【作者】 武丽敏；

【导师】 郑有良；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2005， 博士

【摘要】 本文以中国春性小麦新品种（系）为外植体,进行组织培养和遗传转化参数研究。在此基础上,将含有bar基因和GUS报告基因的质粒pDM803用基因枪法转化小麦,获得可育的转基因小麦植株,从而建立了适于中国春性小麦基因型的基因枪转化体系。主要研究结果如下: 1.本试验首次报道2,4,5-T和NAA对小麦愈伤组织培养反应的影响,特别是详细比较了它们与小麦组织培养试验中广泛应用的生长调节物质2,4-D之间的差异。在添加2,4.5-T的培养基中,幼胚胚性愈伤组织诱导率和愈伤组织鲜重增加量均高于添加2.4-D和NAA的培养基,且2,4,5-T显著抑制外植体萌发率。在添加2mg/L 2,4-D的MS基本培养基上,幼胚胚性愈伤组织诱导率高达33.50%;而成熟胚在添加4 mg/L 2,4-D的N6培养基上反应最好。 2.小麦幼胚愈伤组织分化频率（62%-76.39%）高于成熟胚愈伤组织分化频率（38.57%-47.76%）。对小麦愈伤组织进行干燥处理,适宜时间内可有效地提高外植体分化频率。不同外源激素组合（KT、6-BA、NAA、IAA、TDZ和ZT等）对小麦愈伤组织芽分化频率影响显著,0.5mg/L TDZ可明显提高芽分化频率。IBA0.5+NAA 0.25在生根培养中表现最佳,且加入氯化胆碱和多效唑,幼苗根系生长旺盛、粗壮,有利于移栽。 3.首次探讨在培养基中添加AgNO₃对普通小麦幼胚愈伤组织培养的影响。在培养基中添加适宜浓度硝酸银可显著提高胚性愈伤组织率和愈伤鲜重,且能降低愈伤组织褐化率。基因型和硝酸银浓度间的互作效应显著地影响胚性愈伤组织诱导率和愈伤组织鲜重增加指数。在小麦组织培养不同阶段,添加适宜浓度的硝酸银是十分必要的。更多还原

【Abstract】 New Chinese spring wheat varieties （lines） with desirable agronomic and yield characters were used as explants to investigate the tissue culture and genetic transformation parameters in this study. The plasmid pDM803, carrying the bar gene, conferring resistance to the herbicide, and the screenable uidA gene, was transferred into wheat by particle bombardment. The main results showed as following:1. This is the first report about the effects of 2, 4, 5-T and NAA on wheat tissue culture, particularly in comparison with 2, 4-D in detail. The frequency of embryogenic callus and increment of callus fresh weight on 2, 4, 5-T media were higher than those on 2, 4-D media, and in the presence of 2, 4, 5-T the precocious germination of explants for all genotypes were significantly suppressed. For immature embryos cultured on MS medium, 2 mg/L of 2, 4-D was optimal, and the highest frequency of embryogenic callus （33.50%） was observed. For the mature embryos on N₆ medium, 4 mg/L of 2, 4-D was optimal.2. The results indicated that the differentiation frequency （62-76.39%） of callus from immature was higher than that（38.57-47.76%） of callus from mature embryo. Proper partial desiccation of callus before transferred to regeneration medium could improve the frequency of plant differentiation. Different hormones combinations （KT , IAA , NAA , TDZ , 6-BA and ZT ,etc. ） significant affected the bud differentiation frequency , among which , 0.5mg/L TDZ increased the frequency of shoot formation. Supplementing with 0.5mg/L IBA and 0.25mg/L NAA in rooting medium was optimal. Choline Chloride and Paclobutrazol could facilitate root formation.3. We have first reported that the effects of AgNCh on tissue culture of common wheat. Addition of AgNO3 could significantly improve embryogenic callus frequency and callus growth, whereas it could reduce the callus necrosis and callus induction frequency. Strong genotypic effects were observed on callus induction frequency, embryogenic callus frequency and callus necrosis frequency among the four genotypes. It also was found that there were significant genotype ><AgNO3 concentration interactions on embryogenic cullus frequency and callus growth index. These results suggested that it is important to optimize the AgNC>3 concentration in the different stage of wheat immature embryos culture.4. Growth inhibition of Agrobacterium by Carbenicillin and Cefotaxitin was valuated. Cefotaxitin was significantly more toxic than carbenicillin in induction of callus in wheat. lOOmg/L Cefotaxtin almost inhibited Agrobacterium growth, while Carbenicillin was 250mg/L. The induction frequency of callus was 84.7% supplementing with 250mg/L Carbenicillin in medium. Carbenicillin of low concentration showed auxin effects in tissue culture and disturbed hormone proportion. Thus, the ideal concentration of carbenicillin was 500mg/L when used in genetic transformation of wheat mediated by Agrobacterium-mediated. Wheat callus was more sensitive to hygromycin and G418 than to kanamycin. Hygromycin and G418 as the selection marker in genetic transformation of wheat was optimal, and the ideal selecting pressure was 25mg/L.5. The objective of this experiment was to optimize the major parameters controlling DNA delivery by particle bombardment to immature embryos of elite Chinese spring wheat {Triticum aestivum L.) cultivars. Efficiency of DNA （uidA gene） delivery was assessed by determining transient GUS （p-glucuronidase） expression in bombarded calli. Among the estimated parameters, acceleration pressure, bombardment distance, chamber vacuum pressure, bombardment times and osmotic conditioning of culture had significant effects on the transient gene expression. A bombardment procedure suitable for elite Chinese spring wheat cultivars was developed, allowing a highly efficiency DNA delivery with reduced damage of target calli. A total of 429 resistant calli from cultivar Chuannongl6 were obtained and 59 of them regenerated plants. PAT activity and the bar gene in transformed plants were detected by leaf painting with Basta solutions and PCRamplification, respectively. A total of 13 herbicide-resistant and partially resistant plants were screened out of 59 regenerated plants, among which 6 had the expected fragment corresponding to the bar gene PCR amplification. Thus, the transformation frequency of Chuannongl6 was 1.40% by PCR result.6. The mature seeds were implanted on Petri dish, in which concentration of Basta was 0, 3, 6, 9 and 12mg/L respectively. The sensitive responsibility of 4 genotypes with Basta were estimated. The results showed that seedling could be refrained and the rate of etiolation rate increased with concentration improving of Basta. Effects on seedling height and root length of Basta were significant difference. Etiolated seedlings appeared after sowing 4-5 days when concentration was 9mg/L. The selection effects to wheat with this treatment was appropriate. Spraying the offspring of 6 transformation lines with 0.25% Basta in field, the segregation Basta resistance in Ti was investigated. The results showed the bar was inherited as Mendelian single gene in offspring of all lines contained the bar gene. The segragation of resistance : sensitivity to Basta was 3:1 by X2-test.更多还原