Transformation Of Citrus And Identification Of Transgenic Citrange With PhyB Gene

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, there are many factors such as fungi disease affected the yield and quality of citrus fruits. Cultivation of citrus under dwarf and close planting is the goal of the fruit tree industry. Seedlessness is also important for the fruit tree industry. These goals depend on the breeding program or citrus cultural techniques to solve these problems. Conventional breeding plays a limited role in this program because citrus has characteristics such as polyembryo, 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 disease resistant or dwarf or seedless.In present research work, various factors that affected the adventitious buds regeneration from Dahong sweet orange (C. sinensis (L.) Osbeck) and Shatianyou (C.grandis (L) Osbeck) were investigated. Tissue culture system using mature stem segments derived from Newhall navel orange (C. sinensis (L.) Osbeck) ,Bonanza navel orange (C.sinensis (L.) Osbeck) and Bingtang sweet orange (C. sinensis (L.) Osbeck) were established, which provides a platform for transgenic study using explants derived from mature citrus. rolABC and iaaM gene were successfully transferred into the Shatianyou genome mediated by Agrobacterium, chit42 gene was successfully transferred into the Dahong sweet orange by the same method. The preliminary optimized transformation protocol for Shtianyou and Dahong sweet orange epicotyls were developed. Molecular biological detection and biological characteristic of citranges (P. trifoliata (L) Raf.×C.sinensis (L) Osbeck) transferred with phyB gene were conducted.The main results of our research work are as follows:1) Main factors that affected the Dahong sweet orange and Shatianyou 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 significantly affected adventitious buds regeneration of Dahong sweet orange and Shatianyou. Epicotyl segments were the optimal explants for adventitious buds regeneration. Adventitious buds were regenerated from epicotyl segments in improved MS basal medium, and the addition of 6-BA and IAA in the media influenced the buds regeneration. When 6-BA added to the medium at 3.0 and 5.0mg·L^-1, regeneration frequency of epicotyl segments of Dahong sweet orange and Shatianyou respectively reached 96.7% and 81.5%. 7 d darkness incubation enhanced callus formation .With the application of 1.5 to 2.0 mg·L^-1 IBA the rooting frequency of adventitious shoots of Dahong sweet orange and Shatianyou respectively reached 94.7% and 57.1%.0.2% active carbon promoted rooting of bud.2) An in vitro regeneration system through direct organogenesis from mature nodal and internodal stem segments of Newhall navel orange and intermodal stem segments of Bonanza navel orange and Bingtang sweet orange were preliminarily established. The combination of 6-BA in the culture media significantly affected adventitious buds regeneration. When 6-BA added to the medium at 3.0 mg·L^-1, regeneration frequency of nodal stem segments of Newhall navel orange reached 40% .3) Factors that affected the transformation frequency of Shatianyou epicotyl were investigated. When explants were immersed in the Agrobacterium suspension cells for 15 to 20 min and cocultured 2 to 4 day, the transformation frequency of Shatianyou epicotyl reached a higher level. 200 mM acetosysingone in the coculture media did not significantly enhanced the transformation frequency. Epicotyl segments precultured in darkness for a short period of time (2d) promoted the transformation. The highest transformation frequency amounted to 8.25% in our experiment.The anti-kan buds were micrografted to citrange rootstocks in microtube,then grafted to fructus aurantii rootstocks 30 days later.PCR analysis indicated that rolABC and iaaM gene were successfully transferred into the Shatianyou genome mediated by Agrobacterium, chit42 gene was successfully transferred into the Dahong sweet orange genome. Finally, 31 Shatianyou seedlings transferred by iaaM gene, 9 shatianyou seedlings transferred by rolABC gene and 3 dahong sweet orange seedlings transferred by chit42 gene were obtained.4) Molecular biological detection and biological characteristic of citranges transferred with phyB gene were conducted. PCR, RT-PCT and PCR-Southern blot analysis indicated that phyB gene was successfully integrated into citrange genome and the target gene transcribed in citrange. In comparison with non-transgenic plant , the transgenic plant showed dwarfing characteristics, larger shoots insertion angle , smaller leaf area, whereas higher chlorophyll content, higher net photosynthesis rate, higher light saturation point and specific leaf weigh. Determination of endogenous hormone by HPLC indicated that the content of GA₁, GA₄,ABA and Z of the transgenic plant were 32.7%,50%,121%and 17.8% respectively higher than the non-transgenic plant, while the content of IAA was lower than the non-transgenic plant. (GA₁+ GA₄)/ABA and Z/ ABA ratio of the transgenic plant were 38.4%and 43.2%respectively lower than the control. The microstructure of the transgenic plant leaves has been examined by using optical microscope, SEM and TEM. The result showed that the number of chloroplast in palisade tissue and the phloem section area of the transgenic plant were more than control. Stoma size of the transgenic plant also changed. The number of granum and starch granules in chloroplast were 25.9% and 90% respectively more than the control. The buds of Shatianyou and Dahong sweet orange scion were grafted to the interstock of the transgenic plant and non-transgenic plant.The result showed the scion on the interstocks of the transgenic plant germinated earlier and more rapidly than the control in the initial stage.These results suggest that the active phyB induce expression of many genes such as CAB gene when the transgenic citrange perceive red light.The transgenic plant shows higher chlorophyll content and its chloroplast microstructure appears variations. The content and ratio of endogenous hormone of the transgenic plant also change. As a result, the transgenic plant shows higher net photosynthesis rate and dwarfing characteristics.