PJB-2004-100
FACTORS AFFECTING AGROBACTERIUM TUMEFACIENS MEDIATED GENETIC TRANSFORMATION OF VIGNA RADIATA (L.) WILCZEK
SAIMA TAZEEN AND BUSHRA MIRZA*
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 like 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 ß-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.
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