【作者】 田宇红；

【导师】 王喆之；

【作者基本信息】 陕西师范大学 ， 植物学， 2003， 硕士

【摘要】 喜树(Camptotheca acuminata Decne)是珙桐科旱莲属植物，含有抗肿瘤作用的活性成分喜树碱。丹参(Salvia Miltiorrhiza Bge)是唇形科鼠尾草属植物，是一种治疗心血管系统疾病的重要中草药。分别以喜树、丹参为材料，系统研究其组织细胞培养技术，建立其相应的植株再生系统和细胞培养物繁殖体系，并从其培养物中提取有效成分，无论是对应用生物技术繁育喜树、丹参优良种苗，还是利用细胞培养生产有效活性成分均具有一定的意义。本实验的主要结果如下： 1．系统研究了以喜树顶芽、子叶、胚轴为外植体诱导愈伤组织的培养方法和以顶芽为外植体快速繁殖喜树的方法，获得了大量的愈伤组织和丛生芽。实验结果表明在MS附加2,4-D2.0mg/L、BA1.0mg/L、NAA0.5mg/L的培养基上，子叶愈伤组织的诱导率为87.9％、胚轴的诱导率为82.9％、顶芽的诱导率为6.7％。在附加2,4-D与BA组合的培养基上诱导愈伤组织的效果好，其中在MS附加2,4-D2.0mg/L、BA1.0mg/L的培养基上子叶和胚轴的诱导率都为85％以上，诱导的愈伤组织质地疏松，淡绿色略微带红。在附加NAA与BA组合的培养基上，愈伤组织诱导率低，诱导出的愈伤组织颜色为绿色，质地致密。芽增殖的较佳培养基为MS附加ZT1.0mg/L、NAA0.1mg/L。试管苗生根的较佳培养基为MS附加0.01mg/L的IBA或IAA，生根率可达50％。 2．通过不同种类和浓度激素对喜树愈伤组织中喜树碱积累的研究发现，在以MS为基本培养基，附加2,4-D0.5mg/L、BA1.0mg/L和2,4-D1.0mg/L、BA1.0mg/L时，仅有少量喜树碱积累，当2,4-D浓度增大至2.0mg/L时，有较多的喜树碱积累；褐化的愈伤组织中喜树碱的含量高于绿色愈伤组织中喜树碱的含量，褐化的愈伤组织中喜树碱的含量达0.827‰，接近原植物嫩叶中喜树碱的含量(0.927‰)，远远高于原植物老叶(0.067‰)和种皮(0.220‰)中喜树碱的含量，也高于原植物中喜树碱的平均含量。 3．系统研究了丹参茎段、叶片和花药外植体的培养方法和植株再生技术，获得了大量的试管苗。以MS为基本培养基，附加2,4-D(0.5-2.0)mg/L、BA1.0mg/L适于茎段、叶片诱导愈伤组织，诱导率为100％；附加BA(1.0-2.0)mg/L、NAA0.1mg/L适于茎段、叶片诱导芽和芽的增殖，诱导率为100％；附加IAA、IBA或NAA0.1mg/L或者1/2MS适于根的诱导，诱导率为100％。丹参花药在MS附加BA1.0mg/L、NAA0.1mg/L的培养基上可产生愈伤组织，进而产生不定芽。 4．通过对陕西省安康地区所栽培的12种不同形态丹参的脂溶性成分含量的测定得出：安丹11号脂溶性成分含量最高，其中丹参酮＊。含量为0．7085％、隐丹参酮含量为0．1951％、丹参酮1含量为0．0076％。为选育优良的丹参栽培品种，更好地开发利用丹参药用资源奠定了基础。 5．通过不同种类和浓度激素对丹参愈伤组织中丹参脂溶性成分积累的研究得出：以培养的丹参叶片、茎、根为外植体诱导的丹参愈伤组织中丹参脂溶性成分含量匕较低，而在以晰 为培养基，附加 2，4－D3．omg／L、NAAO．sing／卜的培养基上fll诱导的愈伤组织中，丹参酮II＾含量为0．0142％，高于其它愈伤组织中丹参酮n。的含量。利用丹参愈伤组织牛产其有效成分有待进一步研究。更多还原

【Abstract】 Camptotheca acuminata Decne of Nyssaceae contains Camptothecin (CPT)-a kind of effective component, which is active to cancer. Salvia Miltiorrhiza Bge of Labiatae is an important traditional Chinese herbal drugs, especially in curing cardiovascular disease. A systematic study on the tissue and cell culture of Camptotheca acuminata Decne and Salvia Miltiorrhiza Bge is significant not only in propagating fine plantlets, but also in producing their active components. With Camptotheca acuminata Decne and Salvia Miltiorrhiza Bge as materials, the experiment established and perfected their tissue culture and plant regeneration systems, determined the active components in them. The main results of this experiment were as follows:1 .A systematic study was made on the induction of callus from terminal bud, cotyledon, plumular axis and the regenerated plantlets from terminal bud in Camptotheca acuminata Decne, and a large number of calli and cluster buds were obtained. The experiment result showed: when the medium was MS supplemented with 2,4-D2.0mg/L, BAl.0mg/L and NAA0.5mg/L, the callus induction rate of the cotyledon was 87.9%, the callus induction rate of the plumular axis was 82.9%, and the callus induction rate of the terminal bud was 6.7%. As the medium was MS supplemented with 2,4-D2.0mg/L, BA1.0mg/L, the callus induction rate of cotyledon and plumular axis were more than 85%, the quality of callus was loose, and the color was light green with a slight red. When the medium was MS supplemented with NAA and BA, the induction rate of callus was lower, and the callus obtained was green and hard. The relatively good medium for bud was MS supplemented with ZT1.0mg/L, NAA0.1mg/L. A better medium for root was MS supplemented with IAA or IBA0.01mg/L and the rate of rooting was 50%.2.The content of Camptothecin (CPT) in callus was analyzed. The result was that different kinds and concentrations of hormones had a great effect on the content of CPT in calli. When the medium was MS supplemented with 2,4-D0.5mg/L, BAl.0mg/L and 2,4-D1.0mg/L, BAl.0mg/L, the CPT accumulated only on a smallquantity. As 2,4-D increased to 2.0mg/L, the CPT accumulated more. The content of CPT in brown calli was higher than that in green calli, and it amounted to 0.827 , close to the original plant young leaves in which the content of CPT was (0.927%o), and the content was far higher than that in the original plant old leaves (0.067 ) and plant covers (0.220 ).3.The induction of callus and regenerated plantlets from stem, leaf and anther were systematically studied in Salvia Miltiorrhiza Bge, and a vast amount of shoots were obtained. The MS medium supplemented with 2,4-D (0.5-2.0) mg/L, BAl.0mg/L was suitable for callus induction and the rate was 100%. The MS medium supplemented with BA(1.0-2.0)mg/L, NAA0.1mg/L was good for the induction and propagation of bud and the induction rate was 100%. The MS medium supplemented with IAA or IBA or NAA0.1mg/L or 1/2MS was suitable for the induction of roots and the induction rate was 100%. The anther could produce adventitious bud on MS medium supplemented with BA1.0mg/L, NAA0.1mg/L.4.Determining the active constituents content of 12 kinds of Salvia Miltiorrhiza Bge that were gathered in Ankang, Shaanxi province arrived at the following: The content of liposolable constituents of Andan No.2 was higher than others. Among them the content of Tanshinone IIA was 0.7085%, the content of Cryptotashinone was 0.1951%, and the content of Tanshinone I was 0.0076%. This laid a solid foundation for the fine seed selection of Salvia Miltiorrhiza Bge cultivars, and for developing and utilizing medical resources of Salvia Miltiorrhiza Bge.5.The research of the effect of different kinds and concentrations of hormones on liposolable constituents content of Salvia Miltiorrhiza Bge calli came to such a conclusion: with leaf , stem, root as explants, the liposolable constituents content of Salvia Miltiorrhiza Bge calli was very low. When the medium was MS supplemented with 2,4-D3.0 mg/L, NAA0.5mg/L, and with the更多还原