High Efficient System Establishment Through Thin Cell Layer Culture And Preliminary Studies On Genetic Transformation Application In Citrus Sinensis (L.) Osbeck 'Cutter Valencia'

Citrus (Citrus spp.) is the most important tropic and semi-tropic fruit tree, with the highest yield among all of the fruit in the world. However, the genus Citrus possesses several undesirable characteristics including salt, extreme cold weather sensitivity and fungi and disease susceptibility. With the improvement of our living condition and living habits, the aim of Citrus breeding has been changing simultaneously. Due to the obstacles of polyembryony, sexual incompatibility and male or female sterility, it is hard to satisfy the requirement of cultivating a new variety by conventional breeding method. Genetic transformation is an alternative to overcome these difficulties. For successful transformation of various agronomic and quality characters to Citrus, rapid, reliable and direct in vitro regeneration system is a prerequisite.In this study, an efficient and dynamic in vitro propagation of high quality propagating material by Thin Cell Layers (TCL) technique was established using asetptical seedlings of Citrus sinensis (L.) Osbeck cv. Cutter Valencia as explants. Many aspects afftect the shoot regeneration ability, including seeding pre-culture medium, thin cell layer cutting position and laying orientation, adventitious bud inducing medium were studied. The regeneration process was observed by histological assay. Based on the studies above, preliminary researches on genetic transformation that mediated by Agrobacterium tumefaciens were conducted subsequently. The main results are as follows:(i) Epicotyls of etiolated seedlings were mainly used as explant in traditional genetic transformation regeneration system. In this study, seeds were cultured in 6 different seeding media in dark to obtain etiolated seedlings. Then the epicotyl of etiolated seedlings was cut into 1mm thick thin sections and inoculated in contact with 12 different adventitious bud induction media. By analyzing epicotyls length and thickness of etiolated seedling, the degree of operating difficulty of thin cell layer making and the callus forming ability, the optimal culture medium is MT + 0.5mg/L BA + 0.5mg/L KT + 0.lmg/L NAA + 30g/L Suc, and the pre-culture media is 1/2 MT.(ii) During the research process, the thin cell layer from epicotyls of green seedling that germinated in seed germination medium under 16/8 h light-dark cycles propagated a lot of adventitious shoots. The adventitious bud induction medium which was tested by etiolated seedlings was also suitable for the green seedlings, while the proper pre-culture medium for green seedlings is MT + 0.5mg/L BA + 0.05mg/L IBA + 0.2 mg/L GA₃ + 30g/L Suc.(iii) The illumination was found to have a significant role in adventitious bud forming from epicotyl transversal thin sections. At the initial stage of culture, if exposed to light, the xylem and phloem of the thin sections are likely to be apart, which make the explants dead consequently. Therefore, laying explants in dark for a proper length of time in the early event of adventitious inducing process is essential. However, incubated in dark is not timelessly, the root regeneration ability reduced greatly when incubated in dark more than 28 days. There were many adventitious shoot germinated when cultured in dark 7 to 24 days. Especially with 21 days darkness induction, it can produce the most adventitious shoot, with the highest average number: 21.7 shoots per layer.(iv) In epicotyl transversal thin cell layer culture method, the regeneration ability was not the same from different position of the seedlings. Hypocotyl of green seedling has the highest regeneration rate (67.2%) which was followed by epicotyl of green seedling (59.3%) with the highest average number of adventitious bud per explant was 20.7. The lowest rate was produced from thin cell layer of green seedling root (27.5%) with the highest average number of adventitious bud per explant were 5.3.(v) From the basal to the apical part of epicotyl, the regeneration ability was different. The thin cell layer from the upper cuts has greater regeneration ability than the lower end, and can produce adventitious bud in less time. Also, the placement orientation affects the regeneration ability. If the explants were incubated in an upright orientation on the media, shoots were induced more efficiently, with the highest number: 18.2 shoots per explant, and the explants grew faster and less vitrification was observed than inverted cultural method.(vi) By cytohistological assay, the shoot regenerated from the thin layer comes from a diverse origin: cortex parenchyma cell, vascular parenchyma cell, and xylem cell also has the bud-forming potential after dedifferentiation.(vii) The thin cell layer is sensitive to kanamycine, shoot formation may occur on the medium without kanamycine, while, the layer can not form shoots on the medium with more than 10mg/L kanamycine. The green fluorescence can be observed when inducting Citrus thin cell layer in Agrobacterium with OD₆₀₀ for 10min firstly and then cultured on co-culture medium for 48⁷2h.