IMPROVEMENT OF GENETIC TRANSFORMATION AND PLANT REGENERATION VIA SUSPENSION CULTURES IN Cucurbitaceae FAMILY

Abstract

An improved plant regeneration and Agrobacterium-mediated genetic transformation system was established for squash (Cucurbita pepo L.) and cucumber (Cucumis sativus L.). The effect of 2,4-D and kinetin on callus formation, the effect of liquid and solid MS medium on embryogenic callus proliferation, expression of Gus gene beside its influence by acetosyringone, and regeneration of transformed and non-transformed plant cells were investigated. Cotyledon explants and 2,4-D of 1 mg/L were found to be the best for induction of callus formation giving the highest percentage; 92% and 94% in squash and cucumber, respectively. Transfer of induced callus on liquid MS medium containing 1 mg/L 2,4-D for 4 weeks greatly increased the proliferation of embryogenic callus with an increased percentage of 81.9% and 139.7% in squash and cucumber, respectively. Preculture of embryogenic callus on the liquid MS medium prior to inoculation and cocultivation with Agrobacterium enhanced the genetic transformation efficiency with an increased percentage of 40.6% and 55.7% in the number of blue spots in the absent of acetosyringone in squash and cucumber, respectively, and with an increased percentage of 63.1% and 38.5% in the number of blue spots in the presence of 0.1 mM acetosyringone in squash and cucumber, respectively. Higher percentage of non-transformed regenerates (193.3% and 172.7%) and transformed regenerates (107.1% and 86.4%) were obtained from the embryogenic calli, which were previously cultured on the liquid MS medium in squash and cucumber, respectively. Molecular evidence of transgenic plants was confirmed by polymerase chain reaction (PCR) analysis and demonstrated the effectiveness of the transformation procedure.