【作者】 李焘；

【导师】 王喆之；

【作者基本信息】 陕西师范大学 ， 植物学， 2003， 硕士

【摘要】 山茱萸(Cornus officinalis Sieb.Et Zucc.)为山茱萸科的一种落叶乔木，在我国已有200多年的栽培历史，山茱萸果肉富含甙类、有机酸类、酚类、氨基酸类、维生素类等多种有机化学物质和矿质元素，具有较珍贵的药用价值，开发应用前景十分广阔。本实验以山茱萸为材料，对其组织培养技术进行了系统研究，建立了植株再生体系，并对组织培养过程中超度含水态苗的发生及其形态结构和控制条件等进行了研究，同时对山茱萸愈伤组织中有效成份的含量进行了测定。实验的主要结果如下： 1．分别以具腋芽茎段、叶片、花托、花柄为外植体，对组织培养及其植株再生进行了研究，结果显示： (1) 山茱萸茎段在1／2MS，附加BA1mg／L、IBA0.5mg／L的培养基上经过5-6周的培养，腋芽开始萌发生长，并可少量增殖：在1／2MS，附加BA0.5mg／L、IBA0.05mg／L的培养基上继代培养，幼芽生长较快，并可在幼芽基部形成不定芽丛，芽的增殖倍数达5.3左右。以此建立了山茱萸茎段腋芽增殖的微繁体系，可望在短期内获得较多的山茱萸试管苗。 (2) 适宜山茱萸叶片愈伤组织诱导的培养基组合为：1／2 MS+BA 2-4mg／L+IBA0.5-1.0mg/L及1／2MS+KT2-4mg／L+2，4-D 0.5-1.0mg／L。以山茱萸幼嫩叶片为外植体进行组织培养研究时，BA／IBA、KT／2，4-D的不同组合均可诱导出愈伤组织，当BA的浓度达到2-4mg／L、IBA的浓度为0.5-1.0mg／L时，愈伤组织的诱导率可达到90%以上；当KT和2，4-D的浓度分别达到4.0mg／L和1.0mg／L时，愈伤组织的诱导率可以达到82.2%。所形成的愈伤组织中，有的呈灰白色，质地较为松散，但不易分化；有的为绿色或白色，质地坚硬，可在分化培养基上诱导形成不定芽。 (3) 花托、花柄外植体接种在1／2MS附加BA1.0mg／L、2，4-D0.5mg／L的培养基上时，花托愈伤组织的诱导率可达100%，花柄也可达96.2%。将诱导出的愈伤组织转入1／2MS附加BA0.5mg／L、IBA0.01—0.05mg／L的增殖培养基中，继代培养2-3个月后，愈伤组织可大量增殖，部分可分化出不定芽。 (4) 将由上述外植体诱导出的愈伤组织继代培养在1／2MS附加BA 1-2mg／L、IBA0-0.05mg／L的培养基上，可诱导不定芽的产生。经过2-3次的继代培养，由愈伤组织的表面诱导产生不定芽。其中，叶片愈伤组织不定芽的形成率可达44％，花托和花柄愈伤组织不定芽的形成率为54.8%。 (5) 当不定芽长至1-2cm时将其切下，置于生根培养基上诱导其生根。适于山茱莫幼芽生根的培养基组成为 1／ZMS附加 IBA12g／L。经过一个月左右的培养，山莱荚试管苗可以生根，生根率为2肌左右。 2．山茉萤超度含水态苗与正常苗的形态学结构观察。实验分别以光学显微镜、扫描电子显微镜、透射电子显微镜为观察手段，对山莱莫组织培养过程中的正常苗与超度含水态苗叶的结构进行了观察，结果发现：山茶英正常苗叶的结构基本类似于大田实生苗，而超度含水态茵的叶片结构则在不同观察水平上显示出与正常苗存在看明显的差异，主要结果见下表。 山荣英组织培养正常苗与超度含木态苗叶片结构的观察比较 — ——— A栅栏组织、海绵组织明显； AM细侧卜1紧密整齐A基本无棚栏组织、海绵组织之分 A叶绿体形态大，含量丰畜A叶肉细虚出卜1疏松 W—— A叶脉发育良蚜，成网状分布A叶脉发育不良 A 导管木质化彩度高A导管分子少且木质化程度低 大叶肉细胞胞质浓厚，核大，具明显 可见的核仁 六 稀少，细胞核较小 透射电用田囱怨音果 大叶绿体大、数目多，线粒体、内 大高尔基体，岁水让一和内质网等细胞 质网等细胞盅数目也校多 器稀少而难以烟Q 旁叶表细胞形兢贴，排列整齐、紧 各叶表面细爬用卜1松散，0，细 密，细脾间界线清晰 胞界线不清晰 扫瞄电子显邀烧观察结果 令叶表细胞表面有蜡质分布 旁叶表蜡质少 ＋印LS－F陷，开度小t气jLS明显突起于表皮ie胞上 令叶表被毛表面不先滑 冬叶表具毛表面光滑 3．山茉莫组织赔养中超度含水态苗的发生及防止 门）山莱莫组织培养过程中出现大量的超度含水态苗。当培养基中分裂素浓度较高，培养时间较长时，再生植株中产生的龈含水态苗比例高，当sa赃达到sg／L时，超度含水态苗形成的比率可以达到she以上。在考察不同培养容器的培养效果时发现，透气性好的三角瓶配以纸质瓶盖有利于降低超度含水态苗的发生。在相同的培养条件下，在广口瓶配以聚丙?更多还原

【Abstract】 Cornus officinalis Sieb. Et Zucc.(Cornaceae) is a deciduous arbor with more than 200 years cultivated history in China. Its fruits are used as a precious traditional Chinese medicine which contain abundant chemical componets, such as saponins, phenol constituents, organic acids, amino acid, vitamins and many kinds of ore elements.The experiment with different explants of Cornus officinalis Sieb. Et Zucc. established systematically its tissue culture and plant regeneration systems, investigated the structure of leaf of its normal and hyperhydric shoot, and studied the occurrence and the methods of preventing the hyperhydric shoots from forming In addition, the effective components were determinated.The main results were as follows:1.With different explants including shoots with axillary buds, floral receptacle and floral shoot, the research established their tissue culture and plant regeneration systems, and the main results showed:(1) The regeneration system of Cornus officinalis Sieb. Et Zucc. was set up from shoots with axillary buds. The suitable medium for shoots of Cornus officinalis Sieb. Et Zucc. was 1/2MS+BAlmg/L+IBA0.5mg/L,on which a few buds may multiplied; and 1/2 MS+ BA 0.5mg/L+IBA 0.05mg/L was suitable for buds multiplication on a large scale, on which the buds multiplication rate was about 5.3. According to the result, the micro-propagation system of Cornus officinalis Sieb. Et Zucc. was established, and a lot of test-tube seedling can be obtained.(2) Adventitious buds were regenerated from callus of leaves of Cornus officinalis Sieb. Et Zucc. The suitable medium was l/2MS+BA2-4mg/L+IBA0. 5-1.0mg/L and 1/2MS +KT2-4 mg/L+2,4-D0.5-1.0g/L. When the concentration of BA was 2-4mg/L and IBA was 0.5-1.0mg/L, the induction frequency of callus can be more than 90%; and when the concentration of KT was 4 mg/L and 2,4-D was 1.0 mg/L, the induction frequency of callus can amount to 82.2%.The obtained callus was different, some are fragile, grey and hard to differentiate, while others are hard, white or dark-green and can produce buds.(3) Adventitious buds were regenerated from floral receptacle and floral shoot of Cornus officinalis Sieb. Et Zucc. In this experiment, both floral receptacle and floral shoot were theappropriate explants to study tissue culture and the regeneration system was established. The induction frequency of callus was 100% when the floral receptacle was cultured on the 1/2 MS medium supplemented with BA1.0mg/L and 2,4-D0.5 mg/L; and the induction frequency of callus of floral shoot can be 96.2% on the same medium. The callus can multiply on a large scale and a few adventitious buds can be induced from callus on the medium of 1/2 MS +BA 0.5 mg/L+BA 0.01-0.05 mg/L after 2-3 months.(4)Adventitious buds were induced from callus on the medium of l/2MS+BAl-2mg/L+IBA0.01-0.05mg/L. After 2-3 generations, the adventitious buds can be induced from the surface of the callus. The formation rate of adventitious buds from leaf callus reached 44% and those from floral tissue were 54.8%.(5)The shoots that reached l-2cm were detached and transferred to 1/2MS medium supplemented with l-2mg/L IBA for rooting. About 20% shoots produced roots and formed plantlets.2.The structure of normal and hyperhydric the plantlets was compared by electron scanning microscopy, optics microscopy and transmission electron microscopy. The structure of leaf of the normal plantlets was similar to that of plants found in the field, but the great change occurred in that of hyperhydric plantlets.3.The experiment studied the ways of occurrence of hyperhydric shoots and found out the methods of preventing the normal shoots from forming hyperhydric shoots of Cormis officinalis Sieb. Et Zucc. The main results were as follows:(1) The results showed that many hyperhydric shoots were produced for adding higher concentration of BA in the medium. When the concentration of BA was 5mg/L, the production frequency of hyperhydric shoots can be more than 50%. From the cultural results, it was found t更多还原