【作者】 唐道方；

【导师】 刘德华；

【作者基本信息】 湖南农业大学 ， 茶学， 2008， 硕士

【摘要】 从红豆杉中提取得到的紫杉醇（Taxol）是一种新型抗癌药物。由于资源缺乏,致使其价格昂贵,红豆杉也因此被人们誉为“黄金树”。红豆杉与真菌共生,生长缓慢,种子后熟期长,这给大规模繁殖、栽植造成了一定的困难。目前以大田植株组织或器官为材料,植株再生研究较少,尚无诱导不定胚等的报道。本文以南方红豆杉和曼地亚红豆杉为供试材料,对红豆杉外植体的灭菌、愈伤组织的诱导及其分化、腋芽增殖再生植株体系及根的离体培养、蛋白质分析、PPO和POD酶同工酶的分析等方面进行了较系统的研究。结论如下:1.通过对不同灭菌剂、灭菌方法、灭菌时间、生长年龄及取材部位的外植体材料灭菌研究,使用75%酒精浸泡15 S左右后,0.2%升汞5+5 min的二次灭菌为最适灭菌方案。2.处于生长活跃期,树冠上部或当年生枝条的腋芽、具叶茎段是愈伤组织诱导的最适材料。B₅+NAA 1.0 mg/L+IBA 1.0 mg/L+BA 0.1 mg/L+GA₃ 1.0 mg/L是南方红豆杉和曼地亚红豆杉愈伤组织诱导较适的培养基;GA₃对愈伤组织的增殖及抑制其褐化具有一定的作用。南方红豆杉的愈伤组织在B₅+IBA 0.2 mg/L+BA 0.1 mg/L培养基上培养,获得了假胚珠。3.1个茎段外植体上可萌发多个腋芽,木质化程度高的外植体,形态学最上端的腋芽先萌动,木质化程度较低的,形态学最下端的腋芽先萌动。南方红豆杉腋芽萌动的较适培养基是DCR+IBA 0.1 mg/L+BA 0.05 mg/L,萌动率达97.5%,曼地亚红豆杉较适培养基为DCR+NAA 0.5 mg/L+BA 0.05 mg/L,萌动率达84.9%。4.1/2 DCR+IBA 0.5 mg/L+NAA 0.2 mg/L是南方红豆杉的较适生根培养基,曼地亚红豆杉的较适生根培养基为1/2 DCR+IBA 0.2 mg/L+NAA 0.5 mg/L。5.南方红豆杉和曼地亚红豆杉的小苗扦插至黄泥:细沙=2:1的土壤中,在温度25℃,75%以上湿度下,成活率分别为90.1%、87.5%;解决了红豆杉试管苗难于生根的问题。6.南方红豆杉根离体培养的较适培养基为改良White+IBA 0.2 mg/L,曼地亚红豆杉为改良White+IBA 0.5 mg/L。7.大田栽植的南方红豆杉和曼地亚红豆杉的蛋白质及PPO、POD同工酶谱带存在明显的差异。随着生长发育进行,蛋白质及PPO同工酶的谱带条数呈少-多-少的规律变化。南方红豆杉茎外植体诱导的愈伤组织在衰老的过程中,蛋白质、POD同工酶谱带条数减少:PPO同工酶的谱带条数增加,愈伤组织在培养过程中出现褐化与其基本呈正相关。更多还原

【Abstract】 Taxol is a new anti-cancer drug be extracted from Taxus plant. Taxol has high prices because of lack resources, therefore, Taxus is famous as "Golden tree". Taxus plant is growing with symbiotic fungi and growing slowly. It’s seeds turn ripen need a long period, this take some difficulties for breeding and planting Taxus in large scale. At present, used plant tissues or organs in field as the materials to study on plant regeneration is very less. There is no coverage on induced indefinite embryo. This paper used Taxus.media and Taxus.mairei as the tested materials to make a systematic study on the Taxus explants disinfection, callus induction and differentiation, axillary bud proliferation and regeneration system, root in vitro culture, protein analysis, PPO and POD isozymes analysis. The results are showed as following.1. Study on disinfecting through the disinfectant, disinfecting methods, disinfecting times, growing age and the position of materials. The results showed, the explant materials immerse in 75 percent ethanol after about 15 seconds and then using 0.2 percent mercuric chloride disinfecting 5 by 5 minutes is the best disinfecting program.2. The axillary buds and stems which are in the active growth phase and on the upper canopy branches or growing in this year are the best materials for callus induction. The suitable callus induction medium of Taxus.mairei and Taxus.media is B₅ added with NAA 1.0 mg / L, IBA 1.0 mg / L, BA 0.1 mg / L and GA3 1.0 mg / L. GA3 has a certain role play on callus proliferation and inhibit the browning. The callus of Taxus.mairei cultured on B₅ medium which added with IBA 0.2 mg / L and BA 0.1 mg / L differentiated fake ovule.3. Stem explants can germinate a number of axillary buds. The axillary buds which on the morphology top with a high degree of lignification explants are first germinated. But the axillary buds which on the morphology bottom with a low degree of lignification explants are germinated at first. The optimal medium of axillary buds germination of Taxus.mairei is DCR which added with IBA 0.1 mg / L and BA 0.05 mg / L, the germination rate is 97.5%. The optimal medium of axillary buds germination of Taxus. media is DCR which added with NAA 0.5 mg / L and BA 0.05 mg / L, the germination rate is 84.9%.4. The suitable rooting medium of Taxus.mairei is 1 / 2 DCR which added with IBA 0.5 mg / L and NAA 0.2 mg / L and Taxus. media is 1 / 2 DCR which added with IBA 0.2 mg / L and NAA 0.5 mg/L.5. The seedlings of Taxus.mairei and Taxus.media planted in the yellow soil mixed with sand by 2:1 ratio, at 25 temperature, upon 75 percent humidity, the survival rate were 90.1% and 87.5%.This is resolved the difficult problems of Taxus plantlets rooting.6. The suitable medium of root cultured in vitro of Taxus.mairei is the improved White added with IBA 0.2 mg / L and Taxus.media is the improved White added with 0.5 mg / L.7. The proteins and PPO, POD isozymes bands of Taxus.mairei and Taxus.media have significant differences. The bands number of proteins and PPO isozymes are changed toward less, more and less as following with growth and development of plantlets. The bands number of proteins and POD isozymes reduced, but PPO isozymes bands number increased during the senescenced callus which induced by stem explants of Taxus.mairei. The callus browning is positive correlation to this.更多还原