Abstract
The aim of the present study was to standardize the Agrobacterium tumefaciens mediated transformation protocol for Vigna radiata. For this purpose some important parameters R-e sensitivity of explants to kanamycin, pH of co-culture media, age of explants, types of explants, co-cultivation time and optical density of Agrobacterium Culture medium were studied. Agrobacterium strain C58C1 harboring a binary vector p35SGUSINT containing neomycin phosphotransferase (NPTII) gene as selectable marker and beta-glucuronidase (GUS) as a reporter Gene was used for transformation. Kanamycin at a concentration of 50mg/l was used to select transformed cells. Transient and stable GUS expressions were Studied in transformed explants and regenerated calli respectively. Highest transient GUS (70 %) expression was observed at pH 5.8 after 3 days of co-culturing in 2-days-old explants. Optical density of 560nm=1 was considered optimal to obtain the highest transformation rate. Agrobacterium Culture containing both kanamycin and ampicillin had dramatic effect on transformation efficiency. Primary leaves showed higher transformation efficiency (80 %) than hypocotyl (60 %) or root (40 %) explants. Transformed calli were resistant to up to 800mg/l of kanamycin concentration. Transformed shoot were produced on shoot regeneration medium Containing 50mg/l kanamycin and 500mg/l cefotaxime.