DEVELOPMENT OF YELLOW MOSAIC VIRUS RESISTANT MUNGBEAN [VIGNA RADIATA (L.) WILCZEK] LINE THROUGH GENETIC TRANSFORMATION

Abstract

Mungbean [Vigna radiata (L.) Wilczek] yellow mosaic virus (MYMV) is responsible for the yellow mosaic disease causing maximum yield loss of mungbean in Bangladesh. RNAi-based antiviral strategy has been used with the purpose of generating yellow mosaic virus resistant transgenic mungbean lines through targeting coat protein (CP) and silencing suppressor gene (AC2). MYMV coat protein (CP) and silencing suppressor (AC2) gene specific primers were designed from the conserved regions after alignment of the available CP and AC2 gene sequences in NCBI database. MYMV CP and AC2 genes were amplified through PCR using gene specific primers. Sequence analysis of PCR amplified DNA confirmed the presence of MYMV coat protein and silencing suppressor gene. Amplified CP gene (750 bp) and AC2 gene (450 bp) were cloned in an antisense orientation under CaMV35S promoter of pBI121 vector replacing the GUS gene by using BamHI and SacI restriction recognition sites in antisense orientation resulting pBI121CPAC2 construct. The cloned construct was transferred to Agrobacterium tumefaciens strain LBA4404. The transformation efficiency of this newly developed antiviral gene construct was checked using tobacco as a model plant. Putatively transformed tobacco plants were recovered following Agrobacterium-mediated transformation and the transgene integration in tobacco plants was confirmed by polymerase chain reaction (PCR) analysis. Following these results a protocol for Agrobacterium-mediated genetic transformation was developed for locally grown mungbean varieties (BARI mung-3 and Binamoog-5) using Agrobacterium strain LBA4404 harboring binary plasmid pBI121 containing GUS (β–glucuronidase) and nptII (neomycin phosphotransferase II) genes (Construct I). Among the explants studied cotyledon attached decapitated embryo (CADE) explant of mungbean was found to be suitable for transformation. Best response (80%) towards multiple shoots regeneration from CADE explant was achieved on MS medium containing B5 vitamins supplemented with 5.0 μM BAP following 28 days of culture. Bacterial suspension having an O.D of 0.6 (at 600 nm) in an incubation period of 30 minutes with 3 days of co-cultivation period was found to be optimum for transformation of CADE explants. Transformed shoots were selected using 200 mg/l kanamycin. Nontransformed shoots became albino and died within 5 weeks due to this selection pressure. Using this protocol Agrobacterium-mediated transformation of mungbean was further carried out using the newly developed antiviral gene construct, pBI121CP-AC2 and consequently transformed shoots of mungbean were recovered using CADE explants. However, in vitro regenerated shoots produced low number (26.66 %) of roots on both full and half strength of MS medium supplemented with different concentrations and combinations of auxins. To overcome the problems of in vitro rooting, an alternative approaches of root development i.e., in vitro micrografting technique was applied using regenerated shoots to obtain complete plantlets. The best response (55%) towards successful grafting was obtained when 3.0 cm long scions and 14-days old in vitro mungbean rootstocks were utilized. Following proper hardening successful micro-grafted plants produced flowers and set viable seeds. On the other hand, in a separate set of experiments the putatively transformed shoots developed roots when cultured on half strength of MS medium supplemented with 2μM IBA. Following the development of roots putative plantlets were hardened and successfully acclimatized in soil. Integration of antisense CP-AC2 gene in the transgenic mungbean plants was confirmed by polymerase chain reaction (PCR) using CP forward and AC2 reverse gene specific primers. Following proper hardening the T0 and T1 plants produced flowers and viable seeds.