Improving the transformation efficiency of Cucurbita species: factors and strategy for practical application

Cucurbita species are refractory to transformation. Hence, the only 2 reports published regarding the transformation of Cucurbita species until our successful transformation of C. moschata (cv. Heiankogiku) in 2011. The efficiency was 2.7±1.3% using wounded explants vortexed with whisker suspension. To improve transformation efficiency, transformation experiments were carried out with various ages of cotyledonary explants. The highest efficiency was obtained with 1-day-old (3.2±0.9%) and 2-day-old (3.3±0.8%) explants after germination. Histochemical analysis of GUS activity revealed that wounding allowed Agrobacterium access to the deeper layer of explants. These results suggested that cells having a high ability of shoot organogenesis exist not on the surface but in the deeper layers of explants. We applied vacuum infiltration to wounded explants to enhance Agrobacterium access to the deeper layers, which resulted in improvement of transformation efficiency by 3-fold (9.2±2.9%). To verify the efficacy of our transformation procedure for other Cucurbita species, we attempted to obtain transformants with 3 Cucurbita species: C. maxima (cv. Ebisu), C. pepo (cv. Black Tosca), and C. ficifolia (cv. Kurotanenankin). The average regeneration efficiencies were 54.2±7.2%, 62.5±19.1%, and 72.2±13.4% under our regeneration system. Although the efficiency for production of tansgenic plants was very low (ca. 0.2–0.3%), a transgenic line was obtained from C. maxima and C. pepo, respectively. We discuss recent advances that may help in the development of beneficial applications for the transformation of Cucurbita species.