【作者】 王维飞；

【导师】 韩烈保；

【作者基本信息】 北京林业大学 ， 草业科学， 2007， 硕士

【摘要】 本研究通过单因素和多因素正交实验探讨了多个因素对高羊茅(Festucaarundinacea Schreb.)外植体愈伤组织诱导及生长的影响,建立了高效的离体植株再生体系。在此基础上,通过基因枪轰击法把与抗旱、耐盐有关的外源基因(DREB1A)对高羊茅胚性愈伤组织进行遗传转化,经过潮霉素筛选、PCR扩增与Southern杂交检测,获得了转基因植株。同时,对部分转基因植株进行了干旱胁迫处理,检测了转基因植株在抗旱特性上的实际效果。通过研究,获得了以下结果:1.为了建立高羊茅(Festuca arundinacea Schreb.)的组织培养体系,为转基因研究做准备,本实验对高羊茅的四个品种:猎狗5号(Houndog 5)、交战Ⅱ号(CrossfireⅡ)、凌志(Barlexus)及千年盛世(Millennium),进行了愈伤组织诱导及植株再生的研究。结果显示:①外植体种子的灭菌程序以“次氯酸钠(活性氯≥5%)1h+无菌水搅拌去皮”最佳。其不同处理方式(切口与不切口)对愈伤组织的诱导影响不大。②四个高羊茅品种以千年盛世在愈伤组织诱导及继代培养中优势最突出。③千年盛世的愈伤组织诱导的适宜培养基为:MS基本培养基+9mg/L 2,4-D+0mg/L6-BA+300mg/L水解酪蛋白。④千年盛世的愈伤组织的适宜继代培养基为:MS基本培养基+9mg/L 2,4-D+0.05mg/L 6-BA+0.5mg/L KT+1.25mg/L CuSO4。⑤再生频率较高的愈伤组织状态为:淡黄色、结构致密、生长旺盛。⑥愈伤组织再生培养基(MS基本培养基)中6-BA的适宜浓度为0.05mg/L,KT的适宜浓度为0.2mg/L。2.建立了高羊茅抗性愈伤组织的筛选方案。对于基因枪法,采用延迟筛选的方式,愈伤组织轰击后恢复培养5d,然后转入含有80mg/L潮霉素的愈伤组织继代培养基上,培养2个月,待分化出抗性植株后,将抗性植株转移至再生培养基中,潮霉素浓度降至40mg/L。3.建立了高羊茅基因枪转化体系。其技术要点为:1100psi的轰击压力下,选用直径1μm的金粉,每枪金粉500μg+1μgDNA、6cm的轰击距离、轰击2次的基因枪转化参数,选择高羊茅胚性愈伤组织进行基因枪轰击,靶材料在轰击浅4h至轰击后12h间,培养于含有各0.2M的甘露醇及山梨醇高渗培养基上。4.获得了转入DREB1A基因的高羊茅。通过PCR、Southern分子检测,证明在’千年盛世’品种中获得转DREB1A基因株系1个,共77个单株。5.通过对部分转基因植株进行干旱胁迫处理,对质膜透性、MDA含量、POD活性活性及叶绿素含量四个生理指标进行测定,结果表明,转基因植株比对照抗旱性有不同程度的提高。更多还原

【Abstract】 The influences of varied factors on calls induction and growth from explants of Tall Fescue （Festuca arundinacea Schreb.） were discussed, and plant regeneration system with high frequency was established. Embryonic callus of Tall Fescue were used as material for genetic transformation with the DREB1A gene by particle bombardment. The transgenic plants were obtained. At the same time, partial transgenic plants were treated with drought stress to check the actual results on drought resistance. Through the hard efforts, conclusions are showed as following.Ⅰ. In order to establish the tissue culture system of tall fescue （Festuca arundinacea Schreb.） for the transgenic study,callus induction and plantlet regeneration of four lines of tall fescue,Houndog 5,Crossfire II,Barlexus and Millennium, were studied in this experiment.The results indicated that: ①The best surface sterilization process for seeds is stirred in NaC10（active chlorin≥5%） one hour and then sterilized water for dehusking dissolving the skin of seeds in water without germs in seeds surface sterilization.The effect of different ways of seeds treatment （with a cut or not）on callus induction was not significant. ②Millennium performed better than other lines of tall fescue in both callus induction and subculture. ③The suitable medium for callus induction of Millennium is a basical MS salt mixed with 9mg/L 2,4-D+0mg/L 6-BA+300mg/L Hydrolysate. ④The suitable medium for subculture of Millennium is MS+9mg/L 2,4-D+0.05mg/L 6-BA+0.5mg/LKT+0.5mg/L CuSO₄. ⑤Callus with a higher regeneration are light yellow to green,compact fast-growing and green.⑥The suitable concentration of 6-BA and KT in medium （MS） for plantlet regeneration are 0.05mg/L and 0.2mg/L.Ⅱ. Hygromycin selection system for tall fescue transformants was standardized. Delayed selection was employed in biolistic gene deiver system. Two monthly callus subcultures on medium with 80mg/L hygromycin were followed by one monthly selection on plants regeneration medium containing 40mg/L hygromycin. Once emerged, shoot clusters were transferred to hygromycin-free rooting medium to develop into plants.Ⅲ. Biolistic gene deliver system for Tall Fescue was established. Plates containing the target tissue were placed 6 cm below the stopping mesh. Partice acceleration was done using Biorad PDS-1000/He device at 1100 psi under partial vacuum and each Petri plate was shot twice, using 500 μg gold particle （1μM） and lug DNA per bombardment. This optimized condition requires using embryonic callus of Tall Fescue. Embryonic callus were selected and arranged in the center of osmotic medium （callus induction medium containing 0.2M mannitol and 0.2M sorbitol） 4 h prior to transformation. After 16 h following bombardment, the callus were transferred to osmoticum-free medium. Ⅳ. Transgenic Tall Fescue with foreign DREB1A gene were obtained. The transgenic plants were obtained. Through PCR and Southern blot analysis, 1 DREB1A-gene transgenic lines of cultivar ’Millennium’ obtained. The total number of transgenic plants were 77.Ⅴ. Partial transgenic plants were treated with drought stress, and then the Relative leakage electrolytes, concentration of MDA, POD enzymes activity and concentration of chlorophylls are mensurated. The results showed that, transgenic plants have higher drought resistance than that of the control.更多还原