Asian Journal of Microbiology, Biotechnology & Environmental Sciences Paper

Vol 24, Issue 1: 2022; Page No.(102-107)

ISOLATION, CLONING AND AGROBACTERIUM MEDIATED TRANSFORMATION OF MYMV REPLICASE (REP) GENE IN VIGNA RADIATA (L.) WILCZEK FOR MYMV RESISTANCE

RAMACHANDRA, ANANTAPUR, ROSHNI, M., ANITHA PETER, SOUMYA GANAPATI BHAT AND RAVITEJA, C. R.

Abstract

The binary plant cloning vector pCAMBIA1305.2 carrying the MYMV Replicase (Rep) gene was constructed and transformed into a competent Agrobacterium tumefaciens, strain LBA4404 and was used for co-cultivation of mungbean varieties, KKM-3, IC-39340-1, China mung and LM-1668 for imparting mungbean yellow mosaic virus (MYMV) resistance. The putative transformants (To generation) were selected on shooting media (SM) supplemented with 5 mgl-1 BAP, 50 mgl-1 hygromycin and 250 mgl-1 cefotaxime. The multiple shoots were inoculated on shoot elongation media (SEM) with 0.3 mgl-1 zeatin and 2.5 mgl-1 BAP for elongation and rooted on rooting media (RM), comprising half MS supplemented with 0.2 mgl-1 NAA. The GUS assay and genomic PCR analysis of the To transformants revealed, four positive putative transgenic lines for Rep gene specific primers and had a positive correlation with vector specific hptII primers. Likewise, VirG amplification revealed no Agrobacterium contamination in the apoplast of allthe putative To transformants.