ORIGINAL PAPER Transgenic indica rice cv. ADT 43 expressing a D 1 -pyrroline-5-carboxylate synthetase (P5CS) gene from Vigna aconitifolia demonstrates salt tolerance Alagarsamy Karthikeyan • Shumugiah Karutha Pandian • Manikandan Ramesh Received: 5 April 2011 / Accepted: 8 June 2011 / Published online: 25 June 2011 Ó Springer Science+Business Media B.V. 2011 Abstract To develop salt tolerant rice, the P5CS gene of Vigna aconitifolia, encoding for proline synthesis, was introduced into the popular indica rice cultivar ADT 43. Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pCAMBIA 1301/P5CS, carrying the proline synthesis encoding gene P5CS, was co-cultivated with embryogenic callus of rice. Adding 100 lM acetosyrin- gone to the Linsmaier and Skoog (LS) liquid and solid co-culture medium, along with 30 mg/l hygromycin and 250 mg/l timentin, contributed to significantly higher effi- ciency of transformation. Southern blot analysis of T 1 independent transformants revealed that the copy number of transgene varied between one and three. When trans- genic plants were subjected to salt stress, these plants grew well in the presence of up to 200 mM NaCl, while control plants died within 10 days under these treatment condi- tions. These transgenic plants grew under salt stress for a period of 4 weeks, and were capable of flowering and set seed. T 1 plants segregated into 3:1 ratio suggesting Men- delian segregation pattern of inheritance of the P5CS transgene. Keywords Agrobacterium tumefaciens Á indica rice cv. ADT 43 Á Vigna aconitifolia Á Abiotic sensitive Abbreviations P5CS D 1 -pyrroline-5-carboxylate synthetase BA N6-Benzyladenine LS Linsmaier and Skoog medium MS Murashige and Skoog medium NAA Naphthalene acetic acid (HPT) Hygromycin phosphotransferase 2,4-D 2,4- Dichlorophenoxyacetic acid Introduction Amongst abiotic stresses, drought and salinity restricts rice production primarily in non-irrigated and irrigated rice production systems (Data 2002; Kumar et al. 2009). Var- ious strategies to combat with abiotic stress include transfer of genes that synthesize osmoprotectants, stress protein (e.g. late embryogenesis abundant protein), ion transport- ers, signaling and control of transcription factors (Bajaj et al. 1999). Ectopic expression or over expression of several genes for regulation of osmoprotectants such as proline (Zhu et al. 1998; Somboonwatthanaku et al. 2010), trehalose (Garg et al. 2002) and glycine-betaine (Mohanty et al. 2002) increased osmotolerance in rice. In India and especially in coastal rice fields of Tamil Nadu state, soil salinity is a major stress that reduces the rice productivity largely. Various mechanisms have been reported to be evolved by crop plants, can reduce membrane water per- meability and may encourage cellular water conservation during drought stress. This osmoregulation was realized mostly by accumulation of osmolytes (Bray 1993; Qiao et al. 2010). Osmolytes are synthesized mostly in response to osmotic stress and do not interfere with normal cellular biochemical reaction. Proline, one of the most common compatible osmolytes, is one of the most particular on study related with plant osmoregulation in water-stressed plants. The accumulation of proline in dehydrated plants is caused both by activation of the biosynthesis of proline and by inactivation of the A. Karthikeyan Á S. K. Pandian Á M. Ramesh (&) Department of Biotechnology, Alagappa University, Karaikudi, Tamil Nadu 630 003, India e-mail: maniindica42@yahoo.com 123 Plant Cell Tiss Organ Cult (2011) 107:383–395 DOI 10.1007/s11240-011-9989-4