Development of a Synchronized Germination System and Evaluation of Cotyledon Age and Selection Agents in Large-scale Transformation of a Model Tomato Cultivar ‘Micro-Tom’ Using Functional Genomics

We developed a “synchronized germination system” for large-scale transformation of a model tomato cultivar, ‘Micro-Tom’, and used it to generate large quantities of uniform explants for transformation. Following initial germination inhibition in a medium with high concentrations of sugar and other compounds, over 80% of the selected seeds germinated synchronously after being transferred to a germination-induction medium (GI medium). In addition, we used the “synchronized germination system” to investigate the appropriate cotyledon age and selection agent for efficient transformation. To study the effect of cotyledon developmental stages on transformation, ‘Micro-Tom’ seeds were treated with the “synchronized germination system”. Cotyledons cultured for 1, 2, 4, and 6 days in GI medium were transformed using Agrobacterium-pCAMBIA1305.1 for β-glucuronidase (GUS) reporter expression. The results showed a significantly higher GUS-positive shoot formation rate for cotyledons cultured for 2 days compared to those cultured for 4–6 days. Furthermore, using GUS and the anthocyanin regulatory gene VlmybA1-2 as reporters, the effects of kanamycin, hygromycin, and G-418 on shoot selection were compared using 2-day cultured cotyledons. Selection with hygromycin and G-418 proved more efficient than kanamycin, with significantly lower percentages of shoots lacking GUS or VlmybA1-2 traits under G-418 and hygromycin selection. The “synchronized germination system” developed in this study has been applied to large-scale transformation using activation tagging vectors, and a large number of transformants was successfully obtained.