www.IndianJournals.com Members Copy, Not for Commercial Sale Downloaded From IP - 14.139.62.178 on dated 25-Mar-2014 Indian J. Genet., 71(1): 84-86 (2011) Short Communication Genetic transformation of Vigna unguiculata tissue by Agrobacterium tumifaciens Jitendra Kumar Pal 1 , Sanjeev Kumar and Major Singh* Biotechnology Laboratory, Indian Institute of Vegetable Research, P.B. No. 01, P.O. Jakhini, Varanasi 221 005 (Received: July 2010; Revised: November 2010; Accepted: December 2010) Vigna unguiculata L. (Walp.), commonly known as cowpea is a widely cultivated vegetable legume in tropical countries mainly for its green pods [1]. In India, cowpea pods are an integral part of the diet for a largely vegetarian population. The 100g of raw green seeds typically contain 338 calories, 22-25% protein [2]. Apart from low productivity, diseases are major problem in increasing the cowpea production. Out of many viral diseases golden mosaic disease is the most serious problem caused by cowpea golden mosaic virus (CGMV) incurring losses up to 100 % under epidemic conditions. The farmers have no option but to burn their crop to prevent further spread. To overcome this problem, development of transgenic virus resistant lines using viral gene construct is a vaible option [3] however, development of an efficient transformation and regeneration system for Indian varieties is still remains a major challenge in case of cowpea. Agrobacterium tumefaciens -mediated transformation is an effective and widely used approach to introduce desirable genes into plants. In recent years many attempts have been made to genetically transform cowpea to incorporate genes of different traits [4-6]. Regeneration protocol in leguminous crops has been reported in green gram [7], mungbean, common bean [8], and soybean. The regeneration and transformation report in cowpea is very limited and no protocol is available for the Indian cowpea varieties. In present study, we have optimized a regeneration protocol in Indian cowpea variety Kashi Komal (IVRCP-4), and genetic transformation was achieved using reporter gene nptII. Agrobacterium tumefaciens strain LBA4404 harbouring the binary vector pBinAR was used for transformation. Binary vector pBinAR harbors the neomycin phosphotransferase ( nptII ) gene, which confers resistance to the antibiotic kanamycin as a plant selection marker driven by the cauliflower mosaic virus 35S promoter (CaMV 35S) and nopaline synthase (nos) terminator. For inoculation, one single colony from a fresh bacterial culture plate was grown overnight in liquid Luria-Berteny (LB) medium with the appropriate antibiotics(50 mg l –1 kanamycin and 25 mg l –1 rifampicin) at 28 o C in a rotary shaker (200 rpm). Mature and healthy seeds of cow pea variety Kashi Komal (IVRCP-4) were handpicked and soaked in tap water for 15 min and surface disinfected with 1% (w/v) Cetrimide for 15 min. and finally surface sterilized using 0.1% (w/v) HgCl 2 for 2 min. inside a laminar air flow cabinet. For germination, one seed was inoculated in each culture tube containing 20 ml half-strength MS medium [9] solidified with 0.8% agar. Explants excised from 10-day-old seedlings were used for Agrobacterium co-cultivation. Multiple shoots were induced on MS medium containing 0.5 to 3.0 mg1 –1 BAP with or without 0.5 mg1 –1 IAA. Regenerated plants were rooted on MS basal medium, acclimatized and transferred to pot containing soil in glass house. Eight to ten days old cotyledonary joints were *Corresponding author’s e-mail: singhvns@gmail.com 1 Present Address: National Research Centre on Plant Biotechnology, IARI, New Delhi 110 012 Published by Indian Society of Genetics & Plant Breeding, F2, First Floor, NASC Complex, PB#11312, IARI, New Delhi 110 012 Online management by indianjournals.com