Engineering Non-transgenic Gynoecious Cucumber Using an Improved Transformation Protocol and Optimized CRISPR/Cas9 System

Plant molecular breeding has been boosted tremendously by the development of sequencing technology. However, cucumber is intractable to transformation. The low efficiency of transformation in cucumber makes it a daunting task to apply gene editing tools such as CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated system 9). Here, to find a way to improve the genetic transformation efficiency, GFP (green fluorescent protein) was used as a marker during Agrobacterium infection and in situ hybridization of CsSTM was carried out. The result suggested the regenerated adventitious shoot was originate from cells in deeper layers where immerse infection could barely arrive. To enhance infection, a simple syringe was used for vacuum infiltration. Additionally, hemin was used to promote the rooting of transgenic shoots, which solved the problem that addition of auxin promoted chlorosis of the transgenic shoots. The transformation protocol established in this study was used to perform CRISPR/Cas9-mediated knocking out of three genes (CsVFB1, CsMLO8 and CsGAD1) and the transformation efficiency approached 1.00‰. Besides cucumber, the transgenic and genome-editing approach has been validated in melon, so it is very likely that this approach can be widely used in Cucurbitaceae species.