A Two-Step System for Efficient Agrobacterium tumefaciens–Mediated Transformation of Cucumber (Cucumis sativus L.)

Cucumber ( Cucumis sativus L.), as one of the major vegetable crops in the world, is frequently affected by fungal, bacterial, and viral diseases, as well as environmental stress that can cause serious loss of yield. Although there has been a steady improvement of many traits in cucumber through traditional breeding, the lack of sufficient genetic variability within the cucumber germplasm for disease and abiotic stress resistance remains a major challenge. Genetic transformation has opened new possibilities for the introduction of novel traits through genetic engineering or genome editing of the cucumber genome. However, an efficient and stable regeneration and genetic transformation system in cucumber is lacking. In this study, a two-step culture procedure for shoot regeneration and selection of transformed plants has been developed for highly efficient transformation of cucumber. In step 1, following Agrobacterium transfection, a large number of transformed cells are generated on culture medium using a low concentration of antibiotics. Subsequently, in step 2 the explants are transferred onto medium containing a high concentration of antibiotics in addition to abscisic acid (ABA) and AgNO 3 . Furthermore, a fluorescent maker is used to separate transformants from escapes and chimeric shoots in the early developmental stages, increasing the recovery of transformed shoots. An average transformation frequency of 14.52% was recorded 2 months after infection. Finally, all shoots were rooted and plantlet acclimatized by applying an improved transgenic plantlet propagation, rooting, and acclimatization protocol. This is the first study to establish a cucumber transformation system by using two-step regeneration/chemical selection combined with visual screening of fluorescent marker proteins, a process that also may facilitate enhanced transformation of other plant species.