墨西哥落羽杉组织培养的研究
|
摘要
本研究以墨西哥落羽杉为对象，进行了体细胞胚胎发生和植株再生、器官发生和植株再生、试管无性系快速繁殖等方面的研究，主要结果如下：
1．以墨西哥落羽杉(Taxodium mucronatum)种子萌发后的下胚轴和子叶为外植体，进行墨西哥落羽杉直接体细胞胚胎发生的研究。结果表明：子叶外植体未能诱导出体细胞胚，与子叶相比，下胚轴更有利于体细胞胚胎的发生，但下胚轴培养时间的长短也直接影响体细胞胚胎发生频率。取种子萌发、子叶展开一周后的下胚轴在DCR+BA2.0mg／l+IBA0.1mg／l的培养基上直接体细胞胚发生频率可达24.5％。诱导出的体细胞胚及时转入不添加任何激素的DCR基本培养基中以促进其萌发。
2．以墨西哥落羽杉种子萌发后的下胚轴和子叶为外植体，进行器官发生的研究。结果表明：子叶未能诱导产生不定芽。下胚轴在DCR+BA2.0mg／l+IBA0.1mg／l+TDZ0.01mg／l的培养基上不定芽诱导频率为58.5％，低浓度的TDZ与BA配合使用有利于下胚轴不定芽的诱导与分化。诱导出的不定芽继代在不添加任何激素的DCR基本培养基中伸长培养，在不添加任何激素的1／2DCR基本培养基中进行生根诱导，生根诱导率可达70％以上。
3．通过幼苗和腋芽两种不同外植体来建立墨西哥落羽杉试管无性系。结果表明：幼苗在DCR+BA0.5mg／l+IBA0.2mg／l培养基上诱导分化效果最好，分化频率80％以上，平均每个外植体产芽2-5个。诱导出针叶基潜伏芽后，转入DCR+500mg／lGln中伸长生长。Gln能够促进针叶基潜伏芽的伸长生长。试管苗在3／4DCR+0.05％AC培养基中生根效果最好，生根率可达78％。选择节间较长，生长旺盛，茎干较粗壮的小苗诱导生根过程往往容易完成。根长达2-4cm时，开瓶炼苗3-7天后即可进行移栽。
4．DCR+IBA0.05mg／l+BA0.025mg／l培养基最有利于茎段腋芽的抽梢与伸长生长。腋芽针叶基潜伏芽的诱导以DCR+BA0.3mg／l+NAA0.1mg／l+Gln500mg／l培养基为最佳，加入Gln可明显提高腋芽针叶基潜伏芽的诱导率。TDZ不适宜墨西哥落羽杉腋芽针叶基潜伏芽的诱导。针叶基潜伏芽在DCR+0.1％AC的培养基中伸长效果最好，继代2-3次后即可剪切较长的芽用于生根。以腋芽针叶基潜伏芽增殖方式建立起来的试管无性系诱导生根困难，生根频率较低。
上述研究为发展墨西哥落羽杉器官发生、体细胞胚胎发生诱导技术、以及墨西哥落羽杉无性系的快速繁殖，进而开展遗传转化研究奠定了良好的基础。
Somatic embryogenesis, direct organogenesis and plantlet clone for Taxodium mucronatum were conducted in this research.. The main results as follows:
1. Hypocotyl and cotyledon developed from the seeds of Taxodium mucronatum were cultured as the initiation explants for direct inducement of somatic embryos. Comparing with cotyledon, hypocotyl was better for us to induce somatic embryos, and the hypocotyl culture times directly effect on inducement frequency of somatic embryos also. The hypocotyl direct inducement frequency was 24.5% on the DCR medium with 2.0mg/l BA+0.1mg/lIBA. It is necessary for the development of somatic embryos by transferring the culture materials on hormone-free DCR medium.
2. Hypocotyl and cotyledon developed from the seeds of Taxodium mucronatum were also cultured as the initiation explants for direct organogenesis. It was not able to induce adventitious buds by the explant of cotyledon. With hypocotyl explants, the highest inducement frequency of adventitious buds was 58.5% on the DCR medium with 2.0mg/l BA, 0.1mg/1 IBA and 0.01mg/1 TDZ. The medium with low concentration of TDZ and BA was good to inducement and differentiation of adventitious buds of hypocotyl. Adventitious buds cultured on hormone-free DCR medium for elongation, subculture every four or five weeks, and cut the buds for rooting inducement after subculture two or three times. On hormone-free 1/2DCR medium the rooting frequency could be above 70%.
3. There are two kinds of explants to be used for plantlet clone of Taxodium mucronatum. The first one was the young seedling developed from the seeds. The results were shown that: dormant buds induced are sensitive to the different hormone concentration. Cultured young seedlings on DCR medium supplemented with 0.5 mg/1 BA and 0.2mg/l IBA was the best way of inducement, and with inducement frequency higher than 80%, mean number of dormant buds was around 2-5. The medium should be wiped off hormone when dormant buds induced. For elongation of the dormant buds, the suitable culture medium was DCR+Gln500mg/l. It can advance elongation of dormant buds when DCR basal medium supplemented with 500mg/lGln. The medium of 3/4DCR+0.05%AC is used for rooting, and the highest rooting rate is 78%. Using hormone to induce roots could produce mass of callus on the end top of cuttings and it was not benefit to the plantlets survive after transplant. In addition, it was easily for the plantlets with long internode and vigorous stem to induce root system. When the roots developed to 2-4cm long, the plant could be transplanted after 3-7 days acclimation
4. The second kinds of explants for plantlet clone was axillary buds developed from stem. The best inducement and elongation medium for axillary buds was DCR medium supplemented with 0.05mg/l IBA and 0.025mg/l BA, and inducement frequency up to 100%. Cutting down longer axillary buds from stem was good to other smaller axillary buds developed, and more new axillary buds produced from stem. The proper medium for inducing dormant buds from axillary buds was DCR medium supplemented with 0.3mg/l BA, 0.1 mg/1 NAA and 500mg/l Gln. The frequency of inducement was significant increased by adding Gin. TDZ was not suitable for dormant buds inducing from axillary buds. The best medium for elongating induced dormant buds was DCR+0.1%AC, subculturing 2-3 times, cutting down for rooting. The best medium for rooting in this research is 3/4DCR medium supplemented with 0.1 mg/1 IBA and 0.05mg/l NAA, however, the rooting rate was only 25%.
The frameworks for somatic embryogenesis, organogenesis, and micropropagation as mentioned above were established, and then the genie transformation of Taxodium mucronatum can be conducted on this system also.
1．以墨西哥落羽杉(Taxodium mucronatum)种子萌发后的下胚轴和子叶为外植体，进行墨西哥落羽杉直接体细胞胚胎发生的研究。结果表明：子叶外植体未能诱导出体细胞胚，与子叶相比，下胚轴更有利于体细胞胚胎的发生，但下胚轴培养时间的长短也直接影响体细胞胚胎发生频率。取种子萌发、子叶展开一周后的下胚轴在DCR+BA2.0mg／l+IBA0.1mg／l的培养基上直接体细胞胚发生频率可达24.5％。诱导出的体细胞胚及时转入不添加任何激素的DCR基本培养基中以促进其萌发。
2．以墨西哥落羽杉种子萌发后的下胚轴和子叶为外植体，进行器官发生的研究。结果表明：子叶未能诱导产生不定芽。下胚轴在DCR+BA2.0mg／l+IBA0.1mg／l+TDZ0.01mg／l的培养基上不定芽诱导频率为58.5％，低浓度的TDZ与BA配合使用有利于下胚轴不定芽的诱导与分化。诱导出的不定芽继代在不添加任何激素的DCR基本培养基中伸长培养，在不添加任何激素的1／2DCR基本培养基中进行生根诱导，生根诱导率可达70％以上。
3．通过幼苗和腋芽两种不同外植体来建立墨西哥落羽杉试管无性系。结果表明：幼苗在DCR+BA0.5mg／l+IBA0.2mg／l培养基上诱导分化效果最好，分化频率80％以上，平均每个外植体产芽2-5个。诱导出针叶基潜伏芽后，转入DCR+500mg／lGln中伸长生长。Gln能够促进针叶基潜伏芽的伸长生长。试管苗在3／4DCR+0.05％AC培养基中生根效果最好，生根率可达78％。选择节间较长，生长旺盛，茎干较粗壮的小苗诱导生根过程往往容易完成。根长达2-4cm时，开瓶炼苗3-7天后即可进行移栽。
4．DCR+IBA0.05mg／l+BA0.025mg／l培养基最有利于茎段腋芽的抽梢与伸长生长。腋芽针叶基潜伏芽的诱导以DCR+BA0.3mg／l+NAA0.1mg／l+Gln500mg／l培养基为最佳，加入Gln可明显提高腋芽针叶基潜伏芽的诱导率。TDZ不适宜墨西哥落羽杉腋芽针叶基潜伏芽的诱导。针叶基潜伏芽在DCR+0.1％AC的培养基中伸长效果最好，继代2-3次后即可剪切较长的芽用于生根。以腋芽针叶基潜伏芽增殖方式建立起来的试管无性系诱导生根困难，生根频率较低。
上述研究为发展墨西哥落羽杉器官发生、体细胞胚胎发生诱导技术、以及墨西哥落羽杉无性系的快速繁殖，进而开展遗传转化研究奠定了良好的基础。
Somatic embryogenesis, direct organogenesis and plantlet clone for Taxodium mucronatum were conducted in this research.. The main results as follows:
1. Hypocotyl and cotyledon developed from the seeds of Taxodium mucronatum were cultured as the initiation explants for direct inducement of somatic embryos. Comparing with cotyledon, hypocotyl was better for us to induce somatic embryos, and the hypocotyl culture times directly effect on inducement frequency of somatic embryos also. The hypocotyl direct inducement frequency was 24.5% on the DCR medium with 2.0mg/l BA+0.1mg/lIBA. It is necessary for the development of somatic embryos by transferring the culture materials on hormone-free DCR medium.
2. Hypocotyl and cotyledon developed from the seeds of Taxodium mucronatum were also cultured as the initiation explants for direct organogenesis. It was not able to induce adventitious buds by the explant of cotyledon. With hypocotyl explants, the highest inducement frequency of adventitious buds was 58.5% on the DCR medium with 2.0mg/l BA, 0.1mg/1 IBA and 0.01mg/1 TDZ. The medium with low concentration of TDZ and BA was good to inducement and differentiation of adventitious buds of hypocotyl. Adventitious buds cultured on hormone-free DCR medium for elongation, subculture every four or five weeks, and cut the buds for rooting inducement after subculture two or three times. On hormone-free 1/2DCR medium the rooting frequency could be above 70%.
3. There are two kinds of explants to be used for plantlet clone of Taxodium mucronatum. The first one was the young seedling developed from the seeds. The results were shown that: dormant buds induced are sensitive to the different hormone concentration. Cultured young seedlings on DCR medium supplemented with 0.5 mg/1 BA and 0.2mg/l IBA was the best way of inducement, and with inducement frequency higher than 80%, mean number of dormant buds was around 2-5. The medium should be wiped off hormone when dormant buds induced. For elongation of the dormant buds, the suitable culture medium was DCR+Gln500mg/l. It can advance elongation of dormant buds when DCR basal medium supplemented with 500mg/lGln. The medium of 3/4DCR+0.05%AC is used for rooting, and the highest rooting rate is 78%. Using hormone to induce roots could produce mass of callus on the end top of cuttings and it was not benefit to the plantlets survive after transplant. In addition, it was easily for the plantlets with long internode and vigorous stem to induce root system. When the roots developed to 2-4cm long, the plant could be transplanted after 3-7 days acclimation
4. The second kinds of explants for plantlet clone was axillary buds developed from stem. The best inducement and elongation medium for axillary buds was DCR medium supplemented with 0.05mg/l IBA and 0.025mg/l BA, and inducement frequency up to 100%. Cutting down longer axillary buds from stem was good to other smaller axillary buds developed, and more new axillary buds produced from stem. The proper medium for inducing dormant buds from axillary buds was DCR medium supplemented with 0.3mg/l BA, 0.1 mg/1 NAA and 500mg/l Gln. The frequency of inducement was significant increased by adding Gin. TDZ was not suitable for dormant buds inducing from axillary buds. The best medium for elongating induced dormant buds was DCR+0.1%AC, subculturing 2-3 times, cutting down for rooting. The best medium for rooting in this research is 3/4DCR medium supplemented with 0.1 mg/1 IBA and 0.05mg/l NAA, however, the rooting rate was only 25%.
The frameworks for somatic embryogenesis, organogenesis, and micropropagation as mentioned above were established, and then the genie transformation of Taxodium mucronatum can be conducted on this system also.
引文
Abdul Rahman NN, Diner AM, Skilling DD, Karnosky DF. 1987. In vitro responses conifer adventitious shoots and calli inoculated with Gremmeniella abietina. For Sci., 33:1047-1053
Aitken J, Horgan KJ, Thorpe TH. 1981. Influence of explant selection on the shoot-forming capacity of juvenile tissue of Pinus radiata. Can J For Res., 11:112-117
Attree SM, et al. 1989. Plantlet regeneration from embryogenic protoplasts of white spruce (Picea glauca). Bio/Technology, 7:1060-1062
Becwar M R, Nagmani R, Wann S R. 1990. Initiation of embryogenic cultures and somatic embryo development in loblolly pine (Pinus taeda). Can J For Res., 20:810-817
Bonga JM, Duran DJ. 1982. Tissue Culture in Foresty. Martinus Nijhoff/Dr.W.Junk, The Hague, The Netherlands.
Bornman CH. 1983. Possibilities and constraints in the regeneration of trees from cotyledonary needles of Picea abies in vitro. Physiol Plant, 57:5-16
Bornman CH, Jansson E. 1982. Regeneration of plants from the conifer leaf with special reference to Picea abies and Pinus sylvestris. Colloque international sur la culture in vitro des essences forestiers, IUFRO, Fontainebler, AFOCEL, Nangis, France. 41-53
Bornman CH, Jansson E. 1980. Organogenesis in cultured Pinus sylvestris tissue. Zpflanzenphysiol, 96:1-6
Boulay MP, et al. 1988. Development of somatic embryos from cell suspension cultures of Norway Spruce. PI. Cell. Rep., 7:134-137
Bozhkov PV, Lebedenko LA, Shiryaeva GA. 1992. A pronounced synergistic effect of abscisic acid and 6-benzyladnine on Norway spruce (Picea abies L.Karst) somatic embryo maturation. Plant Cell Rep., 11:386-389
Cheng TY. 1975. Adventitious bus formation in culture of Douglas fir (Pseudotsuga menziesii (Mirb.) Franco). Plant Sci Lett., 5:97-102
Cheliak WM, et al. 1991. Genetic variation in somatic embryogenic response in open-pollinated families of black spruce. Theor. Appl. Genet., 82:185-190
De Fossard RA. 1976. Tissue Culture for plant propagators. The University of New England Printery, Armidale, Australia.
Hakman I, Fowke I C, Arnold Von S et al. 1985. The development of somatic embryos in tissue cultures initiated from immature embryos of Picea abies (Norway spruce). Plant Sci., 38:53-59
Gupta P K. Durzan D J. 1986. Somatic polyembryogenesis from callus of mature sugar pine embryos. Bio/Technology, 4:643-645
Gupta P K, Durzan D J. 1985. Shoot multiplication from mature trees of Douglas-fir (Pseudotsuga menziesii) and sugar pine (Pinus lambertiana ). Plant Cell Rep., 4:
177～179
Gupta P K, Pullman G S. 1991. Method for reproducing coniferous plants by somatic embryogenesis using abscisic acid and osmotic potential variation. U S Patent, No.5, 036, 007
Jwlaska S. 1987. European pines celland tissue culture in forestry. Martinus Nijihoff Publishers, Boston. Vol, 3:42-60
J. M. Favre, H. Tranvan, Y. Arnaud. 1995. F. Bourgkard. Present state of somatic embryogenesis in Sequoia sempervirens (Lamb.) Endl. Somatic Embryogenesis in Woody Plants. Kluwer Academic Publishers. Printed in the Netherlands. Vol. 3, 357-370
Jain SM, Gupta PK, Newton RJ. 1995. Somatic embryogenesis in woody plants. Kuler Acad. Pub., Dordrecht, 1-108
Jain S M, Dong N, Newton R J. 1989. Somatic embryogenesis in slash pine (Pinus elliottii) from immature embryo cultures In vitro. Plant Sci., 65:233-241
Laine E et al. 1992. Recovery of plants from cryopreserved embryogenic cell suspensions of Pinus caribaea. Plant Cell Rep., 11:295-298
Liu G, M. Bao. 2003. Adventitious shoot regeneration from in vitro cultured leaves of London plane tree (Platanus acerifolia Willd.). Plant Cell Rep., 21:640-644
Supriyanto S, Rohr R. 1988. Micropropagation and in vitro mycorrhization of Pinus sylvestris L. 6th FESPP Congress, Split, Yugoslavia, 14-34
Tautorus T E, Fowke L C, Dunstan D I. 1991. Somatic embryogenesis in conifers. Can J Bot., 69:1873-1899
Thorpe TA, Harry IS, Kumar PP. 1991. Application of Micropropagation to Foresty. Kluwar Acad. Pub., Dordrecht, 311-336
Von Arnold S, Eriksson T. 1978. Inducement of adventitious buds on embryos of Norway spruce grown in vitro. Physiol Plant, 44 (2): 283-287
Von Arnold S, Eriksson T. 1979. Bud differentiation on isolated needles of Norway spruce (Picea abies ) grown in vitro. Plant Sci Lett., 15:363～372
Von Arnold S, Eriksson T. 1981. In vitro studies of adventitious shoot formation in Pinus contorta. Can J Bot., 59:870-874
Webb D T, Santiago O D. 1983. Cytokinin-induced bud formation on Caribbean pine ( Pinus caribea morlet ) embryos in vitro. Plant Sci Lett., 32:17～21
Zel J, Gogala N, Camloh M. 1988. Micropropagation of Pinus sylvestris. Plant Cell Tiss Org Cult., 14:169-175
曹福亮，方升佐，唐罗忠等．1995．美国落羽杉种源试验初报．南京林业大学学报，(1)：65-70
陈永辉，王名金，伍寿彭．1988．落羽杉属的引种和选育．江苏林业科技，15(2)：43-47，49
陈龙清，张雨琴，袁芳亭．2000．PP_(333)及矮壮素对地被菊试管苗生根的影响．植物生理学通
讯，36(5)：425-427
陈龙清，鞠庆坤．1995．活性炭对几种植物试管苗生根的影响．华中农业大学学报．14(6)：600-602
崔克明．1997．植物细胞分化的启动控制和分化过程的阶段性．生命科学，9(2)：49-54
广东省林科所林木组织培养组．1981．杉木花药培养试验初报．广东林业科技通讯，(5)：10-13，27
广东省林科所林木组织培养课题组．1982．杉树优株的微型繁殖．广东林业科技通讯，(3)：15-20
谷瑞升，蒋湘宁，郭仲琛．1999．植物离体培养中器官发生调控机制的研究进展．植物学通报，16(3)：238-244
高凤菊，汤忠梅．2002．多效唑在植物组织培养中的应用．植物杂志，3：36-37
韩素英，齐力旺，杨云龙，冯晓燕，杜云开．1995．几种针叶树种离体培养条件的研究．林业科技通讯，(10)：20-22
黄利斌，汪企明，李晓储等．2000．落羽杉属种源研究：扦插生根能力变异．江苏林业科技，27(1)：1-5
黄健秋，卫志明．1995．针叶树体细胞胚胎发生的研究进展．植物生理学通讯，31(2)：85-90
黄健秋，卫志明．1994．松属树种的组织培养和原生质体培养．植物学通报，11(1)：34-42
黄健秋，卫志明，许智宏．1995．马尾松成熟合子胚的体细胞胚胎发生和植株再生．植物学报，37(4)：289-294
蒋立锐．1998．谷氨酰胺在代谢中的作用．氨基酸杂志，4：17-22
阙国宁．1980．杉木组织培养的初步研究．林业科学，16(增刊)：137-140
江如蓝．2000．多效唑对观赏植物的生物学效应及其在组织培养上的应用．仲恺农业技术学院学报，13(1)：50-57
罗建勋．1997．南洋杉组织培养与快速繁殖研究．园艺学报，24(2)：100-12
李映红，郭仲琛．1990．青扦在不同条件下的体细胞胚胎发生及苗的形成．植物学报，32：568-570
李浚明．1992．植物组织培养教程．北京：北京农业大学出版社
林小凡．1999．针叶树种的无性繁殖与无性系林业．江西林业科技，(6)：36-40，52
刘用生，李友勇．1994．植物组织培养中活性炭的使用．植物生理学通讯，30(3)：214-217
陆维玮，沈亚领．1998．谷氨酰胺的研究及开发进展．中国生化药物杂志，19(3)：161-163
卜学贤，陈维伦．1987．试管植物的玻璃化现象．植物生理学通讯，5：13-18
卜学贤，陈维伦．1988．活性炭对培养基中生长调节物质的吸附作用．植物生理学通讯，14(4)：401
沈惠娟．1992．木本植物组织培养技术．北京：中国农业科技出版社
沈惠娟．1989．赤霉素结构与功能及其在木本植物花端中的应用．南京林业大学学报，13(2)：89-93
施季森．2000．迎接21世纪现代林木生物技术育种的挑战．南京林业大学学报，24(1)：1-6
谭文澄．戴策刚．1997．观赏植物组织培养技术．北京：中国林业出版社
唐巍．欧阳藩，郭仲琛．1997．针叶树体细胞无性系研究和应用进展．生物工程进展，17(4)：2-9
唐巍，欧阳藩，郭仲琛．1998．火炬松(Pinus taeda L．)合子胚愈伤组织的器官发生和植株再生．实验生物学报，31(1)：87-91
唐巍，欧阳藩，郭仲琛．1997．火炬松成熟合子胚培养直接器官发生和植株再生．云南植物研究，19(3)：285-288
唐巍．欧阳藩，郭仲琛．1998．火炬松胚性愈伤组织诱导和植株再生的研究．林业科学，34(3)：115-118
唐翠，詹亚光．1997．松属林木组织培养不定芽的发生过程．哈尔滨师范大学自然科学学报，13(5)：68-71
唐巍，欧阳藩．1997．松柏类植物组织培养形态发生研究述评．大自然探索，16(2)：20-23
王明麻．1989．林木育种学概论．北京：中国林业出版社
王关林，方宏筠，那杰．1997．高活性细胞激动素TDZ在植物组织培养中的应用．植物学通报，14(3)：47-53
汪企明，王伟．1999．落羽杉．南京：南京出版社
汪企明，吕祥生，江泽平等．1993．落羽杉属种源研究：种子和苗期变异．江苏林业科技，20(1)：1-4，8
汪企明，王伟，蒋志新等．1998．落羽杉属种源研究：生长和生物量变异．江苏林业科技，25(1)：1-6
汪企明，江泽平，吕祥生等．1995．落羽杉属种源研究：树种生物学特性．江苏林业科技，22(2)：15-20
王凯基，张丕方，倪德祥，包慈华．1981．几种木本植物组织培养的愈伤组织形成和器官再生．植物学报，23(2)：97-101
徐福香．1980．墨西哥落羽杉杂种引种初报．林业科技通讯，(12)：2-3
杨映根，桂耀林，郭仲琛．1995．裸子植物体细胞胚胎发生和人工种子的研究．种子，(3)：25-29
姚洪军，罗晓芳，田砚亭．1999．植物组织培养外植体褐变的研究进展．北京林业大学学报，21(3)：78-83
郑均宝，王进茂，杜克久，李立国，陈正华，谷瑞升．1996．油松体细胞无性系的建立．遗传学报，23(4)：307-314
张绮纹．杨传平，施季森．1998．跨世纪林木高新科技——林木生物工程育种．世界林业研究．1：7-13
张丕方，倪德祥，包慈华．1984．植物组织培养与繁殖上的应用．上海：上海教育出版社
张宝红，李秀兰．1994．TDZ在棉花组织培养中的效应研究．棉花学报，6(2)：78-82
张志宏，景士西，王关林．1997．TDZ对苹果叶片离体再生不定芽的效应．植物生理学通讯，
33(6)：420-423
张军民，高振川．1999．谷氨酰胺营养生理研究进展．氨基酸和生物资源，21(2)：52-55
张艳敏，郭北海．2001．谷氨酰胺在小麦花药培养中的作用之探讨．河北省科学院学报，18(2)：105-108
周俊彦．1981．植物体细胞在组织培养中产生的胚状体(Ⅰ植物体细胞的胚状体发生)．植物生理学报，7(4)：391-394
周俊彦．1982．植物体细胞在组织培养中产生的胚状体(Ⅱ影响植物胚状体发生和发育的因素)．植物生理学报，8(1)：91-95
朱鹿鸣，金韵琴，栾永华．1980．用组织培养法繁殖杉木优树．江苏林业科技，(4)：47-49
Aitken J, Horgan KJ, Thorpe TH. 1981. Influence of explant selection on the shoot-forming capacity of juvenile tissue of Pinus radiata. Can J For Res., 11:112-117
Attree SM, et al. 1989. Plantlet regeneration from embryogenic protoplasts of white spruce (Picea glauca). Bio/Technology, 7:1060-1062
Becwar M R, Nagmani R, Wann S R. 1990. Initiation of embryogenic cultures and somatic embryo development in loblolly pine (Pinus taeda). Can J For Res., 20:810-817
Bonga JM, Duran DJ. 1982. Tissue Culture in Foresty. Martinus Nijhoff/Dr.W.Junk, The Hague, The Netherlands.
Bornman CH. 1983. Possibilities and constraints in the regeneration of trees from cotyledonary needles of Picea abies in vitro. Physiol Plant, 57:5-16
Bornman CH, Jansson E. 1982. Regeneration of plants from the conifer leaf with special reference to Picea abies and Pinus sylvestris. Colloque international sur la culture in vitro des essences forestiers, IUFRO, Fontainebler, AFOCEL, Nangis, France. 41-53
Bornman CH, Jansson E. 1980. Organogenesis in cultured Pinus sylvestris tissue. Zpflanzenphysiol, 96:1-6
Boulay MP, et al. 1988. Development of somatic embryos from cell suspension cultures of Norway Spruce. PI. Cell. Rep., 7:134-137
Bozhkov PV, Lebedenko LA, Shiryaeva GA. 1992. A pronounced synergistic effect of abscisic acid and 6-benzyladnine on Norway spruce (Picea abies L.Karst) somatic embryo maturation. Plant Cell Rep., 11:386-389
Cheng TY. 1975. Adventitious bus formation in culture of Douglas fir (Pseudotsuga menziesii (Mirb.) Franco). Plant Sci Lett., 5:97-102
Cheliak WM, et al. 1991. Genetic variation in somatic embryogenic response in open-pollinated families of black spruce. Theor. Appl. Genet., 82:185-190
De Fossard RA. 1976. Tissue Culture for plant propagators. The University of New England Printery, Armidale, Australia.
Hakman I, Fowke I C, Arnold Von S et al. 1985. The development of somatic embryos in tissue cultures initiated from immature embryos of Picea abies (Norway spruce). Plant Sci., 38:53-59
Gupta P K. Durzan D J. 1986. Somatic polyembryogenesis from callus of mature sugar pine embryos. Bio/Technology, 4:643-645
Gupta P K, Durzan D J. 1985. Shoot multiplication from mature trees of Douglas-fir (Pseudotsuga menziesii) and sugar pine (Pinus lambertiana ). Plant Cell Rep., 4:
177～179
Gupta P K, Pullman G S. 1991. Method for reproducing coniferous plants by somatic embryogenesis using abscisic acid and osmotic potential variation. U S Patent, No.5, 036, 007
Jwlaska S. 1987. European pines celland tissue culture in forestry. Martinus Nijihoff Publishers, Boston. Vol, 3:42-60
J. M. Favre, H. Tranvan, Y. Arnaud. 1995. F. Bourgkard. Present state of somatic embryogenesis in Sequoia sempervirens (Lamb.) Endl. Somatic Embryogenesis in Woody Plants. Kluwer Academic Publishers. Printed in the Netherlands. Vol. 3, 357-370
Jain SM, Gupta PK, Newton RJ. 1995. Somatic embryogenesis in woody plants. Kuler Acad. Pub., Dordrecht, 1-108
Jain S M, Dong N, Newton R J. 1989. Somatic embryogenesis in slash pine (Pinus elliottii) from immature embryo cultures In vitro. Plant Sci., 65:233-241
Laine E et al. 1992. Recovery of plants from cryopreserved embryogenic cell suspensions of Pinus caribaea. Plant Cell Rep., 11:295-298
Liu G, M. Bao. 2003. Adventitious shoot regeneration from in vitro cultured leaves of London plane tree (Platanus acerifolia Willd.). Plant Cell Rep., 21:640-644
Supriyanto S, Rohr R. 1988. Micropropagation and in vitro mycorrhization of Pinus sylvestris L. 6th FESPP Congress, Split, Yugoslavia, 14-34
Tautorus T E, Fowke L C, Dunstan D I. 1991. Somatic embryogenesis in conifers. Can J Bot., 69:1873-1899
Thorpe TA, Harry IS, Kumar PP. 1991. Application of Micropropagation to Foresty. Kluwar Acad. Pub., Dordrecht, 311-336
Von Arnold S, Eriksson T. 1978. Inducement of adventitious buds on embryos of Norway spruce grown in vitro. Physiol Plant, 44 (2): 283-287
Von Arnold S, Eriksson T. 1979. Bud differentiation on isolated needles of Norway spruce (Picea abies ) grown in vitro. Plant Sci Lett., 15:363～372
Von Arnold S, Eriksson T. 1981. In vitro studies of adventitious shoot formation in Pinus contorta. Can J Bot., 59:870-874
Webb D T, Santiago O D. 1983. Cytokinin-induced bud formation on Caribbean pine ( Pinus caribea morlet ) embryos in vitro. Plant Sci Lett., 32:17～21
Zel J, Gogala N, Camloh M. 1988. Micropropagation of Pinus sylvestris. Plant Cell Tiss Org Cult., 14:169-175
曹福亮，方升佐，唐罗忠等．1995．美国落羽杉种源试验初报．南京林业大学学报，(1)：65-70
陈永辉，王名金，伍寿彭．1988．落羽杉属的引种和选育．江苏林业科技，15(2)：43-47，49
陈龙清，张雨琴，袁芳亭．2000．PP_(333)及矮壮素对地被菊试管苗生根的影响．植物生理学通
讯，36(5)：425-427
陈龙清，鞠庆坤．1995．活性炭对几种植物试管苗生根的影响．华中农业大学学报．14(6)：600-602
崔克明．1997．植物细胞分化的启动控制和分化过程的阶段性．生命科学，9(2)：49-54
广东省林科所林木组织培养组．1981．杉木花药培养试验初报．广东林业科技通讯，(5)：10-13，27
广东省林科所林木组织培养课题组．1982．杉树优株的微型繁殖．广东林业科技通讯，(3)：15-20
谷瑞升，蒋湘宁，郭仲琛．1999．植物离体培养中器官发生调控机制的研究进展．植物学通报，16(3)：238-244
高凤菊，汤忠梅．2002．多效唑在植物组织培养中的应用．植物杂志，3：36-37
韩素英，齐力旺，杨云龙，冯晓燕，杜云开．1995．几种针叶树种离体培养条件的研究．林业科技通讯，(10)：20-22
黄利斌，汪企明，李晓储等．2000．落羽杉属种源研究：扦插生根能力变异．江苏林业科技，27(1)：1-5
黄健秋，卫志明．1995．针叶树体细胞胚胎发生的研究进展．植物生理学通讯，31(2)：85-90
黄健秋，卫志明．1994．松属树种的组织培养和原生质体培养．植物学通报，11(1)：34-42
黄健秋，卫志明，许智宏．1995．马尾松成熟合子胚的体细胞胚胎发生和植株再生．植物学报，37(4)：289-294
蒋立锐．1998．谷氨酰胺在代谢中的作用．氨基酸杂志，4：17-22
阙国宁．1980．杉木组织培养的初步研究．林业科学，16(增刊)：137-140
江如蓝．2000．多效唑对观赏植物的生物学效应及其在组织培养上的应用．仲恺农业技术学院学报，13(1)：50-57
罗建勋．1997．南洋杉组织培养与快速繁殖研究．园艺学报，24(2)：100-12
李映红，郭仲琛．1990．青扦在不同条件下的体细胞胚胎发生及苗的形成．植物学报，32：568-570
李浚明．1992．植物组织培养教程．北京：北京农业大学出版社
林小凡．1999．针叶树种的无性繁殖与无性系林业．江西林业科技，(6)：36-40，52
刘用生，李友勇．1994．植物组织培养中活性炭的使用．植物生理学通讯，30(3)：214-217
陆维玮，沈亚领．1998．谷氨酰胺的研究及开发进展．中国生化药物杂志，19(3)：161-163
卜学贤，陈维伦．1987．试管植物的玻璃化现象．植物生理学通讯，5：13-18
卜学贤，陈维伦．1988．活性炭对培养基中生长调节物质的吸附作用．植物生理学通讯，14(4)：401
沈惠娟．1992．木本植物组织培养技术．北京：中国农业科技出版社
沈惠娟．1989．赤霉素结构与功能及其在木本植物花端中的应用．南京林业大学学报，13(2)：89-93
施季森．2000．迎接21世纪现代林木生物技术育种的挑战．南京林业大学学报，24(1)：1-6
谭文澄．戴策刚．1997．观赏植物组织培养技术．北京：中国林业出版社
唐巍．欧阳藩，郭仲琛．1997．针叶树体细胞无性系研究和应用进展．生物工程进展，17(4)：2-9
唐巍，欧阳藩，郭仲琛．1998．火炬松(Pinus taeda L．)合子胚愈伤组织的器官发生和植株再生．实验生物学报，31(1)：87-91
唐巍，欧阳藩，郭仲琛．1997．火炬松成熟合子胚培养直接器官发生和植株再生．云南植物研究，19(3)：285-288
唐巍．欧阳藩，郭仲琛．1998．火炬松胚性愈伤组织诱导和植株再生的研究．林业科学，34(3)：115-118
唐翠，詹亚光．1997．松属林木组织培养不定芽的发生过程．哈尔滨师范大学自然科学学报，13(5)：68-71
唐巍，欧阳藩．1997．松柏类植物组织培养形态发生研究述评．大自然探索，16(2)：20-23
王明麻．1989．林木育种学概论．北京：中国林业出版社
王关林，方宏筠，那杰．1997．高活性细胞激动素TDZ在植物组织培养中的应用．植物学通报，14(3)：47-53
汪企明，王伟．1999．落羽杉．南京：南京出版社
汪企明，吕祥生，江泽平等．1993．落羽杉属种源研究：种子和苗期变异．江苏林业科技，20(1)：1-4，8
汪企明，王伟，蒋志新等．1998．落羽杉属种源研究：生长和生物量变异．江苏林业科技，25(1)：1-6
汪企明，江泽平，吕祥生等．1995．落羽杉属种源研究：树种生物学特性．江苏林业科技，22(2)：15-20
王凯基，张丕方，倪德祥，包慈华．1981．几种木本植物组织培养的愈伤组织形成和器官再生．植物学报，23(2)：97-101
徐福香．1980．墨西哥落羽杉杂种引种初报．林业科技通讯，(12)：2-3
杨映根，桂耀林，郭仲琛．1995．裸子植物体细胞胚胎发生和人工种子的研究．种子，(3)：25-29
姚洪军，罗晓芳，田砚亭．1999．植物组织培养外植体褐变的研究进展．北京林业大学学报，21(3)：78-83
郑均宝，王进茂，杜克久，李立国，陈正华，谷瑞升．1996．油松体细胞无性系的建立．遗传学报，23(4)：307-314
张绮纹．杨传平，施季森．1998．跨世纪林木高新科技——林木生物工程育种．世界林业研究．1：7-13
张丕方，倪德祥，包慈华．1984．植物组织培养与繁殖上的应用．上海：上海教育出版社
张宝红，李秀兰．1994．TDZ在棉花组织培养中的效应研究．棉花学报，6(2)：78-82
张志宏，景士西，王关林．1997．TDZ对苹果叶片离体再生不定芽的效应．植物生理学通讯，
33(6)：420-423
张军民，高振川．1999．谷氨酰胺营养生理研究进展．氨基酸和生物资源，21(2)：52-55
张艳敏，郭北海．2001．谷氨酰胺在小麦花药培养中的作用之探讨．河北省科学院学报，18(2)：105-108
周俊彦．1981．植物体细胞在组织培养中产生的胚状体(Ⅰ植物体细胞的胚状体发生)．植物生理学报，7(4)：391-394
周俊彦．1982．植物体细胞在组织培养中产生的胚状体(Ⅱ影响植物胚状体发生和发育的因素)．植物生理学报，8(1)：91-95
朱鹿鸣，金韵琴，栾永华．1980．用组织培养法繁殖杉木优树．江苏林业科技，(4)：47-49