72 Bioinfo Publications IJBA International Journal of Biotechnology Applications ISSN: 0975–2943 & E-ISSN: 0975–9123, Vol. 3, Issue 2, 2011, pp-72-77 Available online at http://www.bioinfo.in/contents.php?id=22 SOMATIC EMBRYOGENESIS FOR AGROBACTERIUM MEDIATED TRANSFORMATION OF TOMATO- SOLANUM LYCOPERSICUM L. ASHAKIRAN KILANKAJE AND GIRIJA SHANMUGAM* DRDO-BU-Centre for Life Sciences, Bharathiar University, Coimbatore, 46, India *Corresponding author. E-mail: girijabiotech@yahoo.co.in, +919843572999 Received: Received: July 09, 2011; Accepted: August 05, 2011 Abstract Agrobacterium mediated transformation through somatic embryogenesis was achieved using cotyledon explants of tomato cv. Shalimar (Solanum lycopersicum L.). Somatic embryos were induced directly from the cotyledon explants on MS medium supplemented with 2, 4-D ( 5mg/l) for the first time, and also through the callus produced from cotyledon explants on MS liquid medium supplemented with 2, 4-D (2mg/l). The maturation of somatic embryo globular to torpedo was presumed by using sorbitol (0.4 M) and ABA (0.25mg/l). The competency of induced somatic embryos for agrobacterium mediated transformation was confirmed by using GUS reporter gene in pBI121 vector and the npt II with PCR. The transformant was selected using kanamycin (100mg/l). Seventy percentage of transformation efficiency was obtained from the transformed somatic embryo. Keywords: pB1121, Shoot regeneration, transformation, 2, 4-D Introduction Tomato is an important worldwide commercial crop. World tomato trade is growing rapidly (World markets and trade - 2009). Several transgenic traits encoding genes were introduced in to tomato to improve their nutritional quality a path started by Calgene Inc. (Kramer et al, 2004), all these studies were carried out by adopting different regeneration methodology standardised henceforth. Micro propagation through somatic embryogenesis provides an efficient mean of producing large numbers of elite or transgenic plants. The potential use of somatic embryogenesis in developmental studies, crop improvement and genetic transformation has been widely recognized and the number of species displaying the potential for somatic embryogenesis is constantly increasing (Girija et al, 2000). Somatic embryogenesis can probably be achieved for all plant species provided that the appropriate explant, culture media and environmental conditions are employed (Arnold et al, 2002). Somatic embryogenesis in tomato is still at infancy, and substantial research is still required to be undertaken to obtain high quality somatic embryos, this technique will be usefull for crop improvement using in vitro culture and transformation (Bhatia et al, 2004). Agrobacterium mediated transformation have been used as an effective tool for transfer of gene into plant system, while use of somatic embryos as explant in transformation of plants is a recent approach. Somatic embryos consist of embryogenic cells when successfully transferred can produce large number of transgenic plant from a single explant. Hence, Agrobacterium mediated transformation of somatic embryos hold an important role in transgenic plant production. Only few works have been reported for Agrobacterium mediated transformation using somatic embryos, in plants like walnut (Mc Granahan et al, 1988 ), cork oak (Quercus suber L.) (Sanchez et al, 2005), alfalfa (Medicago falcata L.) (Shao et al, 2000) and Vitis rotundifolia (Dhekney et al, 2008) so standardising succeful regeneration protocol for agrobacterium mediated transformation using somatic embryo in tomato would help improve the efficiency of obtaining the stable transformant as the gene has been transformed to the embryonic tissue. . The present study was aimed to develop plant regeneration system for tomato via somatic embryogenesis and optimizing several key factors to utilize tomato somatic embryos for Agrobacterium-mediated genetic transformation using cotyledon explants which not reported earlier. Experimental Procedure Collection and germination of seeds Tomato seeds var. Shalimar was obtained from the Defence Institute of High Altitude Research, Leh, India. Seeds were washed with distilled water for 5 times, followed by treatment with 0.1% HgCl2 for 3 min and rinsed with sterile water 6 times to remove the sterilant. Seeds were germinated in test tubes containing moist, sterile cotton. Seedlings were grown under white fluorescent light (40 μ mol m -2 s -1 ) at a 16 h-day photoperiod and 25 ± 2ºC.