大苞鞘石斛（Dendrobium wardianum）离体培养与快速繁殖的研究
|
摘要
本论文以大苞鞘石斛(Dendrobium wardianum)的种胚为试验材料,对其胚培养和快繁技术进行了系统的研究,建立了大苞鞘石斛的快繁技术体系,为种质资源的保存奠定了基础。
试验结果表明,在开花第5 d时授粉(花瓣完全展开当天)结实率最高,结实率为80.95%,种子有胚率为90.73%。将种胚播于B5+绵白糖20 g?L-1+椰乳20%+ pH 5.4上,培养50 d后萌发率达79%。而后转接于花宝1号3 g?L-1+蛋白胨2 g?L-1+NAA 0.2 mg?L-1+椰乳10% +马铃薯提取液10%+葡萄糖20 g?L-1+pH 5.6上进行分化培养,培养时间70 d。选取分化后的小苗接入CHB+ NAA 0.5 + 6-BA 0.2 +椰乳10% +蔗糖30 g?L-1+pH 5.8进行丛生芽增殖培养,培养150 d后增殖系数为6.68。将增殖后的无菌大苗转入花宝2号3 g?L-1+蛋白胨2 g?L-1 +IBA 1 mg?L-1+ CW 10%+蔗糖30 g?L-1+pH 5.6进行生根诱导,培养45 d后诱导率100%,平均根数2.3条。最后在花宝1号3 g?L-1+蛋白胨2 g?L-1+蔗糖30 g?L-1 +活性炭1 g?L-1+pH 5.6做壮苗生根培养,培养90 d后即可炼苗并移栽于松树皮+珍珠岩+兰花基石(1:1:0.5)中,基质采用高压蒸汽灭菌消毒(121℃,120 min),移栽成活率92 %。
利用拟原球茎增殖的方法:选取植株健康、长势健壮,株高在2㎝以上的无菌苗,每1~2节为一段,接入诱导培养基1/2MS+2.4-D 0.2 mg?L-1+蔗糖30g?L-1+pH 5.8,20 d后,拟原球茎约为1.2~1.5㎜时将拟原球茎剥离,并接入同种培养基继续诱导;15~20 d转瓶增殖培养,最优培养基为1/2MS+NAA 1 mg?L-1+椰乳10%+蔗糖30 g?L-1+pH 5.6。25~30 d需转接1次,增殖系数为7.33。拟原球茎分化最优培养基为1/2MS+NAA 0.5 mg?L-1+蔗糖30g?L-1+椰乳10 %+pH 5.6。
The paper took the seeds of Dendrobium. wardianum as research objects. This thesis had a systematic research on the embryo culture and rapid propagation. It was laid the foundation for the preservation of genetic resources and rapid propagation system establishment.
The results showed that the best pollination time of the female flower(mother) was after blooming of 1 day when not only would the capsule setting rate be reached 80.95% but the plump seed ratio be reached 90.73%.Seeds of Den. wardianum cultured on the media of B5 +Sugar20 g?L-1 + CW20 % + pH 5.4 for 50 days and the germination rate was reached 79%. The optimal differentiation medium of protocorm from former stage was Hyponex No.1 3 g?L-1+Peptone 2 g?L-1+NAA 0.2 mg?L-1+CW 10% + Potato extract 10%+Plucose 20 g?L-1+pH5.6 and the protocorm was cultured 70 days. The proliferation medium for adventitious buds was CHB+ NAA 0.5 + 6-BA 0.2+CW10%+Sugar 30g?L-1 +pH 5.8.It was 150 days for this culture stage and the multiplication coefficient was 6.68.The aseptic seedling which obtained from the former culture stage were cultured on the root induction media of Hyponex No.2 3g?L-1+ Peptone 2 g?L-1 +IBA1 mg?L-1+ CW 10%+ Sugar 30 g?L-1 +pH 5.6 for 45 days ,with the rooting rate at 100%,and the average root number of 2.3. The last stage of in vitro was strong plantlets and rootage. The optimal medium was Hyponex No.1 3 g?L-1+ Peptone 2 g?L-1 +Sugar 30 g?L-1 + Activated carbon 1 g?L-1+pH 5.6. 90 days later the plantlets were hardening-seedling and transplanting in the materials mixed pine bark with perlite and orchid matrix, ,with the survival rate at 92%.The culture substrate was sterilized with high pressure steam(121℃, 120 min).
Aseptic seedling with strong growth vigor and 2㎝ height was adopted for the PLBs induction materials. Shoot segments with 1-2 nodes were excised from Den. wardianum plants raised in vitro. The segments were cultured on the media of 1/2MS+2.4-D 0.2 mg?L-1+Sugar 30g?L-1+pH 5.8 for 20 days.When the shoot bud diameter reached to 1.2-1.5㎜, it must be detached and transferred to the same media for 15-20 days.Then the PLBs subcultured on 1/2MS+NAA 1 mg?L-1+CW10%+Sugar 30 g?L-1 +pH 5.6 for proliferation with the proliferation rate at 7.33.The PLBs transferred to the 1/2MS media within 25-30 days.The optimal PLBs differentiation medium was 1/2MS+NAA 0.5 mg?L-1+Sugar 30 g?L-1+CW 10%+pH 5.6.
试验结果表明,在开花第5 d时授粉(花瓣完全展开当天)结实率最高,结实率为80.95%,种子有胚率为90.73%。将种胚播于B5+绵白糖20 g?L-1+椰乳20%+ pH 5.4上,培养50 d后萌发率达79%。而后转接于花宝1号3 g?L-1+蛋白胨2 g?L-1+NAA 0.2 mg?L-1+椰乳10% +马铃薯提取液10%+葡萄糖20 g?L-1+pH 5.6上进行分化培养,培养时间70 d。选取分化后的小苗接入CHB+ NAA 0.5 + 6-BA 0.2 +椰乳10% +蔗糖30 g?L-1+pH 5.8进行丛生芽增殖培养,培养150 d后增殖系数为6.68。将增殖后的无菌大苗转入花宝2号3 g?L-1+蛋白胨2 g?L-1 +IBA 1 mg?L-1+ CW 10%+蔗糖30 g?L-1+pH 5.6进行生根诱导,培养45 d后诱导率100%,平均根数2.3条。最后在花宝1号3 g?L-1+蛋白胨2 g?L-1+蔗糖30 g?L-1 +活性炭1 g?L-1+pH 5.6做壮苗生根培养,培养90 d后即可炼苗并移栽于松树皮+珍珠岩+兰花基石(1:1:0.5)中,基质采用高压蒸汽灭菌消毒(121℃,120 min),移栽成活率92 %。
利用拟原球茎增殖的方法:选取植株健康、长势健壮,株高在2㎝以上的无菌苗,每1~2节为一段,接入诱导培养基1/2MS+2.4-D 0.2 mg?L-1+蔗糖30g?L-1+pH 5.8,20 d后,拟原球茎约为1.2~1.5㎜时将拟原球茎剥离,并接入同种培养基继续诱导;15~20 d转瓶增殖培养,最优培养基为1/2MS+NAA 1 mg?L-1+椰乳10%+蔗糖30 g?L-1+pH 5.6。25~30 d需转接1次,增殖系数为7.33。拟原球茎分化最优培养基为1/2MS+NAA 0.5 mg?L-1+蔗糖30g?L-1+椰乳10 %+pH 5.6。
The paper took the seeds of Dendrobium. wardianum as research objects. This thesis had a systematic research on the embryo culture and rapid propagation. It was laid the foundation for the preservation of genetic resources and rapid propagation system establishment.
The results showed that the best pollination time of the female flower(mother) was after blooming of 1 day when not only would the capsule setting rate be reached 80.95% but the plump seed ratio be reached 90.73%.Seeds of Den. wardianum cultured on the media of B5 +Sugar20 g?L-1 + CW20 % + pH 5.4 for 50 days and the germination rate was reached 79%. The optimal differentiation medium of protocorm from former stage was Hyponex No.1 3 g?L-1+Peptone 2 g?L-1+NAA 0.2 mg?L-1+CW 10% + Potato extract 10%+Plucose 20 g?L-1+pH5.6 and the protocorm was cultured 70 days. The proliferation medium for adventitious buds was CHB+ NAA 0.5 + 6-BA 0.2+CW10%+Sugar 30g?L-1 +pH 5.8.It was 150 days for this culture stage and the multiplication coefficient was 6.68.The aseptic seedling which obtained from the former culture stage were cultured on the root induction media of Hyponex No.2 3g?L-1+ Peptone 2 g?L-1 +IBA1 mg?L-1+ CW 10%+ Sugar 30 g?L-1 +pH 5.6 for 45 days ,with the rooting rate at 100%,and the average root number of 2.3. The last stage of in vitro was strong plantlets and rootage. The optimal medium was Hyponex No.1 3 g?L-1+ Peptone 2 g?L-1 +Sugar 30 g?L-1 + Activated carbon 1 g?L-1+pH 5.6. 90 days later the plantlets were hardening-seedling and transplanting in the materials mixed pine bark with perlite and orchid matrix, ,with the survival rate at 92%.The culture substrate was sterilized with high pressure steam(121℃, 120 min).
Aseptic seedling with strong growth vigor and 2㎝ height was adopted for the PLBs induction materials. Shoot segments with 1-2 nodes were excised from Den. wardianum plants raised in vitro. The segments were cultured on the media of 1/2MS+2.4-D 0.2 mg?L-1+Sugar 30g?L-1+pH 5.8 for 20 days.When the shoot bud diameter reached to 1.2-1.5㎜, it must be detached and transferred to the same media for 15-20 days.Then the PLBs subcultured on 1/2MS+NAA 1 mg?L-1+CW10%+Sugar 30 g?L-1 +pH 5.6 for proliferation with the proliferation rate at 7.33.The PLBs transferred to the 1/2MS media within 25-30 days.The optimal PLBs differentiation medium was 1/2MS+NAA 0.5 mg?L-1+Sugar 30 g?L-1+CW 10%+pH 5.6.
引文
[1]曾宋君,胡松华.石斛兰[M].广州:广东科技出版社, 2004:3~31, 53-54.
[2]何清正,朱根发.商品洋兰[M].广州:广东科技出版社, 2005:34.
[3]吉占和.中国植物志[M].北京:科学出版社, 1999, 19:75~146.
[4]王燕,李振坚,彭红明.石斛兰资源·生产·应用[M].北京:中国林业出版社, 2007:152~154.
[5]胡松华.洋兰赏培600问[M].北京:中国林业出版社, 2005:78~81.
[6]陈心启,吉占和,罗毅波.中国野生兰科植物彩色图鉴[M].北京:科学出版社, 1999:150~193.
[7]李少球.世界洋兰[M].广州:广东科技出版社, 1996:33~41.
[8]陈宇勒.洋兰欣赏与栽培图说[M].北京:金盾出版社, 2004:37~49.
[9]彭锐,范俊安,张艳.石斛属药用植物种质资源研究进展[J].时珍国医国药, 2001, 12(3):273~274.
[10]范黎,郭顺星,肖培根.密花石斛等六种兰科植物菌根的显微结构研究[J].植物学通报, 2000, 17(1):73~79.
[11]陈瑞蕊,施亚琴,林先贵.杯鞘石斛组培苗接种菌根真菌和细菌肥的研究[J].中草药, 2005, 36(11):1718~1720.
[12]陈瑞蕊,施亚琴,林先贵.兰科菌根真菌对石斛组培苗的接种效应[J].土壤, 2004, 36 (6): 658~661.
[13]田丹青,曹群阳,丁华侨.春石斛低温催花试验初探[J].浙江农业科学, 2007, 1:38~39.
[14]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, 1998:74.
[15] Singh F. Encapsulation of Spathoglottis plicataprotocorms [J]. Lindleyana, 1991, 6: 61~62.
[16] Sharma A, Tandon P, Anjani K. Regeneration of Dendrobium wardianum from synthetic seeds [J]. Indian Journal of Experimental Biology, 1992, 30: 747~748.
[17] Corrie S, Tandon P. Propagation of Cymbidium giganteum through high frequency conservation of encapsulated protocorms underin vivoandin vitroconditions [J]. Indian Journal of Experimental Biology, 1993, 31: 61~64.
[18] Malemnganba H, Ray B K, Bhattacharyya S, et al. Regeneration of encapsulated protocorms of PHaius tankervilliaestored at low temperature [J]. Indian Journal of Experimental Biology, 1996, 34: 802~805.
[19]郭顺星,曹文芩,张集慧.铁皮石斛人工种子制作流程及发芽研究[J].中草药, 1996, 27 (2): 105~107.
[20]张铭,魏小勇,黄华荣.铁皮石斛人工种子固形包埋系统的研究[J].园艺学报, 2001, 28 (5): 435~439.
[21] Saiprasad G V S, Raghuveer Polisetty. Propagataion of three orchid genera using encapsulatedprotocorm-like bodies[J]. In Vitro Cellular and Developmental Biology, 2003, 39:42~48.
[22]李振坚.春石斛规模化繁殖要领[J].中国花卉园艺, 2006, 22:20~22.
[23]裘文达,园艺植物组织培养[M].上海:上海科学技术出版社, 1986:120.
[24]叶秀嶙,程式君.黑节草未成熟种子的形态发育及其在离体培养时的表现[J].云南植物研究, 1988, 10(30):285.
[25]王兰新,曾彩云.齿瓣石斛的胚培养[J].林业调查规划, 2006, 31(5):128~130.
[26]丁长春.齿瓣石斛的胚培养技术及其快速繁殖研究[J].云南农业科技, 2004, 6:20.
[27]宋锡全,龚宁,詹孝慈.金钗石斛种子非共生萌发和种质保存[J].贵州师范大学学报(自然科学版), 2004, 22(2):13~16.
[28]陈之林,曾宋君,段俊.独角石斛的离体繁殖[J].植物生理学通讯, 2004, 40(3):342.
[29]毛秀华,金家兴,刘作易.环草石斛种子萌发培养的研究[J].贵州农业科学, 2004, 32(3):48~49.
[30]常俊,丁小余,保曙琳.喇叭唇石斛组织培养的研究[J].中国中药杂志, 2004, 29(4):313~317.
[31]曾宋君,程式君,张京丽.五种石斛兰的胚培养及其快速繁殖研究[J],园艺学报, 1998, 25(1):75~80.
[32]孔琼,袁盛勇,查应洪.梳唇石斛成熟胚的离体培养和植株再生[J].植物生理学通讯, 2005, 41(5):644.
[33]李小军,刘石泉,路群.香蕉提取物对霍山石斛原球茎增殖的影响[J].上海师范大学学报(自然科学版), 2004 , 33(4):74~77.
[34]杨念,陈之林,段俊.羚羊石斛的离体快速繁殖[J].植物生理学通讯, 2006, 42(2):248.
[35].李小军,刘石泉,潘维陵.香蕉提取物对霍山石斛试管苗壮苗的影响[J],江苏大学学报(自然科学版), 2004, 25(6):469~472.
[36]白音,包英华,王文全.玫瑰石斛的组织培养及快速繁殖[J].植物生理学通讯, 2006, 42(5):903.
[37]黄家平,戴思兰.兰花组织培养研究综述[J].广东园林, 1997(2):29~31.
[38]黄志明,林庆良,佘慧敏.蝴蝶石斛兰工厂化育苗技术的研究[J].莆田学院学报, 2002, 9(3):22~26.
[39]徐桂萍,吕剑涛,柏新富.黄草石斛的组培和快速繁殖[J].烟台教育学院学报, 2004, 10(4):59~62.
[40]罗岚,关仕港,刘建昌.秋石斛兰离体快速繁殖研究[J].佛山科学技术学院学报(自然科学版), 2004, 22(2):69~71.
[41] Sharma A, Tandon P. In vitro culture of Dendrobium Wardianum :MorpHogenetic effects of some nitrogenous adjuvats[J]. Indian Journal Plant Physiol. , 1992, 35(1):80~85.
[42] Jonojit R, Nirmalya B. Induction of callus and plant regeneration from shoot-tip explants of Dendrobium fimbriatum Lindl. var. oculatu Hk. f. [J]. Scientia Horticulturae 2003, 97:333-340.
[43] Ravindra B M, Gangadhar S M, Nataraja K. Micropropagation of Dendrobium nobile from shoot tip sections[J]. Journal of Plant Physiology, 2005, 162:473~478.
[44]李浚明.植物组织培养教程[M].第2版.北京:中国农业大学出版社, 2002: 44.
[45]孙廷.霍山石斛离体培养研究[D].西北农林科技大学, 2004:12~18.
[46]孙廷,杨玉珍,陶杰.金钗石斛的组织培养和快繁技术[J].东农业大学学报(自然科学版), 2005, 36(1):47~50.
[47]乙引,张宇斌.粉花石斛的组织培养和植株再生[J].植物生理学通讯, 2004, 40(1):64.
[48]林泋君.小美石斛茎段培养的研究[J] .中国农学通报, 2006, 22(2):292~294.
[49] Nihar Ranjan Nayak, Shiba Prasad Rath, Satyanarayan Patnaik. In vitro propagation of three epipHytic orchids, Cymbidium aloifolium (L. ) SW. , Dendrobium apHyllum (Roxb. ) Fisch. and Dendrobium moschatum (Buch-Ham) SW. through thidiazuron-induced high frequency shoot proliferation[J]. Scientia Horticultura, 1997, 71:243~250.
[50]林茵,刘伟. PP_(333)对离体培养金钗石斛根生长的影响[A]. 2005年全国植物生长物质研讨会论文摘要汇编[C], 2005:70.
[51]廖飞雄,王恒明,黄群慧.春石斛的工厂化快速繁殖技术研究[J].西南农业学报, 2006, 19(5):943.
[52]周月坤.兰花苗端和茎节离体培养[J].植物学集刊, 1983(1):119~122.
[53] Martin K P, Madassery J. Rapid in vitro propagation of Dendrobium hybrids through direct shoot formation from foliar explants, and protocorm-like bodies[J]. Scientia Horticulturae, 2006, 108:95~99.
[54] Chung H. H., Chen J. T., Chang W. C.. Plant regeneration through direct somatic embryogenesis from leaf explants of Dendrobium[J]. Biologia Plantarum, 2007, 51 (2): 346~350.
[55]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.
[56]赵天榜,陈志秀,陈占宽.石斛组织培养与栽培技术的研究[J].河南大学学报, 1994, 28(2):128~133.
[57]王光远,刘培,许智宏.石斛离体培养中ABA对有到花芽形成的影响[J].植物学报, 1995, 37(5):374~378.
[58]王再花.金钗石斛和西茎石斛试管开花研究[D].华南师范大学, 2005.
[59] Sim G E, Loh C S, Goh C J. High frequency early in vitro flowering of Dendrobium Madame Thong-In (Orchidaceae)[J]. Plant Cell Rep. 2007, 26:383~393.
[60]国家药典委员会.中华人民共和国药典(一部)[M].北京:北京工业出版社, 2005. 62.
[61]王艳,潘扬,马国祥.西双版纳地区的石斛资源调查及鉴定[J].中国野生植物资源, 1995, 2:41~43.
[62]管艳红,马洁,张丽霞.西双版纳石斛属药用植物资源[J].中国野生植物资源, 2004, 23(6):38~39.
[63] Luan V Q, Thien N Q, Khiem D. In Vitro germination capacity and Plant Recovery of Some Native and Rare Orchids[J]. Proceedings of International workshop on biotechnology in agriculture, 2006:1~3.
[64] Sharma A, Tandon P, Anjani Kumar. Regeneration of Dendrobium wardianum Warner(Orchidaceae) from Synthetic Seeds[J]. Indian Journal of Experimental Biology, 1992, 30:747-748.
[65]王素英,宋锡全,蔡瑞.金钗石斛传粉生物学和种子萌发特性研究[J].种子, 2006, 25(6): 23-26.
[66]王学奎主编.植物生理生化试验原理和技术[M].北京:高等教育出版社, 2006: 134~136, 190~192.
[67] PHilip J. Kauth1, Wagner A. In vitro seed culture and seedling development of Calopogon tuberosus[J]. Tissue and Organ Culture, 2006, (85): 91–102.
[68]詹忠根,徐程,张铭.兰科植物原球茎(类原球茎)的形态建成[J].种子, 2002, 5: 36~37.
[2]何清正,朱根发.商品洋兰[M].广州:广东科技出版社, 2005:34.
[3]吉占和.中国植物志[M].北京:科学出版社, 1999, 19:75~146.
[4]王燕,李振坚,彭红明.石斛兰资源·生产·应用[M].北京:中国林业出版社, 2007:152~154.
[5]胡松华.洋兰赏培600问[M].北京:中国林业出版社, 2005:78~81.
[6]陈心启,吉占和,罗毅波.中国野生兰科植物彩色图鉴[M].北京:科学出版社, 1999:150~193.
[7]李少球.世界洋兰[M].广州:广东科技出版社, 1996:33~41.
[8]陈宇勒.洋兰欣赏与栽培图说[M].北京:金盾出版社, 2004:37~49.
[9]彭锐,范俊安,张艳.石斛属药用植物种质资源研究进展[J].时珍国医国药, 2001, 12(3):273~274.
[10]范黎,郭顺星,肖培根.密花石斛等六种兰科植物菌根的显微结构研究[J].植物学通报, 2000, 17(1):73~79.
[11]陈瑞蕊,施亚琴,林先贵.杯鞘石斛组培苗接种菌根真菌和细菌肥的研究[J].中草药, 2005, 36(11):1718~1720.
[12]陈瑞蕊,施亚琴,林先贵.兰科菌根真菌对石斛组培苗的接种效应[J].土壤, 2004, 36 (6): 658~661.
[13]田丹青,曹群阳,丁华侨.春石斛低温催花试验初探[J].浙江农业科学, 2007, 1:38~39.
[14]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, 1998:74.
[15] Singh F. Encapsulation of Spathoglottis plicataprotocorms [J]. Lindleyana, 1991, 6: 61~62.
[16] Sharma A, Tandon P, Anjani K. Regeneration of Dendrobium wardianum from synthetic seeds [J]. Indian Journal of Experimental Biology, 1992, 30: 747~748.
[17] Corrie S, Tandon P. Propagation of Cymbidium giganteum through high frequency conservation of encapsulated protocorms underin vivoandin vitroconditions [J]. Indian Journal of Experimental Biology, 1993, 31: 61~64.
[18] Malemnganba H, Ray B K, Bhattacharyya S, et al. Regeneration of encapsulated protocorms of PHaius tankervilliaestored at low temperature [J]. Indian Journal of Experimental Biology, 1996, 34: 802~805.
[19]郭顺星,曹文芩,张集慧.铁皮石斛人工种子制作流程及发芽研究[J].中草药, 1996, 27 (2): 105~107.
[20]张铭,魏小勇,黄华荣.铁皮石斛人工种子固形包埋系统的研究[J].园艺学报, 2001, 28 (5): 435~439.
[21] Saiprasad G V S, Raghuveer Polisetty. Propagataion of three orchid genera using encapsulatedprotocorm-like bodies[J]. In Vitro Cellular and Developmental Biology, 2003, 39:42~48.
[22]李振坚.春石斛规模化繁殖要领[J].中国花卉园艺, 2006, 22:20~22.
[23]裘文达,园艺植物组织培养[M].上海:上海科学技术出版社, 1986:120.
[24]叶秀嶙,程式君.黑节草未成熟种子的形态发育及其在离体培养时的表现[J].云南植物研究, 1988, 10(30):285.
[25]王兰新,曾彩云.齿瓣石斛的胚培养[J].林业调查规划, 2006, 31(5):128~130.
[26]丁长春.齿瓣石斛的胚培养技术及其快速繁殖研究[J].云南农业科技, 2004, 6:20.
[27]宋锡全,龚宁,詹孝慈.金钗石斛种子非共生萌发和种质保存[J].贵州师范大学学报(自然科学版), 2004, 22(2):13~16.
[28]陈之林,曾宋君,段俊.独角石斛的离体繁殖[J].植物生理学通讯, 2004, 40(3):342.
[29]毛秀华,金家兴,刘作易.环草石斛种子萌发培养的研究[J].贵州农业科学, 2004, 32(3):48~49.
[30]常俊,丁小余,保曙琳.喇叭唇石斛组织培养的研究[J].中国中药杂志, 2004, 29(4):313~317.
[31]曾宋君,程式君,张京丽.五种石斛兰的胚培养及其快速繁殖研究[J],园艺学报, 1998, 25(1):75~80.
[32]孔琼,袁盛勇,查应洪.梳唇石斛成熟胚的离体培养和植株再生[J].植物生理学通讯, 2005, 41(5):644.
[33]李小军,刘石泉,路群.香蕉提取物对霍山石斛原球茎增殖的影响[J].上海师范大学学报(自然科学版), 2004 , 33(4):74~77.
[34]杨念,陈之林,段俊.羚羊石斛的离体快速繁殖[J].植物生理学通讯, 2006, 42(2):248.
[35].李小军,刘石泉,潘维陵.香蕉提取物对霍山石斛试管苗壮苗的影响[J],江苏大学学报(自然科学版), 2004, 25(6):469~472.
[36]白音,包英华,王文全.玫瑰石斛的组织培养及快速繁殖[J].植物生理学通讯, 2006, 42(5):903.
[37]黄家平,戴思兰.兰花组织培养研究综述[J].广东园林, 1997(2):29~31.
[38]黄志明,林庆良,佘慧敏.蝴蝶石斛兰工厂化育苗技术的研究[J].莆田学院学报, 2002, 9(3):22~26.
[39]徐桂萍,吕剑涛,柏新富.黄草石斛的组培和快速繁殖[J].烟台教育学院学报, 2004, 10(4):59~62.
[40]罗岚,关仕港,刘建昌.秋石斛兰离体快速繁殖研究[J].佛山科学技术学院学报(自然科学版), 2004, 22(2):69~71.
[41] Sharma A, Tandon P. In vitro culture of Dendrobium Wardianum :MorpHogenetic effects of some nitrogenous adjuvats[J]. Indian Journal Plant Physiol. , 1992, 35(1):80~85.
[42] Jonojit R, Nirmalya B. Induction of callus and plant regeneration from shoot-tip explants of Dendrobium fimbriatum Lindl. var. oculatu Hk. f. [J]. Scientia Horticulturae 2003, 97:333-340.
[43] Ravindra B M, Gangadhar S M, Nataraja K. Micropropagation of Dendrobium nobile from shoot tip sections[J]. Journal of Plant Physiology, 2005, 162:473~478.
[44]李浚明.植物组织培养教程[M].第2版.北京:中国农业大学出版社, 2002: 44.
[45]孙廷.霍山石斛离体培养研究[D].西北农林科技大学, 2004:12~18.
[46]孙廷,杨玉珍,陶杰.金钗石斛的组织培养和快繁技术[J].东农业大学学报(自然科学版), 2005, 36(1):47~50.
[47]乙引,张宇斌.粉花石斛的组织培养和植株再生[J].植物生理学通讯, 2004, 40(1):64.
[48]林泋君.小美石斛茎段培养的研究[J] .中国农学通报, 2006, 22(2):292~294.
[49] Nihar Ranjan Nayak, Shiba Prasad Rath, Satyanarayan Patnaik. In vitro propagation of three epipHytic orchids, Cymbidium aloifolium (L. ) SW. , Dendrobium apHyllum (Roxb. ) Fisch. and Dendrobium moschatum (Buch-Ham) SW. through thidiazuron-induced high frequency shoot proliferation[J]. Scientia Horticultura, 1997, 71:243~250.
[50]林茵,刘伟. PP_(333)对离体培养金钗石斛根生长的影响[A]. 2005年全国植物生长物质研讨会论文摘要汇编[C], 2005:70.
[51]廖飞雄,王恒明,黄群慧.春石斛的工厂化快速繁殖技术研究[J].西南农业学报, 2006, 19(5):943.
[52]周月坤.兰花苗端和茎节离体培养[J].植物学集刊, 1983(1):119~122.
[53] Martin K P, Madassery J. Rapid in vitro propagation of Dendrobium hybrids through direct shoot formation from foliar explants, and protocorm-like bodies[J]. Scientia Horticulturae, 2006, 108:95~99.
[54] Chung H. H., Chen J. T., Chang W. C.. Plant regeneration through direct somatic embryogenesis from leaf explants of Dendrobium[J]. Biologia Plantarum, 2007, 51 (2): 346~350.
[55]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.
[56]赵天榜,陈志秀,陈占宽.石斛组织培养与栽培技术的研究[J].河南大学学报, 1994, 28(2):128~133.
[57]王光远,刘培,许智宏.石斛离体培养中ABA对有到花芽形成的影响[J].植物学报, 1995, 37(5):374~378.
[58]王再花.金钗石斛和西茎石斛试管开花研究[D].华南师范大学, 2005.
[59] Sim G E, Loh C S, Goh C J. High frequency early in vitro flowering of Dendrobium Madame Thong-In (Orchidaceae)[J]. Plant Cell Rep. 2007, 26:383~393.
[60]国家药典委员会.中华人民共和国药典(一部)[M].北京:北京工业出版社, 2005. 62.
[61]王艳,潘扬,马国祥.西双版纳地区的石斛资源调查及鉴定[J].中国野生植物资源, 1995, 2:41~43.
[62]管艳红,马洁,张丽霞.西双版纳石斛属药用植物资源[J].中国野生植物资源, 2004, 23(6):38~39.
[63] Luan V Q, Thien N Q, Khiem D. In Vitro germination capacity and Plant Recovery of Some Native and Rare Orchids[J]. Proceedings of International workshop on biotechnology in agriculture, 2006:1~3.
[64] Sharma A, Tandon P, Anjani Kumar. Regeneration of Dendrobium wardianum Warner(Orchidaceae) from Synthetic Seeds[J]. Indian Journal of Experimental Biology, 1992, 30:747-748.
[65]王素英,宋锡全,蔡瑞.金钗石斛传粉生物学和种子萌发特性研究[J].种子, 2006, 25(6): 23-26.
[66]王学奎主编.植物生理生化试验原理和技术[M].北京:高等教育出版社, 2006: 134~136, 190~192.
[67] PHilip J. Kauth1, Wagner A. In vitro seed culture and seedling development of Calopogon tuberosus[J]. Tissue and Organ Culture, 2006, (85): 91–102.
[68]詹忠根,徐程,张铭.兰科植物原球茎(类原球茎)的形态建成[J].种子, 2002, 5: 36~37.