Study On The Transformation Of Male Sterility Gene In Citrus Mediated By Agrobacterium Tumefaciens

Citrus is one of the most important fruit crops in the world, its area and yield are both ranked the first among all the fruit crops. However, there are many problems in the citrus industry, such as serious diseases, destructive insects, natural disaster, concentrated maturing season etc. Chinese citrus industry has been challenged by the imported fruits after the nation became the member of WTO. Many of the local varieties such as Ponkan mandarin and Shatianyou pumelo etc. in China are seedy and lack of potential in the market. To solve these problems, breeding the seedless excellent citrus that can challenge with the market is required. However, long juvenility, polyembryony, high genetic heterozygosity and arthenogenesis have been impeding the traditional breeding in citrus. The application of genetic transformation can transfer the foreign gene into present commercial cultivar so as to improve the agronomic traits.In this research, the chimera male sterility gene TA29-barnse is transformed into the epicotyls and cotyledons of several seedy and less seedy citrus by Agrobacterium tumefaciens for the male sterility, aiming at the creation of seedless citrus types. The main results are as follows:1 The effects of the herbicide Barsta on the regeneration and growth of citrus callus, epicotyl and cotyledon were investigated. The suitable concentration for callus, epicotyl and cotyledon was 9.25 mg/L2 Efficient regeneration media for citrus epicotyl were investigated. The high efficient regeneration medium for Murcott tangelo ( Citrus reticulata x C. sinensis ) consisted of MT plus 0.5 mg/L of BA and 0.1 mg/L of IAA with the regeneration frequency 96.8% and the mean number of regenerated shoots 1.7. The medium for Hongjiang sweet orange (C. sinensis) epicotyl is of MT plus 0.5 mg/L of BA and 0.2 mg/L of IAA with the regeneration frequency 54.4% and the mean number of regenerated shoots 1.5.3 In the transformation for citrus callus, the resistant callus of Murcott tangelo (C. reticulata x C. sinensis), Chaxiang (C. reticulata) were obtained, and there were embryos differented from the resistant calluses of Gailiang orange(Citrus sinensis).4 Several factors affecting transformation in citrus epicotyl and cotyledon wereinitially optimized:Pre-cultivation of Hongjiang sweet orange (Citrus sinensis) had negative effect on the transformation in citrus epicotyl and cotyledon, Co-cultivation was the key factor in transformation. The transformation efficiency was higher when the explants be co-cultivated for 3 days than the others treatments.5 PCR amplification verified that four resistant shoots of Hongjiang sweet orange were transformed with T429-barnase gene. Resistant shoots of other varieties including pummelo (C. grandis) are rooting and going to be confirmed later.