title: Optimization of Agrobacterium mediated genetic transformation of cotyledonary node explants of Vigna radiata.
abstract: A reproducible and highly efficient protocol for genetic transformation mediated by Agrobacterium has been established for greengram (Vigna radiata L. Wilczek). Double cotyledonary node (DCN) explants were inoculated with Agrobacterium tumefaciens strain LBA 4404 harboring a binary vector pCAMBIA 2301 containing neomycin phosphotransferase (npt II) gene as selectable marker, β-glucuronidase (GUS) as a reporter (uidA) gene and annexin 1 bj gene. Important parameters like optical density of Agrobacterium culture, culture quantity, infection medium, infection and co-cultivation time and acetosyringone concentration were standardized to optimize the transformation frequency. Kanamycin at a concentration of 100 mg/l was used to select transformed cells. Transient and stable GUS expressions were studied in transformed explants and regenerated putative plants, respectively. Transformed shoot were produced on regeneration medium containing 100 mg/l kanamycin and 250 mg/l cefotaxime and rooted on ½ MS medium. Transient and constitutive GUS expression was observed in DCN explants and different tissues of T(0) and T(1) plants. Rooted T(0) and T(1) shoots confirming Polymerase Chain Reaction (PCR) positive for npt II and annexin 1bj genes were taken to maturity to collect the seeds. Integration of annexin gene into the greengram genome was confirmed by Southern blotting.
authors: Central Research Institute for Dryland Agriculture, Santoshnagar, Hyderabad, 500 059 India.
authors: Sushil Kumar
authors: SK
authors: Yadav
authors: Sweety
authors: S
authors: Katikala
authors: Varalaxmi
authors: V
authors: Yellisetty
authors: Annapurna
authors: A
authors: Kannepalle
authors: Jyothi Lakshmi
authors: JL
authors: Narayana
authors: Vanaja
authors: V
authors: Maddi
authors: Maheswari
authors: M
authors: Mandapaka
authors: Arun Kumar
authors: AK
authors: Shanker
authors: Venkateswarlu
authors: V
authors: Bandi
authors: Kirti Pulugurtha
authors: KP
authors: Bharadwaja
keywords: Agrobacterium mediated transformation; Annexin; Double cotyledonary node; Vigna radiata