蝴蝶兰快繁技术的研究
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摘要
蝴蝶兰具有较高的观赏价值和经济价值。其花形、花色变化无穷，花期可达三个月，长期以来深受消费者的喜爱。近二十年来，台湾的蝴蝶兰产业蓬勃发展，中国内地的蝴蝶兰市场也在迅速扩大。对于国内蝴蝶兰产业化的发展来说，建立和完善快繁技术是当务之急。
通过类原球茎进行快繁是蝴蝶兰商业化生产的一个重要途径，本试验对蝴蝶兰叶片类原球茎的诱导和增殖作了初步探讨。
对于蝴蝶兰叶片类原球茎诱导而言，进行叶片培养时先要经过两个星期的暗处理，然后移到明处可以提高类原球茎诱导率；培养基中6-BA是诱导类原球茎必不可少的因素，添加腺嘌呤10mg／L可以提高类原球茎诱导率；试验还在明确6-BA剂量(10mg/L)的前提下，利用正交设计方法L_(16)(4~5)探讨了培养基中NAA、肌醇、VB_1、VB_6、琼脂粉五种因素对蝴蝶兰叶片诱导类原球茎的影响。结果表明：NAA是影响蝴蝶兰叶片诱导类原球茎的主要因素，VB_1、VB_6及琼脂粉次之，肌醇的影响程度较弱。试验的结果分析表明，蝴蝶兰叶片诱导类原球茎的最优组合是NAA1mg／L，肌醇100mg／L，VB_15mg／L，琼脂粉8g／L。
对于类原球茎的增殖，试验采用二次正交旋转组合设计法探讨了五个因素(NAA、蔗糖、麦芽糖、VB_1、VB_6)对蝴蝶兰类原球茎增殖的影响。结果表明：对于类原球茎增殖来说NAA的浓度最为重要，其次是蔗糖、麦芽糖和VB_1，VB_6对类原球茎增殖没有影响。并且筛选出了蝴蝶兰类原球茎增殖的最优组合是：NAA为0.9mg/L、蔗糖10g／L、麦芽糖15g／L、VB_1 1mg／L，类原球茎的增殖系数可以达到理论最大值7.33。
The genus Phalaenopsis (Orchidaceae) comprises a number of Commercially important species that have produced a wide range of attractive consumers. Phalaenopsis is a kind of popular potted-plant due to its wide variation of flower color and long florescence. In the past 20 years, the industry of Phalaenopsis in Taiwan has grown vigorously, In China, the market for Phalaenopsis has rapidly expanded too, but technique of rapid propagation lag behind.
To the production of Phalaenopsis, induction and multiplication of Protocorm-like-body (PLB) are vital steps.
In this experiment, top leaves of shoots cultured in darkness for two weeks were chosen that could raise the rate of induction. 6-BA is necessary for Phalaenopsis to induce PLBs, which suitable concentration is 10mg/L we have found. And, Adding adenine lOmg/L can raise induction rate too; But other factors also play important roles in induction. In the study, the different effects of NAA, myo-Inositol, Thiamine HC1, Pyridoxine HC1 and agar on protocorm formation from leaves of Phalaenopsis were compared. Experiments were done using the orthogonal design. The Results showed that: (1) NAA played the best important role in inducing. The effects of Thiamine HC1, Pyridxin HC1 and agar were stronger than myo-Inositol. (2) The best combination was NAA Img/L, myo-Inositol 200mg/L, Thiamine HC1 5mg/L, and agar 8g/L.
For multiplication of PLBs, the effects of NAA, Sucrose, Maltose, Thiamine HC1, Pyridoxin HC1 were examined by orthogonal rotation combination design. The results revealed that NAA is the radical factor, and sucrose, maltose, Thamine HC1, also have important effects, but Pyridoxine HC1 is not necessary to multiplication. The optimum
combination of above factors were obtained, which is NAA 0.9mg/L, sucrose 10g/L, maltose 5g/L, Thamine HCI Img/L. The highest ratio of multiplication of PLB is 7.33 in theory.
通过类原球茎进行快繁是蝴蝶兰商业化生产的一个重要途径，本试验对蝴蝶兰叶片类原球茎的诱导和增殖作了初步探讨。
对于蝴蝶兰叶片类原球茎诱导而言，进行叶片培养时先要经过两个星期的暗处理，然后移到明处可以提高类原球茎诱导率；培养基中6-BA是诱导类原球茎必不可少的因素，添加腺嘌呤10mg／L可以提高类原球茎诱导率；试验还在明确6-BA剂量(10mg/L)的前提下，利用正交设计方法L_(16)(4~5)探讨了培养基中NAA、肌醇、VB_1、VB_6、琼脂粉五种因素对蝴蝶兰叶片诱导类原球茎的影响。结果表明：NAA是影响蝴蝶兰叶片诱导类原球茎的主要因素，VB_1、VB_6及琼脂粉次之，肌醇的影响程度较弱。试验的结果分析表明，蝴蝶兰叶片诱导类原球茎的最优组合是NAA1mg／L，肌醇100mg／L，VB_15mg／L，琼脂粉8g／L。
对于类原球茎的增殖，试验采用二次正交旋转组合设计法探讨了五个因素(NAA、蔗糖、麦芽糖、VB_1、VB_6)对蝴蝶兰类原球茎增殖的影响。结果表明：对于类原球茎增殖来说NAA的浓度最为重要，其次是蔗糖、麦芽糖和VB_1，VB_6对类原球茎增殖没有影响。并且筛选出了蝴蝶兰类原球茎增殖的最优组合是：NAA为0.9mg/L、蔗糖10g／L、麦芽糖15g／L、VB_1 1mg／L，类原球茎的增殖系数可以达到理论最大值7.33。
The genus Phalaenopsis (Orchidaceae) comprises a number of Commercially important species that have produced a wide range of attractive consumers. Phalaenopsis is a kind of popular potted-plant due to its wide variation of flower color and long florescence. In the past 20 years, the industry of Phalaenopsis in Taiwan has grown vigorously, In China, the market for Phalaenopsis has rapidly expanded too, but technique of rapid propagation lag behind.
To the production of Phalaenopsis, induction and multiplication of Protocorm-like-body (PLB) are vital steps.
In this experiment, top leaves of shoots cultured in darkness for two weeks were chosen that could raise the rate of induction. 6-BA is necessary for Phalaenopsis to induce PLBs, which suitable concentration is 10mg/L we have found. And, Adding adenine lOmg/L can raise induction rate too; But other factors also play important roles in induction. In the study, the different effects of NAA, myo-Inositol, Thiamine HC1, Pyridoxine HC1 and agar on protocorm formation from leaves of Phalaenopsis were compared. Experiments were done using the orthogonal design. The Results showed that: (1) NAA played the best important role in inducing. The effects of Thiamine HC1, Pyridxin HC1 and agar were stronger than myo-Inositol. (2) The best combination was NAA Img/L, myo-Inositol 200mg/L, Thiamine HC1 5mg/L, and agar 8g/L.
For multiplication of PLBs, the effects of NAA, Sucrose, Maltose, Thiamine HC1, Pyridoxin HC1 were examined by orthogonal rotation combination design. The results revealed that NAA is the radical factor, and sucrose, maltose, Thamine HC1, also have important effects, but Pyridoxine HC1 is not necessary to multiplication. The optimum
combination of above factors were obtained, which is NAA 0.9mg/L, sucrose 10g/L, maltose 5g/L, Thamine HCI Img/L. The highest ratio of multiplication of PLB is 7.33 in theory.
引文
尤崇魁．蝴蝶兰栽培技术[M]．台湾园艺世界出版社．1988，7．
王介元等．应用回归旋转设计确定最佳施肥量的探讨[J]．土壤通报，1987(2)：87-89
王怀宇．蝴蝶兰的快速无性繁殖[J]．园艺学报，1989，16(1)：74-77
刘荣维，梅庆超，崔元方等．丛生芽蝴蝶兰无性快繁的新途径[J]．热带作物，1993，14(2)：105-167．
何子育．蝴蝶兰的组织培养[J]．热带作物科技，1999，(5)：26-27．
张秀清，王志武．蝴蝶兰实生苗不同器官的离体培养[J]．植物学通报，1996，13(1)：50．
张秀清，王志武．蝴蝶兰实生苗原球茎诱导研究[J]．莱阳农学院学报，1995，12(1)：44-46．
张国治，严霄，王凤仙．正交设计在组织培养研究中的应用[J]．植物生理学报，1985，21(5)：214-216．
张治国，严霄，王风仙，等．正交设计在植物组织培养基研究中的应用[J]．植物生理学通讯．1985，(5)：46—48．
张剑斌，吕庆茹，刘金生．应用正交旋转设计确定银中扬最佳施肥量的试验[J]．防护林科技，1998(3)：25-28．
李曙辉，万其发，管怀骥等．V_B对番茄根尖离体培养的效应[J]．安徽农业科学，2002，30(3)：418-4
杨美纯．外部因子对蝴蝶兰叶片原球茎状体发生的影响[J]．广西植物，2000，20(1)：42-46．
肖兵．农业多因素试验设计与统计分析[M]．长沙：湖南科技出版社，1985：216-280．
孟玉玲，谷祝乎．正交设计在诱导植物体细胞胚胎发生中的应用[J]．西北植物学报，1995，13(1)：10-15
胡松华．热带兰花[M]．北京：中国林业出版社，2002，45．
唐启义，冯光明．实用统计分析及其计算机处理平台[M]．北京：中国农业出版社，1997，77-91．
奚元龄，颜昌敬．植物细胞培养手册．北京：科学出版社，1983：57-58．
徐位力，罗涣亮，范恩友．二次正交旋转组合设计对马占相思组培增殖培养基的优化[J]．广西植物，2002(11)：517-520．
秦贺兰，孙红梅．蝴蝶兰研究进展[J]．河南职业技术师范学院学报，2002，30(2)：31-35．
彭立新，王妹．蝴蝶兰组织培养快繁研究[J]．天津农业科学，1999，5(2)：27-29．
彭宇，钟昌珍，宗良炳．二次正交旋转组合设计在家蝇幼虫人工养殖上的应用研究[J]．湖北大学学报(自然科学版)，1997(3)：83-88．
曾宋君，彭晓明．蝴蝶兰的组织培养和快速繁殖[J]，武汉植物学研究，2000，18(4)：344-346
熊丽，吴丽芳．观赏花卉的组织培养与大规模生产[M]．北京：化学工业出版社，2003，71-76．
蔡平里．图解兰花繁殖最新技术．台湾：淑馨出版社．1996，28．
蔡培思，张志诚，李彦生．2次正交旋转设计统筹上下两茬作物的试验研究简报．耕作与栽培，1993(5)：7-8．
谭文澄，戴策刚．观赏植物组织培养技术[M]．北京：中国林业出版社，1991，247-257．
谭文澄，戴策刚．观赏植物组织培养技术[M]．北京：中国林业出版社，1991．237-247
广东省植物研究所遗传室，广东师范学院数学系．用正交与不完全区组试验法提高籼稻花药培养成功率[J]．广东师范学院学报，1976，(1)：100-112
Chang,C and Chang WC.Plant regeneration from callus of Cymbidium ensifolium var "miserrcors". Plant Cell Rep, 1998(17):251-255.
Chen Y.C.,Chang C.,Chang.W.C.A reliable protocol for plant regeneration from callus of Phalaenopsis.In Vitro Cellular & Developmental Biology-Plant,2000(36):420-423.
Cho UH,Kasha KJ.Ethylene production and embryogenesis from anther cultures of
barley (Hordeum vulgare). Plant Cell Rep. 1989(8):415-419.
Fu FML.Studies on the tissue culture of orchids.I.Clonal propagation of Phalaenopsis by lateral buds from flower stems.Orchid Reciew, 1978(86):308-310.
Griesbaeh R J.The use of indoleacetylamino acids in the in vitro propagation of Phalaenopsis. Scientia Horticulture, 1983(19):363-366.
Ishii Y, Takamura T, Goi M, et al.Callus induction and somatic embryogenesis of Phalaenopsis Plant [J]. Cell Reports, 1998 (17): 446-450.
Ken Tokuhara, Masahiro Mii. Induction of embryogenic callus and cell suspension culture from shoot tips excised from flower stalk buds of Phalaenopsis(Orchidaceae). In Vitro Cell. Dev.Biol.-Plant.2001(37):457-461.
Lam T.W, Ernst R, Arditi J, et al. The effects of complex additives and 6-(-dimethylal-lylamino)-purine on the proliferation of Phalaenopsis protocorms.Lindeyana, 1991(6):24-26.
Lin CC. In-vitro cultue of flower stalk internodes of Phalaenopsis and Doritaenopsis. Lindleyana, 1986(1): 158-163.
Park SY, Murthy HN, Pack KY. Rapid propagation of Phalaenopsis from floral stalk-derived leaves.In Vitro Cellular & Developmental Biology-Plant,2002(38): 168-172.
Park SY,.Yeung EC, hakrabary D.An efficient direct induction of protocorm-like-bodies from leaf subepidermal cells of Doritaenopsis hybrid using thin-section culture.Plant Cell Rep,2000(21):46-51.
Reynolds TH.A possible role for ethylene during IAA induced pollen embryogenesis in anther cultures of Solanum carolinense L.Am J Bot 1987(74):967-969.
Tanaka M.,Hasegaw A,Goi M.Studies on the clonal propagation of monopodial orchid by tissue eulture.I.Formation of protocorm-like bodies from leaf tissue in Phalaen-opsis and Vanda. Journal of the Japanese Society for Horticultural Science, 1975(44):47-58.
Tanaka M, Senada Y, Hasegawa A. Plantle formation by root-tip culture in Phalaen-
opsis.American Ochid Society Bulletin, 1976(45): 1022-1024.
Thomes TH,Hare PD,Van Staden J,Phytochrome and cytokinin responses.Plant Growth Regulation, 1997(23): 105-122.
王介元等．应用回归旋转设计确定最佳施肥量的探讨[J]．土壤通报，1987(2)：87-89
王怀宇．蝴蝶兰的快速无性繁殖[J]．园艺学报，1989，16(1)：74-77
刘荣维，梅庆超，崔元方等．丛生芽蝴蝶兰无性快繁的新途径[J]．热带作物，1993，14(2)：105-167．
何子育．蝴蝶兰的组织培养[J]．热带作物科技，1999，(5)：26-27．
张秀清，王志武．蝴蝶兰实生苗不同器官的离体培养[J]．植物学通报，1996，13(1)：50．
张秀清，王志武．蝴蝶兰实生苗原球茎诱导研究[J]．莱阳农学院学报，1995，12(1)：44-46．
张国治，严霄，王凤仙．正交设计在组织培养研究中的应用[J]．植物生理学报，1985，21(5)：214-216．
张治国，严霄，王风仙，等．正交设计在植物组织培养基研究中的应用[J]．植物生理学通讯．1985，(5)：46—48．
张剑斌，吕庆茹，刘金生．应用正交旋转设计确定银中扬最佳施肥量的试验[J]．防护林科技，1998(3)：25-28．
李曙辉，万其发，管怀骥等．V_B对番茄根尖离体培养的效应[J]．安徽农业科学，2002，30(3)：418-4
杨美纯．外部因子对蝴蝶兰叶片原球茎状体发生的影响[J]．广西植物，2000，20(1)：42-46．
肖兵．农业多因素试验设计与统计分析[M]．长沙：湖南科技出版社，1985：216-280．
孟玉玲，谷祝乎．正交设计在诱导植物体细胞胚胎发生中的应用[J]．西北植物学报，1995，13(1)：10-15
胡松华．热带兰花[M]．北京：中国林业出版社，2002，45．
唐启义，冯光明．实用统计分析及其计算机处理平台[M]．北京：中国农业出版社，1997，77-91．
奚元龄，颜昌敬．植物细胞培养手册．北京：科学出版社，1983：57-58．
徐位力，罗涣亮，范恩友．二次正交旋转组合设计对马占相思组培增殖培养基的优化[J]．广西植物，2002(11)：517-520．
秦贺兰，孙红梅．蝴蝶兰研究进展[J]．河南职业技术师范学院学报，2002，30(2)：31-35．
彭立新，王妹．蝴蝶兰组织培养快繁研究[J]．天津农业科学，1999，5(2)：27-29．
彭宇，钟昌珍，宗良炳．二次正交旋转组合设计在家蝇幼虫人工养殖上的应用研究[J]．湖北大学学报(自然科学版)，1997(3)：83-88．
曾宋君，彭晓明．蝴蝶兰的组织培养和快速繁殖[J]，武汉植物学研究，2000，18(4)：344-346
熊丽，吴丽芳．观赏花卉的组织培养与大规模生产[M]．北京：化学工业出版社，2003，71-76．
蔡平里．图解兰花繁殖最新技术．台湾：淑馨出版社．1996，28．
蔡培思，张志诚，李彦生．2次正交旋转设计统筹上下两茬作物的试验研究简报．耕作与栽培，1993(5)：7-8．
谭文澄，戴策刚．观赏植物组织培养技术[M]．北京：中国林业出版社，1991，247-257．
谭文澄，戴策刚．观赏植物组织培养技术[M]．北京：中国林业出版社，1991．237-247
广东省植物研究所遗传室，广东师范学院数学系．用正交与不完全区组试验法提高籼稻花药培养成功率[J]．广东师范学院学报，1976，(1)：100-112
Chang,C and Chang WC.Plant regeneration from callus of Cymbidium ensifolium var "miserrcors". Plant Cell Rep, 1998(17):251-255.
Chen Y.C.,Chang C.,Chang.W.C.A reliable protocol for plant regeneration from callus of Phalaenopsis.In Vitro Cellular & Developmental Biology-Plant,2000(36):420-423.
Cho UH,Kasha KJ.Ethylene production and embryogenesis from anther cultures of
barley (Hordeum vulgare). Plant Cell Rep. 1989(8):415-419.
Fu FML.Studies on the tissue culture of orchids.I.Clonal propagation of Phalaenopsis by lateral buds from flower stems.Orchid Reciew, 1978(86):308-310.
Griesbaeh R J.The use of indoleacetylamino acids in the in vitro propagation of Phalaenopsis. Scientia Horticulture, 1983(19):363-366.
Ishii Y, Takamura T, Goi M, et al.Callus induction and somatic embryogenesis of Phalaenopsis Plant [J]. Cell Reports, 1998 (17): 446-450.
Ken Tokuhara, Masahiro Mii. Induction of embryogenic callus and cell suspension culture from shoot tips excised from flower stalk buds of Phalaenopsis(Orchidaceae). In Vitro Cell. Dev.Biol.-Plant.2001(37):457-461.
Lam T.W, Ernst R, Arditi J, et al. The effects of complex additives and 6-(-dimethylal-lylamino)-purine on the proliferation of Phalaenopsis protocorms.Lindeyana, 1991(6):24-26.
Lin CC. In-vitro cultue of flower stalk internodes of Phalaenopsis and Doritaenopsis. Lindleyana, 1986(1): 158-163.
Park SY, Murthy HN, Pack KY. Rapid propagation of Phalaenopsis from floral stalk-derived leaves.In Vitro Cellular & Developmental Biology-Plant,2002(38): 168-172.
Park SY,.Yeung EC, hakrabary D.An efficient direct induction of protocorm-like-bodies from leaf subepidermal cells of Doritaenopsis hybrid using thin-section culture.Plant Cell Rep,2000(21):46-51.
Reynolds TH.A possible role for ethylene during IAA induced pollen embryogenesis in anther cultures of Solanum carolinense L.Am J Bot 1987(74):967-969.
Tanaka M.,Hasegaw A,Goi M.Studies on the clonal propagation of monopodial orchid by tissue eulture.I.Formation of protocorm-like bodies from leaf tissue in Phalaen-opsis and Vanda. Journal of the Japanese Society for Horticultural Science, 1975(44):47-58.
Tanaka M, Senada Y, Hasegawa A. Plantle formation by root-tip culture in Phalaen-
opsis.American Ochid Society Bulletin, 1976(45): 1022-1024.
Thomes TH,Hare PD,Van Staden J,Phytochrome and cytokinin responses.Plant Growth Regulation, 1997(23): 105-122.