【作者】 张倩；

【导师】 刘国锋；

【作者基本信息】 华中农业大学 ， 园林植物与观赏园艺， 2013， 硕士

【摘要】 百合花大而美丽,寓意百年好合,可用于鲜切花、盆栽以及花境的布置。虽然百合应用广泛,但百合也有很多性状需要改良,如花粉污染,某些品种抗性较差等。由于百合远源杂交不亲和,传统的杂交育种方法很难对百合的品种进行快速改良,而分子育种的手段如转基因的方法为百合性状的改良提供了可行的方法。但百合在遗传转化方面也存在一些问题。如百合转化效率较低,PCR检测假阳性率高等。本研究以麝香百合为实验材料,进行了胚性愈伤的诱导和遗传转化的研究,为提高百合的转化效率打下基础。主要研究结果如下：1、以麝香百合鳞片薄层切片为材料,诱导愈伤组织的最佳培养基为MS+60g/L蔗糖+7.5g/L琼脂+0.5mg/L NAA+0.4mg/L TDZ,诱导率可达到95.25%。诱导胚性愈伤组织的最佳培养基为：MS+60g/L蔗糖+7.5g/L琼脂+2.0mg/L PIC+0.1mg/L TDZ,诱导率可达到94.36%。对诱导的四种不同形态的愈伤组织进行石蜡切片,观察到PIC诱导的愈伤组织中存在较多的胚性细胞,因此PIC为诱导胚性愈伤的良好试剂。2、通过对鳞片薄层切片、鳞茎盘和愈伤组织三种不同受体材料的比较,得出最佳的转化受体材料为麝香百合愈伤组织；通过对不同类型的农杆菌菌株EHA105、 AGLO、GV3101的比较得出,菌株EHA105的侵染能力最强,AGL0和GV3101侵染能力较弱；通过对卡那霉素抗性标记基因和潮霉素抗性标记基因的比较得出,用潮霉素筛选的百合愈伤组织转化率明显高于用卡那霉素筛选的愈伤组织,因此在构建载体时尽量选用潮霉素抗性标记基因。通过对百合鳞片薄层切片的不同处理(划伤/不划伤)比较,得出筛选后划伤处理的鳞片薄层切片出芽率仅为1.5%,而未划伤处理的鳞片出芽率为25.2%,划伤处理对鳞片细胞造成了一定损害,不利于筛选过程中植株的生长。3、在遗传转化体系优化的基础上,以麝香百合愈伤组织为受体材料,EHA105为侵染菌株,潮霉素抗性基因为选择标记,进行了目的基因LlDREB1的转化,筛选后共得到12株抗性苗,经过PCR检测,其中3株呈阳性,转化效率为3.4%。更多还原

【Abstract】 As a kind of ornamental plant with big and beautiful flower, lily symbolized eternal love. It is usually used for cut flower, pot culture and flower border. With so many advantages, lily still has some features that need to be improved, such as pollen pollution, stress resistance and so on. But due to the interspecific hybridization incompatibility, its features are hard to be changed rapidly by traditional breeding methods. Molecular breeding such as genetic transformation can easily overcome the shortage of traditional breeding. But some difficulties in genetic transformation of Lilium plants are still exist. The results are as following:1. The optimal culture medium for embryonic callus was investigated, and the results showed that while taking the bulb transverse thin cell layer as explants, proper callus induction medium is MS+60g/L sucrose+7.5g/L agarose+0.5mg/L NAA+0.4mg/L TDZ, and the induction rate is95.25%. The better embryonic callus induction medium is MS+60g/L sucrose+7.5g/L agarose+2mg/L PIC+O.lmg/L TDZ, and the callus induction rate is94.36%. Moreover, a higher rate of embryonic callus was found from these calli. This research also observed4kinds of calli with different morphologies by paraffin section, and find out there are more embryonic cell in PIC induction callus. So PIC is appropriate to embryonic callus induction.2. The Agrobacterium-mediated transformation rate of Lilium plant is low, so mass production of transgenic plants is difficult. Callus is a better material for transformation than bulb scale and bulb base; bacterial strain of EHA105has a stronger ability to infect target material than AGLO and GV3101; screening the transformed explants with Hyg is better than Km resistance screening system. The bulb scales being scratched show lower rate of bud regeneration (1.5%) than the normal bulb scales (25.2%). It is probably because scratching damaged the bulb cell.3. Target gene transformation:PIC induction callus are used as transformation materials, the target gene is LIDREB1,87calli are infected and three shoots derived from them are positive by PCR analysis. So the transformation rate is3.4%.更多还原