盾叶薯蓣离体培养技术的优化及其同源四倍体的诱导
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摘要
盾叶薯蓣(Dioscorea zingiberensis C.H. Wright)是薯蓣科薯蓣属一种重要的药用植物，为我国所特有。其根状茎内含有的薯蓣皂甙元是合成甾体激素药物的重要原料，被医药界称之为“药用黄金”。随着野生资源的逐渐枯竭，寻找新的药用资源，特别是利用现代生物技术手段迅速繁殖和培育出更优良的新种质资源为当务之急。为了探索出盾叶薯蓣快速繁殖有效的途径，优化培养条件，并且选育出皂素含量和生物学产量有可能得到提高的同源四倍体新品系，本实验用盾叶薯蓣二倍体种子为原始材料，以种子苗的胚轴等外植体建立无菌实验材料；而后以试管苗的叶片、茎段和圆球茎为外植体诱导了致密愈伤组织，经分化培养得到植株；探索了盾叶薯蓣试管苗的生根和移栽问题，建立了快繁体系，并对培养条件进行了优化筛选；通过化学诱变得到了同源四倍体植株。获得了以下主要结果：
1．探索了提高二倍体盾叶薯蓣种子萌发率的有效途径，并用实生种子苗建立了无菌材料：
先用35℃温水浸泡种子15-20小时软化种皮，随后经20-50ppm的GA_3等激素浸泡30分钟，再种于细沙中，可明显提高萌发率，萌发率达到62％。
用实生种子苗的胚轴经消毒后接种于添加6-BA2-4mg／l的MS培养基上可诱导产生大量致密的愈伤组织，继代培养和增殖可产生大员植株，建立了无菌起始材料。
2．二倍体盾叶薯蓣再生繁殖技术的优化：
在附加6-BA的MS培养基上，叶片、茎段、圆球茎、胚轴外植体均能诱导出致密的愈伤组织，但不同外植体的诱导率有很大差异：叶片和茎段的诱导率较低，分别为26.7％和33.3％；而圆球茎、胚籼外植体可诱导出大量的致密愈伤组织，诱导率分别为100％和85％，而且生长较快。致密愈伤组织不需转瓶，直接就能分化产生圆球茎和植株，因而实现了诱导与分化一步完成的目的，节约了时间和成本。
探讨了致密愈伤组织生长最快的激素组合和碳源种类：致密愈伤组织在以麦芽糖为碳源，添加了6-BA2mg／l，KT0.5mg/l的MS培养基上生长最快；而在以蔗糖为碳源，添加了6-BA2mg／l+KT 0.5mg／l的MS分化培养基上培养时，能很快分化出圆球茎突起，产生丛状芽，继而得到枝、叶茂盛的植株，而且致密愈伤组织的生长也比较快。
切取带枝条的圆球茎接种在MS+NAA0.5mg／l+IAA0.5mg／l生根培养基上培养时，生根率可达87.5％。
探讨了盾叶薯蓣试管苗的移栽问题，提高了成活率：研究发现在栽培基质(沙土)中
添加少量生根培养基，可显著提高成活率。移栽成活率可达53．7％。
因此，以茎段、圆球茎、胚轴作外植体，在附加 6丑 Amg／l的 MS培养基上诱导
出致密愈伤组织，是盾叶薯猿离体再生、快速繁殖的最有效的途径。
3．化学诱变获得了同源四倍体植株：
用浓度为0刀5％的秋水仙素溶液浸泡带有丛生芽的致密愈伤组织72小时，得到33．3％
的诱变植株的变异率。变异植株经过细胞学鉴定，染色体数目为 40（2 n—4x＝40，x—10），
而未经过诱变的正常H倍体植株染色体数目为20c习－20，x叫）；变异植株的叶片气
孔和保卫细胞明显比正常二倍体的大：叶片大，颜色深绿；茎粗壮；根茎生长加快。变
异植株为同源四倍体盾叶薯渍新品系。
此外，本文对盾叶薯预致密愈伤组织、圆球茎和根状茎以及试管苗的根与茎交接处
进行了石蜡切片观察，以揭示它们之间的内在联系。
Dioscorea zingiberensis C. H. Wright of dioscoreaceae is an important medical plant growing only in our country by reason that Diosgenin in its rhizome is one of the most important sources for steroid hormonal medicines, which is called "Pharmacentical Gold" by medical field. In order to find out the efficient path to rapidly propagate D.zingiberensis, optimize the nurture conditions and assortatively breed out the new varieties of polyploid, whose content of sapogenin and biological production may be higher than diploid, the embryonal axis of seedlings, rhizome of the highly content species and leaves, internode segments and protocorms of test-tube seedlings are used as explants in this experiment to induce callus. Consequently, regeneration system was established and complete plants were also produced from callus in differentiation culture. Furthermore, by chemical-inducing mutation, autotetraploid plants were attained. In addition, the protocol for transplanting test-tube plantlet of D.zingiberensis was a
lso discussed in this paper. The following is the major results of this experiment.
1. The valid pathway to enhance the germination rate of the seeds of diploid D. zingiberensis was studied and the steri le materials with the satus seedl ings were also gained.
The germination rate of seeds was apparently raised by the following means: firstly, we immersed seeds for 15-20 hours in the warm water of 35癈 to soften the seed coat, then, to immerse the seeds for 30 minutes with such hormones as GA3 whose concentration is about 20-50 ppm and finally, when we plant the seeds in fine sands, the germination rate amounted to 62%.
A large number of compact calli can be attained with the caulicles of satus seedlings being inoculated to the medium of MS added 6-BA2-4mg/l, and then a large number of plants can also be gotten after subculture.
2. The regeneration system of D. zingiberensis has been established and the technical conditions were also been optimized.
The explants of leaves, internode segments and protocorms can all be induced to loose calluses on the medium of MS added 2,4-D, but different explants have different rates of induction. The induction rate of leaves is very low, 25% at the best; Internode segments can be induced 50% loose callus on the medium of MS+2,4-D6mg/l, and yet protocorms can be induced above 93% loose calli on the medium of MS+2,4-Dl-6mg/l. However, loose calli
only produce a few of regeneration seedlings on differentiation medium and thus, not fit for establishing tachytelic propagation system.
The explants from leaves, internode segments, protocorms and caulides can all be induced to compact calli on the medium of MS added 6-B A, but different explants also have different rates of induction. The rates of induction of leaves and internode segments are comparatively low amount to 26.7% and 33.3% respectively; protocorms and caulicles can produce a large number of compact calli on the medium of MS+6-BA2mg/l. And the callus can produce plants whose branches and leaves are flourish on the differentiation medium of MS+6-BA2mg/l+KT0.5mg/l. Splitting branches which cling to protocorms and transplanting them onto the rooting medium of MS+NAA0.5mg/l+IAA0.5mg/l can get a high rooting rate of 87.5%, and transplanting survival rate can get to 53.7%.
Our research results indicated that the survival rate of transplanting of test-tube seedlings can be apparently enhanced when add a little of rooting medium to soil.
In short, the pathway that the explants of protocorms and caulicles are induced compact
calluses on the medium of MS added 6-BA2mg/l is the most efficient in isolated culturing
and rapid propagation of D.zingiberensis.
3. Autotetraploid plants were attained through chemical induction.
By immersing the compact calluses with contagious buds into 0.05% colchicines for 72 hours, mutation rate of 33,3% were obtained. The muted plants have been proved to be autotetraploid D.zingiberensis with fine traits through cytology identification and field culti
1．探索了提高二倍体盾叶薯蓣种子萌发率的有效途径，并用实生种子苗建立了无菌材料：
先用35℃温水浸泡种子15-20小时软化种皮，随后经20-50ppm的GA_3等激素浸泡30分钟，再种于细沙中，可明显提高萌发率，萌发率达到62％。
用实生种子苗的胚轴经消毒后接种于添加6-BA2-4mg／l的MS培养基上可诱导产生大量致密的愈伤组织，继代培养和增殖可产生大员植株，建立了无菌起始材料。
2．二倍体盾叶薯蓣再生繁殖技术的优化：
在附加6-BA的MS培养基上，叶片、茎段、圆球茎、胚轴外植体均能诱导出致密的愈伤组织，但不同外植体的诱导率有很大差异：叶片和茎段的诱导率较低，分别为26.7％和33.3％；而圆球茎、胚籼外植体可诱导出大量的致密愈伤组织，诱导率分别为100％和85％，而且生长较快。致密愈伤组织不需转瓶，直接就能分化产生圆球茎和植株，因而实现了诱导与分化一步完成的目的，节约了时间和成本。
探讨了致密愈伤组织生长最快的激素组合和碳源种类：致密愈伤组织在以麦芽糖为碳源，添加了6-BA2mg／l，KT0.5mg/l的MS培养基上生长最快；而在以蔗糖为碳源，添加了6-BA2mg／l+KT 0.5mg／l的MS分化培养基上培养时，能很快分化出圆球茎突起，产生丛状芽，继而得到枝、叶茂盛的植株，而且致密愈伤组织的生长也比较快。
切取带枝条的圆球茎接种在MS+NAA0.5mg／l+IAA0.5mg／l生根培养基上培养时，生根率可达87.5％。
探讨了盾叶薯蓣试管苗的移栽问题，提高了成活率：研究发现在栽培基质(沙土)中
添加少量生根培养基，可显著提高成活率。移栽成活率可达53．7％。
因此，以茎段、圆球茎、胚轴作外植体，在附加 6丑 Amg／l的 MS培养基上诱导
出致密愈伤组织，是盾叶薯猿离体再生、快速繁殖的最有效的途径。
3．化学诱变获得了同源四倍体植株：
用浓度为0刀5％的秋水仙素溶液浸泡带有丛生芽的致密愈伤组织72小时，得到33．3％
的诱变植株的变异率。变异植株经过细胞学鉴定，染色体数目为 40（2 n—4x＝40，x—10），
而未经过诱变的正常H倍体植株染色体数目为20c习－20，x叫）；变异植株的叶片气
孔和保卫细胞明显比正常二倍体的大：叶片大，颜色深绿；茎粗壮；根茎生长加快。变
异植株为同源四倍体盾叶薯渍新品系。
此外，本文对盾叶薯预致密愈伤组织、圆球茎和根状茎以及试管苗的根与茎交接处
进行了石蜡切片观察，以揭示它们之间的内在联系。
Dioscorea zingiberensis C. H. Wright of dioscoreaceae is an important medical plant growing only in our country by reason that Diosgenin in its rhizome is one of the most important sources for steroid hormonal medicines, which is called "Pharmacentical Gold" by medical field. In order to find out the efficient path to rapidly propagate D.zingiberensis, optimize the nurture conditions and assortatively breed out the new varieties of polyploid, whose content of sapogenin and biological production may be higher than diploid, the embryonal axis of seedlings, rhizome of the highly content species and leaves, internode segments and protocorms of test-tube seedlings are used as explants in this experiment to induce callus. Consequently, regeneration system was established and complete plants were also produced from callus in differentiation culture. Furthermore, by chemical-inducing mutation, autotetraploid plants were attained. In addition, the protocol for transplanting test-tube plantlet of D.zingiberensis was a
lso discussed in this paper. The following is the major results of this experiment.
1. The valid pathway to enhance the germination rate of the seeds of diploid D. zingiberensis was studied and the steri le materials with the satus seedl ings were also gained.
The germination rate of seeds was apparently raised by the following means: firstly, we immersed seeds for 15-20 hours in the warm water of 35癈 to soften the seed coat, then, to immerse the seeds for 30 minutes with such hormones as GA3 whose concentration is about 20-50 ppm and finally, when we plant the seeds in fine sands, the germination rate amounted to 62%.
A large number of compact calli can be attained with the caulicles of satus seedlings being inoculated to the medium of MS added 6-BA2-4mg/l, and then a large number of plants can also be gotten after subculture.
2. The regeneration system of D. zingiberensis has been established and the technical conditions were also been optimized.
The explants of leaves, internode segments and protocorms can all be induced to loose calluses on the medium of MS added 2,4-D, but different explants have different rates of induction. The induction rate of leaves is very low, 25% at the best; Internode segments can be induced 50% loose callus on the medium of MS+2,4-D6mg/l, and yet protocorms can be induced above 93% loose calli on the medium of MS+2,4-Dl-6mg/l. However, loose calli
only produce a few of regeneration seedlings on differentiation medium and thus, not fit for establishing tachytelic propagation system.
The explants from leaves, internode segments, protocorms and caulides can all be induced to compact calli on the medium of MS added 6-B A, but different explants also have different rates of induction. The rates of induction of leaves and internode segments are comparatively low amount to 26.7% and 33.3% respectively; protocorms and caulicles can produce a large number of compact calli on the medium of MS+6-BA2mg/l. And the callus can produce plants whose branches and leaves are flourish on the differentiation medium of MS+6-BA2mg/l+KT0.5mg/l. Splitting branches which cling to protocorms and transplanting them onto the rooting medium of MS+NAA0.5mg/l+IAA0.5mg/l can get a high rooting rate of 87.5%, and transplanting survival rate can get to 53.7%.
Our research results indicated that the survival rate of transplanting of test-tube seedlings can be apparently enhanced when add a little of rooting medium to soil.
In short, the pathway that the explants of protocorms and caulicles are induced compact
calluses on the medium of MS added 6-BA2mg/l is the most efficient in isolated culturing
and rapid propagation of D.zingiberensis.
3. Autotetraploid plants were attained through chemical induction.
By immersing the compact calluses with contagious buds into 0.05% colchicines for 72 hours, mutation rate of 33,3% were obtained. The muted plants have been proved to be autotetraploid D.zingiberensis with fine traits through cytology identification and field culti
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43．刘承来等．小花盾叶薯蓣中甾体皂甙的分离和鉴定．植物学报，1985，27(6)：635-639
44．单文典．用双波长薄层色谱扫描仪测定盾叶薯蓣皂甙元的含量，中草药，1981，12(1)：15-16
45．顾生明，唐世蓉等．薯蓣皂甙元的气液色谱分析．植物学报，1980，22(2)：204-206
46．陈建国等．薯蓣皂甙元的分光光度法测定．分析化学，1996，24(2)：227-229
47．徐成基．植物激素对盾叶薯蓣皂素收率的影响．植物生理学通讯，1981，(1)：38-40
48．薯蓣植物资源开发利用前景新进展．中国植物学会五十五周年年会学术论文摘要汇编．中国植物学会，1988，654-655
49．舒璞．中国薯蓣属花粉形态的初步研究，植物分类学报，1987，25(5)：357-364
50．秦慧贞，张美珍等．中国薯蓣属细胞分类的研究—染色体数与该属起源和演化．1985，23(1)：11-18
51．中国薯蓣属根茎组植物的分类和染色体数的研究．江苏省植物研究所薯蓣课题研究组，1976，14(1)：65-72
52．奚镜清等．浙江省薯蓣科植物的数量分类研究，植物分类学报，1990，28(6)：442-451
53. Jayanti Sengupta et al. Organogenesis and tuberization in cultures of D.floribunda. Plant Cell,Tissue and Organ Culture. 3:325-331,1984
54. Chatarveli, H.C. et al, Morphogenesis, micropropagation, and germplasm preservation of some economic plants by tissue cultures. Plant Tissue Culture. 1982,687-688
55. Tal B. Rokem j et al, The effect of chlorphyll-bleaching herbicides on growth carotenoid and dosgenin levels in cell suspension cultures of D.delt0id, Phytochem, 1984,23:13-23
56．刘鹏，郭良水，浙江省薯蓣属植物资源的开发利用，63-65
57．裴鉴，丁志遵等．中国薯蓣属根茎组系统分类的初步研究．植物分类学报，1979，17(3)：61-71
58．任建伟，白云等．盾叶薯蓣培养细胞的生长及薯蓣皂甙元产生的变化规律．中草药，1994，25(2)：93-94
59．李伯刚，周瑾．黄山药不同物候期薯蓣皂甙元含量变化规律研究．天然产物研究与开发，1997，11(2)：23-26
60．徐成基．中国薯蓣资源-甾体激素药源植物的研究与开发．四川科学技术出版社，2000，10
61．李伯刚，周瑾．栽培黄山药薯蓣皂甙元含量年度变化与适宜采挖期研究．天然产物研究与开发，1998，11(6)：6-9
62．李子先，刘东旭编著．植物细胞遗传学的研究方法与技术．四川科学技术出版社，1992，3
63．江苏省植物研究所，影响盾叶薯蓣皂甙元含量的有关因素，中草药，1991，12(6)：34-36
64．中国医学科学院药物研究所，薯蓣属植物中的甾体皂甙元，中草药，1981，12(8)：41-48
65．藤井胜也等．药学杂志(日)，56：408，1936
66．江苏省植物研究所，盾叶薯蓣引种栽培研究，中草药，1989，20(10)：35-37
67．周振起，封玉贤．薯蓣属植物提取皂素的工艺研究，中草药，1985，16(7)：15-17
68．丁志遵．天然甾体药物原料开拓利用的回顾与展望，中草药，1985，16(6)：28-36
69. Dirk R. Vuylsteke, Rony L. Swennen &Rodomiro Ortiz, Development and performance of black sigatoka-resistant tetraploid hybrids of plantain( Musa spp., AAB group) Euphytica 65: 33-42, 1993
2．杨明河．薯蓣属植物中的甾体皂甙元．中草药，1981，8：41-48
3．秦天才，张友德，张君芝．湖北黄姜资源的现状及开发利用．自然资源，1996，3：58-61
4．秦天才等．重要的甾体激素药源—黄姜．植物杂志，1997，1：8
5．李忌等．天然甾体皂甙化合物的抗肿瘤活性．天然产物研究与开发，1999，11(1)：14-17
6．张天义，张丽红等．盾叶薯蓣灭钉螺活性成分的研究．武汉植物学研究，1998，16(1)：65-68
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9．蒋震涛，范昆华等．水稻体细胞同源四倍体无性系诱导及遗传特性，上海农业学报，1993，9(1)：6-10
10. Xiao Shuan-Suo,Bao Wen-Kui,One-step Induction Method of Test-tube Seedlings for Autotetraploid Rice, 植物生理学报, 1989, 15 (3): 329-332
11．刘金香．秋水仙素对水稻诱变效果的初步研究．生物学杂志，1993，55(5)：19-21
12．李佩芬，卢炳芝等．葡萄细胞悬浮培养及诱变研究初报，园艺学报，1993，20(3)：301-302
13. Zhang Chenghe, Shen Shuxing, Wang Yanhua, Shang Aiqin, Liu Shixiong, Inducement of Autotetraploid Chinese Cabbage (Brassica var. communis Tsen et Lee) and Its Cytological Study, Journal of Agriculture University of Hebei, 1994, 22(4): 53-56
14. M.Van Duren, R.Morpurgo, J.Dolezel&R. Afza,Induction and vertifition of antotetraplids in diploid banana(Musa acuminata) by in vitro techniques, Euphytica, 1996,88 (1): 25-34
15. Ammirato,P.V. Induction maintainance,and manipulation of development in embryogenic cell suspension cultures. Cell Culture and Somatic Cell Genetics of Plants. 1984; 139-151
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17. Forsyth. C. Van, Staden. J. Tuberization of D.bulbifera stem nodes in culture, J. Plant physiol, 1984,115:79-83
18. Shou Yong Choy NG. In vitro tuberization in white yam (D.rotundata Poir). Plant Cell,Tissue and Organ Culture 14:121-128,1988
19．周朴华，何立珍，刘选明．组织培养中用秋水仙素诱发黄花菜同源四倍体的研究．中国农业科学，1995，28(1)：49-55
20．周朴华，刘选明，何立珍．黄花菜圆球茎经秋水仙素处理后再生苗加倍效果观察．湖南农学院学报，1991，17(2)：158-165
21．刘选明．同源四倍体龙牙百合诱导及其形态发生的细胞学与生理生化特性的研究．博士学位论文，1995，7
22．谭素英，黄秀强，刘济伟．提高西瓜四倍体诱导率的研究．华北农学报，1993，8(4)：12-15
23．纪凤高，邓景扬．秋水仙素与二甲基亚枫混合水溶液处理小麦-黑麦杂交一代苗进行染色体加倍的实验．遗传，1987，9(3)：1-4，10
24．向齐君，吴兰佩．离体条件下用秋水仙素处理小麦-黑麦杂种细胞的研究．遗传，1194，16(1)：35-37
25．蒋华仁，刘润山．同源四倍体大麦的产量性状和蛋白质含量的研究．四川农业大学学报，1994，12(3)：418-422
26．张绍南，余建华等．人工诱导四倍体大麦研究初报．作物学报，1990，16(4)：373-376
27．张成合，宋长志，王淑芳多倍体育种综述，河北农业大学学报，1988，11(2)：136-139
28．奚元龄，颜昌敬编译．植物细胞培养手册，农业出版社，1992，408-415
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30. Mantell, S.H. Apical meristrm-tip culture for eradication of flexous rod viruses in yan(Dioscorea alata), Tropocal Pest Management, 1980,26: 170-179
31．四川生物研究所—室体细胞组．盾叶薯蓣组织培养研究初报．植物学报，1978，20(3)：279-280
32．江苏植物所．盾叶薯蓣试管植株的诱导．植物生理学通报，1985，(5)：31
33．毕世荣等．高含量薯蓣皂素植株的细胞克隆．天然产物研究与开发，1997，9(4)：1-6
34．张宗勤等．叉蕊薯蓣的微繁殖及微型薯蓣的离体诱导．生物技术，1998，8(1)：18-20
35. Martine Jean & Marlo Cappadocia, In vitro tuberization in Dioscoarea alata L. Brazo fuerte and Florido and D.abyssinica Hoch. Plant Cell, Tissue and Organ Culture 26:147-152,1991
36. Bernard Malaurie et al. Effect of growth regulators concerntrations on morphological development of meristem-tip in D.cayenensis-D.rotundata complex and D.praehensilis. Plant Cell, Tissue and Organ Culture. 41:229-235,1995
37. Bernard Malaurie et al. Influrance of meristem-tip size and location on morphological development in D.cayensis-D.rotundata complex Grosse Caille and genotype of D.praehensilis.Plant Cell, Tisse and Organ Culture. 42:215-218,199
38．秦慧贞等．盾叶薯蓣的胚胎发育及其在演化上的意义．生物技术，1988，307-308
39．谢德容．薯蓣根茎的发育形态学研究．中国植物学会五十周年年会学术论文摘要汇编，中国植物学会，1988，307
40．刘秦来等．盾叶薯蓣中甾体皂甙的分离和鉴定，植物学报，1984，26(3)：283-289
41．刘秦来，陈延镛．鲜盾叶薯蓣中原始皂甙的分离与鉴定．植物学报，1985，27(1)：68-74
42．唐世蓉，张涵庆等．薯蓣科植物甾体皂甙元的含量和鉴定，植物学报，1979，21(2)：171-175
43．刘承来等．小花盾叶薯蓣中甾体皂甙的分离和鉴定．植物学报，1985，27(6)：635-639
44．单文典．用双波长薄层色谱扫描仪测定盾叶薯蓣皂甙元的含量，中草药，1981，12(1)：15-16
45．顾生明，唐世蓉等．薯蓣皂甙元的气液色谱分析．植物学报，1980，22(2)：204-206
46．陈建国等．薯蓣皂甙元的分光光度法测定．分析化学，1996，24(2)：227-229
47．徐成基．植物激素对盾叶薯蓣皂素收率的影响．植物生理学通讯，1981，(1)：38-40
48．薯蓣植物资源开发利用前景新进展．中国植物学会五十五周年年会学术论文摘要汇编．中国植物学会，1988，654-655
49．舒璞．中国薯蓣属花粉形态的初步研究，植物分类学报，1987，25(5)：357-364
50．秦慧贞，张美珍等．中国薯蓣属细胞分类的研究—染色体数与该属起源和演化．1985，23(1)：11-18
51．中国薯蓣属根茎组植物的分类和染色体数的研究．江苏省植物研究所薯蓣课题研究组，1976，14(1)：65-72
52．奚镜清等．浙江省薯蓣科植物的数量分类研究，植物分类学报，1990，28(6)：442-451
53. Jayanti Sengupta et al. Organogenesis and tuberization in cultures of D.floribunda. Plant Cell,Tissue and Organ Culture. 3:325-331,1984
54. Chatarveli, H.C. et al, Morphogenesis, micropropagation, and germplasm preservation of some economic plants by tissue cultures. Plant Tissue Culture. 1982,687-688
55. Tal B. Rokem j et al, The effect of chlorphyll-bleaching herbicides on growth carotenoid and dosgenin levels in cell suspension cultures of D.delt0id, Phytochem, 1984,23:13-23
56．刘鹏，郭良水，浙江省薯蓣属植物资源的开发利用，63-65
57．裴鉴，丁志遵等．中国薯蓣属根茎组系统分类的初步研究．植物分类学报，1979，17(3)：61-71
58．任建伟，白云等．盾叶薯蓣培养细胞的生长及薯蓣皂甙元产生的变化规律．中草药，1994，25(2)：93-94
59．李伯刚，周瑾．黄山药不同物候期薯蓣皂甙元含量变化规律研究．天然产物研究与开发，1997，11(2)：23-26
60．徐成基．中国薯蓣资源-甾体激素药源植物的研究与开发．四川科学技术出版社，2000，10
61．李伯刚，周瑾．栽培黄山药薯蓣皂甙元含量年度变化与适宜采挖期研究．天然产物研究与开发，1998，11(6)：6-9
62．李子先，刘东旭编著．植物细胞遗传学的研究方法与技术．四川科学技术出版社，1992，3
63．江苏省植物研究所，影响盾叶薯蓣皂甙元含量的有关因素，中草药，1991，12(6)：34-36
64．中国医学科学院药物研究所，薯蓣属植物中的甾体皂甙元，中草药，1981，12(8)：41-48
65．藤井胜也等．药学杂志(日)，56：408，1936
66．江苏省植物研究所，盾叶薯蓣引种栽培研究，中草药，1989，20(10)：35-37
67．周振起，封玉贤．薯蓣属植物提取皂素的工艺研究，中草药，1985，16(7)：15-17
68．丁志遵．天然甾体药物原料开拓利用的回顾与展望，中草药，1985，16(6)：28-36
69. Dirk R. Vuylsteke, Rony L. Swennen &Rodomiro Ortiz, Development and performance of black sigatoka-resistant tetraploid hybrids of plantain( Musa spp., AAB group) Euphytica 65: 33-42, 1993