Optimization of Agrobacterium mediated genetic transformation of cotyledonary node explants of Vigna radiata.

Sushil Kumar Yadav,Kirti Pulugurtha Bharadwaja,Jyothi Lakshmi Narayana,Arun Kumar Shanker,Venkateswarlu Bandi,Annapurna Kannepalle,Sweety Katikala,Maheswari Mandapaka,Vanaja Maddi,Varalaxmi Yellisetty

doi:10.1186/2193-1801-1-59

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 T0 and T1 plants. Rooted T0 and T1 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.

Highlights

Grain legumes constitute an important dietary constituent for humans and animals
We describe a reproducible and efficient Agrobacterium mediated genetic transformation protocol for greengram using double cotyledonary node (DCN) explants derived from three day old seedlings with binary vector pCAMBIA 2301 containing annexin gene
Earlier, hypocotyl and primary leaves excised from 2-day-old in-vitro grown seedlings produced transgenic calli on B(5) basal medium supplemented with 5 × 10(−6) M Benzlaminopurine (BAP), 2.5 × 10(−6) M each of 2,4-Dichlorophenoxyacetic Acid (2,4-D) and 1-Napthaleneacetic acid (NAA) and 50 mg l(−1) kanamycin after co-cultivation with Agrobacterium tumefaciens strains, LBA4404, EHA105 and C58C1 all containing beta-glucuronidase and neomycin phosphotransferase II marker genes at a frequency of 0.9% (Jaiwal et al 2001)

Summary

Introduction

Grain legumes constitute an important dietary constituent for humans and animals. They associate with nitrogen fixing bacteria and play an important role in low input agricultural production systems; small and marginal farm holdings. We describe a reproducible and efficient Agrobacterium mediated genetic transformation protocol for greengram using double cotyledonary node (DCN) explants derived from three day old seedlings with binary vector pCAMBIA 2301 containing annexin gene. We in our study for the first time, transformed green shoots showing strong GUS activity regenerated directly from cotyledonary node explants cultured after co-cultivation with LBA4404 (pTOK233) on B(5) medium containing 6-benzylaminopurine (5 × 10(−7) M) and 100 mg l(−1)

Results

Conclusion