红豆杉资源遗传多样性分析及南方红豆杉愈伤组织诱导体系的建立
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摘要
红豆杉是国家一级保护植物，由于红豆杉含有具有重要的抗癌物质紫杉醇，红豆杉资源的保护面临着诸多困难。本研究从南方红豆杉资源的有效保护出发，以福建省武平县梁野山国家自然保护区的万亩南方红豆杉自然种群为主要材料，研究南方红豆杉群落的遗传多样性及其与其它类型红豆杉资源的关系，并建立其愈伤组织诱导技术体系，主要结果如下：
(1) 南方红豆杉侧芽是诱导愈伤组织的最佳外植体。(2) 南方红豆杉愈伤组织诱导的最佳培养基为：B_5+2，4-D 2.0mg／L +NAA 1.0mg／L +IAA0.2mg／L+LH 2.0g／L+AC 0.5g/L。(3) 红豆杉愈伤组织增殖的培养基：B'_5+NAA 4.0mg／L+2，4-D 0.5mg／L+GA_3 0.2mg／L+AC 0.5g／L+LH 2.0g／L，AC、LH和GA_3三种物质一起使用，基本遏制了褐化的发生，可取得较好的效果。(4) 南方红豆杉RAPD分析的最佳技术体系为：25μl反应体积中，含200μmol／L dNTP，0.2μmol／L随机引物，30～100ng模板DNA，1.5U Taq DNA聚合酶。反应程序为：首先94℃预变性3.0 min；94℃变性1.0 min；36℃退火2.0 min，72℃延伸2.0 min，45个循环后，72℃延伸10.0 min。最后于4℃保存直至取出。(5) 在11个供试材料中，9个RAPD引物共扩增出398条带，多态性条带比率(PPB)接近27％，表明供试红豆杉材料之间的遗传基础较窄。(6) 依据RAPD分析数据，对红豆杉属植物品种间及种内的供试材料进行了聚类分析。在D=0.52时可将供试材料分为2类：第一类是栽培的西藏红豆杉和栽培的曼地亚红豆杉，第二类包括栽培的南方红豆杉和其他一系列野生的南方红豆杉；在D=0.67时将供试材料分成3个类群，进一步细分几个变种间的遗传差异，将栽培的南方红豆杉和野生的南方红豆杉区分开来；第三次等级划分(D=0.80)也突出了几个野生的南方红豆杉间(来自不同海拔)的差异。
Taxus L. is the first Class preserved plant in China and
it contains taxol, an important anticancer, which made it very difficult to preserve Taxus L. resources. In order to utilize and preserve Taxus L. resources effectively, the genetic diversity of Taxus var. mairei from Liangye National Natural Protection District was analysed and was compared with Taxus wallichiana and Taxus Chinensis var. mairei. The callus induction system of Taxus var. mairei was also established in the expriment, the main results were as following:
1. Buds was the best explant for callus induction. 2. Medium B5 adding 2. Omg/L 2, 4-D , 1. Omg/L NAA . 1. Omg/L IAA, 2. Og/L LH and 0. 5g/L AC was the most optimized medium for callus induction. 3. Medium B5' containing 4. Omg/L NAA. 0. 5mg/L 2, 4-D. 0. 2mg/L GA3. 0. 5g/L AC and 2. Og/L LH was the most favorable for callus growth. The brownness of callus was avoided or mitigated to some degree when AC. LH and GA3 were added to the culture medium together. 4. The parameters of RAPD analysis were optimized for Taxus L, the total volume of 25M-1 containing 200Wnol/L dNTP, 0. 2Pmol/L random primers. 30- 100ng template DNA and 1. 5 unit Taq DNA enzyme. The program for the PCR amplification was that first denatured the template DNA at 94癈for 3.0 min and then denatured the DNA at 94癈 for 1.0 min, annealed at 36癈 for 2.0 min, extended at 72癈 for 2. 0 min, 45 cycles, then extended at 72癈 for
10. 0 min and kept the reaction at 4癈 until it was taken out. 5. The total of 398 DNA bands were amplified in the eleven Taxus L. resources by the above PCR procedure using 9 random primers, and about 27% of the bands were polymorphic, indicating that the genetic background was narrow in Taxus L. involved in this experiment. 6. An cluster analysis was made by using the RAPD data of Taxus L . involved in the experiment, and the results showed that the Taxus L. resources were divided into 2 subclasses when the value of D was 0. 52:the first subclass was the cultivars of Taxus Media var. and Taxus wallichiana , the second was Taxus Chinensis var.mairei; Taxus L. resources were divided into 3 subclasses when the value of D was 0.67,apart from the above first subclass , the second subclass was further distinguished between cultivars of Taxus Chinensis var.mairei and wild varieties of Taxus Chinensis var.mairei, indicating that genetic differences among the subclasses of Taxus Chinensis var.mairei; when the value
of D was 0. 80, some genetic differences were showed among Taxus Chinensis var.mairei.
(1) 南方红豆杉侧芽是诱导愈伤组织的最佳外植体。(2) 南方红豆杉愈伤组织诱导的最佳培养基为：B_5+2，4-D 2.0mg／L +NAA 1.0mg／L +IAA0.2mg／L+LH 2.0g／L+AC 0.5g/L。(3) 红豆杉愈伤组织增殖的培养基：B'_5+NAA 4.0mg／L+2，4-D 0.5mg／L+GA_3 0.2mg／L+AC 0.5g／L+LH 2.0g／L，AC、LH和GA_3三种物质一起使用，基本遏制了褐化的发生，可取得较好的效果。(4) 南方红豆杉RAPD分析的最佳技术体系为：25μl反应体积中，含200μmol／L dNTP，0.2μmol／L随机引物，30～100ng模板DNA，1.5U Taq DNA聚合酶。反应程序为：首先94℃预变性3.0 min；94℃变性1.0 min；36℃退火2.0 min，72℃延伸2.0 min，45个循环后，72℃延伸10.0 min。最后于4℃保存直至取出。(5) 在11个供试材料中，9个RAPD引物共扩增出398条带，多态性条带比率(PPB)接近27％，表明供试红豆杉材料之间的遗传基础较窄。(6) 依据RAPD分析数据，对红豆杉属植物品种间及种内的供试材料进行了聚类分析。在D=0.52时可将供试材料分为2类：第一类是栽培的西藏红豆杉和栽培的曼地亚红豆杉，第二类包括栽培的南方红豆杉和其他一系列野生的南方红豆杉；在D=0.67时将供试材料分成3个类群，进一步细分几个变种间的遗传差异，将栽培的南方红豆杉和野生的南方红豆杉区分开来；第三次等级划分(D=0.80)也突出了几个野生的南方红豆杉间(来自不同海拔)的差异。
Taxus L. is the first Class preserved plant in China and
it contains taxol, an important anticancer, which made it very difficult to preserve Taxus L. resources. In order to utilize and preserve Taxus L. resources effectively, the genetic diversity of Taxus var. mairei from Liangye National Natural Protection District was analysed and was compared with Taxus wallichiana and Taxus Chinensis var. mairei. The callus induction system of Taxus var. mairei was also established in the expriment, the main results were as following:
1. Buds was the best explant for callus induction. 2. Medium B5 adding 2. Omg/L 2, 4-D , 1. Omg/L NAA . 1. Omg/L IAA, 2. Og/L LH and 0. 5g/L AC was the most optimized medium for callus induction. 3. Medium B5' containing 4. Omg/L NAA. 0. 5mg/L 2, 4-D. 0. 2mg/L GA3. 0. 5g/L AC and 2. Og/L LH was the most favorable for callus growth. The brownness of callus was avoided or mitigated to some degree when AC. LH and GA3 were added to the culture medium together. 4. The parameters of RAPD analysis were optimized for Taxus L, the total volume of 25M-1 containing 200Wnol/L dNTP, 0. 2Pmol/L random primers. 30- 100ng template DNA and 1. 5 unit Taq DNA enzyme. The program for the PCR amplification was that first denatured the template DNA at 94癈for 3.0 min and then denatured the DNA at 94癈 for 1.0 min, annealed at 36癈 for 2.0 min, extended at 72癈 for 2. 0 min, 45 cycles, then extended at 72癈 for
10. 0 min and kept the reaction at 4癈 until it was taken out. 5. The total of 398 DNA bands were amplified in the eleven Taxus L. resources by the above PCR procedure using 9 random primers, and about 27% of the bands were polymorphic, indicating that the genetic background was narrow in Taxus L. involved in this experiment. 6. An cluster analysis was made by using the RAPD data of Taxus L . involved in the experiment, and the results showed that the Taxus L. resources were divided into 2 subclasses when the value of D was 0. 52:the first subclass was the cultivars of Taxus Media var. and Taxus wallichiana , the second was Taxus Chinensis var.mairei; Taxus L. resources were divided into 3 subclasses when the value of D was 0.67,apart from the above first subclass , the second subclass was further distinguished between cultivars of Taxus Chinensis var.mairei and wild varieties of Taxus Chinensis var.mairei, indicating that genetic differences among the subclasses of Taxus Chinensis var.mairei; when the value
of D was 0. 80, some genetic differences were showed among Taxus Chinensis var.mairei.
引文
1．陈际伸．红豆杉资源与紫杉醇研究进展[J]．江西林业科技，1998(4)：32～36．
2．陈大明，张上隆，金庸韦．一种木本果树基因组DNA提取方法研究[J]．浙江农业大学学报，1997，23(6)：621～624．
3．陈永勤，朱蔚华．红豆杉属植物的组织、细胞及胚培养[J]．植物生理学通讯，1997，33(3)：213～219．
4．仇燕，贾宁，王丽，王刚．继代时间对南方红豆杉细胞生长和产生紫杉醇的影响[J]．河北师范大学学报，2002，26(3)：301～304．
5．邓联东，孙多元，董岸杰．紫杉醇注射剂的研究[J]．药学进展，2002，26(3)：162～165．
6．F．奥斯伯，R 布伦特，R．E．金斯敦．精编分子生物学手册[M]．科学出版社，1999．
7．方起程，韩锐，陈未名等．抗肿瘤新药紫杉醇及其注射液的研究与开发[J]．中国医学科学院学报，1996，18(4)：251．
8．甘烦远，郑光植．红豆杉细胞培养的研究[J]．云南植物研究，1996，18(2)：134～138．
9．桂耀林，胡玉熹．红豆杉属叶子的表皮特征与分类的关系[J]．植物分类学报，1974，12(3)：329～333．
10．高山林．东亚和加拿大产红豆杉属七种植物中紫杉醇及短叶醇的含量[J]．药科大学学报，1995，(26)：8～10．
11．甘烦远，沈月毛，赫小江．红豆杉生物工程的研究进展[J]．武汉植物学研究，1999，17(1)：65～74．
12．顾志建，周其兴，岳中枢．中国红豆杉属和白豆杉属的核形态学研究[J]．云南植物研究，1998，3．
13．Glark M S主编，顾红雅，瞿礼嘉等主译．植物分子生物学手册[M]．高等教育出版社，1998．
14．何水林．植保素代谢与植物防御反应[M]．广东科技出版社，2002．
15．黄浩．红豆杉细胞培养中抗褐变剂的筛选[J]．华中理工大学学报，1999，27(4)：18～22．
16．黄浩．活性炭及VC对红豆杉细胞培养的影响[J]．甘肃教育学院学报(自然科学版)，2001，15(1)：34～36．
17．贾继增．分子标记种质资源鉴定和分子标记育种[J]．中国农业科学，1996，29(4)：1～10．
18．李伯林，赵群华，庐山，曹日强．红豆杉植物愈伤组织的培养及其紫杉醇形成的初探[J]．南京大学学报，1995，31(3)：424～429．
19．李家儒，智志勇，刘曼西，吴振斌，王君健．Cu2+对红豆杉培养细胞中紫
杉醇形成的影响[J]．华中农业大学学报，1999，18(2)：117～120．
20．梁珍海，刘根林，徐锋．红豆杉属植物组织培养及其快速繁殖研究综述[J]．江苏林业科技，2001，28(3)：45～48．
21．林风等．纤维素酶的生物化学和分子生物学研究新进展[J]．生命科学，1994，6(1)：18～23．
22．梅兴国，董妍玲，潘学武．红豆杉细胞继代培养防褐变措施的研究[J]．自然产物研究与开发，2001，13(4)：8～11．
23．马忠武，何关福，印万芳．双黄酮成分在红豆杉科各属中的分析[J]．植物分类学报，1985，23(3)：192～195．
24．苏湘鄂，梅兴国，龚伟，张周宁．红豆杉细胞时相与其次生代谢强度的关系[J]．生物技术，2000，10(4)：14～16．
25．汪小全，邹喻苹，张大明等．RAPD应用于遗传多样性和系统学研究中的问题[J]．植物学报，1996，38(12)：954～962．
26．王艇，苏应娟，黄超，朱建明．红豆杉科植物RAPD分析及其系统学意义[J]．西北植物学报，2000，20(2)：243～249．
27．王关林，方宏筠主编．植物基因工程原理技术[M]．科学出版社．1998：426．
28．吴丽圆，陈少谕，项伟．云南红豆杉自然群体内同工酶遗传变异的研究[J]．遗传，2001(3)：237～242．
29．王玲．辣椒遗传多样性的RAPD分析．福建农林大学硕士学位论文集，2002．
30．徐铮奎．天然植物抗癌药国内外研究开发新进展[J]．中国医药情报，2002，8(4)：36～38．
31．谢秋玲，郭勇．刺激剂在植物细胞培养次生代谢物中的应用[J]．广西植物，1999，19(2)：142～146．
32．席以珍．中国红豆杉科花粉形态的研究[J]．植物分类学报，1986，24(4)：247～252．
33．席以珍．穗花杉属划分外壁的超微结构及其分类意义[J]．植物分类学报，1986，24(6)：439～442．
34．夏铭，吴绛云，张丽梅．红豆杉组织培养中褐变问题的研究[J]．生物技术，1996，6(3)：18～20．
35．邢建民，查丽杭，李佐虎．植物细胞培养生产紫杉醇的研究进展[J]．植物学通报，1997，14(3)：282～292．
36．周金，姜笑梅．木材构造特征在裸子植物系统学中的意义[J]．植物分类学报，1992，30(5)：405～414．
37．项伟，沅德春，张宏杰等．不同产地云南红豆杉紫杉醇的含量分析[J]．云南林业科技，1996，18(2)：74～76．
38．岳琴．紫杉醇及其相关化合物的研究进展[J]．国外医学药学分册，1996 23(6)：332～334．
39．周波，姜虎生．红豆杉及其产物紫杉醇研究进展[J]．松辽学刊(自然科学
版)，2001，2(1)：78～81．
40．张长平，李春，元英进等．真菌诱导子对悬浮培养南方红豆杉细胞态势及紫杉醇合成的影响[J]．生物工程学报，2001，17(4)；436～440．
41．张恒庆，安利佳，祖元刚等．红松RAPD实验中各组成成份含量对实验结果的影响[J]．植物研究，1990，19(2)：183～188．
42．周东新．黄麻遗传多样性的RAPD和ISSR分析．福建农林大学硕士学位论文集，2001．
43. Burte J N. Taxus Le Bon Jardinier. Paris:La Maison Rustique, 1992, :2588～2589.
44. Becker J, Vos P, Kuiper M et al. Gombined mapping of AFLP and RFLP markers in barley[J]. J Mol. Genet. 1995, 246: 65～73.
45. Christen A A, Gibson D M, Bland J,et al, 1991. production of taxol or taxol-like compounds with Taxus brevifolia callus cell culture[J]. U S Patent, 5019504.
46. Chee P P. In vitro culture of zygotic embryos of Taxus species[J]. HortScience, 1994,29:695～697.
47. Durzan D J,Ventimiglia F. Free taxanes and the release of bound compounds having taxane antibody reactive by xylanase in female, haploid-derived cell suspension culture of taxusd brevifolia[J]. IN Vitro Cell Dev Biol, 1994,30:219～227.
48. Denis J N, Greene A E, Guenard D et al. A highly efficient practical approach to natural taxol[J]. J Am Chem Soc, 1998,110:5917.
49. Fett-Neto A.G.,F. DiCosmo,W.F.R eynolds et al. Cell culture of Taxus as a source of the antinecoplastic drug taxol and related taxanes[J]. J Biotechnolgy, 1992 (10):1572～1575.
50. Fett-Neto A G, Zhang W Y, Dicosom F. Kinetics of taxol production, growth, and nutrient uptake in cell suspensions of Taxus cuspidate[J]. j Biotechnol Bioengineer, 1994,44:205.
51. Fett-Neto A G, Melanson S J, Sakata K et al. Improved growth and taxol yield in developing calli of Taxus cusoidate by medium composition modification[J]. Bio/Technology, 1993,11:731.
52. Fritsch P, Rieseberg L H. The use of random amplified polymorpHic DNA(RAPD) in conservation genetics. Smith T B,Wayne R K. Molecular Genetic Approaches in conservartion. London[J].Oxford University.
53. Hezari M, Croteau R. Taxol biosynthesis[J]. J an update. J Plant Medica. 1997,63: 291～295.
54. Hezari M,,Ketchum R E B, Gibson D M et al. Lew is N G, Croteau R. Purification and characterization of taxa-4(5), 11(12)-diene synthase from Pacific
yew(Taxus brevifolia) that catalyzes the first step of taxol biosynthesis[J]. J Arch Biochem BiopHys, 1995,322: 437～444.
55. Hezari M, ,Ketchum R E B, Gibson D M et al. taxol production and taxadiene synthase activity in Taxus Canadensis cell suspension cultures[J]. J Arch BiopHys, 1997,337: 185～190.
56. Jaziri M, Zhiri A, Guo Y W et al. Taxus sp. Cell, tissue and organ culture as alternative sources for taxoids production: a literature survey[J]. Plant Cell Tiss Organ Culture, 1996,45:59～75.
57. Koepp A E, Hezari M, Croteau R et al. CyClization of geranylgeranyl dipHospHate to taxa-4(5), 11(12)-diene is the committed step of taxol biosynthesis in Pacific yew[J]. J Bio Chem, 1995, 270:: 8686～8690.
58. Lin X Y, Hezari M, Koepp A E et al. Mechanism of taxadiene synthase,a diterpene cyClase that catalyzes the first step of taxol biosynthesis in Pacific yew[J]. Biochemistry, 1997,337: 2968～2977.
59. Ma W W, park G L, Gomez G A et al. New bioactive taxoids from cell cultures of Taxus baccata[J]. J Nat Prod, 1994,57: 116～122.
60. Mirjilili N, Linden J C. Gas pHase composition effects on suspension cultures of Taxus cuspidate[J]. J Biotechnol Bioeng, 1995,48: 123～132.
61. R.E.B. Ketchum, D.M. Gibson&L. Greenspan Gallo. Media optimization for maximum biomass production in cell cultures of pacific yew[J]. J Biotechnol Bioeng,1995,42: 185～193.
62. Thomas J. Hirasuna, Luis J. Pestchanker. Taxol production in suspension cultures of Taxus baccata[J], plant cell, Tissue and Organ Culture, 1996,44: 95～102.
63. Vidensek N. et al:Taxol content in bark, wood, root, leaf, twig and seedling from several Taxus species[J] J. Nat. Prod. 1995,56(10): 1657.
64. Vidensek N, Lim. P, Campbell A. et al. JNAT PROD(LLOYDIA),1990,53(6): 1609～1610.
65. Williams J G K, Kubelik A R,Livak J, et al. DNA polymorpHisma amplified by arbitrary primers are useful as genetic markers[J]. J VuCl Acids Res, 1990,18:6531～6535.
66. Wani MC, Taylor HL,Wall M E,et al. Plant antitumor agents. Ⅵ. The isolation and structure of taxol, a novel antileuke mic and antitumor agent from taxus brevifolia. [J]. J Am Chem Soc, 1971,27(7): 433～437.
67. Wick re mesinhe E R M, Arteca R N. Taxus Callus Cultures:Initiation, Growth Optimization, Characterixation and Taxol Production[J]. J Plant Cell Tissue Organ Cult, 1993,38: 181～193.
68. Wildung M R. A cDNA Clone for taxadiene synthase, the diterpene cyClass
that catalyzes the committed step of taxol biosynthesis[J]. J Bio Chem, 1996, 271, 9201～9204.
69. Wickremesinhe E R M. Arteca R N. Taxus callus: Initiation, growth optimization, characterization and taxol production [J]. Plant Cell Tissue Culture, 38:181～185.
70. Yukimune Y, Tabata H, Higashi Y et al. Methyl jasmonate-induced overproduction of paClitaxel and baccatin Ⅲ in Taxus cell suspension cultures[J]. J Nature Biotech, 1996, 14: 1129～1136.
2．陈大明，张上隆，金庸韦．一种木本果树基因组DNA提取方法研究[J]．浙江农业大学学报，1997，23(6)：621～624．
3．陈永勤，朱蔚华．红豆杉属植物的组织、细胞及胚培养[J]．植物生理学通讯，1997，33(3)：213～219．
4．仇燕，贾宁，王丽，王刚．继代时间对南方红豆杉细胞生长和产生紫杉醇的影响[J]．河北师范大学学报，2002，26(3)：301～304．
5．邓联东，孙多元，董岸杰．紫杉醇注射剂的研究[J]．药学进展，2002，26(3)：162～165．
6．F．奥斯伯，R 布伦特，R．E．金斯敦．精编分子生物学手册[M]．科学出版社，1999．
7．方起程，韩锐，陈未名等．抗肿瘤新药紫杉醇及其注射液的研究与开发[J]．中国医学科学院学报，1996，18(4)：251．
8．甘烦远，郑光植．红豆杉细胞培养的研究[J]．云南植物研究，1996，18(2)：134～138．
9．桂耀林，胡玉熹．红豆杉属叶子的表皮特征与分类的关系[J]．植物分类学报，1974，12(3)：329～333．
10．高山林．东亚和加拿大产红豆杉属七种植物中紫杉醇及短叶醇的含量[J]．药科大学学报，1995，(26)：8～10．
11．甘烦远，沈月毛，赫小江．红豆杉生物工程的研究进展[J]．武汉植物学研究，1999，17(1)：65～74．
12．顾志建，周其兴，岳中枢．中国红豆杉属和白豆杉属的核形态学研究[J]．云南植物研究，1998，3．
13．Glark M S主编，顾红雅，瞿礼嘉等主译．植物分子生物学手册[M]．高等教育出版社，1998．
14．何水林．植保素代谢与植物防御反应[M]．广东科技出版社，2002．
15．黄浩．红豆杉细胞培养中抗褐变剂的筛选[J]．华中理工大学学报，1999，27(4)：18～22．
16．黄浩．活性炭及VC对红豆杉细胞培养的影响[J]．甘肃教育学院学报(自然科学版)，2001，15(1)：34～36．
17．贾继增．分子标记种质资源鉴定和分子标记育种[J]．中国农业科学，1996，29(4)：1～10．
18．李伯林，赵群华，庐山，曹日强．红豆杉植物愈伤组织的培养及其紫杉醇形成的初探[J]．南京大学学报，1995，31(3)：424～429．
19．李家儒，智志勇，刘曼西，吴振斌，王君健．Cu2+对红豆杉培养细胞中紫
杉醇形成的影响[J]．华中农业大学学报，1999，18(2)：117～120．
20．梁珍海，刘根林，徐锋．红豆杉属植物组织培养及其快速繁殖研究综述[J]．江苏林业科技，2001，28(3)：45～48．
21．林风等．纤维素酶的生物化学和分子生物学研究新进展[J]．生命科学，1994，6(1)：18～23．
22．梅兴国，董妍玲，潘学武．红豆杉细胞继代培养防褐变措施的研究[J]．自然产物研究与开发，2001，13(4)：8～11．
23．马忠武，何关福，印万芳．双黄酮成分在红豆杉科各属中的分析[J]．植物分类学报，1985，23(3)：192～195．
24．苏湘鄂，梅兴国，龚伟，张周宁．红豆杉细胞时相与其次生代谢强度的关系[J]．生物技术，2000，10(4)：14～16．
25．汪小全，邹喻苹，张大明等．RAPD应用于遗传多样性和系统学研究中的问题[J]．植物学报，1996，38(12)：954～962．
26．王艇，苏应娟，黄超，朱建明．红豆杉科植物RAPD分析及其系统学意义[J]．西北植物学报，2000，20(2)：243～249．
27．王关林，方宏筠主编．植物基因工程原理技术[M]．科学出版社．1998：426．
28．吴丽圆，陈少谕，项伟．云南红豆杉自然群体内同工酶遗传变异的研究[J]．遗传，2001(3)：237～242．
29．王玲．辣椒遗传多样性的RAPD分析．福建农林大学硕士学位论文集，2002．
30．徐铮奎．天然植物抗癌药国内外研究开发新进展[J]．中国医药情报，2002，8(4)：36～38．
31．谢秋玲，郭勇．刺激剂在植物细胞培养次生代谢物中的应用[J]．广西植物，1999，19(2)：142～146．
32．席以珍．中国红豆杉科花粉形态的研究[J]．植物分类学报，1986，24(4)：247～252．
33．席以珍．穗花杉属划分外壁的超微结构及其分类意义[J]．植物分类学报，1986，24(6)：439～442．
34．夏铭，吴绛云，张丽梅．红豆杉组织培养中褐变问题的研究[J]．生物技术，1996，6(3)：18～20．
35．邢建民，查丽杭，李佐虎．植物细胞培养生产紫杉醇的研究进展[J]．植物学通报，1997，14(3)：282～292．
36．周金，姜笑梅．木材构造特征在裸子植物系统学中的意义[J]．植物分类学报，1992，30(5)：405～414．
37．项伟，沅德春，张宏杰等．不同产地云南红豆杉紫杉醇的含量分析[J]．云南林业科技，1996，18(2)：74～76．
38．岳琴．紫杉醇及其相关化合物的研究进展[J]．国外医学药学分册，1996 23(6)：332～334．
39．周波，姜虎生．红豆杉及其产物紫杉醇研究进展[J]．松辽学刊(自然科学
版)，2001，2(1)：78～81．
40．张长平，李春，元英进等．真菌诱导子对悬浮培养南方红豆杉细胞态势及紫杉醇合成的影响[J]．生物工程学报，2001，17(4)；436～440．
41．张恒庆，安利佳，祖元刚等．红松RAPD实验中各组成成份含量对实验结果的影响[J]．植物研究，1990，19(2)：183～188．
42．周东新．黄麻遗传多样性的RAPD和ISSR分析．福建农林大学硕士学位论文集，2001．
43. Burte J N. Taxus Le Bon Jardinier. Paris:La Maison Rustique, 1992, :2588～2589.
44. Becker J, Vos P, Kuiper M et al. Gombined mapping of AFLP and RFLP markers in barley[J]. J Mol. Genet. 1995, 246: 65～73.
45. Christen A A, Gibson D M, Bland J,et al, 1991. production of taxol or taxol-like compounds with Taxus brevifolia callus cell culture[J]. U S Patent, 5019504.
46. Chee P P. In vitro culture of zygotic embryos of Taxus species[J]. HortScience, 1994,29:695～697.
47. Durzan D J,Ventimiglia F. Free taxanes and the release of bound compounds having taxane antibody reactive by xylanase in female, haploid-derived cell suspension culture of taxusd brevifolia[J]. IN Vitro Cell Dev Biol, 1994,30:219～227.
48. Denis J N, Greene A E, Guenard D et al. A highly efficient practical approach to natural taxol[J]. J Am Chem Soc, 1998,110:5917.
49. Fett-Neto A.G.,F. DiCosmo,W.F.R eynolds et al. Cell culture of Taxus as a source of the antinecoplastic drug taxol and related taxanes[J]. J Biotechnolgy, 1992 (10):1572～1575.
50. Fett-Neto A G, Zhang W Y, Dicosom F. Kinetics of taxol production, growth, and nutrient uptake in cell suspensions of Taxus cuspidate[J]. j Biotechnol Bioengineer, 1994,44:205.
51. Fett-Neto A G, Melanson S J, Sakata K et al. Improved growth and taxol yield in developing calli of Taxus cusoidate by medium composition modification[J]. Bio/Technology, 1993,11:731.
52. Fritsch P, Rieseberg L H. The use of random amplified polymorpHic DNA(RAPD) in conservation genetics. Smith T B,Wayne R K. Molecular Genetic Approaches in conservartion. London[J].Oxford University.
53. Hezari M, Croteau R. Taxol biosynthesis[J]. J an update. J Plant Medica. 1997,63: 291～295.
54. Hezari M,,Ketchum R E B, Gibson D M et al. Lew is N G, Croteau R. Purification and characterization of taxa-4(5), 11(12)-diene synthase from Pacific
yew(Taxus brevifolia) that catalyzes the first step of taxol biosynthesis[J]. J Arch Biochem BiopHys, 1995,322: 437～444.
55. Hezari M, ,Ketchum R E B, Gibson D M et al. taxol production and taxadiene synthase activity in Taxus Canadensis cell suspension cultures[J]. J Arch BiopHys, 1997,337: 185～190.
56. Jaziri M, Zhiri A, Guo Y W et al. Taxus sp. Cell, tissue and organ culture as alternative sources for taxoids production: a literature survey[J]. Plant Cell Tiss Organ Culture, 1996,45:59～75.
57. Koepp A E, Hezari M, Croteau R et al. CyClization of geranylgeranyl dipHospHate to taxa-4(5), 11(12)-diene is the committed step of taxol biosynthesis in Pacific yew[J]. J Bio Chem, 1995, 270:: 8686～8690.
58. Lin X Y, Hezari M, Koepp A E et al. Mechanism of taxadiene synthase,a diterpene cyClase that catalyzes the first step of taxol biosynthesis in Pacific yew[J]. Biochemistry, 1997,337: 2968～2977.
59. Ma W W, park G L, Gomez G A et al. New bioactive taxoids from cell cultures of Taxus baccata[J]. J Nat Prod, 1994,57: 116～122.
60. Mirjilili N, Linden J C. Gas pHase composition effects on suspension cultures of Taxus cuspidate[J]. J Biotechnol Bioeng, 1995,48: 123～132.
61. R.E.B. Ketchum, D.M. Gibson&L. Greenspan Gallo. Media optimization for maximum biomass production in cell cultures of pacific yew[J]. J Biotechnol Bioeng,1995,42: 185～193.
62. Thomas J. Hirasuna, Luis J. Pestchanker. Taxol production in suspension cultures of Taxus baccata[J], plant cell, Tissue and Organ Culture, 1996,44: 95～102.
63. Vidensek N. et al:Taxol content in bark, wood, root, leaf, twig and seedling from several Taxus species[J] J. Nat. Prod. 1995,56(10): 1657.
64. Vidensek N, Lim. P, Campbell A. et al. JNAT PROD(LLOYDIA),1990,53(6): 1609～1610.
65. Williams J G K, Kubelik A R,Livak J, et al. DNA polymorpHisma amplified by arbitrary primers are useful as genetic markers[J]. J VuCl Acids Res, 1990,18:6531～6535.
66. Wani MC, Taylor HL,Wall M E,et al. Plant antitumor agents. Ⅵ. The isolation and structure of taxol, a novel antileuke mic and antitumor agent from taxus brevifolia. [J]. J Am Chem Soc, 1971,27(7): 433～437.
67. Wick re mesinhe E R M, Arteca R N. Taxus Callus Cultures:Initiation, Growth Optimization, Characterixation and Taxol Production[J]. J Plant Cell Tissue Organ Cult, 1993,38: 181～193.
68. Wildung M R. A cDNA Clone for taxadiene synthase, the diterpene cyClass
that catalyzes the committed step of taxol biosynthesis[J]. J Bio Chem, 1996, 271, 9201～9204.
69. Wickremesinhe E R M. Arteca R N. Taxus callus: Initiation, growth optimization, characterization and taxol production [J]. Plant Cell Tissue Culture, 38:181～185.
70. Yukimune Y, Tabata H, Higashi Y et al. Methyl jasmonate-induced overproduction of paClitaxel and baccatin Ⅲ in Taxus cell suspension cultures[J]. J Nature Biotech, 1996, 14: 1129～1136.