuswami Balasubramanian
Ponnuswami Balasubramanian
Tamil Nadu Agricultural University (TNAU)
India

World J Microbiol Biotechnol 2013 Apr 27;29(4):589-96. Epub 2012 Nov 27.

Department of Plant Molecular Biology and Biotechnology, Centre for Plant Molecular Biology, Tamil Nadu Agricultural University (TNAU), Coimbatore, 641 003 Tamil Nadu, India.

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 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.

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http://dx.doi.org/10.1007/s11274-012-1214-zDOI Listing
April 2013
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