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Optimization of the pollen-tube pathway method of plant transformation using the Yellow Cameleon 3.6 calcium sensor in Solanum lycopersicum
- Wang, Rong, Li, Ruizhen, Xu, Tao, Li, Tianlai
- Biológia 2017 v.72 no.10 pp. 1147-1155
- Solanum lycopersicum, Western blotting, calcium, fluorescence, fluorescence microscopy, fruit set, genotype, glufosinate, image analysis, plasmids, polymerase chain reaction, resistance genes, screening, sucrose, tissue culture, tomatoes, transgenic plants, yellow fluorescent protein
- The effects of different plasmid DNA densities, different varieties, and transformation solution components on the pollen-tube pathway method of transformation were investigated in tomato Solanum Lycopersicum varieties Zhongshu 6, Liaoyuanduoli, and Jinguan 9 using the transformation indicator Yellow Cameleon 3.6 (YC3.6) vector carrying the phosphinothricin (PPT) resistance gene and a YELLOW FLUORESCENT PROTEIN (YFP) fluorescence gene. Firstly, PPT resistance screening was conducted at the seed stage, and in vivo fluorescence imaging and fluorescence microscopy analysis were carried out to select positive transformants. Transgenic tomato plants were then confirmed by PCR and Southern and western blot analyses. The reliability of the reporting system was further confirmed by examining calcium ion dynamics in transgenic plants. The results showed that plasmid DNA density and transformation solution components significantly correlated with the fruit set ratio but genotype did not. A transformation solution with a plasmid DNA concentration of 600 ng μL⁻¹ and containing 5% sucrose and 0.05% Silwet-L-77 was found to be the optimal combination, resulting in the highest fruit set ratio and transformation efficiency. Using the YC3.6 vector as an indicator we have developed a system to quickly and accurately screen transgenic plants using in vivo imaging and fluorescence microscopy. These advancements, combined its tissue-culture independent nature, make the pollen-tube pathway an effective and affordable method of transferring exogenous genes into tomato plants.