Optimization and overexpression of pyrroline-5-carboxylate synthetase to increase cold stress tolerance in three commercial petunias

Pyrroline-5-carboxylate synthetase is a bifunctional enzyme catalyzing two steps of proline biosynthesis in plants. The potential benefit of this gene is to confer water stress resistance (drought, salt, cold) due to the increased intracellular levels of proline that work like an osmoprotectant. As an attempt to examine the relationship between proline and cold stress in petunia, the p5cs gene was transferred to petunia plants through Agrobacterium-mediated transformation. Agrobacterium strain LBA4404 harboring a plasmid pBI121 was used as the binary vector system for transformation. pBI121 contained β-glucuronidase gus gene as a reporter gene, ∆¹-pyrroline-5-carboxylate synthetase p5cs gene, and neomycin phosphotransferase nptII gene as a selectable marker. GUS and proline assays confirmed the presence of the gus and p5cs genes in the genome of all transformants when under stress and non-stress conditions, and the shoot apex was the best explant for the transformation of petunia. This is also the first report showing the efficiency of transformation from the shoot apex (73-83%)is greater than from leaf discs (0-15%). When under stress conditions transgenic plants of cultivar ‘Super Cascade’ accumulated up to 13.2 times more proline than the wild type under non-stress conditions. Moreover, the transformed plants were more tolerant than the wild types when under stress conditions. This is also the first report showing the effect of genotype differences on cold tolerance after transformation, wherein P. violacea produced the highest amount of proline content compared with the other cultivars, and subsequently the highest amount of resistance under cold stress.