【作者】 李晓娟；

【导师】 马小军； 赖家业；

【作者基本信息】 广西大学 ， 生态学， 2008， 硕士

【摘要】 本论文探索了黄花蒿（Artemisia annua L.）组织培养技术及其与化学方法诱导染色体变异相结合的途径,目的在于对黄花蒿组织培养进行方法优化,并通过秋水仙碱溶液诱导染色体变异的方法提高黄花蒿体细胞无性系后代的变异频率,扩大变异谱,为以后的种质资源研究提供基础,为黄花蒿组织培养及染色体的观察提供方法优化,并为青蒿素代谢的分子机理及细胞学理论提供依据。试验结果如下:1.黄花蒿种子的最佳灭菌组合是:0.05%升汞（HgCl₂）10min+3%次氯酸钠（NaClO）10min。外植体的最佳灭菌组合是:0.05%升汞（HgCl₂）5min+饱和次氯酸钠（NaClO）15min。2.茎段诱导试验结果表明,6-BA、NAA对外植体的诱导和分化有促进作用,最佳诱导培养基为6-BA0.05mg.L^-1+NAA0.01mg.L^-1,芽增殖系数最高,为4.08。在6-BA0.05 mg.L^-1+NAA0.01mg.L^-1培养基下可以诱导黄花蒿叶片不经愈伤组织一步成芽。外植体的部位对芽分化也有一定的影响,以茎段部位增殖系数最高。3.黄花蒿继代试验结果表明,最佳黄花蒿继代培养基为6-BA1.0mg.L^-1NAA0.2mg.L^-1+KT0.5mg.L^-1,平均增殖倍数为6.65。添加蔗糖浓度为30g.L^-1时最有利于黄花蒿继代。添加椰汁对黄花蒿继代影响不大。pH为5.8时最有利于黄花蒿继代增殖。4.黄花蒿组培苗较易生根,但以添加IBA0.5 mg.L^-1的组合为最佳,平均生根率达到100%,根长约为2.75cm,根数约为6.04,苗高为4.44cm。5.不同基质对黄花蒿试管苗移栽有不同影响,以腐殖土:泥炭:珍珠岩:河沙为1:1:1:1效果最佳。6.不同取材时间和预处理方式对黄花蒿染色体制片有一定影响,以在上午10点28分,用饱和对二氯苯预处理4.5-6h或者用a-溴萘置于4℃冰箱中整体处理24h,解离8-9min,解离后用45%的冰醋酸对材料进行处理为黄花蒿染色体制片的最佳组合。8.不同处理方式,处理材料和处理时间对黄花蒿染色体诱导有一定影响。对于黄花蒿浸泡秋水仙碱溶液处理,种子以处理浓度0.1%-0.5%,处理时间5-7天,诱导效果较好;茎段以处理浓度0.25%和0.5%,处理时间达到4-5天时,诱导效果较好。对于固体培养基添加秋水仙碱溶液培养,处理浓度为60mg.L^-1时,诱导效果较好。长时间较高浓度的处理方式有利于诱导染色体变异。黄花蒿染色体数量为:2n=18,4n=36。更多还原

【Abstract】 In this paper we studied the tissue culture method of Artemisia annua L. and the combination of tissue culture method and variation induction of chromosome of Artemisia annua L.by chemical method,aiming at choosing the best method for tissue culture of Artemisia annua L.,and improving variation frequency of somaclones of Artemisia annua L.by using colchicine solution,enlarging variation spectrum,providing basic data for germplasm resource research in the further,optimizing the method of tissue culture and chromosome observation for Artemisia annua L.,and providing evidence for molecular mechanism and cytology theory of metabolism of artemisinin.The results are as follows:1.0.05%HgCl₂ 10min+3%NaClO10min was the best combination for the sterilization of seeds of Artemisia annua L.,and 0.05%HgCl₂ 5min+ Saturated NaClO15min was the best combinations for the sterilization of explant of Artemisia annua L..2.The result of stem induction experiment indicated that 6-BA,NAA had promoting effect for induction and differentiation of explant.6-BA 0.05mg.L^-1+NAA0.01mg.L^-1was the best medium for induction,with the hightest proliferation coefficient of 4.08.Bud formation from leaf of Artemisia annua L.can be induced in 6-BA0.05mg.L^-1+NAA0.01mg.L^-1medium without callus period.The organ types of explant are important for inducing bud different-tiation.Stem was the best position for induction3.The result of bud subculture experiment indicated that 6-BA1.0mg.L^-1 +NAA0.2mg.L^-1+KT0.5 mg.L^-1was the best medium for subculture,with the average proliferation coefficient of 6.65.It was good for subculture of Artemisia annua L.when the sucrose concentration was 30g.L^-1in the medium.Adding coconut in the medium has little effect on the subculture of Artemisia annua L.The best pH for subculture of Artemisia annua L.was pH 5.8.4.Root formation is easy for the tissue culture seedling of Artemisia annua L.,and IBA0.5mg.L^-1was the best concentration for root formation, with average rooting rate of 100%,root length of 2.75cm,root number of 6.04,and seedling height of 4.44cm.5.There were different effects for test-tube seedling transplanting with different medium,and the best ratio of muck:peat:perlite:riversand for the medium was 1:1:1:1.6.There were different effects for chromosome preparation of Artemisia annua L.with different sampling time and pretreatment methods, and the best combination for chromosome preparation of Artemisia annua L. was drawing materials at 10:28am,pretreatmenting with saturated p- Dichlorobenzene for 4.5-6h or with a-bromonaphthalene at 4℃for 24h, dissociating for 8-9min,and treated with 45%acetic acid after dissociation.7.There were different effects for chromosome preparation of Artemisia annua L.with different treatment methods,materials,and time.For soaking treatment with colchicine solution,the best treatment concentration was 0.1%-0.5%,and the best treating time was 5-7days for seed,and the best treatment concentration was 0.25%and 0.5%,and the best treating time was 4-5 days for stem.For solid medium added with colchicine solution,the best treatment concentration was 60mg.L^-1.Treatment with long time and high concentration was better for the induction of chromosome variation of Artemisia annua L..Chromosome number of Artemisia annua L.is 2n=18, 4n=36.更多还原