| Tea plant (Camellia sinensis (L.) O. Kuntze) is one of the most important economic plants in China. To breed and popularize the improved tea cultivars is a very important aspect of science and technology to show their power on tea production. Nowadays, the traditional breeding methods can not meet the great demand of the modern tea production any more. It becomes urgent to take advantage of the findings and productivity of the modern science and technology to breed tea cultivars with high quality. Plant transformation technology, as an important part of plant engineering, not only offers a perfect platform for research on gene regulation and expression but also provide an effective way for plant directional upgrade and plant breeding on a molecular level.In this research, tea plant genetic transformation systems, including Agrobacterium-mediated system and particle bombardment-mediated system, have been optimized.In Agrobacterium-mediated system, if tea explants were pre-cultured on medium containing PVP 16g/L for 2-3d before infiltrated by Agrobacterium tumefaciens, transformation frequency was increased. However, pre-cultured on the medium containing CaCl2 can only make the transformation frequency lower.Furthermore, a micro-projection protocol using particle delivery system and GUS report gene was developed for tea plant callus. Result of the comparison among several pre-bombardment culture media showed that AgNO3 had no significant effect on the tea plant transformation. In addition, if tea calli were cultured on the AgNO3-containing medium more than 30 d, they would become browned. When AgNO3 and CaCl2 were used in integration with PVP, the media always yielded the highest GUS gene activity. Even pre-cultured on the callus inducing medium (1/2MS plus 2, 4-D and KT) without AgNO3 and CaCl2, the bombarded targets could also show relatively higher transient GUS expression rates, only if PVP was added. Instead of making contribution to improving GUS expression, osmotic pre-treatment wouldbombardment micro-particle were that: 10# 1 tungsten suspension (60mg/ml), 1.6 # 1 plasmid DNA (1 # g/# 1), 4# 1 spermidin (0.1M) and 15 # 1 CaCl2 (12.5M). Ethanol was used to dilute the final volume up to 48 # 1. The loading volume for each bombardment was 8-10# 1. Bombarding tea callus one time, at 7MPa with a target distance of 5cm and vacuum at -0.095MPa was suitable for both GUS activity and regeneration. The survival rate of resistant calli was 5.0%-12.1%, when the bombarded explants were cultured on selection media with kanamycin or hygromycin.Besides Agrobacterium-mediated transformation ( AGR ) system and particle bombardment (BOM) system, several combinations of the two systems (BTA, BPA and BOA), have also been tested and compared with each other. Based on the results of GUS expression and survival rates of the resistant calli, the method combining Agrobacterium and particle bombardment systems is suitable to apply for tea plant transformationIn terms of the transformation frequency, there were no significant differences among Wuniuzao, Jinfeng and Zisun, three tea cultivars employed in the experiment. According to this, the differences of cell competent ability among different cultivars have no great influence on tea transformation. However, the regeneration capability of the cultivar would play a more critical role in tea plant transformation. |
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