仙茅组织培养和人工种子研究

发布时间：2018-06-15 11:18

  本文选题：仙茅 + 组织培养　； 参考：《中南林业科技大学》2017年硕士论文

【摘要】：仙茅是我国一味使用了千年的中药,主要治疗阳痿、筋骨萎软、腰膝冷痹、阳虚冷泻等症状,是非常宝贵的中药材。野生仙茅因人为和牲畜的破坏,导致数量急剧减少;但其作为中药材和保健品原料,市场需求却越来越大,野生仙茅数量已经无法满足人们的需要;目前人工方法栽培仙茅已经表现出极大的现实经济价值。传统培育仙茅的方式工作量大,成活率低,且浪费了大量优质仙茅。本论文研究了仙茅更高效、便捷的繁育方式,利用植物组织培养育苗和人工种子技术育苗,在一定程度上解决了仙茅产量不足的问题,具有较大的现实意义。本论文来源于国家林业公益性行业科研专项项目《仙茅、鸡血藤资源培育及林下种植技术研究》,项目编号201304807。目前市场上仙茅的品种很多,本研究基于的是栽培于广州从化的四川宜宾野生仙茅。通过设置单因素试验、正交试验等探究了快速得到仙茅植株的三条途径。以仙茅幼叶诱导愈伤组织得到完整植株;由不定芽制作人工种子得到完整植株;利用仙茅的细胞悬浮培养,诱导出胚状体制作人工种子,以得到完整植株。实验结果证明三种方式均具有可行性:正交试验和单因素试验结果表明,MS基本培养基中加入0.1mg/L的2,4-D、1.5mg/L的KT,3%的多菌灵和8mg/L的柠檬酸,诱导出的仙茅愈伤组织效果最好,可达96%。方差分析得知2,4-D、KT、多菌灵的浓度对仙茅出愈率的影响差异极显著,6-BA浓度和NAA浓度对出愈率影响差异极显著。继代培养基为MS加入0.3mg/L的NAA,2mg/L的6-BA,0.3mg/L的2,4-D,继代效果最好,增殖率最高,愈伤组织的质量最好,较易诱导出不定芽。在培养基中添加0.3mg/L的NAA有利于生根,移栽至营养土中的成活率可达到90%以上。在人工胚乳中加入0.05 mg/L6-BA,0.8mg/L NAA制作的人工种子萌发率可达到93%,添加3mg/L多菌灵时人工种子的染菌率最低。对比试验表明包两层膜的人工种子比包一层膜的人工种子,保水性和机械性都更好。当海藻酸钠浓度为3%时,制作的人工种子质量最好,大小相同,软硬合适。以2%的CaCl2为固化剂,反应两次,第一次固化5分钟,第二次固化10分钟,制成的人工种子形态状况最好,颗粒均匀,软硬适中,呈球形,透明有弹性。愈伤组织继代时,培养基蔗糖浓度为15g/L,6-BA浓度为2mg/L、NAA浓度为0.5mg/L时,可得到疏松易碎适合做悬浮细胞的愈伤组织。正交试验探究非激素水平对细胞悬浮培养影响结果表明:蔗糖影响悬浮细胞形态,MES对悬浮培养液的PH值影响较大,对数据进行方差分析得知,不同浓度的蔗糖、水解酪蛋白、MES、脯氨酸对仙茅细胞悬浮培养影响的差异极显著。探究激素对细胞悬浮培养的影响时发现,2,4-D影响悬浮细胞结构,当2,4-D浓度为0.5mg/L时,胚性细胞数量达到最大值。细胞的生长随着培养时间的变化呈现“S”形曲线,接种量对悬浮培养的影响最大,接种量为15%是仙茅细胞悬浮培养的最适接种量。本论文以组织培养育苗,替代传统的根茎繁殖,成功建立了仙茅组织培养繁殖体系,可将其应用于仙茅的大规模生产,解决目前仙茅市场紧缺的状况。并且以此为基础,利用仙茅不定芽,成功研制出仙茅双层膜人工种子,在一定程度上解决了传统人工种子抗压能力弱和保水性能差的问题。成功建立仙茅植物细胞悬浮体系,为今后在细胞水平上研究仙茅奠定理论基础。
[Abstract]:Chinese herb is a Chinese herbal medicine used for thousands of years. It mainly treats impotence, soft bone, cold arthralgia, cold and diarrhea of the waist and knees, Yang deficiency and diarrhea and so on. It is a very valuable Chinese herbal medicine. Wild imfairy is the destruction of people and livestock, resulting in a sharp decrease in quantity. But as a raw material of Chinese medicine and health care products, the demand for the market is getting bigger and bigger, the number of wild imfairy has already been found. It is impossible to meet the needs of people. The cultivation of curculia is of great practical economic value at present. The traditional cultivation method of the Chinese curculus is great work, low survival rate and a large number of high quality curl. This paper studies the more efficient and convenient breeding methods of the grass, the cultivation of seedling and the technology of artificial seed cultivation with plant tissue culture. To a certain extent, it has solved the problem of insufficient yield of curl, which is of great practical significance. This paper comes from the special project of the national forestry public welfare industry, "imimu", the cultivation of the resources of the Caulis, and the research on the planting technology under the forest. The project No. 201304807. is at present a lot of varieties of the green grass on the market. This study is based on the cultivation in Guangzhou. The wild centith in Yibin, Sichuan, Conghua. Through the setting of single factor test and orthogonal experiment, three ways to quickly get the plant of centip were explored. The whole plant was obtained by the induction of the callus from the young leaves of the centian; the artificial seeds were made from the adventitious buds, and the artificial seeds were induced by the suspension culture of the centip. The results of the experiment showed that the three methods were all feasible. The orthogonal test and single factor test showed that the MS basic medium added 0.1mg/L 2,4-D, 1.5mg/L KT, 3% Carbendazim and 8mg/L citric acid, and the induced callus effect was the best, and the 96%. variance analysis showed that 2,4-D, KT, and carbendazim were strong. There are very significant differences in the effect of degree on the recovery rate of curculus. The effect of 6-BA concentration and NAA concentration on the recovery rate is very significant. The subculture medium is MS adding 0.3mg/L NAA, 2mg/L 6-BA, 0.3mg/L is the best, the highest proliferation rate, the best quality of the callus, and the easier induction of adventitious buds. The NAA favorable for adding 0.3mg/L in the medium is favorable. Yu Shenggen, the survival rate of transplanted into the nutrient soil could reach 90%. The germination rate of artificial seeds in artificial endosperm was 0.05 mg/L6-BA, and the germination rate of artificial seeds produced by 0.8mg/L NAA could reach 93%. The rate of infection of artificial seeds was the lowest when adding 3mg/L carbendazim. When the concentration of sodium alginate is 3%, the quality of the artificial seeds is the best, the size is the same, the soft and hard is suitable. With 2% CaCl2 as the curing agent, two times, the first curing 5 minutes, and the second curing 10 minutes, the artificial seeds have the best shape, the particles are uniform, the soft and hard, the transparent and elastic. On the other hand, when the concentration of sucrose is 15g/L, the concentration of 6-BA is 2mg/L and the concentration of NAA is 0.5mg/L, the callus which is loose and suitable for suspension cells can be obtained. The results of the effect of the non hormone level on the cell suspension culture show that the sucrose affects the morphology of the suspension cell, the MES has great influence on the pH value of the suspension culture, and the data are carried out. The variance analysis showed that different concentrations of sucrose, casein hydrolysate, MES, proline had a very significant difference in suspension culture of curculus. When the effect of hormone on cell suspension culture was found, 2,4-D affected the structure of suspension cells. When the concentration of 2,4-D was 0.5mg/L, the number of embryogenic cells reached the maximum. The change showed "S" shape curve, the influence of inoculation on suspension culture is the most, the inoculation amount is 15% is the optimum inoculation quantity of the suspension culture of curculus. This paper successfully established the culture and breeding system of curculus curculera by tissue culture and breeding, instead of the traditional rhizome propagation. On the basis of the market, and using the adventitious bud of the curl, the artificial seed of the bilayer film of the curl grass has been successfully developed. To a certain extent, the problem of the weak compression ability and poor water retention of the traditional artificial seeds is solved. The suspension system of the plant of the plant is successfully established to lay a theoretical foundation for the study of the Curca on the cell level in the future.
【学位授予单位】：中南林业科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S567.239
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本文编号：2021828