Creating New Self-Compatibility Germplasm In Pear Using Transgenic Method

Pear shows a typical gametophyte self-incompatibility in Rosaceae. The production of pear fruits would be associated with pollination trees or artificial pollination. Pear has a long juvenile period, high heterozygous genome and their most traits are controlled by muti-alleles, so that the efficiency of traditional method in improving pear traits is relatively low, and it is difficult to meet the needs of industrial modernization. With the development of molecular biology technology and genomics research for pears, using the genetic engineering to improve target agronomic traits has becoming an important way to pear cultivars. In this study, self-incompatibility alleles S3-RNase and S34-RNase alleles are cloned from’Fengshui’and’Yali’, whose antisense expression vector and RNAi vector are constructed to silence S-RNase gene for creating self-compatibility new pear germpalsm. Our specific reseaches are as follows:1. The full-length cDNA sequences of S3-RNase, S34-RNase alleles were obtained from’Fengshui’ and ’Yali’ styles, which were used to constuct antisense expression vectors (pGSA1285-S3 and pGSA1285-S34) and RNAi vectors (pHGRV-S3 and PHGRV-S34) based on the methods of double digestion and Gateway.2. Four factors including ultrasonic processing power, processing time, interval time and the processing times were optimized basing on the central composite-orthogonal design; the efficiency of exogeneous gene transfomation to pollen were detected by expressive vector of pCAMBIA 1304, which contains the GFP (green fluorescent protein) gene. The optimal parameters of the ultrasonic treatment of pollen were determined as follows: ultrasonic processing power 160-170 W, processing time 6-7 S, interval time 7 S, processing times 7.3. The pollen grains of’huang hua1 pear were treated with the established ultrasonic treatment of pollen-mediated transformation system, which would be as pollinator to cross with’Feng shui1,’Cuiguan’ and ’Dangshan su’ pears. The hybrid seeds were cultivated. Three seedlings containing pGSA1285-S3 antisense expression vector had been identified from the progeny of fengshui’×’huanghua’.4. After the plasmid containg pHGRV-S3 and pHGRV-S34 were introduced into A. tumefaciens strain GV3101, Agrobacterium-medisted transformation of leaf discs was used to get transgenic pear, and then obtained ’Yali’ regeneration buds, which layed foundation for creating self-compatibility pear germplasm.