【作者】 蒋卫东；

【导师】 陈放；

【作者基本信息】 四川大学 ， 植物学， 2007， 硕士

【摘要】 伞形科植物川芎（Ligusticum chuanxiong Hort．）是我国四川省著名的道地中药材。川芎在全国有着较为广泛的分布，主产四川省都江堰市。川芎以其干燥的根状茎入药，在临床上可用于治疗多种疾病。川芎的根状茎中富含大量的药用成份，而在川芎的代用品研究中证实，川芎根中挥发油含量及有效成份与根状茎基本相同。故以其根为外植体诱导生成的愈伤组织中也很有可能含有药用有效成分，在生产和临床上可能有一定的参考价值。本实验以川芎的根为外植体进行组织培养，通过器官发生途径获得了大量的组培苗。川芎根的愈伤组织诱导的最佳培养基配方为MS+6-BA 0.8 mg／L（单位下同）+NAA 1.2；根愈伤组织分化不定芽的最佳培养基为MS+6-BA 2.2+IAA0.3；生根培养基为1／2 MS+IBA 1.0+NAA 0.7。以川芎根为外植体诱导出的愈伤组织中是否含有药用成份，还需要进一步的实验探索和证实。在组织培养研究中，同时以川芎的茎段和叶片为外植体作为对照。因为在川芎组织培养过程中很容易出现黄化苗和玻璃苗，故在实验中筛选出壮苗培养基（MS+GA₃ 1.2+6-BA 1.8+IAA 0.3），可以使小苗在较短时间内（14d左右）成长健壮，并且使黄化苗和玻璃苗的发生率大大降低。以上培养基均选用pH 5.8，琼脂0.6％～0.7％。经过炼苗后，组培苗移栽成活率达98％。在农杆菌介导转化法进行植物遗传转化的研究中，抗生素种类和浓度的选择是获得转化植株的一个重要前提。植物转基因中常用的抗生素根据其用途分为两大类：一类是抑菌剂，用于植物受体材料感染农杆菌共培养后的脱菌（去除农杆菌）和防止杂菌污染；另一类是选择剂，用于筛选转化体和检测转基因后代。本实验以川芎（Ligusticum chuanxiong Hort．）的根、茎段、叶片及其对应的愈伤组织作为实验材料，研究了四种抗生素对其生长的影响。在MS培养基中添加不同浓度和不同种类的抗生素，比较其对川芎不同外植体及其对应愈伤组织生长的影响。方差分析和总体观察的结果表明，当筛选转化植株的抗生素为卡那霉素时，其最佳抗性筛选浓度为20-40mg／L；当筛选转化植株的抗生素为利福平时，其最佳抗性筛选浓度约为200mg／L。氨苄青霉素和特美汀是两种抑菌性抗生素。抑菌剂的使用原则是达到抑菌目的的同时使抗生素对植物愈伤组织形成和分化的影响最小，即尽量降低其浓度。在本实验中，特美汀对川芎外植体的毒害作用较大，因此不选择它作为抑菌剂；氨苄青霉素对川芎外植体的生长影响较小，因此可以考虑选择它作为川芎遗传转化过程中的抑菌剂，其使用浓度约为200mg／L。更多还原

【Abstract】 Ligusticum chuanxiong Hort. of Umbelliferae is truebom Chinese traditional medicine in Sichuan province in China. Ligusticum chuanxiong Hort. is planted in many regions in our country, and it is mainly planted in Du Jiangyan in Sichuan province. We make use of its dry rhizome clinically and it can cure a good deal of disease. The officinal effective components of Ligusticum chuanxiong Hort. are mainly in their rhizomes. Studies proved that the contents of naphtha and effective components in their rhizomes were generally the same as in their roots. Therefore, calli induced from roots probably have many kinds of officinal effective components. It is very important for production and medicine studies.The tissue culture and plant regeneration of Ligusticum chuanxiong Hort. were studied when the explants were roots, and a large number of regenerated plantlets were obtained. When the explants were roots, the optimal compounding of medium that induced calli, formed shoots and roots were the MS medium supplemented with 6-BA 0.8 mg/L and NAA 1.2 mg/L, the MS medium supplemented with 6-BA 2.2 mg/L and IAA 0.3 mg/L and the 1/2 MS medium supplemented with IBA 1.0 mg/L and NAA 0.7 mg/L, respectively. We are going to do further experiments to test the components in calli induced from Ligusticum chuanxiong Hort.’s roots.Comparing with root explants in tissue culture, leaf explants and stem explants were also studied in plant regeneration.Vitrification plantlets and etioiation plantlets appeared easily in tissue culture of Ligusticum chuanxiong Hort., so we chose and obtained the optimal compounding of medium for shoots growth: the MS medium supplemented with GA₃ 1.2 mg/L, 6-BA 1.8 mg/L and IAA 0.3 mg/L. The shoots growth medium could make the plantlets grow healthy and strong in a short time （about 14 days）, and vitrification plantlets and etiolation plantlets rarely appeared.0.6%-0.7% agar and pH 5.8 were optimal for mediums above. The tube seedling could be successfully transplanted.In Agrobacterium mediated transformation of plant genetic transformation, how to choose an antibiotic and the concentration is very important. In Agrobacterium mediated transformation, antibiotics can be divided into two kinds according to their effects: one kind of antibiotics is used to inhibit Agrobacterium and other bacteria; the other kind of antibiotics is used to select transformers and test their offspring.The stems, leaves, roots and the corresponding calli of Ligusticum chuanxiong Hort. were selected to be experimental materials to carry out research on effects of four antibiotics on their growth. Different concentrations of various antibiotics were added into MS culture media with hormone combinations and then their influences on the growth of the explants and calli were compared. After analysis of variance and overall observation of tissue culture, it was concluded that when we chose kanamycin to select transformers, the best resistance selection concentration was between 20～40mg/L; when we chose rifampicin to select transformers, the best resistance selection concentration was about 200mg/L.Ampicillin and tementin were two ablastins. The concentrations of them varied with different bacteria and we should take the both effects of inhibiting bacteria and negative influence of inducing calli by antibiotics into consideration. We should choose the lowest concentration that inhibited bacteria and had the least negative influence of explant and callus culture by antibiotics. As a result, tementin was very harmful for explant and callus culture ofLigusticum chuanxiong Hort., so we were not able to choose tementin to be the ablastin; ampicillin had less influence on explant and callus culture of Ligusticum chuanxiong Hort., so we could choose ampicillin to be an ablastin, and its inhibition concentration was 200mg/L.更多还原