Cold Tolerance Gene Cloning And Agrobacterium-mediated ICE1 Gene Transfer In Citrus

Citrus is one of the most important fruit trees in the world, and it also holds an important position in the fruit tree industry in China, especially in the southern China. However, periodic freezing injury seriously affected the yield and quality of citrus fruits. It depends on the breeding program to develop cold tolerant citrus cultivars to solve this problem. Conventional breeding plays a limited role in this program because citrus has characteristics such as poly embryo, complex inheritance background as well as male or female sterility. Tissue culture and transgenic research shortened the breeding program cycle, thus provided a promising way to obtain elite citrus germplasm that are cold tolerant.In present research work, various factors that affected the adventitious buds regeneration from cold sensitive "Eureka" lemon (C. limon (L.) Burm. f. cv. Eureka) were investigated. Tissue culture system using mature stem segments derived from Newhall navel orange (C. sinensis (L.) Osbeck cv. Newhall) was established, which provides a platform for transgenic study using explants derived from mature citrus. Plant expression vector was constructed using ICE1 that was cloned from Arabidopsis, and this gene was successfully transferred into the lemon genome mediated by Agrobacterium, and the preliminary optimized transformation protocol for lemon epicotyls was developed.The main results of our research work are as follows:1) Main factors that affected the lemon (C. limon (L.) Burm. f. cv. Eureka) adventitious buds regeneration were investigated. The results showed that some factors such as explant types, the combination of the kinds and concentration of plant growth regulators in the culture media and the time of dark treatment, significantly affected lemon adventitious buds regeneration. Epicotyl segments were the optimal explants for adventitious buds regeneration. Adventitious buds were regenerated from epicotyl segments in MT basal medium, and the addition of BAP and NAA in the media influenced the buds regeneration. When BAP and NAA added to the medium at 1.0 and 0.1 mg L~-1 respectively, regeneration frequency of epicotyl segments reached 76.6%, at the same time, 9.25 buds per explant were obtained. 20 d darkness incubation enhanced the adventitious bud regeneration, which produced 13.46 adventitious buds per epicotylsegment under optimal plant growth regulator agenda. With the application of either IBA or NAA, the adventitious shoots developed roots, with IBA elongated the root length and NAA increased the root number.2) An efficient in vitro regeneration system through direct organogenesis from mature nodal and intemodal stem segments of Newhall navel orange (C. sinensis (L.) Osbeck cv. Newhall) was established. Illuminating conditions together with plant growth regulators affected the adventitious bud regeneration frequency and efficiency. The initial 15 d darkness inoculation increased the adventitious bud regeneration, and the highest regeneration frequency (85.2%) and bud formation efficiency (3.7 per responsive internodal stem segment) were obtained when 1.0 mg L~-1 BAP and 0.5 mg L~-1 NAA was supplemented. The elongated adventitious buds were successfully rooted in 1/2 MT media supplemented with 1.0 mg L~-1 NAA and transferred to the soil.3) Based on the GPAT gene sequences deposited in Genebank or their analogues ESTs, a pair of degenerate primers were designed, and subsequently a fragment about 500 bp was amplified using Poncirus genome as template. Bioinformatic analysis indicated that the cloned fragment was not the target sequence for its sequence similarity with other GPAT genes is pretty low. Possible reasons were discussed and preliminary methods to solve the problem were proposed. A pair of primers was designed for the Arabidopsis ICEl gene, and subsequent RT-PCR amplified the fragment of ICEl gene with its coding sequence region. Based on the plant expression vector pMV, another vector nominated pMVICEl was constructed, in which ICEl gene was regulated by CaMV 35S promoter and NOS terminator. This binary vector was successfully transferred to Agrobacterium tumafaciency strain EHA105.4) Factors that affected the transformation frequency of lemon epicotyl were investigated. The results showed that younger explants are more readily to be transformed than older ones. The time (7.5-30 min) that explant immersed in the Agrobacterium suspension cells did not significantly affected the transformation frequency. 100 uM or 200 uM acetosysingone in the coculture media enhanced the transformation frequency when lemon epicotyls were used as explants. Epicotyl segments precultured in darkness for a short period of time enhanced transformation frequency, however, prolonged dark treatment inhibited it. The results also demonstrated that 19℃ coculture temperature, 5 d coculture in the media as well as tobacco feeder plate and the addition of 2,4-D in coculture media improved the lemon epicotyltransformation frequency. The highest transformation frequency amounted to 15.4% in our experiment, which was greatly enhanced compared with previous reports that used lemon stem segment as explants. PCR and Southern blot analysis indicated that ICE1 gene was successfully integrated into lemon genome, and the target gene was inserted into more than one place in lemon genome. Finally, 7 transgenic lemon seedlings were obtained and 5 of them were successfully transferred to soil.