【作者】 桑庆亮；

【导师】 赖钟雄； 潘东明；

【作者基本信息】 福建农林大学 ， 果树学， 2002， 硕士

【摘要】 本研究以从荔枝(Litchi chinensis Sonn.)“下番枝”品种幼胚诱导并长期继代保持的胚性愈伤组织(Embryogenic Calli 简称EC)为试验材料，采用基因枪法进行荔枝EC转化系统的建立与优化研究。主要试验结果如下： ①优化了荔枝体胚发生条件，建立了荔枝EC高效再生系统。在附加5mg.L玉米素(ZT)的高糖(6％蔗糖)高琼脂(1.0％)MS培养基上诱导荔枝体胚高频率(100％)形成；在附加10％椰子汁(CW)的高糖(6％蔗糖)的MS培养基中诱导体胚正常成熟并在无激素MS培养基中诱导体胚萌发。 ②建立了荔枝基因转化的适宜受体系统。采用生长量测定的方法确定了继代培养5-10d的荔枝EC处于快速生长分裂期，适宜用作遗传转化受体；采用生长量测定结合定性观察的方法测定了荔枝EC对卡那霉素和潮霉素的敏感性，发现荔枝EC对卡那霉素不敏感，而潮霉素对荔枝EC有致死效应，且显著抑制其增殖，并确定50mg/L为筛选工作浓度。 ③确定了采用基因枪轰击荔枝EC的最佳条件。以含有标记基因gus和潮霉素抗性基因hpt的质粒pCAMB101301，采用基因枪轰击的方法转化荔枝EC，并对轰击条件进行了优化。结果表明，选择预培养5-10d的荔枝EC，轰击前用附加甘露醇0.25mol/L的高渗培养基前处理4h，质粒DNA用量为1.0μg/枪，金粉用量为10μl/枪，在氦气压力为1100psi、轰击距离为6cm条件下轰击1次，并在轰击后在同一高渗培养基上后高渗处理16h，可以获得GUS瞬时表达的最佳效果。 ④建立了荔枝抗性愈伤组织筛选、体胚发生、再生植株与转基因检测的技术体系。采用附加50mg/L潮霉素的筛选培养基对轰击后的荔枝EC筛选，共获得大约80个抗性细胞系，经进一步筛选和对培养条件的优化，保留了7个抗性细胞系(其中4个稳定表达GUS)，经PCR检测，gus和hpt基因已整合进荔枝基因组；以所保持的抗性细胞系为试验材料进行体胚发生和再生植株的研究，共获得了再生植株30余株，其中20株来自稳定表达GUS的抗性细胞系，经GUS组织化学检测为转基因植株，其余来自不表达GUS的抗性细胞系，经检测为不表达GUS的植株。 此外，本研究还采用根癌农杆菌感染的方进行了荔枝EC转化的初步研究，经筛选共获得了4个抗性细胞系，最终2个得以保留并获得表达GUS的转基因植株。 本研究对转基因技术在荔枝品种改良上有重要意义，并为今后的荔枝转基因研究的发展提出了问题。更多还原

【Abstract】 In the present study, a high efficient transgenic system via particle bombardment was established and optimized using the long-term maintained embryogenic calli (EC) induced from immature embryos of Litchi chinensis Sonn. cv. Xiafanzhi. The main results were described as follows:1) A high efficient plant regeneration system was established through optimization of parameters affecting somatic embryogenesis in litchi. The optimal medium for inducing somatic embryogenesis was MS medium supplemented with 5mg/L zeatin (ZT), 6% sucrose and 1.0% agar. Somatic embryos matured on MS medium supplemented with 6% sucrose, 1.0% agar and 10% coconut water (CW), and germinated on MS medium without plant growth regulators but supplemented with 2% sucrose and 0.7 % agar.2) An appropriate recipient system for litchi genetic transformation was established. Calli pre-cultured for 4-5 days were proved at their rapid-growth stage through the measurement of EC fresh weight increase and suitable for transgenics. The sensitivities of litchi EC to hygromycin B and kanamycin were evaluated through the method of the measurement of fresh weight increase combined with qualitative observation, which showed that litchi EC were not sensitive to kanamycin but sensitive to hygromycin B. Hygromycin B obviously depressed the growth of litchi EC. which was mortal to them. The concentration of 50 mg/L for hygromycin B was proved to be suitable for litchi EC transgenic selection through further experiments.3) The optimal parameters used in litchi EC bombardment were obtained. Litchi EC were bombarded using the plastmd pCAMBI01301 including gus and hpt genes, and the parameters affecting bombardment were optimized. The results showed that the best transient expression of GUS was obtained when litchi EC were bombarded with the following procedures: litchi EC pre-cultured for 5-10 days were transferred onto the osmotic medium supplemented with 0.25 mol/L mannitol for 4 hour-long pre-treatment; and then they were bombarded one time after the preparation of particle-plasmid compounds was performed using 0.5 Ugplasmid DNA and 600 ug gold microparticies per shoot under the conditions of 1100 psi helium pressure and 6 cm shooting distance; and finally cultured on the same osmotic medium as above for another 16 hours.4) A whole technical system of litchi resistant calli selection, somatic embryogenesis from resistant cell lines, transgenic plant regeneration and transgenic assay was established. After the bombarded litchi EC were selected on MS medium supplemented with 50 mg/L hygromycin B, some 80 resistant cell lines were obtained, and finally 7 cell lines were maintained for further studies through the optimization of culture conditions and further selecting procedures. Gus and hpt gene had both integrated into the genome of the 7 resistant cell lines proved by PCR assays and among them, 4 resistant cell lines steadily expressed the GUS enzyme proved by GUS histochemical assays. About 30 plantlets regenerated via somatic embryogenesis from theresistant cell lines. Among them, about 20 plantlets regenerated from the maintained resistant cell lines steadily expressing the GUS enzyme, and they were proved to be transgemc plantlets by GUS histocherrucal assay; and the other were non-GUS expressing plantlets which regenerated from the resistant cell lines without GUS expression.Additionally, a preliminary study onAgrobacterium-mediated litchi EC transformation was also performed, from which 4 resistant cell lines were obtained, and finally 2 resistant cell lines were maintained for further studies, from which transgenic plantlets expressing the GUS enzyme were obtained.All the above 9 resistant cell lines were well maintained in our laboratory.The establishment and optimization of EC transformation system in litchi would be of great importance for genetic improvement in litchi; and finally some problems about litchi genetic transformtion to be solved were put forward in this thesis.更多还原