铁皮石斛组培及次生代谢物积累规律的研究
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摘要
论文以铁皮石斛无菌苗幼嫩组织（茎段、叶片、茎尖和幼芽）为外植体，经过对不同组培方式、激素种类与浓度、最适环境条件优化等系列研究，最终建立了经丛生芽诱导途径和拟原球茎诱导途径两套较为完整的组织培养再生体系。并针对在不同光照、不同培养方式和不同有机添加物条件下拟原球茎的次生代谢产物含量变化规律进行了初步研究。主要试验结果如下：
1.铁皮石斛快繁技术
（1）丛生芽途径
铁皮石斛组培快繁的最佳基本培养基为MS，以铁皮石斛无菌苗的带腋芽茎段为材料，诱导丛生芽，最佳激素浓度配比为NAA0.1mg/L+6-BA2.0mg/L，30d增殖系数为2.82，60d增殖系数为4.8。
（2）PLBs途径
分别以铁皮石斛幼嫩茎段、叶片、茎尖和幼芽为外植体进行拟原球茎（PLBs）诱导，结果显示，茎段和幼芽的诱导率较高。以茎段为外植体，PLBs最佳诱导培养基为MS+6-BA0.5mg/L+NAA1.0mg/L+2,4-D2.0mg/L，以幼芽为外植体，PLBs的最佳诱导培养基为MS+6-BA1.0mg/L+NAA1.0mg/L+2,4-D2.0mg/L。PLBs增殖最佳培养基为MS+NAA0.1mg/L; PLBs分化最佳培养基为MS+NAA0.1mg/L+6-BA1.0mg/L。
（3）壮苗生根
铁皮石斛幼苗壮苗的最佳培养基为MS+0.5mg/LIBA+0.5mg/L NAA。同时，单独使用0.5mg/L NAA的培养基中小苗生根快，根数多。香蕉泥能促进幼苗根系的生长。组培苗移栽到纯树皮的基质中，成活率最高。
2.拟原球茎次生代谢产物积累规律研究
对PLBs的生长规律及其次生代谢产物（总生物碱含量和多糖含量）积累动态进行了研究。结果表明，PLBs的生长大致呈“S”曲线，在第40d左右达到了生长高峰。此时，总生物碱含量和多糖含量也达到了最大值。在PLBs的分化过程中，当PLBs分化出叶时，PLBs的次生代谢产量均达到分化期最高，之后的幼苗期次生代谢产物含量均略有下降。
黑暗条件下PLBs鲜重增殖率明显低于光照条件下，且铁皮石斛PLBs的次生代谢产物积累受到抑制。
液体培养下的PLBs生长曲线均大致呈“S”型曲线，液体培养下PLBs的鲜重增殖率高于固体培养方式。液体培养中多糖含量和生物碱含量均高于固体培养。
不同有机添加物对PLBs次生代谢产物积累产生不同的影响。其中土豆培养基的PLBs鲜重增殖率最大，香蕉汁培养基中的PLBs生物碱含量最高，而苹果汁培养基中的的多糖含量最高。
An effective method for rapid propagation of Dendrobium officinale was established toinduct cluster shoots after a series of culture study. took young tissue of aseptic seedling wereexplants, through Stem axillary bud induction pathway and Young tissue intended protocorminduction pathway, two more complete tissue culture regeneration system were established. Apreliminary study for the proposed protocorm in a different light, different training methods anddifferent natural additives medium conditions secondary metabolites content were couducted.
1.Tissue culture and rapid propagation of Dendrobium officinale
(1).Buds ways
Dendrobium officinale plantlet grew best in MS medium. The proliferation rate of clustershoots reached the highest in MS medium with NAA0.1mg/L and6-BA2.0mg/L．30dproliferation coefficient is2.82,60d proliferation coefficient is4.8.
(2).PLBs ways
Respectively to Dendrobium officinale tender stem segments, leaf, stem tip and buds asexplants of PLBs induced, the results showed, the stem segments and bud induction rate wasbest.The stems as explants, the best PLBs induction culture medium for MS+6-BA0.5mg/L+NAA1.0mg/L+2,4-D2.0mg/L, with buds as explants, PLBs optimal induction medium wasMS+6-BA1.0mg/L+NAA1mg/L+2,4-D2.0mg/L. The best medium for proliferation of PLBsMS+NAA0.1mg/L, PLBs differentiation optimum medium for MS+NAA0.1mg/L+6-BA1.0mg/L.
(3). Rooting and transplantation
The most suitable medium for strengthening plantlet and taking roots was MS+0.5mg/LIBA+0.5mg/LNAA.The plantlet rooted rapidly and got more roots with0.5mg/L NAA alonein addition. The banana juice could promote growth of plantlet roots．The highest survival ratewere raised, when Seedlings were transplanted into the matrix of pure bark.
2. Studies on Induction of Protocorm-like Bodies and Accumulation of Production ofSecondary Metabolites of Dendrobium officinale
The Protocorm-like bodies were inducted with the young stem section of Dendrobiumofficinale as explant using the method of orthogonal test of three factors at three different levels.The relationship of growth rhythm and its secondary metabolites (total alkaloid content and polysaccharide) accumulation were researched. The results showed that the best inducedmedium was MS+6-BA0.5mg/L+NAA1.0mg/L+2,4-D2.0mg/L.Simultaneously the growth ofProtocorm-like bodies appeard in "S" curve in one semester (60d),reached the growth peak at the40d around. At that time, the total alkaloid content and polysaccharide also reached the peakvalue. The results also showed that the correlation coefficients between the total alkaloid contentand polysaccharide and the growth of Protocorm-like bodies were highly significant. When theleaf were divided from Protocorm-like bodies,the Secondary metabolism production ofProtocorm-like bodies were highest in differentiation phase, and then declined slightly in periodof young seedling.
Fresh weight of the proliferation rate of PLBs were significant lower exposed in lightconditions than in dark conditions. The accumulation of Secondary metabolites of Dendrobiumofficinale candidum PLBs were inhibited in various degree.
Simultaneously the growth of Protocorm-like bodies appeard in "S" curve in dark conditions.Fresh weight of the proliferation rate of PLBs were significant lower exposed in Solid culturethan in Liquid culture. Polysaccharide content and alkaloid content were much higer in Liquidculture.
The accumulationof PLBs secondary metabolite were influencede by different naturaladditives. PLBs fresh weight of the proliferation rate got largest by Potato medium, alkaloid ofPLBs got highest by Banana juice medium. polysaccharide content got largest by apple juicemedium.
1.铁皮石斛快繁技术
（1）丛生芽途径
铁皮石斛组培快繁的最佳基本培养基为MS，以铁皮石斛无菌苗的带腋芽茎段为材料，诱导丛生芽，最佳激素浓度配比为NAA0.1mg/L+6-BA2.0mg/L，30d增殖系数为2.82，60d增殖系数为4.8。
（2）PLBs途径
分别以铁皮石斛幼嫩茎段、叶片、茎尖和幼芽为外植体进行拟原球茎（PLBs）诱导，结果显示，茎段和幼芽的诱导率较高。以茎段为外植体，PLBs最佳诱导培养基为MS+6-BA0.5mg/L+NAA1.0mg/L+2,4-D2.0mg/L，以幼芽为外植体，PLBs的最佳诱导培养基为MS+6-BA1.0mg/L+NAA1.0mg/L+2,4-D2.0mg/L。PLBs增殖最佳培养基为MS+NAA0.1mg/L; PLBs分化最佳培养基为MS+NAA0.1mg/L+6-BA1.0mg/L。
（3）壮苗生根
铁皮石斛幼苗壮苗的最佳培养基为MS+0.5mg/LIBA+0.5mg/L NAA。同时，单独使用0.5mg/L NAA的培养基中小苗生根快，根数多。香蕉泥能促进幼苗根系的生长。组培苗移栽到纯树皮的基质中，成活率最高。
2.拟原球茎次生代谢产物积累规律研究
对PLBs的生长规律及其次生代谢产物（总生物碱含量和多糖含量）积累动态进行了研究。结果表明，PLBs的生长大致呈“S”曲线，在第40d左右达到了生长高峰。此时，总生物碱含量和多糖含量也达到了最大值。在PLBs的分化过程中，当PLBs分化出叶时，PLBs的次生代谢产量均达到分化期最高，之后的幼苗期次生代谢产物含量均略有下降。
黑暗条件下PLBs鲜重增殖率明显低于光照条件下，且铁皮石斛PLBs的次生代谢产物积累受到抑制。
液体培养下的PLBs生长曲线均大致呈“S”型曲线，液体培养下PLBs的鲜重增殖率高于固体培养方式。液体培养中多糖含量和生物碱含量均高于固体培养。
不同有机添加物对PLBs次生代谢产物积累产生不同的影响。其中土豆培养基的PLBs鲜重增殖率最大，香蕉汁培养基中的PLBs生物碱含量最高，而苹果汁培养基中的的多糖含量最高。
An effective method for rapid propagation of Dendrobium officinale was established toinduct cluster shoots after a series of culture study. took young tissue of aseptic seedling wereexplants, through Stem axillary bud induction pathway and Young tissue intended protocorminduction pathway, two more complete tissue culture regeneration system were established. Apreliminary study for the proposed protocorm in a different light, different training methods anddifferent natural additives medium conditions secondary metabolites content were couducted.
1.Tissue culture and rapid propagation of Dendrobium officinale
(1).Buds ways
Dendrobium officinale plantlet grew best in MS medium. The proliferation rate of clustershoots reached the highest in MS medium with NAA0.1mg/L and6-BA2.0mg/L．30dproliferation coefficient is2.82,60d proliferation coefficient is4.8.
(2).PLBs ways
Respectively to Dendrobium officinale tender stem segments, leaf, stem tip and buds asexplants of PLBs induced, the results showed, the stem segments and bud induction rate wasbest.The stems as explants, the best PLBs induction culture medium for MS+6-BA0.5mg/L+NAA1.0mg/L+2,4-D2.0mg/L, with buds as explants, PLBs optimal induction medium wasMS+6-BA1.0mg/L+NAA1mg/L+2,4-D2.0mg/L. The best medium for proliferation of PLBsMS+NAA0.1mg/L, PLBs differentiation optimum medium for MS+NAA0.1mg/L+6-BA1.0mg/L.
(3). Rooting and transplantation
The most suitable medium for strengthening plantlet and taking roots was MS+0.5mg/LIBA+0.5mg/LNAA.The plantlet rooted rapidly and got more roots with0.5mg/L NAA alonein addition. The banana juice could promote growth of plantlet roots．The highest survival ratewere raised, when Seedlings were transplanted into the matrix of pure bark.
2. Studies on Induction of Protocorm-like Bodies and Accumulation of Production ofSecondary Metabolites of Dendrobium officinale
The Protocorm-like bodies were inducted with the young stem section of Dendrobiumofficinale as explant using the method of orthogonal test of three factors at three different levels.The relationship of growth rhythm and its secondary metabolites (total alkaloid content and polysaccharide) accumulation were researched. The results showed that the best inducedmedium was MS+6-BA0.5mg/L+NAA1.0mg/L+2,4-D2.0mg/L.Simultaneously the growth ofProtocorm-like bodies appeard in "S" curve in one semester (60d),reached the growth peak at the40d around. At that time, the total alkaloid content and polysaccharide also reached the peakvalue. The results also showed that the correlation coefficients between the total alkaloid contentand polysaccharide and the growth of Protocorm-like bodies were highly significant. When theleaf were divided from Protocorm-like bodies,the Secondary metabolism production ofProtocorm-like bodies were highest in differentiation phase, and then declined slightly in periodof young seedling.
Fresh weight of the proliferation rate of PLBs were significant lower exposed in lightconditions than in dark conditions. The accumulation of Secondary metabolites of Dendrobiumofficinale candidum PLBs were inhibited in various degree.
Simultaneously the growth of Protocorm-like bodies appeard in "S" curve in dark conditions.Fresh weight of the proliferation rate of PLBs were significant lower exposed in Solid culturethan in Liquid culture. Polysaccharide content and alkaloid content were much higer in Liquidculture.
The accumulationof PLBs secondary metabolite were influencede by different naturaladditives. PLBs fresh weight of the proliferation rate got largest by Potato medium, alkaloid ofPLBs got highest by Banana juice medium. polysaccharide content got largest by apple juicemedium.
引文
[1]中国科学院中国植物志编辑委员会.中国植物志:第19卷[M].北京:科学出版社,1999.
[2]江苏新中医学院.中药大药典[M].上海:上海科学技术出版社,1985.
[3]586.贵州省中医研究所.贵州石斛的调查研究[M].1976.
[4]郑光植,梁峥.药用植物组织培养的研究[J].植物学报,1976,18(2):16.
[5]中华人名共和国药典编写委员会.中华人民共和国药典:人民卫生出版社,2000．
[6]国家药典委员会编.中国药典[M].北京:化学工业出版社,2005年版,62-63.
[7]俞群,王海燕.铁皮石斛在经口气管插管患者口腔溃疡中的应用[J].护士进修,2006,21(4):347-349.
[8]王康正,高文远.石斛属药用植物研究进展[J].中草药1997,28(10):633-635.
[9]张中建,阎小伟,铁皮石斛制剂免疫调节作用的实验研究[J].食品研究与开发,2004,25(2):34-35.
[10]王蜀秀,温远影.我国利用植物组织和细胞培养产生药用成分的研究概况[J].植物学报,1995,12(1):33-37.
[11]王宪楷,赵同芳.石斛属植物的化学成分与中药石解斛.药学通报,1986,2l(ll):666-669.
[12]陈晓梅,郭顺星.石斛属植物的化学成分与药理作用的研究进展[J].天然产物研究与开发,2001,13(1):70.
[13]郭洪波.铁皮石斛组培快繁及其抗癌作用的研究[D].沈阳:农业大学.2008.
[14]张光浓,毕志明,王峥涛,等.石斛属植物化学成分研究进展[J].中草药,2003,34(6):5-8.
[15]邓银华,徐康平,谭桂山.石解斛属植物化学成分与药理活性研究进展[J].中药材,2002,25(9):677-680.
[16]陈晓梅,郭顺星.石斛属植物化学成分和药理作用的研究进展[J].天然产物研究与开发,2002,13(l):70-75.
[17]郭孟璧,封良燕,田茂军等,人工培养铁皮石斛营养成分分析研究[J].云南化工,2006;33(2):15-16,43.
[18]夏鸿西,张明.石斛属植物化学成份研究进展[[J].重庆中草药研究,199939:54-56.
[19]叶庆华,赵维民,秦国伟.石斛属植物化学成分及生物活性研究进展[J]·药物化学进展,2004(3):113.
[20]陈照荣,来平凡,林巧,等.不同炮制方法对石斛中石斛碱和多糖溶出率的影响[J].浙江中医学院学报,2002,26(4):79.
[21] Chen K K,Chen A L.The alkaloid of chin-shiHu[J]. J Biol Chem,1935,11:653.
[22]金蓉鸾,孙继军,张远名.11种石斛的总生物碱的测定.南京药学院学报,1981,1:9-13.
[23]黄文魁.石解碱与新石解碱的构型[J].化学学报,1965,31:333-335.
[24]丁亚平,杨道麒,吴庆生,等.安徽霍山三种石斛总生物碱的测定及其分布规律研究[J].安徽农业大学学报,1994,21(4):503-506.
[25]李满飞,平田义正.中药石斛类多糖的含量测定[[J].中草药,1990,21(10):10-12.
[26]于力文,蔡永萍,张鹤英,等.安徽霍山三种石斛营养成分分析及其分布规律[J].安徽农业科学,1996,24(4):369-370.
[27]孙卓然,刘园,李晓云,等.石斛不同种、不同药用部位中多糖含量测定[J].时珍国医医药.2009,20(8):1886-1888.
[28]陈云龙,张铭,华九芬,等.细茎石斛不同部位有效成分及分布规律研究[J].中国中药杂志,2001,26(10):709-710.
[29]马国祥,菊池彻.鼓槌石斛中一新的联苄类化合物—-鼓槌石斛素[[J].药学学报,1996,31(3):222-225.
[30] Lee YH.石斛地上部份的体内外抗肿瘤菲类化合物[J].国外医学:中医中药分册,1996,18(2):42.
[31]李俊明.植物组织培养教程（第二版）[M].北京:中国农业大学出版社,2002.
[32] Chen Y H, Tsai Y J, Huang J Z,et al.Transcription analysis of peloric mutants of phalaenopsis orchids derived from tissueculture[J].Cell Research,2005,15(8):639-657.
[33]涂红艳.细茎石斛组织培养和提高拟原球茎有效成分积累的研究[D].广西:华南师范大学.2003.
[34] Chen J T, Chang C. Direct somatic embryogenesis on leaf explants of Oncidium Gower Ramsey and subsequent plantregeneration [J].Plant Cell Report,1999,19:143-149.
[35] Potor G..Method of vegetative propagation of Phalaenopsis stem cuttings [J]. Amer Orchid Soc Bull,1949,3(18):739-742.
[36]王凯基.植物生物学词典.上海:上海科技教育出版社1994.
[37]周俊辉,钟雪锋,蔡丁稳.铁皮石斛的组织培养与快速繁殖研究[J].仲恺农业技术学院学报,2005.18(1):23-26.
[38]张启香,方炎明.2005铁皮石斛组织培养及试管苗营养器官和原球茎的结构观察[J].西北植物学报,25.(9):1761-1765.
[39] Sheelavan S S,Murthy H N,Hema B P,et al.High frequency of protocom like bodies(PLBs)induction and plant regenerationfrom protocom and leaf sections of Aerides crispum[J].ScientiaHorticulturae,2005106:395-401.
[40]张艳,范俊安,李泉森,等.金钗石斛培养初步研究[J].时珍国医国药,2001,12(2):189-190.
[41]蒋波,杨存亮,黄捷,等.铁皮石斛原球茎生长分化及生根壮苗研究[J].玉林师范学院学报（自然科学）,2005,26(3):66-69.
[42]林丛发,钟爱清,林云斌,等.铁皮石斛类原球茎增殖和分化的研究[J].江西农业学报,2007,19(1):84-86,91.
[43]秦廷豪.铁皮石斛的组织培养与快速繁殖[J].热带农业科学,2008,28(1):25-29.
[44]詹忠根.铁皮石斛根尖诱导丛生芽研究[J].中草药,2006,37(6):928-931.
[45]洪香娇,喻晚之,熊正葵.铁皮石斛的组织培养与快速繁殖[J].现代园艺,2008（7）:9-13.
[46]魏小勇.铁皮石斛种胚原球茎液体悬浮培养研究[J].中国现代应用药学杂志,2005,22(6):471-473.
[47] Lam CLT,Lee SM.escence Culture of Some TropicalOrchid Varidties Singapore[J].Journal of PrimaryIndustries,996,24:35-41.
[48]王莲辉,姜运力,杨春华,等.铁皮石斛的组织培养与快速繁殖[J].中国林副特产,2007,3(88):47.
[49]高培元,陈健妙,甘铨,等.金钗石斛的茎段组织培养与植株再生[J].中草药,2002,33(11):1031-1033.
[50] Devi J,Borthakur B,Deka P C.Clonal Propagation of DenelrobiumMoschatum and Cymbidium Aloifolium ThroughShoot-tip Culture[J]. Journal of the Orchid Society of India,1997,11(1~2):19.
[51]陈媛,谢吉荣,铁皮石斛试管苗培养技术的研究[J].北方园艺,2009,(7):122-123.
[52]侯丕勇,郭顺星.悬浮培养的铁皮石斛原球茎在液体培养基上生长和分化的研究[J].中国中药杂志,2005,30(10):729-732.
[53]张启香,付素静,方炎明,等.铁皮石斛拟原球茎的发生过程[J].浙江林学院学报.2009,26(3):444-448
[54]王小菁,李玲.植物生长调节剂在植物组织培养中的应用[M].北京:化学工业出版社,2002.
[55] Loshiavo F, Filipini F, Cozzani F.Modulation of auxin-binding protein in cell suspersion I.Different responses of carrotembryo cultures[J].Plant physial,1991,97:60-64.
[56]冯昌林,吴天贵,罗敦,等.铁皮石斛组培快繁技术的研究[J].科技园地,2008,(11):9-10
[57]林江波,戴艺民,邹晖,等.铁皮石斛组培快繁技术研究进展[J].福建农业科技,2010,(1):25-26.
[58]莫昭展,贝学军,韦江萍,等.不同培养条件对铁皮石斛原球茎增殖的影响[J].安徽农业科学,2007,35(22):35-36.
[59]刘骅,张治国.铁皮石斛试管壮苗培养基的研究[J].中国中药杂志,1998,23(1):654-655
[60]蒋林,丁平,郑迎冬.添加剂对铁皮石斛组织培养和快速繁殖的影响[J].中药材,2003,26(8):539.
[61]唐桂香,黄福灯,周伟军.铁皮石斛的种胚萌发及其离体繁殖研究[J].中国中药杂志,2005,30(20):1583-1586.
[62]倪德祥译.光对植物组织培养的影响[M].国外植物组织培养,1985,1-21.
[63]李俊明.植埴物组织培养教程[M].北京:北京农业大学出版社,1992,347.
[64]梁钾贤,陈彪,张志胜光温互作和培养基中的附加成分对石斛试管苗生长的影响[J].植物生理学通讯,2006,42(3):401-405.
[65]倪勘武,来平凡,陈益平.阜阳野生石斛资源调查及栽培研究[J].中医正骨,2005,17(2):21-22.
[66]斯金平.诸燕,朱玉球.铁皮石斛人工栽培技术研究与应用进展[J].浙江林业科技,2009,29(6):66-70.
[67]黄勇.铁皮石斛组培苗移栽实验[J].广东农业科学,2008,12:66.
[68]李进进,廖俊杰,许继勇,等.铁皮石斛试管苗栽培技术研究[J].中药材,2006,29(11):1133-1134.
[69]蒙平,张向军,何新民.铁皮石斛组培苗移栽新技术[J].中国热带农业,2007(4):52-53．
[70]黄民权,黄步汉,蔡体育,等.铁皮石斛多糖的提取、分离和分析[J].中草药,1994,25(3):128-129.
[71]顾慧芬,晓君,周文婷,等.铁皮石斛试管苗快速生长与栽培研究及多糖含量测定[J].中成药,1999,21(12):658-659.
[72]高建平,金若敏,吴耀平,等.铁皮石斛原球茎与原药材免疫调节作用的比较研究[J].中药材,2002,23(7):487.
[73]罗建平,查学强,姜绍通.药用霍山石解原球茎的液体悬浮培养[J].中国中药杂志.2003,28(7):611-613.
[74]贾书华.光照对霍山石斛试管苗生长特性及有效成分积累的研究[D].安徽:安徽农业大学.2007.
[75]陈世峰.铁皮石斛的快速繁殖及其次生代谢产物的研究[D].湖北武汉:华中农业大学,2006.
[76]胡之璧,周吉燕,刘涤.不同培养方式对三尖杉培养细胞中生物碱组成和含量的影响[J].上海中医药大学学报,1999,13(l):60-61.
[77]唐建军,项田夫,等.植物次生代谢、离体培养条件下次生代谢物积累及其调控研究进展[J].中国野生植物资源.1998,17(4):l-6.
[78]冷佳奕.激素,培养方式与条件对铁皮石斛类原球茎有效成分影响的研究[D].广西:华南师范大学.2002.
[79]郑勇平,王春,俞继英,等.铁皮石斛试管苗移栽技术[J].林业科技开发,2006,20(6):56-58．
[80]李亚芳,张晓华,孙国明,石斛中生物碱和多糖含量的测定[J].中国药事,2002,16(7):426-428.
[81]陈颖,将世熙,周淡宜.鲜石斛口服液的工艺研究[J].中草药,1996,27(12):724-725
[82]唐金刚.金钗石斛快速繁殖及其生物碱积累的研究[D].贵州师范大学,2005.
[83]张晓申,王慧俞,杨录军,等.蝴蝶兰组织培养的研究[J].河北林果研究,2005,20(1):48-49.
[84]叶秀粦,程式君,王伏雄等.黑节草未成熟种子的形态发育及其在离体培养的表现[J].云南植物研究，1988,10(3):285-290.
[85]姚丽娟,徐晓薇,林绍生,等.蝴蝶兰原球茎增殖分化影响因子探讨[J].亚热带植物科学,2004,33(3):42-44.
[86]徐晓薇,柴明良,姚丽娟,等.蝴蝶兰类原球茎增殖和脱分化的研究[J].浙江农业科学,2006(4):401-403.
[87]马杰,何微红,崔波,等.蝴蝶兰原球茎状体的诱导及增殖[J].河南科学,2005,23(1);51-53.
[88]陈薇,寸守铣.铁皮石斛茎段离体快繁[J].植物生理学通讯,2002,38(2):145.
[89]张书萍,白石,陈丽静.铁皮石斛组织培养与快速繁殖[J].辽宁农业科学,2008(6):12-15.
[90]郑宽瑜,邓君浪,赵辉.铁皮石斛组培快繁技术体系研究[J].云南农业科技,2009:57-59.
[91]张治国,刘骅,王黎,等.铁皮石斛原球茎增殖的培养条件研究[J].中草药,1992,23(8):431-433.
[92]李进进,廖俊杰,许继勇,等.铁皮石斛试管苗栽培技术研究[J].中药材,2006,29(11):1133-1134.
[93]赵天榜,陈志秀,陈占宽,等.石斛组织培养与栽培技术的研究[J].河南农业大学学报,1994(2):128-133.
[94]王春.石斛兰组织培养及快繁技术研究[J].浙江林业科技,2002,22(2):77.
[95]尚杨娟.蝴蝶兰组培及器官分化的生理生化基础研究[D].南京林业大学.2009.
[96]洪香娇,喻晚之,熊正葵.铁皮石斛的组织培养与快速繁殖[J].现代林业,2008,7:9-13.
[97]薛庆善,肖渝平,林娟.体外培养的原理与技术[M].术出版社,2001.
[98]陈庭,叶庆生,刘伟.金钗石斛类原球茎诱导及增殖的正交试验[J].华南农业大学学报2005,26(3):60-63
[99]陈兆贵,谭俊.不同激素配比对铁皮石斛组织培养的影响研究[J].惠州学院学报,2006,26(3):11-14
[100] Kerns H R, Meyer M R. Tissue culture propagtion of Acer X freemanii using thidiazuron to stimulate shoot tipprolif-eration[J]. Hortsci,1986,21(5):1209-1210.
[101]王丽萍,梁淑云.铁皮石斛原球茎诱导和增殖研究[J].中国农学通报,2010,26(01):265-268.
[102]谌红辉,冯昌林,吴天贵,等.铁皮石解组培快繁技术研究[J].科技园地,2008,11:9-10.
[103] BrucemiI, Zwar J A. Cytokinin activity of some substi-tuted urea and thioureas[J]. Proc Roy Soc,1966,165:245-265.
[104]诸燕,张爱莲,何伯伟.铁皮石斛总生物碱含量变异规律[J].中国中药杂志,2010,35(18);2388-2390.
[105]吴刚,季祥彪,康冀川.石斛中多糖和生物碱含量的测定[J].山地农业生物学报,2008,27(3);274-278.
[106]张西玉.三种川产人工栽培石斛的多糖含量测定[J].乐山师范学院学报,2004,19(5);88-89.
[107] Yum J,Meng Q,Huang D Y, et al.Advancesin study on active components and pharmacology of Dendrobium[J]. Chin TraditPatentMed,2003,25(11):9l8-921.
[108]胡之璧,周吉燕,刘涤.不同培养方式对三尖杉培养细胞中生物碱组成和含量的影响[J].上海中医药大学学报,1999,13(l):60-61.
[109] Lam C L T, Lee S. Inflorescence Culture of some TropicalOrchid Varieties Singapore. Journal of PrimaryIndustries[J]1996,24:35
[110]李文安,植物细胞组织培养技术[M].北京科学出版社.1988.
[111]何铁光,苏江,王灿琴,等.铁皮石斛不同来源材料多糖和氨基酸含量的比较[J].广西农业科学,2007,38(1):32-34.
[112]宋经元,郭顺星,肖培根.铁皮石斛原球茎液体悬浮培养的研究[J].中草药.2004,35(9):1042-1046.
[113] Huang M Q, Lu Y J. Prospect on the callus culture of Dendrobium cadidumused as drugs [J].J Chin Med Mater,1998,21(11):543-545.
[114]许大全.光合速率、光合效率与作物产量[J],生物学通报,1999,8:11-13.
[115] Bucher D. Applied and fundamental aspects of plant cell, tissue and organ culture[J].Spinger-verlag,1977,668.
[116]赵德修,李茂寅,刑建民,等.光质、光强和光周期对水母雪莲愈伤组织生长和黄酮生物合成的影响[J].植物生理学报,1999,25(2):127-132.
[2]江苏新中医学院.中药大药典[M].上海:上海科学技术出版社,1985.
[3]586.贵州省中医研究所.贵州石斛的调查研究[M].1976.
[4]郑光植,梁峥.药用植物组织培养的研究[J].植物学报,1976,18(2):16.
[5]中华人名共和国药典编写委员会.中华人民共和国药典:人民卫生出版社,2000．
[6]国家药典委员会编.中国药典[M].北京:化学工业出版社,2005年版,62-63.
[7]俞群,王海燕.铁皮石斛在经口气管插管患者口腔溃疡中的应用[J].护士进修,2006,21(4):347-349.
[8]王康正,高文远.石斛属药用植物研究进展[J].中草药1997,28(10):633-635.
[9]张中建,阎小伟,铁皮石斛制剂免疫调节作用的实验研究[J].食品研究与开发,2004,25(2):34-35.
[10]王蜀秀,温远影.我国利用植物组织和细胞培养产生药用成分的研究概况[J].植物学报,1995,12(1):33-37.
[11]王宪楷,赵同芳.石斛属植物的化学成分与中药石解斛.药学通报,1986,2l(ll):666-669.
[12]陈晓梅,郭顺星.石斛属植物的化学成分与药理作用的研究进展[J].天然产物研究与开发,2001,13(1):70.
[13]郭洪波.铁皮石斛组培快繁及其抗癌作用的研究[D].沈阳:农业大学.2008.
[14]张光浓,毕志明,王峥涛,等.石斛属植物化学成分研究进展[J].中草药,2003,34(6):5-8.
[15]邓银华,徐康平,谭桂山.石解斛属植物化学成分与药理活性研究进展[J].中药材,2002,25(9):677-680.
[16]陈晓梅,郭顺星.石斛属植物化学成分和药理作用的研究进展[J].天然产物研究与开发,2002,13(l):70-75.
[17]郭孟璧,封良燕,田茂军等,人工培养铁皮石斛营养成分分析研究[J].云南化工,2006;33(2):15-16,43.
[18]夏鸿西,张明.石斛属植物化学成份研究进展[[J].重庆中草药研究,199939:54-56.
[19]叶庆华,赵维民,秦国伟.石斛属植物化学成分及生物活性研究进展[J]·药物化学进展,2004(3):113.
[20]陈照荣,来平凡,林巧,等.不同炮制方法对石斛中石斛碱和多糖溶出率的影响[J].浙江中医学院学报,2002,26(4):79.
[21] Chen K K,Chen A L.The alkaloid of chin-shiHu[J]. J Biol Chem,1935,11:653.
[22]金蓉鸾,孙继军,张远名.11种石斛的总生物碱的测定.南京药学院学报,1981,1:9-13.
[23]黄文魁.石解碱与新石解碱的构型[J].化学学报,1965,31:333-335.
[24]丁亚平,杨道麒,吴庆生,等.安徽霍山三种石斛总生物碱的测定及其分布规律研究[J].安徽农业大学学报,1994,21(4):503-506.
[25]李满飞,平田义正.中药石斛类多糖的含量测定[[J].中草药,1990,21(10):10-12.
[26]于力文,蔡永萍,张鹤英,等.安徽霍山三种石斛营养成分分析及其分布规律[J].安徽农业科学,1996,24(4):369-370.
[27]孙卓然,刘园,李晓云,等.石斛不同种、不同药用部位中多糖含量测定[J].时珍国医医药.2009,20(8):1886-1888.
[28]陈云龙,张铭,华九芬,等.细茎石斛不同部位有效成分及分布规律研究[J].中国中药杂志,2001,26(10):709-710.
[29]马国祥,菊池彻.鼓槌石斛中一新的联苄类化合物—-鼓槌石斛素[[J].药学学报,1996,31(3):222-225.
[30] Lee YH.石斛地上部份的体内外抗肿瘤菲类化合物[J].国外医学:中医中药分册,1996,18(2):42.
[31]李俊明.植物组织培养教程（第二版）[M].北京:中国农业大学出版社,2002.
[32] Chen Y H, Tsai Y J, Huang J Z,et al.Transcription analysis of peloric mutants of phalaenopsis orchids derived from tissueculture[J].Cell Research,2005,15(8):639-657.
[33]涂红艳.细茎石斛组织培养和提高拟原球茎有效成分积累的研究[D].广西:华南师范大学.2003.
[34] Chen J T, Chang C. Direct somatic embryogenesis on leaf explants of Oncidium Gower Ramsey and subsequent plantregeneration [J].Plant Cell Report,1999,19:143-149.
[35] Potor G..Method of vegetative propagation of Phalaenopsis stem cuttings [J]. Amer Orchid Soc Bull,1949,3(18):739-742.
[36]王凯基.植物生物学词典.上海:上海科技教育出版社1994.
[37]周俊辉,钟雪锋,蔡丁稳.铁皮石斛的组织培养与快速繁殖研究[J].仲恺农业技术学院学报,2005.18(1):23-26.
[38]张启香,方炎明.2005铁皮石斛组织培养及试管苗营养器官和原球茎的结构观察[J].西北植物学报,25.(9):1761-1765.
[39] Sheelavan S S,Murthy H N,Hema B P,et al.High frequency of protocom like bodies(PLBs)induction and plant regenerationfrom protocom and leaf sections of Aerides crispum[J].ScientiaHorticulturae,2005106:395-401.
[40]张艳,范俊安,李泉森,等.金钗石斛培养初步研究[J].时珍国医国药,2001,12(2):189-190.
[41]蒋波,杨存亮,黄捷,等.铁皮石斛原球茎生长分化及生根壮苗研究[J].玉林师范学院学报（自然科学）,2005,26(3):66-69.
[42]林丛发,钟爱清,林云斌,等.铁皮石斛类原球茎增殖和分化的研究[J].江西农业学报,2007,19(1):84-86,91.
[43]秦廷豪.铁皮石斛的组织培养与快速繁殖[J].热带农业科学,2008,28(1):25-29.
[44]詹忠根.铁皮石斛根尖诱导丛生芽研究[J].中草药,2006,37(6):928-931.
[45]洪香娇,喻晚之,熊正葵.铁皮石斛的组织培养与快速繁殖[J].现代园艺,2008（7）:9-13.
[46]魏小勇.铁皮石斛种胚原球茎液体悬浮培养研究[J].中国现代应用药学杂志,2005,22(6):471-473.
[47] Lam CLT,Lee SM.escence Culture of Some TropicalOrchid Varidties Singapore[J].Journal of PrimaryIndustries,996,24:35-41.
[48]王莲辉,姜运力,杨春华,等.铁皮石斛的组织培养与快速繁殖[J].中国林副特产,2007,3(88):47.
[49]高培元,陈健妙,甘铨,等.金钗石斛的茎段组织培养与植株再生[J].中草药,2002,33(11):1031-1033.
[50] Devi J,Borthakur B,Deka P C.Clonal Propagation of DenelrobiumMoschatum and Cymbidium Aloifolium ThroughShoot-tip Culture[J]. Journal of the Orchid Society of India,1997,11(1~2):19.
[51]陈媛,谢吉荣,铁皮石斛试管苗培养技术的研究[J].北方园艺,2009,(7):122-123.
[52]侯丕勇,郭顺星.悬浮培养的铁皮石斛原球茎在液体培养基上生长和分化的研究[J].中国中药杂志,2005,30(10):729-732.
[53]张启香,付素静,方炎明,等.铁皮石斛拟原球茎的发生过程[J].浙江林学院学报.2009,26(3):444-448
[54]王小菁,李玲.植物生长调节剂在植物组织培养中的应用[M].北京:化学工业出版社,2002.
[55] Loshiavo F, Filipini F, Cozzani F.Modulation of auxin-binding protein in cell suspersion I.Different responses of carrotembryo cultures[J].Plant physial,1991,97:60-64.
[56]冯昌林,吴天贵,罗敦,等.铁皮石斛组培快繁技术的研究[J].科技园地,2008,(11):9-10
[57]林江波,戴艺民,邹晖,等.铁皮石斛组培快繁技术研究进展[J].福建农业科技,2010,(1):25-26.
[58]莫昭展,贝学军,韦江萍,等.不同培养条件对铁皮石斛原球茎增殖的影响[J].安徽农业科学,2007,35(22):35-36.
[59]刘骅,张治国.铁皮石斛试管壮苗培养基的研究[J].中国中药杂志,1998,23(1):654-655
[60]蒋林,丁平,郑迎冬.添加剂对铁皮石斛组织培养和快速繁殖的影响[J].中药材,2003,26(8):539.
[61]唐桂香,黄福灯,周伟军.铁皮石斛的种胚萌发及其离体繁殖研究[J].中国中药杂志,2005,30(20):1583-1586.
[62]倪德祥译.光对植物组织培养的影响[M].国外植物组织培养,1985,1-21.
[63]李俊明.植埴物组织培养教程[M].北京:北京农业大学出版社,1992,347.
[64]梁钾贤,陈彪,张志胜光温互作和培养基中的附加成分对石斛试管苗生长的影响[J].植物生理学通讯,2006,42(3):401-405.
[65]倪勘武,来平凡,陈益平.阜阳野生石斛资源调查及栽培研究[J].中医正骨,2005,17(2):21-22.
[66]斯金平.诸燕,朱玉球.铁皮石斛人工栽培技术研究与应用进展[J].浙江林业科技,2009,29(6):66-70.
[67]黄勇.铁皮石斛组培苗移栽实验[J].广东农业科学,2008,12:66.
[68]李进进,廖俊杰,许继勇,等.铁皮石斛试管苗栽培技术研究[J].中药材,2006,29(11):1133-1134.
[69]蒙平,张向军,何新民.铁皮石斛组培苗移栽新技术[J].中国热带农业,2007(4):52-53．
[70]黄民权,黄步汉,蔡体育,等.铁皮石斛多糖的提取、分离和分析[J].中草药,1994,25(3):128-129.
[71]顾慧芬,晓君,周文婷,等.铁皮石斛试管苗快速生长与栽培研究及多糖含量测定[J].中成药,1999,21(12):658-659.
[72]高建平,金若敏,吴耀平,等.铁皮石斛原球茎与原药材免疫调节作用的比较研究[J].中药材,2002,23(7):487.
[73]罗建平,查学强,姜绍通.药用霍山石解原球茎的液体悬浮培养[J].中国中药杂志.2003,28(7):611-613.
[74]贾书华.光照对霍山石斛试管苗生长特性及有效成分积累的研究[D].安徽:安徽农业大学.2007.
[75]陈世峰.铁皮石斛的快速繁殖及其次生代谢产物的研究[D].湖北武汉:华中农业大学,2006.
[76]胡之璧,周吉燕,刘涤.不同培养方式对三尖杉培养细胞中生物碱组成和含量的影响[J].上海中医药大学学报,1999,13(l):60-61.
[77]唐建军,项田夫,等.植物次生代谢、离体培养条件下次生代谢物积累及其调控研究进展[J].中国野生植物资源.1998,17(4):l-6.
[78]冷佳奕.激素,培养方式与条件对铁皮石斛类原球茎有效成分影响的研究[D].广西:华南师范大学.2002.
[79]郑勇平,王春,俞继英,等.铁皮石斛试管苗移栽技术[J].林业科技开发,2006,20(6):56-58．
[80]李亚芳,张晓华,孙国明,石斛中生物碱和多糖含量的测定[J].中国药事,2002,16(7):426-428.
[81]陈颖,将世熙,周淡宜.鲜石斛口服液的工艺研究[J].中草药,1996,27(12):724-725
[82]唐金刚.金钗石斛快速繁殖及其生物碱积累的研究[D].贵州师范大学,2005.
[83]张晓申,王慧俞,杨录军,等.蝴蝶兰组织培养的研究[J].河北林果研究,2005,20(1):48-49.
[84]叶秀粦,程式君,王伏雄等.黑节草未成熟种子的形态发育及其在离体培养的表现[J].云南植物研究，1988,10(3):285-290.
[85]姚丽娟,徐晓薇,林绍生,等.蝴蝶兰原球茎增殖分化影响因子探讨[J].亚热带植物科学,2004,33(3):42-44.
[86]徐晓薇,柴明良,姚丽娟,等.蝴蝶兰类原球茎增殖和脱分化的研究[J].浙江农业科学,2006(4):401-403.
[87]马杰,何微红,崔波,等.蝴蝶兰原球茎状体的诱导及增殖[J].河南科学,2005,23(1);51-53.
[88]陈薇,寸守铣.铁皮石斛茎段离体快繁[J].植物生理学通讯,2002,38(2):145.
[89]张书萍,白石,陈丽静.铁皮石斛组织培养与快速繁殖[J].辽宁农业科学,2008(6):12-15.
[90]郑宽瑜,邓君浪,赵辉.铁皮石斛组培快繁技术体系研究[J].云南农业科技,2009:57-59.
[91]张治国,刘骅,王黎,等.铁皮石斛原球茎增殖的培养条件研究[J].中草药,1992,23(8):431-433.
[92]李进进,廖俊杰,许继勇,等.铁皮石斛试管苗栽培技术研究[J].中药材,2006,29(11):1133-1134.
[93]赵天榜,陈志秀,陈占宽,等.石斛组织培养与栽培技术的研究[J].河南农业大学学报,1994(2):128-133.
[94]王春.石斛兰组织培养及快繁技术研究[J].浙江林业科技,2002,22(2):77.
[95]尚杨娟.蝴蝶兰组培及器官分化的生理生化基础研究[D].南京林业大学.2009.
[96]洪香娇,喻晚之,熊正葵.铁皮石斛的组织培养与快速繁殖[J].现代林业,2008,7:9-13.
[97]薛庆善,肖渝平,林娟.体外培养的原理与技术[M].术出版社,2001.
[98]陈庭,叶庆生,刘伟.金钗石斛类原球茎诱导及增殖的正交试验[J].华南农业大学学报2005,26(3):60-63
[99]陈兆贵,谭俊.不同激素配比对铁皮石斛组织培养的影响研究[J].惠州学院学报,2006,26(3):11-14
[100] Kerns H R, Meyer M R. Tissue culture propagtion of Acer X freemanii using thidiazuron to stimulate shoot tipprolif-eration[J]. Hortsci,1986,21(5):1209-1210.
[101]王丽萍,梁淑云.铁皮石斛原球茎诱导和增殖研究[J].中国农学通报,2010,26(01):265-268.
[102]谌红辉,冯昌林,吴天贵,等.铁皮石解组培快繁技术研究[J].科技园地,2008,11:9-10.
[103] BrucemiI, Zwar J A. Cytokinin activity of some substi-tuted urea and thioureas[J]. Proc Roy Soc,1966,165:245-265.
[104]诸燕,张爱莲,何伯伟.铁皮石斛总生物碱含量变异规律[J].中国中药杂志,2010,35(18);2388-2390.
[105]吴刚,季祥彪,康冀川.石斛中多糖和生物碱含量的测定[J].山地农业生物学报,2008,27(3);274-278.
[106]张西玉.三种川产人工栽培石斛的多糖含量测定[J].乐山师范学院学报,2004,19(5);88-89.
[107] Yum J,Meng Q,Huang D Y, et al.Advancesin study on active components and pharmacology of Dendrobium[J]. Chin TraditPatentMed,2003,25(11):9l8-921.
[108]胡之璧,周吉燕,刘涤.不同培养方式对三尖杉培养细胞中生物碱组成和含量的影响[J].上海中医药大学学报,1999,13(l):60-61.
[109] Lam C L T, Lee S. Inflorescence Culture of some TropicalOrchid Varieties Singapore. Journal of PrimaryIndustries[J]1996,24:35
[110]李文安,植物细胞组织培养技术[M].北京科学出版社.1988.
[111]何铁光,苏江,王灿琴,等.铁皮石斛不同来源材料多糖和氨基酸含量的比较[J].广西农业科学,2007,38(1):32-34.
[112]宋经元,郭顺星,肖培根.铁皮石斛原球茎液体悬浮培养的研究[J].中草药.2004,35(9):1042-1046.
[113] Huang M Q, Lu Y J. Prospect on the callus culture of Dendrobium cadidumused as drugs [J].J Chin Med Mater,1998,21(11):543-545.
[114]许大全.光合速率、光合效率与作物产量[J],生物学通报,1999,8:11-13.
[115] Bucher D. Applied and fundamental aspects of plant cell, tissue and organ culture[J].Spinger-verlag,1977,668.
[116]赵德修,李茂寅,刑建民,等.光质、光强和光周期对水母雪莲愈伤组织生长和黄酮生物合成的影响[J].植物生理学报,1999,25(2):127-132.