【作者】 陈琴芳；

【导师】 戴良英；

【作者基本信息】 湖南农业大学 ， 植物病理学， 2004， 硕士

【摘要】 草莓（Fragaria ananassa Duch）属于蔷薇科（Rosaceae）多年生草本植物。其果实营养丰富，鲜美多汁，具有很高的营养价值和经济价值。但草莓不耐贮运，草莓果实采摘后的贮运保鲜仍是目前生产实践中亟待解决的一大难题。另外，在草莓生产中多采用无性繁殖，经多年栽培后，很容易感染多种病毒，致使种性严重退化、产量降低、品质变劣和抗性降低等。因此，如何改善草莓品质和提高其抗病性已越来越受到人们的关注。近年来飞速发展的现代生物技术为草莓品种遗传改良提供了新途径。 本研究以目前国内主栽品种丰香和章姬为试材，较为系统地研究了影响草莓离体再生的诸多因素，如外植体类型、外源激素种类、外植体生理状态等，建立了草莓叶片高效离体再生体系，从而为草莓的遗传转化研究与应用奠定了基础。试验结果表明：不同激素组合及配比对草莓叶盘不定芽再生有不同的影响。TDZ对芽再生的效果明显比6-BA强，丰香叶盘在MS+TDZ2.0mg／L+2，4-D0.1mg／L+IBA0.2mg／L分化培养基上芽再生频率可达83.2％，平均每叶盘再生芽数达1.4个；章姬则在MS+TDZ4.0mg／L+NAA0.1mg／L+IBA0.2mg／L分化培养基上芽再生频率可达65.7％，平均每叶盘再生芽数为1.3个。同一基因型草莓叶盘再生效果比叶柄好，而其中又以10d左右叶龄的叶片再生能力最高。8.0mg／L AgNO₃能较好地控制外植体的褐化，促进芽的分化。再生芽在1／2MS+IBA0.1mg／L生根培养基中，生根率达100％。本文还对草莓花药离体培养进行了研究，筛选出适合草莓赵屯一号品种花药培养最佳诱导愈伤组织培养基，即F+6-BA1.0mg／L+NAA0.5mg／L+CH300mg／L，并成功诱导出再生芽获得再生植株。这为草莓花药脱毒培养和将来利用花药进行目的基因遗传转化研究提供了理论依据。 为了研究β-1,4-葡聚糖酶基因在草莓果实成熟过程中的作用及探讨利用β-1，4-葡聚糖酶反义基因培育耐贮草莓新品种，从草莓果实中提取总RNA，根据Gen Bank中β-1，4-葡聚糖酶基因FaEG1和FaEG3序列设计合成特异性引物，利用RT一PcR技术分别扩增了两个基因1 491bp和1863bp的cDNA，反向插入到植物表达载体PROKZ中，将该载体转入农杆菌AGL一I中，构建了石hEGI和卢h百G了的反义基因的表达载体。通过农杆菌介导法转化草墓品种丰香和章姬后，从104块抗卡那霉素转化愈伤中随机抽取24块经PCR检测，其中17块扩增出目的带，PCR阳性率为70.8%，初步证实这两个基因整合到草葛染色体基因组中，抗Kan愈伤现已分化形成12个转化芽。试验通过对农杆菌转化前外植体预培养、农杆菌侵染时间及转化后外植体共培养等方面的探索发现，农杆菌介导的草荀基因遗传转化的最佳条件为:预培养1d一共培养3d，农杆菌侵染时间为巧一ZOmin效果最好。更多还原

【Abstract】 Strawberry (Fragaria ananassa Duch) , which is a kind of herbage and belongs to the rose family in the plant kingdom. The fruit of Strawberry contains plentiful nutrient materials and richer sweet juice, and therefore has maximum nutritive value and significant market prospect. However, the present problem which severely affects strawberry production is how to keep the fruit green or to retard softening for longer time after harvesting. In addition, the vegetative propagation may result in many negative effects such as virus attacks, strain degeneration, and both yield and quality reductions. Recently, more and more technics of molecular and cell biology including high efficient plant regeneration, plant genetic transformation, have been used in the studies of improvement of the quality and disease resistance in Strawberry.The present primary cultivars of strawberry ’Fengxiang’ and ’Zhangji’ were used as experimental materials. To establish a reliable, rapid and effective in vitro regeneration system and to carry the strawberry genetic transformation out successfully, the factors affecting adventitious shoots regeneration were studied. The results indicated that the frequencies and amounts of shoot organogenesis were significantly affected by the hormone concentrations for a given genotype. Compared with 6-BA, TDZ had a better effect on inducing shoot regeneration. The regeneration frequency of cultivar ’Fengxiang’ reached the highest level of 83.2% in MS+TDZ2.0mg/L+2,4-D 0.1mg/L+IBA0.2mg/L, while cultivar ’Zhangji’ obtained the highest level of 65.7% in MS+TDZ4.0mg/L + NAA0.1mg/L+IBA0.2mg/L medium. The corresponding numbers of shoots regeneration were 1.4 and 1.3 per leaf disc, respectively. The regeneration of the leaf disc was better than that of petiole in the same genotype and the 10-day-old leaf had higher regeneration ability. Our data also showed that AgNO3 played an important role in controlling the browning of explants, and the concentration of 8.0mg/L was recommended. While using the rooting medium of l/2MS+IBA0.1mg/L, the rooting rate achieved nearly 100%. In addition, the anther culture of ’Zhaotunl’ was studied and the regeneration plants were obtained when cultured with F+6-BA1.0mg/L+NAA0.5mg/L+CH300mg/L medium.To investigate the role of endo-l,4-glucanase gene in strawberry fruit ripening, total RNAs were isolated from the pink strawberry fruits. By using the specific primers designed from the FaEGl and FaEGS cDNAs, 1491bp and 1863bp fragments were amplified by reverse transcriptional polymerase chain reaction (RT-PCR). The FaEGl and FaEG3 cDNAs were inserted into expressional vector pROK2 in reverse orientations.The antisense clonings pROK2-FaEGl and pROK2-FaEG3 were therefore mobilized into Agrobacterium tumefaciens AGL-1 by electroporation. The strawberry transgenic plants were developed by Agrobacterium-mediated plant transformation with kanamycin resistance selection. Among the twenty-four Kan-resistant calli, seventeen of them showed PCR positive. At the same time, we have further studied the factors such as the pre-culture of explants, the time of infection with Agrobacterium and co-cultivation to demonstrate the appropriate conditions for genetic transformation in Strawberry. The results have clearly shown that the conditions of pre-cultured for 1 day, co-cultured for 3 day and infected for 15-20min were favourable for the transformation!.更多还原