【作者】 陈晓玲；

【导师】 江昌俊；

【作者基本信息】 安徽农业大学 ， 遗传学， 2011， 硕士

【摘要】 茶树（Camellia sinensis）是世界上最重要的生产非酒精类饮料的木本植物之一。它生长周期长，自然结实率和杂交结实率都较低，使得常规茶树育种需要20-25年[1]。植物组织培养技术是加快优质品种创新的重要手段，是现代生物技术应用于遗传育种研究的基础。为建立快速、高效的茶树离体再生体系，本研究分别以茶树平阳特早[C. sinensis cv.pingyangtezao]7月下旬枝条和茶树舒茶早[C. sinensis cv. shuchazao]10月中旬的种子子叶为外植体，在离体培养条件下，首先建立了茶树子叶离体再生体系，比较了其器官发生途径和体细胞胚发生途径；其次建立了茶树腋芽离体快繁体系；第三采用微型嫁接的方式，解决了茶树组培苗生根难，耗时长的问题，为茶树组织培养与工业化生产的连接提供技术参考。主要结论如下：1.茶树子叶离体再生的器官发生途径不带胚子叶愈伤组织的诱导率随着2,4-D浓度的升高而增加，细胞分裂素配合生长素使用对愈伤组织的产生有增效作用。在培养基MS+2.0mg L^-12,4-D+0.5mg L^-1 KT中，岀愈时间最短，岀愈率最高，达83.6%。转到芽诱导培养基MS+2.0mg L^-16-BA+0.2mg L^-1 NAA中继代培养，带胚子叶的出芽率、芽增殖倍数以及体细胞胚发生率分别为36.5%、12.3和38.2%，均高于不带胚子叶。2.茶树子叶离体再生的体细胞胚发生途径在培养基MS+2.0mg L^-16-BA+0.2mg L^-1 NAA+1.0mg L^-1 GA3中，带胚子叶和不带胚子叶可迅速脱分化产生胚性愈伤组织，进而产生体细胞胚和次级体细胞胚，并萌发出芽。带胚子叶的体细胞胚发生率为85.7%，体细胞胚萌发率79.4%，芽增殖倍数为17.0，均高于不带胚子叶。3.茶树腋芽离体快繁体系的建立以腋芽为外植体，在MS+2.0mg L^-16-BA+0.2mg L^-1 NAA培养基中腋芽迅速萌发，培养60d，成苗率可达82.3%。将得到的小苗按枝条、去顶芽和部分叶片的枝条、腋芽带茎和部分叶柄三种处理接种于1/2MS+1.0mg L^-16-BA+0.1mg L^-1 NAA培养基中，腋芽带茎和部分叶柄的处理萌发率和增殖倍数最高，分别达到71.4%和5.5；去顶芽和部分叶片的处理生长的最健壮；整个枝条的处理在部分茎段底端出现体细胞胚。4.胚性愈伤组织的继代培养继代培养中，体细胞胚的发生、萌发以及次级胚性愈伤组织的产生同时进行。胚性愈伤组织的体细胞胚萌发率在M（MS不添加任何激素）培养基中最高，为58.3%，在P(MS+2.0mg L^-16-BA+0.2mg L^-1N AA+1.0mg L^-1 GA3)中最低；而次级体细胞胚发生率以及体细胞胚增殖倍数在后者中最高。5．茶树体细胞胚的发生茶树体细胞胚在子叶柄处直接发生，或在子叶处产生胚性愈伤组织间接发生。带胚子叶以直接和间接两种方式产生体细胞胚，而不带胚子叶只以间接方式产生。在体细胞胚间接发生途径中，子叶表面隆起是发生脱分化的标志，随后细胞团膨大、颜色变绿转化成胚性愈伤组织，其表面长出半透明的颗粒状突起即为体细胞胚。体细胞胚的石蜡切片表明，此处细胞小且排列紧密，细胞质浓，有若干个分生区域，具有分生能力。6．组培苗的微型嫁接选取生长健壮的茶树组培苗，剪取1.5-3cm长作为接穗。茶树种子苗长至一芽二叶，离基部3-5cm剪去嫩梢作为砧木。采用劈接法嫁接，将接穗切口处浸入200mg L^-1的2,4-D溶液中3-5min，嫁接于砧木上胚轴处，用石蜡膜固定。20d后成活率为64%。更多还原

【Abstract】 Tea（Camellia sinensis） is one of the most important woody plants which product non-alcoholic beverage drinks worldwide. It’s long growth cycle, low natural seed set and hybrid seed set make the need for conventional breeding and propagation20-25years. Plant tissue culture technology is an important means of innovating high quality varieties, is the basis of modern biotechnology applying to genetic and breeding research. To establish a rapid and effective tea regeneration system, we take axillary buds [C. sinensis cv.pingyangtezao] in late July and cotyledons [C. sinensis cv. shuchazao] in mid-October as explants. In the condition of in vitro culture, first, established a system of in vitro propagation of tea cotyledons, and compared the organogenesis and somatic embryogenesis regeneration systems, then established a system of in vitro propagation of tea axillary buds，third sloved the problem of rooting hard and time-consuming by micrografting. We hope to provide technical reference on the connection of tea plant tissue culture and industrial production. The main conclusions are shown as follows:1. Organogenesis pathway in the system of in vitro propagation from tea cotyledonsInduction rate of cotyledons without embryos increases with the increase of the concentration of2,4-D. In the medium MS+2.0mg L^-12,4-D+0.5mg L^-1 KT, induction rate is highest to83.6%. Ture to buds induction medium MS+2.0mg L^-16-BA+0.2mg L^-1N AA, seedling rate, proliferation multiple and the incidence of somatic embryo in cotyledons with embryos are higher than which in cotyledons without embryos, reach36.5%、12.3and38.2%respectively.2. Somatic embryogenesis pathway in the system of in vitro propagation from tea cotyledonsIn medium MS+2.0mg L^-16-BA+0.2mg L^-1 NAA+1.0mg L-1GA3, cotyledons with embryos and cotyledons without embryos can dedifferentiation to embryogenic callus,and then to somatic embryos and secondary somatic embryos, and germinate. The incidence of somatic embryo in cotyledons with embryos is85.7%, germination rate of somatic embryo is79.4%, proliferation multiple is17.0, are all higher than that of cotyledons without embryos.3. Establishment of in vitro propagation system of tea axillary budsTake axillary buds as explants, axillary buds germinated rapidly in the medium MS+2.0mg L^-16-BA+0.2mg L^-1N AA, after in vitro culture60days, seedling rate can reach82.3%. The plantiets will be divided into three treatments: branches, branches without terminal buds and some leaves, axillary buds with nodal segments and some petioles, then inoculated into1/2MS+1.0mg L^-16-BA+0.1mg L^-1N AA medium, the seedling rate and proliferation multiple of axillary buds with nodal segments and some petioles is highest, reach71.4%and5.5respectively. The treatment of branches without terminal buds and some leaves grow strongest there are somatic embryos in the bottom of branches.4. Subculture of embryogenic callusIn subculture of embryogenic callus, the incidence of somatic embryo, germination and the incidence of secondary somatic embryo are happen in the same time. The incidence of somatic embryo in embryogenic callus can reach the highest58.3%in M（MS without any hermone） medium, and lowest in P(MS+2.0mg L^-16-BA+0.2mg L^-1N AA+1.0mg L-1GA3) medium, but the incidence rate of secondary somatic embryo and the germination rate of somatic embryo is highest in the latter.5. Incidence of somatic embryoSomatic embryo occurred in cotyledon petiole directly, or occurred in cotyledon indirectly. Cotyledons with embryos have both directly and indirectly pathways, cotyledons without embryos have only indirectly pathway. In the indirectly pathway of somatic embryo, strumae occurred on cotyledon surface is a sign of dedifferentiation.6. Micrografting of regenerated microshootsSelect the well-growth regenerated microshoots, take1.5-3cm long as scions.when the seedings grow to two leaves and a bud, cut the shoots at3-5cm away from bottom, take the rest as root stocks. Cut ends of scions were pretreated with200mg L-12,4-D for3-5min, after20days survival rate can reach64%.更多还原