Improved Agrobacterium tumefaciens-mediated transformation using antibiotics and acetosyringone selection in cucumber

Cucumber (Cucumis sativus) is one of the most important vegetable crop species in the world. As conventional breeding of cucumber is very challenging, genetic engineering is an alternative option for introducing important traits such as enhanced stress resistance and nutritional value. However, the efficiency of the transformation system depends on genotypes, transformation conditions, etc. This study aims to optimize the transformation parameters of Agrobacterium-mediated transformation of cucumber. ‘Xintaimici’, which is a very popular and typical northern Chinese cucumber variety, was transformed with Agrobacterium GV3101. The strain carried the pCAMBIA2300s plasmid, which is a binary vector containing the marker gene neomycin phosphotransferase II (npt II). The results indicated that cefotaxime sodium was suitable for inhibiting Agrobacterium in the screening and bud elongation stages. Timentin was best used during the rooting stage. Furthermore, 25 mg/L kanamycin was used in the early stage of screening and increased to 50 mg/L for further screening. At the bud elongation and rooting stages, 75 and 100 mg/L kanamycin was used, respectively, to improve the screening efficiency. To obtain the highest regeneration frequency of resistant buds, 50, 150, and 100 μM acetosyringone was added to the preculture medium, infection solution, and coculture medium, respectively. To confirm the presence of the transgenes, DNA from npt II transformed cucumber plants was analyzed by polymerase chain reaction after transplanting resistant regenerated plants. We ultimately achieved an 8.1% transformation efficiency, which is among the highest values reported to date using the cucumber variety ‘Xintaimici’. Thus, an effective protocol for Agrobacterium tumefaciens-mediated genetic transformation of cucumber was optimized.