Study On Regeneration And Agrobacterium-mediated Genetic Transformation Of Cucumber (Cucumis Sativus L.)

Cucumber (Cucumis sativus L.) is grown worldwide as an important vegetable. Improving the biotic or abiotic resistance of cucumber through conventional breeding is limited by its narrow genetic basis and incompatibility barriers to wild Cucumis species . Therefore, it would be desirable to apply genetic engineering to expand the germplasm base of this crop. In this study, a European genotype EP-6 and a Northern China genotype Jinchun 4 were selected to study the plant regeneration and transformation technology of cucumber.1. The regeneration systems: Cotyledon is a suitable explant for in vitro shoot induction of EP-6, while for Jinchun 4, highest shoot induction rate was obtained if stem node was used as explant. The optimal culture medium of shoot induction of EP-6 cotyledons was MS+0.5 mg/L ABA+2.0 mg/L 6-BA+2.0 mg/L AgNO3, while the best culture medium for Jinchun 4 shoot induction was MS+0.5 mg/L 6-BA. The highest root induction rate can be observed when the regenerated shoots were cultured on the medium 1/2 MS+15 g/L sucrose +0.6 mg/L IBA.2. The transfomation system of EP-6 cotyledons: After 2 days of pre-culture on the optimized shoot induction medium, the cotyledons were incubated with the suspension of pCAMBIA2201/ EHA105(Agrobacterium tumefacious stains/binary vector) for 20 min, then co-cultured for 2 days on the same medium containing 50μmol/L AS in the dark. Explants were further transferred to the plant selection medium(optimal shoot induction medium 100mg/L Kan +400 mg/L Cef) for about 25 d, the antibiotics-resistant shoots were transferred to shoot elongation medium until they were 2 cm long. Shoots were excised from the explants and placed on the optimal root induction medium. Both shoot elongation medium and rooting-culture medium contained 60 mg/L Kan and 400 mg/L Cef.3.The transfomation system of Jinchun 4 stem nodes: After 2 days of pre-culture on the optimized shoot induction medium, the stem nodes were incubated with the suspension of pCAMBIA2201/ EHA105(Agrobacterium tumefacious stains/binary vector) for 20 min, then co-cultured for 2 days on the same medium containing 50μmol/L AS in the dark. Explants were further transferred to the plant selection medium(optimal shoot induction medium 120mg/L Kan +400 mg/L Cef) for about 12 d, the antibiotics-resistant shoots were transferred to shoot elongation medium until they were 2 cm long. Shoots were excised from the explants and placed on the optimal root induction medium. Both shoot elongation medium and rooting-culture medium contained 80 mg/L Kan and 400 mg/L Cef.From explant co-culture to well rooted putative transgenic plantlets, the transfomation systems of Jinchun 4 stem nodes and EP-6 cotyledons needed 42 d and 59 d respectively. The transformation efficiency can be evaluated as 0.83% for the Jinchun 4 stem node system and 0.36% for the EP-6 cotyledon system by RT-PCR positive plantlets.