【作者】 杨莉；

【导师】 陈昆松； 徐昌杰；

【作者基本信息】 浙江大学 ， 果树学， 2006， 博士

【摘要】 金柑（Fortunella crassifolia Single）为芸香科柑桔亚科金柑属植物,原产中国。与其他柑桔类植物相比,金柑童期较短,实生繁殖后约2-3年便可开花结实,是用作研究果实性状或利用转基因体系研究基因功能的较理想的多年生木本植物材料。 本研究以我国金柑主栽品种‘金弹’为试材,建立了高效的再生体系、遗传转化体系和检测技术体系,获得了转BCH反义基因及叶绿素酶基因启动子的转基因植株,并对转基因材料进行了分子鉴定。主要研究结果如下。 （一） 金柑高效再生体系的建立 以‘金弹’成熟种子为试材,研究不同外植体类型及放置方式、激素配比、糖浓度等因素对不定芽或根再生的影响,建立一套高效稳定的组织培养再生体系。结果表明:上胚轴为最佳外植体,其再生不定芽频率为68%,下胚轴和子叶的不定芽再生频率均低于10%;外植体在培养基中的放置方式以形态学上端向上最好,不定芽的再生频率高出平放一倍以上;上胚轴在诱导不定芽的最佳培养基中,每外植体再生不定芽数可高达1.48个,且出芽时间相对较早,生长健壮,无玻璃化现象;培养基中蔗糖浓度为25 gL^-1时最有利于不定芽再生,随蔗糖浓度提高,不定芽再生频率降低,而不能分化的愈伤组织大大增加;再生芽在最佳生根培养基中生根30 d可再生3-5条不定根,平均根长度达1.8 cm;长成的完整植株在移苗以前需经7 d以上时间炼苗,直接移栽易导致再生植株萎蔫死亡。 （二） 金柑遗传转化体系的建立 建立了一套基于Kan筛选的遗传转化体系:以培养约40 d无菌苗的上胚轴为外植体;将刻伤的上胚轴在已稀释至OD值为0.5的农杆菌菌液中浸泡30 min,接种到不含抗生素的培养基中共培养3-4 d;共培养后的外植体先转移至Kan浓度较低的培养基(25 mg L^-1)中筛选1个月,再继代到Kan浓度较高的培养基(50-100 mg L^-1)中筛选;4-5个月后,将长至0.5-1.0 cm的抗性芽嫁接到本砧上,嫁接成活后再移至光照培养箱及温室培养,最终转化率平均为3.5%。更多还原

【Abstract】 Kumquat {Fortunella crassifolia Swingle) also called kinkan, are small evergreen shrubs of Rutaceae, which are closely related to plants of Citrus genus. Ripe fruits of kumquats are quite similar to cherry tomatoes in size and shape;the color ranges from orange yellow to orange red. Kumquats fruits are rich in fiber, vitamins A and C, and contain traces of calcium and iron, offering many nutritional or clinical benefits. The most notable aspect of kumquat fruit, however, is its deliciously sweet rind, providing a special taste that other citrus fruit can not. As a result, kumquats are commercially cultivated as important fruits just as other citrus fruits in China as well as in Japan, United States, etc. Furthermore, Kumquats have a much shorter juvenile stage than other common citrus species, taking only about 2-3 years to grow a flower from a seed, while 5 to 12 years are needed for other citrus. Therefore, kumquats are most suitable among various citrus or even woody plants for evaluating function of genes related to fruit characteristics or studying expression patterns of fruit specific promoters by genetic transformation since fruits can be obtained in a much shorter time.According to the developments of molecular biology and plant genetic engineering, more and more people got new varieties and idioplasm resources by using molecular biotecniques. However, the application of biotechniques in moelcuar biology is along with the development of plant tissue cultures. Consequently, an efficient regeneration and genetic transformation protocols are prerequisite for new varieties of kumquats, and understanding the functions of foreign genes and transcriptional sequences in plant genome by introducing beneficial genes such as genes related caroteniod synthesis for improving fruits qualities.In this paper, using seeds from ripening Fortunella crassifolia fruits as plant materials, we set up an efficient regeneration system, genetic transformation system and transformants detection system, and applicated these systems on genetic transforamation of a promoter of chlorophyllase and a carotene β-ring hydroxylase gene. The results were summerized below:1. Establishment of a high frequency plant regeneration system for kumquats.Here, several important factors influencing the efficiency of plant regeneration of ’Jindan’ kumquat (F. crassifolia Swingle), including explant types and orientation,concentrations and combinations of plant growth regulators, were evaluated for their influences on efficiency of plant regeneration. It was found that the optimum explant and its orientation was epicotyl planted vertically with upper part upward, and a shoot regeneration frequency of 1.48 shoots per explant was obtained on medium Mn. More shoots were regenerated from Mn supplemnted with 25g I"1 sucrose. A rooting percentage as high as 74 % was obtained on the optium rooting medium.2. Establishment of an efficient genetic transformation system for kumquats Epicotyl segments of kumquat were firstly transformed with Agrobacteriumtumefaciens GV3101 harboring neomycin phosphotransferase gene (npt II)-containing plant expression vectors in darkness at 25 °C on kanamycin free Mi i for 3 d, then cultured in darkness on Mn supplemented with 25 mg dm"3 kanamycin and 300 mg dm’3 Cefotaxime for 20 d, and finally subcultured to fresh Mn containing 50-100 mg dm"3 kanamycin monthly and cultured under 16-h photoperiod. Sixty five kanamycin resistant shoots were regenerated from 500 epicotyl explants after four-month selection, and 20 strong shoots of them were shoot tip grafted to 50-day-old kumquat seedlings with a survival rate of 55 %. A transformation efficiency of 3.6% was achieved using the optimized transformation procedure.3. Development of rapid, reliable and simple assays for early detection of transgenic plant materials.3.1.MPCRMultiplex PCR is a variant of conventional PCR which includes two or more pairs of primers in a single reaction to amplify several genes of interest simultaneously. In this study, a reliable multiplex PCR analysis method was established for simple and fast detection of transgenic in plant materials. Two pairs of primers, corresponding to neomycin phosphotransferase and 1-aminocyclopropane-l-carboxylate synthase (ACS) gene, or nptll gene and ChvA gene (special gene in Agrobacterium tumefaciens) were selected as for target and resident gene, respectively. The method bypasses routine DNA extraction, requires only very little amount of plant tissue, and produces reliable results as shown by successful discrimination of transformed and nontransformed tobacco, tomato, and kumquat materials. This method facilitates early identification of transgenic buds when they are still quite small.3.2 T-DNA/plant sequence analysis.On the basis of Zhou et al. (1997), we improved on this method to identify 20 plants successfully, and 16 lines have been integrated with foreign genes, even if twoor more copies presence in 5 transgenic lines. The results suggest that the method of analyzing T-DNA/plant junction sequences can not detect the resistant plants early and quickly, but also can detect the number of copy of those transgenes. Therefore, the method should be an ideal way to detect the resistant plants according the procedure is easy and feasible.4. Application the systems of regeneration, transforamtion and detection on kumquats.4.1. Agrobaterium-mediated transformation of antisense orange p-carotene hydroxylase gene75 resistant shoots with BCH antisense gene were obtained through a succession of Kanamycin selection. The results of the MPCR, T-DNA/sequence analysis, and Southern hybridization analysis showed that the BCH gene was integrated into kumquat genomes.4.2. Agrobaterium-mediated transformation of orange chlorophyllase gene promoter.39 resistant plants incorporating GUS gene under an orange chlorophyllase gene promoter from genomic library were obtained to evaluate the expression pattern of the promoter. After microgafting, 9 transgenic lines grow vigorously in the greenhouse.更多还原