Transformation of the elongation factor eIF(iso) 4E gene and plant regeneration in cucumber (Cucumis sativus L.)

Cucumber (Cucumis sativus L.) is one of the most produced vegetables in Turkey after tomato. However, biotic and abiotic stresses affect the life cycle of the cucumber negatively and cause a decrease in yield and quality. The interaction mechanisms with eIF (iso) 4E, one of the eukaryotic translation initiation factors of viral plant pathogens, have been elucidated by studies in some species, including C. sativus. However, the regeneration frequencies of the obtained transgenic plants are low. Although high transformation efficiency is closely related to plant regeneration capacity, optimizations for both plant regeneration and transformation are required for an effective transformation efficiency in different genotypes to be studied. In this study, silencing of eIF (iso) 4E in two cucumber genotypes (G27 and G247) was planned using CRISPR/Cas9 gene editing technology. For this, first of all, plant regeneration optimization was performed in genotypes before transformation, and the most suitable explant (out of 3 explants) and culture medium (out of 50 media including control) content were determined for adventitious shoot organogenesis.Then, by trying to optimize the important factors (genotype, bacterial strain, seedling age, explant type, pre-incubation, and antibiotic type and brand) that may affect the transformation efficiency, the designed guide RNAs (sgRNA1 and sgRNA2) and Cas9 were transferred to the explants of both genotypes through Agrobacterium tumefaciens. After the optimization studies for both plant regeneration and transformation, the average number of shoots / 100 explants obtained in 8 transformations under equal conditions was determined as 13.6 and 18 for G27 and G247, respectively. Some randomly selected cucumber lines from the obtained ex vitro candidate transgenic plants (T0) were tested by PCR analysis in terms of carrying the transferred genes. In this study carried out within the scope of the project numbered TAGEM/18/AR-GE/03, sequence analyzes will be performed on the T1 plants that will be obtained by the ongoing cultivation and hybridization studies in greenhouse conditions in the plants with positive PCR test results.