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

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 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.

Banana bunchy top disease (BBTD) symptom expression in banana and strategies for transgenic resistance: A review

“…BBTV transmission studies through aphids under greenhouse conditions. Bioassay to screen the transgenic banana and symptom expression observation made on transgenic and non-transgenic bananas (Borth et al, 2011;Elayabalan et al, 2013).…”

“…'Silk') did not develop symptoms when exposed to aphids carrying BBTV. The transformation of the Indian hill banana 'Virupakshi' (AAB genome, Pome subgroup) has also been reported for RNAi technology to impart BBTV resistance in banana (Elayabalan et al, 2013).…”

“…Elayabalan et al (2013) reported on 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 Tamil Nadu India, the hill bananas and Sirumalai AAB-(G.I 126] were grown in multitier system (Figure 1) was highly susceptible to BBTV (Figure 2). The virus has been the main cause for drastic reduction in hill banana cultivation from 18,000 ha in 1970's to 2,000 ha at present and showed the incidence of 14-74% percentage (Kesavamoorthy, 1980;Selvarajan et al, 2010;Elayabalan et al, 2013). Vishnoi et al (2009) reported that BBTD incidence occurred between 20-30% between 2006 to 2007 in India northern zones of Lucknow, Barabanki, Bahraich, Kanpur, and Etawah districts of Uttar Pradesh.…”

Genetic engineering in Banana and Plantain

“…There are several reports now available on the genetic transformation of banana [5,[11][12][13] even then still it is far from routine in most of the labs due to long incubation time in the callus induction, difficulty in developing regenerable embryogenic cell suspension, prolonged incubation in the selection medium with very low potential to regenerate in the presence of a selection agent. Agrobacterium-mediated transformation is a method of choice for banana transformation.…”