【作者】 张丽；

【导师】 柴明良；

【作者基本信息】 浙江大学 ， 风景园林（专业学位）， 2013， 硕士

【摘要】 本文以继代了7年的沟叶结缕草愈伤组织为材料,对其进行了抗稀禾定体细胞无性系变异筛选和抗草甘膦遗传转化两方面的初步研究。沟叶结缕草抗稀禾定体细胞无性系变异筛选以沟叶结缕草7年龄的愈伤组织为材料,分别研究了不同浓度稀禾定对愈伤组织继代生长和再生的影响,并选择10μM作为大批量愈伤组织筛选浓度,总共对大约七千多粒愈伤组织进行了为期至少12周的筛选。筛选出的愈伤组织再生获得了少量抗性植株,并对其进行了田间抗性测试。研究结果表明,愈伤组织在添加稀禾定的继代培养基上培养过程中,相同稀禾定添加浓度下,培养第二个周期时愈伤组织的存活率相比第一个周期显著下降。当稀禾定添加浓度至10μM时,培养8周后愈伤组织的存活率下降至11.5%。当稀禾定浓度为3.0μM时,再生8周后愈伤组织的再生率仅为3.75%。这表明,与继代过程相比,愈伤组织的再生过程对稀禾定非常敏感。因此,与通过再生途径筛选抗稀禾定愈伤组织突变体相比,通过继代途径筛选抗稀禾定愈伤组织突变体的可行性更高。经过筛选并再生后,第一批共有5棵植株参加田间抗性测试,其中3棵植株是经过长期继代筛选出来的,另两棵是普通植株,作为对照。经过田间抗性测试后,植株SV2的田间抗性低于CK2,植株SVl和SV3的田间抗性高于普通植株CK2,其中SV3的抗性最强。沟叶结缕草抗草甘膦遗传转化是以沟叶结缕草7年龄的愈伤组织为材料,通过农杆菌介导法将目的基因转入愈伤组织,从而使愈伤组织及其再生后的植株具有草甘膦抗性。沟叶结缕草愈伤组织分为A、B、C三组,每组各800粒,侵染农杆菌。侵染后,根据愈伤组织生长情况,A组先后在含有2mM和5mM草甘膦的愈伤组织筛选培养基上各筛选4周。B组在含有2mM、3mM和5mM草甘膦的愈伤组织筛选培养基上各筛选4周。C组在含5mM草甘膦的愈伤组织筛选培养基上筛选8周。每组设置两种对照CK1和CK2,CK1与上述愈伤组织一同进行筛选,CK2在不含草甘膦的培养基上培养。将存活下来的愈伤组织转移到不含草甘膦的再生培养基上再生。实验过程中,我们发现部分农杆菌侵染后的愈伤组织,在筛选和再生过程中出现了快速扩繁。对照组CK1中未出现该现象。同时,与CK2相比,农杆菌侵染过的沟叶结缕草愈伤组织再生时生根所需要的时间比较长。再生16周后,试验组共有54粒经过农杆菌侵染的愈伤组织再生,另有少部分愈伤组织存在再生力。未经过农杆菌侵染的对照组CK1中无愈伤组织再生或具有再生力。更多还原

【Abstract】 With the embryogenic calli induced from the stolons of Manilagrass, selection of sethoxydim-tolerant variants in vitro and cultivation of transgenic plants with glyphosate resistance by means of agrobacterium-mediated transformation were studied preliminarily in this paper.Effects of different concentration of sethoxydim solution on callus growth and regeneration were studied. More than seven thousand calli were suffered with10μM sethoxydim stress for at least12weeks. A small number of calli survived after long-term selection and were regenerated into plantlets. Spraying those plantlets regenerated from the survival callus with0.25%commercial sethoxydim solution, we found that the survival rate of callus in the second round selection on the subculture medium supplemented with sethoxydim solution was lower than in the first round selection. Compared to the effect of sethoxydim on the growth of callus, the regeneration of callus was more sensitive to the sethoxydim. The regeneration rate of callus was decreased to3.75%with the concentration of sethoxydim increased to3.0μM. In the first field test, three of the five plantlets subjecting to test came from sehoxydim selection and two were ordinary as controls. After foliar spraying with0.25%commercial sethoxydim solution, SV1and SV3showed less injury than CK2, while SV2showed more injury than CK2. In contrast to all the plantlets sprayed with commercial sethoxydim solution, SV3showed a higher resistance to sethoxydim than SV1.To obtain the glyphosate tolerant Manilagrass, Three groups of calli were infected by agrobacterium carrying target gene and selected on the subculture medium containing with different concentrations of glyphosate solution. Calli of group A were cultivated on mediums containing2mM glyphosate for4weeks, and then were transferred to mediums containing5mM glyphosate for4weeks. Calli of group B were cultivated on mediums containing different concentration (2mM,3mM and5mM) of glyphosate for12weeks. Group C were cultivated on mediums containing2mM glyphosate for8weeks. Then the survival calli were transferred onto the regeneration medium lacking of glyphosate solution. During the experiment we found that comparing with CK1, a few of the infected calli propagated rapidly. And the calli without infection rooted earlier than the infected calli. After16weeks, fifty-four calli were regenerated into plantlets. Besides the regenerated plantlets, a small number of the calli still have the regeneration ability. All of the calli without infection lost the regeneration ability.更多还原