Genetic improvement of tomato (Solanum lycopersicum) with ATDREB1A gene for cold stress tolerance using optimized Agrobacterium-mediated transformation system

Abstract

Cold is a serious threat that limits the productivity of tomato as it is a cold-sensitive crop. The cold tolerant gene DREB1A plays crucial roles in the survival of plants under cold stress. We have developed cold stress tolerant transgenic plants of tomato by the overexpression of an Arabidopsis DREB1A gene. They were obtained by using EHA-105 strain of Agrobacterium harboring pBIH binary vector that contained DREB1A controlled by an inducible promoter Lip9 and hygromycin phosphotransferase gene (hpt) as a plant selectable marker. Sequential 15 day old in vitro seedlings, 48 h pre-culture period, Agrobacterium density (OD600 nm = 0.2), 3 min infection period and 60 μM acetosyringone yielded 15-18% TE. T0 transformants were identified by PCR and then they were self-pollinated to generate T1 progeny that was evaluated by gene expression and cold tolerance analyses. AtDREB1A transcript expression was detected by RT-PCR and stable integration of AtDREB1A gene was confirmed by Southern blotting. Morphological analyses of T2 generations demonstrated that there were no significant differences among transgenic and NT plants under normal growth conditions. The T2 transgenic lines exhibited more cold stress tolerance when exposed to 4°C as compared to their NT counterparts. These findings clearly indicate that transgenic tomato plants over-expressing Arabidopsis DREB1A gene enhanced protection and provided cold tolerance under controlled conditions in transgenic containment.