科罗拉多冷杉组织培养技术研究
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摘要
科罗拉多冷杉组织培养技术的研究为其以后的组培快繁和其它生物技术奠定技术的基础。本文对科罗拉多冷杉组织培养技术进行了研究，包括外植体的表面灭菌、最适宜外植体的筛选和最佳启动培养基、继代培养基、生根培养基等的筛选。
研究结果表明：(1) 本试验以70％酒精和0.1％升汞为消毒剂，筛选适宜的外植体灭菌时间。种子(去除外种皮)用0.1％升汞表面处理8分钟，污染率较低(1.11％)；苗尖用0.1％升汞表面处理2分钟，污染率(1.11％)和褐化率较低(1.11％)。
(2) 以各种源科罗拉多冷杉成熟胚、子叶、子叶—下胚轴、下胚轴、苗尖、带子叶顶芽为外植体，只有成熟胚、苗尖、带子叶顶芽获得了无菌培养体系。
(3) 成熟胚不定芽诱导的培养基为1／2MS+2.7mg／L6-BA，种源间存在差异，只有4号种源成熟胚有不定芽发生，诱导率为23.33％；苗尖启动培养基为MS或MS+0.1mg／L6-BA，种源间差异显著，各种源苗尖启动率在80.0％～93.3％范围之内；胚萌发培养基为DCR，各种源成熟胚萌发率在83.33％～98.33％范围之内；成熟胚愈伤组织诱导培养基为SH+1.8mg／L2，4-D+1.2mg／L6-BA。愈伤组织诱导需要黑暗培养。
(4) 不定芽不易伸长，随着培养时间的延长易干枯或褐化死亡；愈伤组织在继代2～3次后，褐化死亡。这是本试验遇到的困难，也将是今后研究过程中要解决的难点。苗尖的腋芽诱导培养基为MS+0.3mg／L6-BA+0.1 mg／LIBA，从诱导出的腋芽数来看，8号＞12号；从苗高看，12号＞10号。带子叶顶芽的分化培养基为SH+0.4～0.5mg／L6-BA+0.3mg／LIBA，各种源带子叶顶芽的分化率在40.00％～76.67％范围内。诱导出的腋芽和针叶基潜伏芽均可在MS培养基上进行伸长培养，2～3月后，可以长成1～2cm无根苗。此时健壮的无根苗可以转入生根培养基中进行诱导生根。
(5) 科罗拉多冷杉无根试管苗生根困难，经过一年多的研究，目前仅513号种源有根发生，生根培养基为SH+0.5mg／L6-BA+0.5mg／LIBA+0.5mg／LNAA。
以上培养基均添加蔗糖30g／L(生根培养基20g／L)，琼脂6.5g／L，pH值5.8左右。培养温度24℃±2℃，每天光照12～14小时，光照强度1200～1600lx。
本试验采用科罗拉多冷杉成熟胚、苗尖、带子叶顶芽为外植体，成功地得到了无根试管苗，但是诱导无根试管苗生根很困难，需要进一步研究。
The techniques research about the tissue culture of Abies concolor will lay a foundation for its vegetative propagation and application of other biotechnology. In this thesis, the technology about tissue culture of Abies concolor was studied in detail. The whole technique system included sterilization, optimal explants selection and the optimal media selection for initiation, proliferation, rooting.
The results were as follows:
(1) Different disinfectant times were tested with the 70% alcohol and 0.1% HgCl2. For seeds, disinfested in 0.1% HgCl2 for 8 minutes was the best and its pollution rate was the lowest (1.11%). For shoot tips, disinfested in 0.1% HgCl2 for 2 minutes was the best and its pollution rate and browning rate were the lowest.
(2) Mature embryos, cotyledons, cotyledon-hypocotyls, hypocotyls, shoot tips, terminal buds from mature embryos of different provenances of Abies concolor were taken as explants. Mature embryos, shoot tips and terminal buds were the suitable explant materials.
(3) The optimal induction medium of adventitious buds consisted of one-half strength MS medium and 2.7mg/L 6-BA. The results indicated that there were differences among various provenances. Adventitious buds had achieved from mature embryos of the provenance 4 of which induction frequency reached 23.33%. The suitable initiation media of shoot tips were MS medium or MS medium containing 0.1mg/L 6-BA.
Significant differences were detected among provenances, the initiation frequency ranged from 80.0% to 93.3%. The best germination medium of mature embryos was DCR medium. The germination frequency ranged from 83.33% to 98.33%. The callus induction was successfully taken place in darkness on the SH medium containing 1.8mg/L 2,4-Dand 1.2mg/L6-BA.
(4) Adventitious buds elongate difficultly and browned to death with the culture time gone by. It is a difficult problem that callus would browned to death, after it was
transferred 2 to 3 times. The selected induction medium of axillary buds was MS medium containing 0.3mg/L 6-BA and 0.1mg/L IBA. The shoot tips of provenance 8 produced more axillary buds than provenance 12. The height of shoot tips of provenance 12 was higher than that of provenance 8. The differentiation medium of terminal buds was SH medium containing 0.4~0.5mg/L 6-BA and 0.3mg/L IBA. The differentiation frequency ranged from 40.00% to 76.67%. The axillary buds and dormant buds could elongate 1-2 cm height on MS medium.
(5) The shoots of Abies concolor were difficult to root. At present, the roots were only achieved from shoots of provenance 513. The rooting medium was SH medium containing 0.5mg/L 6-BA, 0.5mg/L IBA and 0.5mg/L NAA.
All media contained 30g/L sucrose (rooting medium contained 20g/L) and 6.5g/L agar. The pH of all media was adjusted to 5.8 prior to autoclaving. The temperature in the tissue culture room was adjusted at 24C +2C, with an illumination time of 12-14 hours and illumination intensity 1200~1600Lx.
Shoots were achieved from mature embryos, shoot tips, terminal buds, but which were difficult to root and further work was needed.
研究结果表明：(1) 本试验以70％酒精和0.1％升汞为消毒剂，筛选适宜的外植体灭菌时间。种子(去除外种皮)用0.1％升汞表面处理8分钟，污染率较低(1.11％)；苗尖用0.1％升汞表面处理2分钟，污染率(1.11％)和褐化率较低(1.11％)。
(2) 以各种源科罗拉多冷杉成熟胚、子叶、子叶—下胚轴、下胚轴、苗尖、带子叶顶芽为外植体，只有成熟胚、苗尖、带子叶顶芽获得了无菌培养体系。
(3) 成熟胚不定芽诱导的培养基为1／2MS+2.7mg／L6-BA，种源间存在差异，只有4号种源成熟胚有不定芽发生，诱导率为23.33％；苗尖启动培养基为MS或MS+0.1mg／L6-BA，种源间差异显著，各种源苗尖启动率在80.0％～93.3％范围之内；胚萌发培养基为DCR，各种源成熟胚萌发率在83.33％～98.33％范围之内；成熟胚愈伤组织诱导培养基为SH+1.8mg／L2，4-D+1.2mg／L6-BA。愈伤组织诱导需要黑暗培养。
(4) 不定芽不易伸长，随着培养时间的延长易干枯或褐化死亡；愈伤组织在继代2～3次后，褐化死亡。这是本试验遇到的困难，也将是今后研究过程中要解决的难点。苗尖的腋芽诱导培养基为MS+0.3mg／L6-BA+0.1 mg／LIBA，从诱导出的腋芽数来看，8号＞12号；从苗高看，12号＞10号。带子叶顶芽的分化培养基为SH+0.4～0.5mg／L6-BA+0.3mg／LIBA，各种源带子叶顶芽的分化率在40.00％～76.67％范围内。诱导出的腋芽和针叶基潜伏芽均可在MS培养基上进行伸长培养，2～3月后，可以长成1～2cm无根苗。此时健壮的无根苗可以转入生根培养基中进行诱导生根。
(5) 科罗拉多冷杉无根试管苗生根困难，经过一年多的研究，目前仅513号种源有根发生，生根培养基为SH+0.5mg／L6-BA+0.5mg／LIBA+0.5mg／LNAA。
以上培养基均添加蔗糖30g／L(生根培养基20g／L)，琼脂6.5g／L，pH值5.8左右。培养温度24℃±2℃，每天光照12～14小时，光照强度1200～1600lx。
本试验采用科罗拉多冷杉成熟胚、苗尖、带子叶顶芽为外植体，成功地得到了无根试管苗，但是诱导无根试管苗生根很困难，需要进一步研究。
The techniques research about the tissue culture of Abies concolor will lay a foundation for its vegetative propagation and application of other biotechnology. In this thesis, the technology about tissue culture of Abies concolor was studied in detail. The whole technique system included sterilization, optimal explants selection and the optimal media selection for initiation, proliferation, rooting.
The results were as follows:
(1) Different disinfectant times were tested with the 70% alcohol and 0.1% HgCl2. For seeds, disinfested in 0.1% HgCl2 for 8 minutes was the best and its pollution rate was the lowest (1.11%). For shoot tips, disinfested in 0.1% HgCl2 for 2 minutes was the best and its pollution rate and browning rate were the lowest.
(2) Mature embryos, cotyledons, cotyledon-hypocotyls, hypocotyls, shoot tips, terminal buds from mature embryos of different provenances of Abies concolor were taken as explants. Mature embryos, shoot tips and terminal buds were the suitable explant materials.
(3) The optimal induction medium of adventitious buds consisted of one-half strength MS medium and 2.7mg/L 6-BA. The results indicated that there were differences among various provenances. Adventitious buds had achieved from mature embryos of the provenance 4 of which induction frequency reached 23.33%. The suitable initiation media of shoot tips were MS medium or MS medium containing 0.1mg/L 6-BA.
Significant differences were detected among provenances, the initiation frequency ranged from 80.0% to 93.3%. The best germination medium of mature embryos was DCR medium. The germination frequency ranged from 83.33% to 98.33%. The callus induction was successfully taken place in darkness on the SH medium containing 1.8mg/L 2,4-Dand 1.2mg/L6-BA.
(4) Adventitious buds elongate difficultly and browned to death with the culture time gone by. It is a difficult problem that callus would browned to death, after it was
transferred 2 to 3 times. The selected induction medium of axillary buds was MS medium containing 0.3mg/L 6-BA and 0.1mg/L IBA. The shoot tips of provenance 8 produced more axillary buds than provenance 12. The height of shoot tips of provenance 12 was higher than that of provenance 8. The differentiation medium of terminal buds was SH medium containing 0.4~0.5mg/L 6-BA and 0.3mg/L IBA. The differentiation frequency ranged from 40.00% to 76.67%. The axillary buds and dormant buds could elongate 1-2 cm height on MS medium.
(5) The shoots of Abies concolor were difficult to root. At present, the roots were only achieved from shoots of provenance 513. The rooting medium was SH medium containing 0.5mg/L 6-BA, 0.5mg/L IBA and 0.5mg/L NAA.
All media contained 30g/L sucrose (rooting medium contained 20g/L) and 6.5g/L agar. The pH of all media was adjusted to 5.8 prior to autoclaving. The temperature in the tissue culture room was adjusted at 24C +2C, with an illumination time of 12-14 hours and illumination intensity 1200~1600Lx.
Shoots were achieved from mature embryos, shoot tips, terminal buds, but which were difficult to root and further work was needed.
引文
1．曹孜义、刘国民．实用植物组织培养技术教程．甘肃科学技术出版社，1999
2．陈金慧，王洪云，诸葛强等．林木体胚发生技术进展．林业科技开发，2000，14(3)：9～11
3．陈永勤，戴均贵，朱蔚华．红豆杉成熟胚的离体培养(简报)．植物生理学通讯，1998，34(3)：191～193
4．程广有，杨振国．红松组织培养技术的初步研究．吉林林业科技，2000，29(5)：8～9
5．程广有，王明启，东北红豆杉的微繁技术．东北林业大学学报，2000，28(2)：13～16
6．成小飞，花晓梅，李文钿．马尾松离体培养条件下的微繁殖和菌根的形成．林业科学研究，1995，8(3)：241～246
7．成小飞，李文钿．秃杉的组织培养．林业科学研究，1991，4(2)：147～152
8．杜晓光，袁巧平，李玲．兴安落叶松组织培养中丛生芽的形成研究．林业科技通讯，1992(2)：15～17
9．辜夕容，石大兴．圆柏组织培养繁殖研究．亚热带植物通讯，2000,29(2)：40～42
10．韩素英，齐力旺，杨云龙等．几种针叶树种离体培养条件的研究．林业科技通讯，1995，(10)：20～22
11．韩秀慧．微型月季工厂化离体快速繁殖技术的研究[学位论文]．北京林业大学，2003
12．何月秋．湿地松抗病家系组培繁殖技术研究[学位论文]．南京林业大学，2003
13．黄健秋，卫志明．松属树种的组织培养和原生质体培育．植物学通讯，1994,11(1)：34～42
14．黄健秋，卫志明．针叶树体细胞胚胎发生的研究进展．植物生理学通讯，1995，31(2)：87～89
15．黄学林，李筱菊．高等植物组织离体培养的形态建成及其调控．科学出版社，1995
16．江玲，管晓春．植物激素与不定根的形成．生物学通报，2000,35(11)：17～19
17．康明，田建强，郑均宝．樟子松成熟胚的离体培养与不定芽的形成．河北林学院学报，1993，8(4)：273～277
18．雷泽勇．针叶树无性繁殖研究进展．防护林科技，2001，46(1)：55～57
19．雷泽勇，周凤艳，周莉．关于离体胚组织培养在植物育种中的应用进展．防护林科技，2001，48(3)：57～59
20．李科友，唐德瑞，朱海兰等．美国黄松组织培养不定根诱导的研究．西北植物学报，2003，23(3)：464～467
21．李科友，唐德瑞，刘永红等．美国黄松成熟胚的离体培养与不定芽的形成．西北植物学报，2001，21(6)：1223～1227
22．梁玉堂，邢世岩．几种影响黑松针叶束离体培养再生植株的因子(简报)．植物生理学通讯，1990，3：31～33
23．梁玉堂，邢世岩．北美圆柏组织培养不定芽形成及生长研究．林业科技通讯，1989，234(11)：1～3
24．刘玉喜，唐翠，詹亚光．红松成熟胚离体培养器官发生组织学研究．植物研究，1992，12(2)：189～193
25．刘用生，李友勇．植物组织培养中活性炭的使用．植物生理学通讯，1994，30(3)：214～217
26．罗建勋．南洋杉组织培养与快速繁殖研究．园艺学报，1997，24(1)：100～102
27．普晓兰，林萍，李乡旺．五针白皮松离体胚培养初步研究．西南林学院学报，1997，17(1)：17～20
28．齐力旺，杨云龙，韩素英等．油松封顶芽的组织培养．植物生理学通讯，1995，31(1)：40～41
29．齐力旺，杨云龙，韩素英等．油松的无性繁殖与离体培养研究．林业科技通讯，1996，11：12～14
30．齐力旺，杨云龙，韩素英等．侧柏的组织培养和植株再生．植物生理学通讯(上海)，1995，31(4)：284～285
31．阙国宁，房建军，葛万川等．火炬松、湿地松、晚松组培繁殖的研究．林业科学研究，1997，10(3)：227～232
32．唐翠，詹亚光．松属林木组织培养不定芽的发生过程——红松胚培养不定芽发生．哈尔滨师范大学自然科学学报，1997,13(5)：68～71
33．唐巍，欧阳藩，郭仲琛．针叶树体细胞无性系研究和应用进展．生物工程进展，1997，17(4)：2～9
34．唐巍，欧阳藩，郭仲琛．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，1997，19(3)：285～288
35．唐巍，欧阳藩，郭仲琛．火炬松胚性愈伤组织诱导和植株再生的研究．林业科学(北京)，1998，34(3)：115～119
36．唐巍，欧阳藩，郭仲琛．火炬松合子胚愈伤组织的器官发生与植株再生．实验生物学报(上海)，1998，31(1)：87～93
37．王水，贾勇炯，魏峰等．云南红豆杉的组织培养及植株再生．云南植物研究，1997，19(4)：407～410
38．吴克贤，李伟，徐妙珍等．长白落叶松组织培养的研究．林业科技(北京)，1996，32(2)：125～133
39．吴耀溪．杉木组织培养繁殖体系建立的研究．福建林学院学报，1991，11(1)：67～74
40．吴若菁．马尾松离体组织培养初报．福建林学院学报，1993，13(2)：98～100
41．邢世岩．松属树种细胞，组织和器官培养名录．植物生理学通讯．1990，1：75～79，41
42．邢子琢，康忠铭．大别山五针松子叶和下胚轴离体培养成苗．植物生理学通讯，1990，(1)：52～53
43．张华通，翟应昌，周志坚等．火炬松胚性愈伤组织的诱导及体胚的形成．广东林业科技，1994，(4)：33～35
44．张守英，杜小光，李玲．兴安落叶松组织培养不定根诱导的研究．林业科技通讯(北京)，1993，5：8～10
45．张宇．马尾松组织培养的研究[学位论文]．南京林业大学，2003
46．郑均宝，潘冬梅，陈正华．油松离体胚子叶的组织分化和无根试管苗的形成．河北林学院学报，1994，9(2)：97～101
47．周微，黄健秋，卫志明等．云南松成熟胚的不定芽诱导及植株再生(简报)．植物生理学通讯(上海)，1995，31(5)：351～353
48．朱广廉．植物组织培养中的外植体灭菌．植物生理学通讯，1996，32(6)：444～449
49．祝朋芳，罗凤霞，陈长卿等．落叶松属树种的组织培养研究概述．辽宁农业科学，2000(5)：29～32
50. Astrid Schuller, Gerhard Reuther. Response of Abies alba embryonal-suspensor mass to various carbohydrate treatments. Plant Cell Reports, 1993,12:199～202
51. A.V. Ramarosandratana, J. Van Staden. Tissue position, explant orientation and naphthaleneacetic acid (NAA) affect initiation of somatic embryos and callus proliferation in Norway spruce (Picea abies). Plant Cell Tissue and Organ Culture, 2003,74:249～255
52. B. VOOKOV, A. GAJDOOV, R.MATOV. Somatic embryogenesis in Abies alba × Abies alba and Abies alba × Abies nordmanniana hybrids. Biologia Plantarum, 1997/98,40(4): 523～530
53. B. VOOKOV, A. KORMUK. Effect of sucrose concentration, charcoal, and indole-3-butyric acid on germination of Abies numidica somatic embryos. Biologia Plantarum, 2001,44(2): 181～184
54. B. VOOKOV, R.MATOV, A. KORMUK. Secondary somatic embryogenesis in Abies numidica. Biologia Plantarum, 2003,46(4): 513～517
55. Célia Miguel, Snia Goncalves, Susana Tereso et al. Somatic embryogenesis from 20 open-pollinated families of Portuguese plus trees of maritime pine. Plant Cell Tissue and Organ Culture, 2004,76: 121～130.
56. Dembny H, Zoglauer K. Rooting of in vitro cloned lawch shoots. Naturwissenschafiliche, 1992,41(3):107～114
57. Dhundy R. Bastola, V. P. Agrawal, Nirmal Joshee. In Vitro Propagation of a Himalayan pine P. wallichiana A.B. Jacks. Current Science, 2000,78(3):338～341
58. Doris Prehn, Carolina Serrano, Ana Mercado et al. Regeneration of whole plants from apical meristems of Pinus radiata. Plant Cell Tissue and Organ Culture, 2003,73:91～94
59. Dragana Stojii, Snezana Budimir, Ljubinka ulafi. Micropropagation of Pinus heldreichii. Plant Cell Tissue and Organ Culture, 1999,59:147～150
60. Garcia-Ferriz L, Serrano L, Pardos J. In vitro shoot organogenesis from excised immature cotyledons and microcuttings production in stone pine. Plant Cell Tissue and Organ Culture, 1994,36:135～140
61. Guevin T G, Micah V, Kirby E G. Somatic embryogenesis in cultured mature zygotic embryos of Abies balsamea. Plant Cell Tissue and Organ Culture, 1994,37(2):205～208
62. Hristoforoglu K, Schmidt J, Bolhar Nordenkampf H. Development and germination of Abies alba somatic embryos. Plant Cell Tissue and Organ Culture, 1995,40(3):277～284
63. Jain SM, Gupta PK, Newton RJ. Somatic embryogenesis in woody plants. Kuler Acad. Pub., Dordrecht, 1995,1～108
64. J.M. Bonga. The effect of various culture media on the formation of embryo-like structures in cultures derived from explants taken from mature Larix deciduas. Plant Cell Tissue and Organ Culture, 2004,77:43～48
65. Katharina Hristoforoglu, Josef Schmidt, Harald Bolhar-Nordenkampf. Development and germination of Abies alba somatic embryos. Plant Cell Tissue and Organ Culture, 1995,40:277～284
66. Konstantin A. Schestibratov, Roman V. Mikhailov, Sergey V. Dolgov. Plantlet regeneration from
subculturable nodular callus of Pinus radiata. Plant Cell Tissue and Organ Culture, 2003,72:139～146
67. L.John Frampton, JR., Henry V. Amerson et al. Tissue culture method affects ex vitro growth and development of Ioblolly pine. New Forests, 1998,16:125～138
68. Norgaard J V. Somatic embryo maturation and plant regeneration in Abies nordmanniana Lk. Plant Science, 1997,124(2):211～221
69. P.M.W.Drake, A.John, J.B.Power et al. Cytokinin pulse-mediated shoot organogenesis from cotyledons of Sitka spruce [Picea sitchensis (Bong.)Carr.] and high frequency in vitro rooting of shoots. Plant Cell Tissue and Organ Culture, 1997,50:147～151
70. Rajbhandari N, Stomp A M. Embryogenic callus induction in Fraser fir. Hortscience, 1997,32(4):737～738
71. Rancillac M, Faye M, David A. In Vitro Rooting of Cloned Shoots in Pinus Pinaster. Physiol Plant, 1982,56:97
72. Salajova T, Jasik J, Kormutak A et al. Embryogenic culture initiation and somatic embryo development in hybrid firs (Abies alba × Abies cephalonica and Abies alba × Abies numidica). Plant Cell Reports, 1996,15(7):527～530
73. Saravitz, C. H., E A. Blazich, H. V. Amerson. In vitro-propagation of Fraser Fir from embryonic explants. Canadian Journal of Forest Research, 1991,21 :(3) 404～409
74. Sen S, Newton RJ, Fong F. Abscisis acid: a role in shoot enhancement from loblolly pine (Pinus taeda L.) cotyledon explants. Plant Cell Reports, 1989, 8:191～194
75. Sharon L, Bishop-Hurley, Richard C. Gardner et al. Isolation and molecular characterization of genes expressed during somatic embryo development in Pinus radiata. Plant Cell Tissue and Organ Culture, 2003,74:267～281
76. Tang Wei, Guo Zhongchen. In vitro propagation of loblolly pine via direct somatic organogenesis from mature cotyledons and hypocotyls. Plant Growth Regulation, 2001,33:25～31
77. Tang Wei, Ouyang Fan, Guo Zhongchen. Factors affecting adventitious bud differentiation of Loblolly pine from mature zygotic embryos cultured in vitro. Developmental and Reproductive Biology, 1998,7(1):49～54
78. Tang Wei, Ouyang Fan, Guo Zhongchen. Plant regeneration through organogenesis from callus induced from mature zygotic embryos loblolly pine. Plant Cell Reports, 1998,17:557～560
79. Tang Wei, Ouyang Fan. Plant regeneration via organogenesis from six families of loblolly pine. Plant Cell Tissue and Organ Culture, 1999,58: 223～226.
80. Todd G. Guevin, Edward G. Kirby. Induction of embryogenesis in cultured mature zygotic embryos of Abies fraseri (Pursh) Poir. Plant Cell Tissue and Organ Culture, 1997,49:219～222
81. T.SALAJ, J.SALAJ. Somatic embryo formation on mature Abies alba × Abies ecphalonica zygotic embryo explants. Biologia Plantarum, 2003/4,47(1):7～11
2．陈金慧，王洪云，诸葛强等．林木体胚发生技术进展．林业科技开发，2000，14(3)：9～11
3．陈永勤，戴均贵，朱蔚华．红豆杉成熟胚的离体培养(简报)．植物生理学通讯，1998，34(3)：191～193
4．程广有，杨振国．红松组织培养技术的初步研究．吉林林业科技，2000，29(5)：8～9
5．程广有，王明启，东北红豆杉的微繁技术．东北林业大学学报，2000，28(2)：13～16
6．成小飞，花晓梅，李文钿．马尾松离体培养条件下的微繁殖和菌根的形成．林业科学研究，1995，8(3)：241～246
7．成小飞，李文钿．秃杉的组织培养．林业科学研究，1991，4(2)：147～152
8．杜晓光，袁巧平，李玲．兴安落叶松组织培养中丛生芽的形成研究．林业科技通讯，1992(2)：15～17
9．辜夕容，石大兴．圆柏组织培养繁殖研究．亚热带植物通讯，2000,29(2)：40～42
10．韩素英，齐力旺，杨云龙等．几种针叶树种离体培养条件的研究．林业科技通讯，1995，(10)：20～22
11．韩秀慧．微型月季工厂化离体快速繁殖技术的研究[学位论文]．北京林业大学，2003
12．何月秋．湿地松抗病家系组培繁殖技术研究[学位论文]．南京林业大学，2003
13．黄健秋，卫志明．松属树种的组织培养和原生质体培育．植物学通讯，1994,11(1)：34～42
14．黄健秋，卫志明．针叶树体细胞胚胎发生的研究进展．植物生理学通讯，1995，31(2)：87～89
15．黄学林，李筱菊．高等植物组织离体培养的形态建成及其调控．科学出版社，1995
16．江玲，管晓春．植物激素与不定根的形成．生物学通报，2000,35(11)：17～19
17．康明，田建强，郑均宝．樟子松成熟胚的离体培养与不定芽的形成．河北林学院学报，1993，8(4)：273～277
18．雷泽勇．针叶树无性繁殖研究进展．防护林科技，2001，46(1)：55～57
19．雷泽勇，周凤艳，周莉．关于离体胚组织培养在植物育种中的应用进展．防护林科技，2001，48(3)：57～59
20．李科友，唐德瑞，朱海兰等．美国黄松组织培养不定根诱导的研究．西北植物学报，2003，23(3)：464～467
21．李科友，唐德瑞，刘永红等．美国黄松成熟胚的离体培养与不定芽的形成．西北植物学报，2001，21(6)：1223～1227
22．梁玉堂，邢世岩．几种影响黑松针叶束离体培养再生植株的因子(简报)．植物生理学通讯，1990，3：31～33
23．梁玉堂，邢世岩．北美圆柏组织培养不定芽形成及生长研究．林业科技通讯，1989，234(11)：1～3
24．刘玉喜，唐翠，詹亚光．红松成熟胚离体培养器官发生组织学研究．植物研究，1992，12(2)：189～193
25．刘用生，李友勇．植物组织培养中活性炭的使用．植物生理学通讯，1994，30(3)：214～217
26．罗建勋．南洋杉组织培养与快速繁殖研究．园艺学报，1997，24(1)：100～102
27．普晓兰，林萍，李乡旺．五针白皮松离体胚培养初步研究．西南林学院学报，1997，17(1)：17～20
28．齐力旺，杨云龙，韩素英等．油松封顶芽的组织培养．植物生理学通讯，1995，31(1)：40～41
29．齐力旺，杨云龙，韩素英等．油松的无性繁殖与离体培养研究．林业科技通讯，1996，11：12～14
30．齐力旺，杨云龙，韩素英等．侧柏的组织培养和植株再生．植物生理学通讯(上海)，1995，31(4)：284～285
31．阙国宁，房建军，葛万川等．火炬松、湿地松、晚松组培繁殖的研究．林业科学研究，1997，10(3)：227～232
32．唐翠，詹亚光．松属林木组织培养不定芽的发生过程——红松胚培养不定芽发生．哈尔滨师范大学自然科学学报，1997,13(5)：68～71
33．唐巍，欧阳藩，郭仲琛．针叶树体细胞无性系研究和应用进展．生物工程进展，1997，17(4)：2～9
34．唐巍，欧阳藩，郭仲琛．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，1997，19(3)：285～288
35．唐巍，欧阳藩，郭仲琛．火炬松胚性愈伤组织诱导和植株再生的研究．林业科学(北京)，1998，34(3)：115～119
36．唐巍，欧阳藩，郭仲琛．火炬松合子胚愈伤组织的器官发生与植株再生．实验生物学报(上海)，1998，31(1)：87～93
37．王水，贾勇炯，魏峰等．云南红豆杉的组织培养及植株再生．云南植物研究，1997，19(4)：407～410
38．吴克贤，李伟，徐妙珍等．长白落叶松组织培养的研究．林业科技(北京)，1996，32(2)：125～133
39．吴耀溪．杉木组织培养繁殖体系建立的研究．福建林学院学报，1991，11(1)：67～74
40．吴若菁．马尾松离体组织培养初报．福建林学院学报，1993，13(2)：98～100
41．邢世岩．松属树种细胞，组织和器官培养名录．植物生理学通讯．1990，1：75～79，41
42．邢子琢，康忠铭．大别山五针松子叶和下胚轴离体培养成苗．植物生理学通讯，1990，(1)：52～53
43．张华通，翟应昌，周志坚等．火炬松胚性愈伤组织的诱导及体胚的形成．广东林业科技，1994，(4)：33～35
44．张守英，杜小光，李玲．兴安落叶松组织培养不定根诱导的研究．林业科技通讯(北京)，1993，5：8～10
45．张宇．马尾松组织培养的研究[学位论文]．南京林业大学，2003
46．郑均宝，潘冬梅，陈正华．油松离体胚子叶的组织分化和无根试管苗的形成．河北林学院学报，1994，9(2)：97～101
47．周微，黄健秋，卫志明等．云南松成熟胚的不定芽诱导及植株再生(简报)．植物生理学通讯(上海)，1995，31(5)：351～353
48．朱广廉．植物组织培养中的外植体灭菌．植物生理学通讯，1996，32(6)：444～449
49．祝朋芳，罗凤霞，陈长卿等．落叶松属树种的组织培养研究概述．辽宁农业科学，2000(5)：29～32
50. Astrid Schuller, Gerhard Reuther. Response of Abies alba embryonal-suspensor mass to various carbohydrate treatments. Plant Cell Reports, 1993,12:199～202
51. A.V. Ramarosandratana, J. Van Staden. Tissue position, explant orientation and naphthaleneacetic acid (NAA) affect initiation of somatic embryos and callus proliferation in Norway spruce (Picea abies). Plant Cell Tissue and Organ Culture, 2003,74:249～255
52. B. VOOKOV, A. GAJDOOV, R.MATOV. Somatic embryogenesis in Abies alba × Abies alba and Abies alba × Abies nordmanniana hybrids. Biologia Plantarum, 1997/98,40(4): 523～530
53. B. VOOKOV, A. KORMUK. Effect of sucrose concentration, charcoal, and indole-3-butyric acid on germination of Abies numidica somatic embryos. Biologia Plantarum, 2001,44(2): 181～184
54. B. VOOKOV, R.MATOV, A. KORMUK. Secondary somatic embryogenesis in Abies numidica. Biologia Plantarum, 2003,46(4): 513～517
55. Célia Miguel, Snia Goncalves, Susana Tereso et al. Somatic embryogenesis from 20 open-pollinated families of Portuguese plus trees of maritime pine. Plant Cell Tissue and Organ Culture, 2004,76: 121～130.
56. Dembny H, Zoglauer K. Rooting of in vitro cloned lawch shoots. Naturwissenschafiliche, 1992,41(3):107～114
57. Dhundy R. Bastola, V. P. Agrawal, Nirmal Joshee. In Vitro Propagation of a Himalayan pine P. wallichiana A.B. Jacks. Current Science, 2000,78(3):338～341
58. Doris Prehn, Carolina Serrano, Ana Mercado et al. Regeneration of whole plants from apical meristems of Pinus radiata. Plant Cell Tissue and Organ Culture, 2003,73:91～94
59. Dragana Stojii, Snezana Budimir, Ljubinka ulafi. Micropropagation of Pinus heldreichii. Plant Cell Tissue and Organ Culture, 1999,59:147～150
60. Garcia-Ferriz L, Serrano L, Pardos J. In vitro shoot organogenesis from excised immature cotyledons and microcuttings production in stone pine. Plant Cell Tissue and Organ Culture, 1994,36:135～140
61. Guevin T G, Micah V, Kirby E G. Somatic embryogenesis in cultured mature zygotic embryos of Abies balsamea. Plant Cell Tissue and Organ Culture, 1994,37(2):205～208
62. Hristoforoglu K, Schmidt J, Bolhar Nordenkampf H. Development and germination of Abies alba somatic embryos. Plant Cell Tissue and Organ Culture, 1995,40(3):277～284
63. Jain SM, Gupta PK, Newton RJ. Somatic embryogenesis in woody plants. Kuler Acad. Pub., Dordrecht, 1995,1～108
64. J.M. Bonga. The effect of various culture media on the formation of embryo-like structures in cultures derived from explants taken from mature Larix deciduas. Plant Cell Tissue and Organ Culture, 2004,77:43～48
65. Katharina Hristoforoglu, Josef Schmidt, Harald Bolhar-Nordenkampf. Development and germination of Abies alba somatic embryos. Plant Cell Tissue and Organ Culture, 1995,40:277～284
66. Konstantin A. Schestibratov, Roman V. Mikhailov, Sergey V. Dolgov. Plantlet regeneration from
subculturable nodular callus of Pinus radiata. Plant Cell Tissue and Organ Culture, 2003,72:139～146
67. L.John Frampton, JR., Henry V. Amerson et al. Tissue culture method affects ex vitro growth and development of Ioblolly pine. New Forests, 1998,16:125～138
68. Norgaard J V. Somatic embryo maturation and plant regeneration in Abies nordmanniana Lk. Plant Science, 1997,124(2):211～221
69. P.M.W.Drake, A.John, J.B.Power et al. Cytokinin pulse-mediated shoot organogenesis from cotyledons of Sitka spruce [Picea sitchensis (Bong.)Carr.] and high frequency in vitro rooting of shoots. Plant Cell Tissue and Organ Culture, 1997,50:147～151
70. Rajbhandari N, Stomp A M. Embryogenic callus induction in Fraser fir. Hortscience, 1997,32(4):737～738
71. Rancillac M, Faye M, David A. In Vitro Rooting of Cloned Shoots in Pinus Pinaster. Physiol Plant, 1982,56:97
72. Salajova T, Jasik J, Kormutak A et al. Embryogenic culture initiation and somatic embryo development in hybrid firs (Abies alba × Abies cephalonica and Abies alba × Abies numidica). Plant Cell Reports, 1996,15(7):527～530
73. Saravitz, C. H., E A. Blazich, H. V. Amerson. In vitro-propagation of Fraser Fir from embryonic explants. Canadian Journal of Forest Research, 1991,21 :(3) 404～409
74. Sen S, Newton RJ, Fong F. Abscisis acid: a role in shoot enhancement from loblolly pine (Pinus taeda L.) cotyledon explants. Plant Cell Reports, 1989, 8:191～194
75. Sharon L, Bishop-Hurley, Richard C. Gardner et al. Isolation and molecular characterization of genes expressed during somatic embryo development in Pinus radiata. Plant Cell Tissue and Organ Culture, 2003,74:267～281
76. Tang Wei, Guo Zhongchen. In vitro propagation of loblolly pine via direct somatic organogenesis from mature cotyledons and hypocotyls. Plant Growth Regulation, 2001,33:25～31
77. Tang Wei, Ouyang Fan, Guo Zhongchen. Factors affecting adventitious bud differentiation of Loblolly pine from mature zygotic embryos cultured in vitro. Developmental and Reproductive Biology, 1998,7(1):49～54
78. Tang Wei, Ouyang Fan, Guo Zhongchen. Plant regeneration through organogenesis from callus induced from mature zygotic embryos loblolly pine. Plant Cell Reports, 1998,17:557～560
79. Tang Wei, Ouyang Fan. Plant regeneration via organogenesis from six families of loblolly pine. Plant Cell Tissue and Organ Culture, 1999,58: 223～226.
80. Todd G. Guevin, Edward G. Kirby. Induction of embryogenesis in cultured mature zygotic embryos of Abies fraseri (Pursh) Poir. Plant Cell Tissue and Organ Culture, 1997,49:219～222
81. T.SALAJ, J.SALAJ. Somatic embryo formation on mature Abies alba × Abies ecphalonica zygotic embryo explants. Biologia Plantarum, 2003/4,47(1):7～11