Genetic transformation of lipid transfer protein encoding gene in <Emphasis Type="Italic">Phalaenopsis amabilis</Emphasis> to enhance cold resistance

Embryogenic callus of Phalaenopsis amabilis derived from leaf tissue was cocultivated with Agrobacterium tumefaciens strain LBA4404 harboring a plant cloning vector. The vector carried the lipid transfer protein (LTP) encoding gene cloned from cold tolerant Brazilian upland rice cv. IAPAR 9. The highest transformation efficiency (12.16%) was obtained when 1–2 mm calli were infected and cocultivated with 0.4 (OD₆₀₀) A. tumefaciens for 20 min. Transgene integration of kan-resistant plants was confirmed through polymerase chain reaction analysis and Southern hybridization. Four hundred seventy transgenic plants, each derived from an independent protocorm-like body, were obtained. The expression of rice cold-inducible LTP gene in transgenic P. amabilis improved its adaptive responses to cold stress. The examination of transgenic plants revealed that enhanced cold tolerance was most likely due to the increased accumulation of several compatible solutes such as total soluble sugars, proline, antioxidant superoxide dismutase, decreased accumulation of malondialdehyde, and maintained electrolytes within the membrane compared with controls.