Improvement of Agrobacterium-mediated transformation of cucumber (Cucumis sativus L.) by combination of vacuum infiltration and co-cultivation on filter paper wicks

Abstract

An improved method for genetic transformation of cucumber (<i>Cucumis sativus</i> L. cv. Shinhokusei No. 1) was developed. Vacuum infiltration of cotyledonary explants with <i>Agrobacterium</i> suspension enhanced the efficiency of <i>Agrobacterium</i> infection in the proximal regions of explants. Co-cultivation on filter paper wicks suppressed necrosis of explants, leading to increased regeneration efficiency. Putative transgenic plants were screened by kanamycin resistance and green fluorescent protein (GFP) fluorescence, and integration of the transgene into the cucumber genome was confirmed by genomic polymerase chain reaction (PCR) and Southern blotting. These transgenic plants grew normally and T₁ seeds were obtained from 7 lines. Finally, stable integration and transmission of the transgene in T₁ generations were confirmed by GFP fluorescence and genomic PCR. The average transgenic efficiency for producing cucumbers with our method was 11.9 ± 3.5 %, which is among the highest values reported until date using kanamycin as a selective agent.