Agrobacterium tumefaciens-Mediated Genetic Transformation of Green Gram [Vigna radiata (L.) Wilczek] - A Recalcitrant Grain Legume

Abstract

Background: Green gram is grown in many parts of India as a source of dietary protein (21-25%). It is an important nitrogen fixing crop which fixes atmospheric nitrogen (119-140 kg/ha) to soil and enhance the soil productivity. In the present investigation, efficient Agrobacterium-mediated genetic transformation of Vigna radiata L. (Wilczek) has been achieved with VrTIP1 gene for abiotic stress resistance i.e. moisture and salinity stress. Methods: Four days old shoot tip and cotyledonary node were used for in vitro regeneration with MS medium supplemented with BAP 2.0 mg/l, kinetin 0.5 mg/l and 50 mg/l kanamycin for co-cultivation with Agrobacterium tumefaciens strains, LBA 4404. The modified binary vector pCXSN, EHA105 containing hygromycin phosphotransferase II (hpt II) marker genes and a synthetic TIP1 gene under a constitutive CaMV35S promoter were used for transformation of Vigna radiata L. cotyledonary node explants. Putative transformants selected from hygromycin resistant shoots were subsequently rooted on MS medium supplemented with 1.0 mg/l NAA and later transferred to sterile vermiculite followed by transfer to the transgenic green house. Result: The T1 plants were produced from PCR positive T0 plants and analysed for presence and integration of transgenes in putative T1 plants were confirmed by polymerase chain reaction (PCR) amplification of 752 bp of hpt II fragment. This protocol can be effectively used for transferring new traits in greengram and other legumes for their quantitative and qualitative improvements.