【作者】 李光明；

【导师】 易自力； 刘选明；

【作者基本信息】 湖南农业大学 ， 植物学， 2002， 硕士

【摘要】 盾叶薯蓣（Dioscorea zingiberensis C.H. Wright）是薯蓣科薯蓣属一种重要的药用植物，为我国所特有。其根状茎内含有的薯蓣皂甙元是合成甾体激素药物的重要原料，被医药界称之为“药用黄金”。随着野生资源的逐渐枯竭，寻找新的药用资源，特别是利用现代生物技术手段迅速繁殖和培育出更优良的新种质资源为当务之急。为了探索出盾叶薯蓣快速繁殖有效的途径，优化培养条件，并且选育出皂素含量和生物学产量有可能得到提高的同源四倍体新品系，本实验用盾叶薯蓣二倍体种子为原始材料，以种子苗的胚轴等外植体建立无菌实验材料；而后以试管苗的叶片、茎段和圆球茎为外植体诱导了致密愈伤组织，经分化培养得到植株；探索了盾叶薯蓣试管苗的生根和移栽问题，建立了快繁体系，并对培养条件进行了优化筛选；通过化学诱变得到了同源四倍体植株。获得了以下主要结果： 1．探索了提高二倍体盾叶薯蓣种子萌发率的有效途径，并用实生种子苗建立了无菌材料： 先用35℃温水浸泡种子15-20小时软化种皮，随后经20-50ppm的GA₃等激素浸泡30分钟，再种于细沙中，可明显提高萌发率，萌发率达到62％。 用实生种子苗的胚轴经消毒后接种于添加6-BA2-4mg／l的MS培养基上可诱导产生大量致密的愈伤组织，继代培养和增殖可产生大员植株，建立了无菌起始材料。 2．二倍体盾叶薯蓣再生繁殖技术的优化： 在附加6-BA的MS培养基上，叶片、茎段、圆球茎、胚轴外植体均能诱导出致密的愈伤组织，但不同外植体的诱导率有很大差异：叶片和茎段的诱导率较低，分别为26.7％和33.3％；而圆球茎、胚籼外植体可诱导出大量的致密愈伤组织，诱导率分别为100％和85％，而且生长较快。致密愈伤组织不需转瓶，直接就能分化产生圆球茎和植株，因而实现了诱导与分化一步完成的目的，节约了时间和成本。 探讨了致密愈伤组织生长最快的激素组合和碳源种类：致密愈伤组织在以麦芽糖为碳源，添加了6-BA2mg／l，KT0.5mg/l的MS培养基上生长最快；而在以蔗糖为碳源，添加了6-BA2mg／l+KT 0.5mg／l的MS分化培养基上培养时，能很快分化出圆球茎突起，产生丛状芽，继而得到枝、叶茂盛的植株，而且致密愈伤组织的生长也比较快。 切取带枝条的圆球茎接种在MS+NAA0.5mg／l+IAA0.5mg／l生根培养基上培养时，生根率可达87.5％。 探讨了盾叶薯蓣试管苗的移栽问题，提高了成活率：研究发现在栽培基质（沙土）中 添加少量生根培养基，可显著提高成活率。移栽成活率可达53．7％。 因此，以茎段、圆球茎、胚轴作外植体，在附加 6丑 Amg／l的 MS培养基上诱导 出致密愈伤组织，是盾叶薯猿离体再生、快速繁殖的最有效的途径。 3．化学诱变获得了同源四倍体植株： 用浓度为0刀5％的秋水仙素溶液浸泡带有丛生芽的致密愈伤组织72小时，得到33．3％ 的诱变植株的变异率。变异植株经过细胞学鉴定，染色体数目为 40（2 n—4x＝40，x—10）， 而未经过诱变的正常H倍体植株染色体数目为20c习－20，x叫)；变异植株的叶片气 孔和保卫细胞明显比正常二倍体的大：叶片大，颜色深绿；茎粗壮；根茎生长加快。变 异植株为同源四倍体盾叶薯渍新品系。 此外，本文对盾叶薯预致密愈伤组织、圆球茎和根状茎以及试管苗的根与茎交接处 进行了石蜡切片观察，以揭示它们之间的内在联系。更多还原

【Abstract】 Dioscorea zingiberensis C. H. Wright of dioscoreaceae is an important medical plant growing only in our country by reason that Diosgenin in its rhizome is one of the most important sources for steroid hormonal medicines, which is called "Pharmacentical Gold" by medical field. In order to find out the efficient path to rapidly propagate D.zingiberensis, optimize the nurture conditions and assortatively breed out the new varieties of polyploid, whose content of sapogenin and biological production may be higher than diploid, the embryonal axis of seedlings, rhizome of the highly content species and leaves, internode segments and protocorms of test-tube seedlings are used as explants in this experiment to induce callus. Consequently, regeneration system was established and complete plants were also produced from callus in differentiation culture. Furthermore, by chemical-inducing mutation, autotetraploid plants were attained. In addition, the protocol for transplanting test-tube plantlet of D.zingiberensis was also discussed in this paper. The following is the major results of this experiment.1. The valid pathway to enhance the germination rate of the seeds of diploid D. zingiberensis was studied and the steri le materials with the satus seedl ings were also gained.The germination rate of seeds was apparently raised by the following means: firstly, we immersed seeds for 15-20 hours in the warm water of 35癈 to soften the seed coat, then, to immerse the seeds for 30 minutes with such hormones as GA3 whose concentration is about 20-50 ppm and finally, when we plant the seeds in fine sands, the germination rate amounted to 62%.A large number of compact calli can be attained with the caulicles of satus seedlings being inoculated to the medium of MS added 6-BA2-4mg/l, and then a large number of plants can also be gotten after subculture.2. The regeneration system of D. zingiberensis has been established and the technical conditions were also been optimized.The explants of leaves, internode segments and protocorms can all be induced to loose calluses on the medium of MS added 2,4-D, but different explants have different rates of induction. The induction rate of leaves is very low, 25% at the best; Internode segments can be induced 50% loose callus on the medium of MS+2,4-D6mg/l, and yet protocorms can be induced above 93% loose calli on the medium of MS+2,4-Dl-6mg/l. However, loose callionly produce a few of regeneration seedlings on differentiation medium and thus, not fit for establishing tachytelic propagation system.The explants from leaves, internode segments, protocorms and caulides can all be induced to compact calli on the medium of MS added 6-B A, but different explants also have different rates of induction. The rates of induction of leaves and internode segments are comparatively low amount to 26.7% and 33.3% respectively; protocorms and caulicles can produce a large number of compact calli on the medium of MS+6-BA2mg/l. And the callus can produce plants whose branches and leaves are flourish on the differentiation medium of MS+6-BA2mg/l+KT0.5mg/l. Splitting branches which cling to protocorms and transplanting them onto the rooting medium of MS+NAA0.5mg/l+IAA0.5mg/l can get a high rooting rate of 87.5%, and transplanting survival rate can get to 53.7%.Our research results indicated that the survival rate of transplanting of test-tube seedlings can be apparently enhanced when add a little of rooting medium to soil.In short, the pathway that the explants of protocorms and caulicles are induced compactcalluses on the medium of MS added 6-BA2mg/l is the most efficient in isolated culturingand rapid propagation of D.zingiberensis.3. Autotetraploid plants were attained through chemical induction.By immersing the compact calluses with contagious buds into 0.05% colchicines for 72 hours, mutation rate of 33,3% were obtained. The muted plants have been proved to be autotetraploid D.zingiberensis with fine traits through cytology identification and field culti更多还原