大蒜离体保存技术初步研究
|
摘要
我国是世界上大蒜栽培的第一大国，经过世世代代劳动人民的精心种植、培育，已经形成许多各具特色的地方类型或品种。保存这些类型和品种对我国大蒜业的可持续发展具有重要的战略意义。大蒜属无性繁殖植物，传统的保存方法需要每年进行种质更新，不仅需要耗费大量的人力、财力，还容易受到病虫害和自然灾害的影响。为了克服传统种质资源保存所面临的问题，我们以山东苍山大蒜为材料，进行了大蒜离体保存技术的初步研究，丰要结果如下：
(1) 进行了以微繁为基础的大蒜试管苗保存研究。多芽分化试验表明，大蒜茎尖在含有NAA0.1mg/L+6-BA0.1mg/L的培养基中诱导多芽分化的效果最好。将诱导出的芽分离培养成试管苗，接种在15种不同培养基中，在四种不同的温度下保存。结果显示：试管苗在0±2℃和4±2℃，黑暗保存保存6个月，各种处理无死亡现象：15±2℃，光照强度为1080Lux(100FC)(8小时/天)条件生长的试管苗保存6个月，各种处理都生长非常瘦弱，且苗色偏淡，不适于继续保存；20±2℃，光照强度为6130Lux(560FC)(8小时/天)条件下保存6个月，除加有甘露醇和脱落酸的培养基中保存的试管苗更换新鲜培养基可恢复生长外，其他处理的试管苗已不能成活；培养基中添加甘露醇或ABA对试管苗的生长有抑制作用，两者相比，ABA的抑制作用更强烈。
(2) 对大蒜茎尖玻璃化超低温保存的基本条件进行了研究。大蒜茎尖超低温保存的基本程序为，大蒜茎尖经含高浓度蔗糖的培养基预培养后，室温下用60％PVS2预处理一段时间，换用PVS2再处理一段时间，然后将材料直接投入液氮中保存。通过对预处理时间、预处理培养基、材料大小、冰冻保护剂处理时间等的初步研究，结果表明，在大蒜的超低温保存中，当长度为3.0～3.5mm的大蒜茎尖经含0.7mol/L蔗糖的MS培养基预培养5～7天，室温下60％PVS2预处理60min，再用PVS2在0℃下处理30min，冻存无论是2天还是1个月，成活率均达到最大(100％)。
Garlic is planted widely in China and has come into being lots of special local types or cultivars by people. It is strategic for the garlic industry sustainable development to keep those garlic types and cultivars. Garlic is one of the vegetative plant and conserved with the traditional method which needs renewing every year. The traditional conservation not only needs labor and money very much, but also is affected by diseases and insect pests, natural disasters. In order to settle those problems, we initiated the study garlic germplasm conservation in vitro using Cangshan garlic. The major results were as follows:
(1) The conservation of garlic plantlets that based on micro-propagation was studied. The results showed that medium with 0.1mg/L NAA and 0.1mg/L 6-BA was best for multi-proliferation of garlic shoot-tips. The plantlets that come from the induced buds cultured on 15 different media and conserved at 4 different temperatures for 6 months. The results showed that, conserved at 0±2℃ and 4±2℃, in darkness for 6 months, the plantlets all survived and could continue to be conserved; conserved at 15±2℃ with a 8h/d photoperiod and 1080 Lux for 6 months, the plantlets were slim . light green and not suitable to conservation: conserved at 20±2℃ with a 8h/d photoperiod and 6130 Lux for 6 months, the plantlets all could't live after being re-cultured except those plantlets which cultured on the media with mannitol and abscisic acid: conserved on media with mannitol or ABA. the plantlets could be restained grow, but the effect of ABA was stronger than mannitol.
(2) The basic condition for garlic shoot-tips cryopreservation by vitrification was researched. The procedure obtained was as follows: the shoot-tips of garlic were pre-cultured on medium with high concentration of sucrose, then pretreated with 60% PVS2 at room temperature and treated with PVS2 at 0℃ . at last plunged into liquid nitrogen. The results based on the preliminary studies on pre-culture media, pre-culture time, material size and treatment time of cryprotectants showed that garlic shoot-tips with 3.0 to 3.5mm length that pre-cultured on MS medium with 0.7mol/L sucrose for 5 to 7 days, pre-treated with 60% PVS2 for 60min and treated with PVS2 for 5 to 60min could get highest survival rate after being cryopreserved in liquid nitrogen for 2 dsys or 30 days.
(1) 进行了以微繁为基础的大蒜试管苗保存研究。多芽分化试验表明，大蒜茎尖在含有NAA0.1mg/L+6-BA0.1mg/L的培养基中诱导多芽分化的效果最好。将诱导出的芽分离培养成试管苗，接种在15种不同培养基中，在四种不同的温度下保存。结果显示：试管苗在0±2℃和4±2℃，黑暗保存保存6个月，各种处理无死亡现象：15±2℃，光照强度为1080Lux(100FC)(8小时/天)条件生长的试管苗保存6个月，各种处理都生长非常瘦弱，且苗色偏淡，不适于继续保存；20±2℃，光照强度为6130Lux(560FC)(8小时/天)条件下保存6个月，除加有甘露醇和脱落酸的培养基中保存的试管苗更换新鲜培养基可恢复生长外，其他处理的试管苗已不能成活；培养基中添加甘露醇或ABA对试管苗的生长有抑制作用，两者相比，ABA的抑制作用更强烈。
(2) 对大蒜茎尖玻璃化超低温保存的基本条件进行了研究。大蒜茎尖超低温保存的基本程序为，大蒜茎尖经含高浓度蔗糖的培养基预培养后，室温下用60％PVS2预处理一段时间，换用PVS2再处理一段时间，然后将材料直接投入液氮中保存。通过对预处理时间、预处理培养基、材料大小、冰冻保护剂处理时间等的初步研究，结果表明，在大蒜的超低温保存中，当长度为3.0～3.5mm的大蒜茎尖经含0.7mol/L蔗糖的MS培养基预培养5～7天，室温下60％PVS2预处理60min，再用PVS2在0℃下处理30min，冻存无论是2天还是1个月，成活率均达到最大(100％)。
Garlic is planted widely in China and has come into being lots of special local types or cultivars by people. It is strategic for the garlic industry sustainable development to keep those garlic types and cultivars. Garlic is one of the vegetative plant and conserved with the traditional method which needs renewing every year. The traditional conservation not only needs labor and money very much, but also is affected by diseases and insect pests, natural disasters. In order to settle those problems, we initiated the study garlic germplasm conservation in vitro using Cangshan garlic. The major results were as follows:
(1) The conservation of garlic plantlets that based on micro-propagation was studied. The results showed that medium with 0.1mg/L NAA and 0.1mg/L 6-BA was best for multi-proliferation of garlic shoot-tips. The plantlets that come from the induced buds cultured on 15 different media and conserved at 4 different temperatures for 6 months. The results showed that, conserved at 0±2℃ and 4±2℃, in darkness for 6 months, the plantlets all survived and could continue to be conserved; conserved at 15±2℃ with a 8h/d photoperiod and 1080 Lux for 6 months, the plantlets were slim . light green and not suitable to conservation: conserved at 20±2℃ with a 8h/d photoperiod and 6130 Lux for 6 months, the plantlets all could't live after being re-cultured except those plantlets which cultured on the media with mannitol and abscisic acid: conserved on media with mannitol or ABA. the plantlets could be restained grow, but the effect of ABA was stronger than mannitol.
(2) The basic condition for garlic shoot-tips cryopreservation by vitrification was researched. The procedure obtained was as follows: the shoot-tips of garlic were pre-cultured on medium with high concentration of sucrose, then pretreated with 60% PVS2 at room temperature and treated with PVS2 at 0℃ . at last plunged into liquid nitrogen. The results based on the preliminary studies on pre-culture media, pre-culture time, material size and treatment time of cryprotectants showed that garlic shoot-tips with 3.0 to 3.5mm length that pre-cultured on MS medium with 0.7mol/L sucrose for 5 to 7 days, pre-treated with 60% PVS2 for 60min and treated with PVS2 for 5 to 60min could get highest survival rate after being cryopreserved in liquid nitrogen for 2 dsys or 30 days.
引文
1．陈礼光，郑郁善。锥栗种子离体胚超低温保存脱氢酶活性研究。福建林学院学报，2000，21(1)：32～35
2．陈品良。植物组织培养物的超低温保存。武汉植物学研究，1989，7(4)：390～398
3．陈箐英，兰贺胜。不同培养条件对菊花试管苗继代存活的影响。林业科技开发，2000，14(3)：18～20
4．陈世儒。蔬菜育种学。北京：农业出版社，1980
5．陈勇，王君晖，黄纯农。胡萝卜悬浮培养细胞和原生质体的玻璃化法超低温保存。浙江大学学报(理学版)，2002，29(1)：94～98
6．陈勇，王君晖，黄纯农。铁皮石斛种质资源的玻璃化法超低温保存。浙江大学学报(农业与生命科学版)，2001，27(4)：436～438
7．陈一华，陈正华。植物离体培养细胞的类减数分裂。遗传，1996，18(6)：32～36
8．陈学军，邢国明。大蒜组织培养进展与展望。长江蔬菜，1997(6)：1～3
9．邓才生。大蒜脱毒培养与试管快繁技术初探。江西农业科技，2002(4)：33～34
10．高山林，金雍安。大蒜分生组织培养脱病毒和快速繁殖技术。植物资源与环境学报，2000，9(3)，15～18
11．管正学，王建立，张学予。我国大蒜资源及其开发利用研究。自然资源，1994，(5)，54～58
12．郭延平，李嘉瑞。甘露醇对猕猴桃试管苗生长与保存的影响。落叶果树，1993(3)：24～27
13．郭延平，李嘉瑞，吉爱梅．ABA对猕猴桃种质离体保存的生理效应。西北农业学报，1994，3(4)：84～87
14．郝玉金。柑橘和苹果等果树种质资源的离体保存及其遗传变异。[博士学位论文]，武汉，华中农业大学，2000
15．黄菊辉。生姜种质资源的离体繁殖和保存。中国农业学，1995，28(2)：24～30
16．李昌华，李小川，赵美华，赵军良，罗春香。大蒜茎尖脱毒技术及组织培养研究。华北农学报，1995，10(3)：20～25
17．李锡香，戚春章，朱德蔚，王素，沈嫡。中国蔬菜种质资源保存和利用现状、问题与对策。高卫东主编，《中国植物遗传资源保存和利用》(中国植物遗传资源保存和利用战略研讨会论文集)，国际植物遗传资源研究所，2001。
18．刘高琼，李式军，张学军。大蒜试管鳞茎微繁技术研究。南京农业大学，1996，19(3)：31～36
19．刘高琼，李式军，张学军。温度和光照对大蒜试管鳞茎形成的影响(简报)。植物生理通讯，1997．33(2)：106～109
20．栾非时，陈典。大蒜茎尖愈伤组织诱导、植株分化及变异的研究。北方园艺，1993，(4)：23～25
21．马兵钢，崔辉梅，马连营．葡萄体细胞无性系变异中过氧化物酶同工酶的变化。石河子大学学报，2001，5(4)：299～301
22．彭民璋，邓九生，甘霖．油梨种质的离体低温保存。果树科学，1996，13(2)：96～98
23．沈嫡，漆小泉。大白菜超含水量种子保存研究。中国蔬菜，1996，(4)：19～22。
24．史永忠，潘瑞炽，王小箐，叶庆生，郭丽荣。铁皮石斛种质室温离体保存。华南师范大学学报(自然科学版)，1999(4)：73～77
25．史永忠，邓秀新，万蜀渊，薛光荣。苹果种质资源的离体保存。作物品种资源，1996(4)：42～44
26．王金龙。利用大豆硬实种子保存大豆种质的研究[J]。大豆科学，1999，18(4)：351～354。
27．王君晖，黄纯农。玻璃化法——园艺作物茎尖和分生组织超低温保存的新途径——文献综述。园艺学报，1994，21(3)：277～282
28．汪良驹，周夔。高等植物脱落酸的生物合成及调节。植物生理学通讯，1989，(2)：7～12
29．王善广，张华云。大蒜品种资源与分类。保鲜与加工，2002，2(3)：35
30．王子成。柑橘种质资源离体保存研究。[博士学位论文]，武汉，华中农业大学，2002
31．吴信民，陈秀兰，马鸿翔。草莓休眠芽离体培养与丛生芽低温保存实验。中国果树，1999(2)：19
32．辛淑英，谢欣。影响甘薯离体种质存活率的若干因素初探。作物品种资源，1996，(2)：36～37
33．臧新，梅兴国，龚伟，常俊丽．中国红豆杉悬浮培养细胞的超低温保存。生物技术，2002，12(2)：13～14
34．张金文，曹孜义，崔建垣。继代十七年的玉米花粉胚性细胞系的异常核和异常分裂现象。实验生物学报，1997，30(4)：407～415
35．张文海，李伯寿，黄红弟。莴苣种子贮藏条件研究。中国蔬菜，1999，(2)：18～20
36．张玉进，张兴国，刘佩瑛．魔芋花粉的低温和超低温保存。园艺学报，2000，27(2)：139～140
37．章志宏，胡中立。水稻单倍体不定芽的超低温保存和植株再生及其遗传稳定性。武汉植物学研究，2000，18(3)：169～173
38．赵树仁，武丽英，姚民昌，程爱华。番茄花粉超低温保存的研究。园艺学报，1993，20(1)：66～70
39．周小梅，王国英，敖光明，赵云云。玉米组培材料的玻璃化法超低温保存及冻后植株再生。农业生物技术学报，2001，9(4)：355～358
40．徐刚标。植物种质离体保存研究。中南林学院学报，2000，20(4)：81～87
41．徐刚标，李美娥，郑从义。银杏愈伤组织超低温保存的研究。林业科学，2001，37(3)：30～34
42．徐培文，曲士松，刘恒英。中国大蒜种质资源离体保存初步研究。中国农业科学，2002，35(3)：314～319
43．徐培文，孙慧生，介瑞杰，张培歧，张崇义，张春仁。大蒜茎尖培养脱毒及增产效果的研究。山东农业科学，1991，(6)：6～9
44. Angel, F, Barney V, Tohme J, Roca W. Stability of cassava plants at the DNA level after retrieval from 10 years of in vitro storage. Euphytica, 1996, 90(3): 307～313
45. Ashmore S E. Status reports on the development and application of in vitro techniques for the conservation and use of plant genetic resources. International Plant Genetic Resources Institute, Rome, 1997
46. Bernard F, Shaker-Bazarnow H, Kaviani B. Effects of salicylic acid on cold preservation and cryopreservation of encapsulated embryonic axes of Persian lilac (Melia azedarach L.). Euphytica, 2002, 123(1): 85～88
47. Bertrand—Desbrunais A, Noirot M, Charrier A. Minimal growth in vitro conservation of coffee (Coffea spp) 1. Influence of low concentrations of 6—benzyladenine. Plant Cell, Tissue and Organ Culture, 1991, 27: 333～339
48. Bertrand—Desbrunais A, Noirot M, Charrier A. Slow growth in vitro conservation of Coffea (Coffea Spp). Plant Cell, Tissue and Oragan Culture, 1992, 31: 105～110
49. Bessembinder J. J. E., Staritsky G, Zandvoort E A. Long term in vitro storage of Colocasia esculenta under minimal growth conditions. Plant Cell, Tissue and Organ Culture, 1993, 33: 121～127
50. Blakesley D, Kiernan R J. Cryopreservation of axillary buds of a Eucalyptus grandis × Eucalyptus camaldulensis hybrid. Cryo-Letters, 2001, 22(1): 13～18
51. Bouman H, De Klerk G J. Measurement of the extent of somaclonal variation in begonia plants regenerated under various conditions: Comparison of three assays. Theor Appl genet, 2001, 102: 111～117
52. Caplin S M. Mineral oil overlay for conservation of plant tissue cultures. Amer. Jour. Bot, 1959, 46(5): 324～329
53. Decruse S W, Seeni S. Ammonium nitrate in the culture medium influences regeneration potential of cryopreseved shoot-tips of Holostemma annulare. Cryo-letters, 2002, 23(1): 55～60
54. Desbrunais B A, Noirot M, Charrier A. Slow growth in vitro conservation of coffee (Cofffea spp.) 2: Influences of reduced concentrations of sucrose and low temperature. Plant Cell, Tissue and Organ Culture, 1992, 31: 105～110
55. Dolezel J, Lucretti S, Novak F J. The influence of 2,4-dichlorophenoxyacetic acid on cell cycle kinetics and sister-chromatid exchange frequency in garlic (Allium
sativa L.) meristem cells. Biolo plant, 1987, 29: 253～257
56. Eun G C, Yue LH, Haeng H K, Ramanatha Rao V, Englmann F. Cryopreservation of Citrus madurensis zygotic embryonic axes by vitrification: Importance of pregrowth and preculture conditions. Cryo-Letters, 2001, 22(6): 391～396
57. Gagliardi R F, Pacheco G P, Valls J F M, Mansur E. Cryopreservation of cultivated and wild Arachis species embryonic axes using desiccation and vitrification methods. Cryo-Letters, 2002, 23(1): 61～68
58. Gonzalez-Benito M E, Perez—Garcia F. Cryopreservation of lipid-rich seeds: Effect of moisture content and cooling rate on germination. Cryo-Letters, 2001, 22(2): 135～140
59. Harding K. Molecular stability of the ribosomal RNA gene in Solanum tuberosum plants recovered from slow growth and cryoconservation. Euphytica, 1991, 55: 141～146
60. Hashi G. Huettel R, Meyer R, Krusberg L, Hammerschlag F. RAPD analysis of somaclonal variants derived from embryo callus cultures of peach. Plant Cell Reports, 1997, 16: 624～627
61. Henshow G G. Tissue cultures and germplasm storage. Plant Cell Cultures: Results and Perspectives. Elsevier/North Holland Biomed, 1980. 227～282
62. Heo J W, Kubota C, Kozai T. Effects of CO_2 concentration, PPFD and sucrose concentration on Cymbidium plantlet growth in vitro. Acta Horticulturae, 1996, 440: 560～565
63. Hirai D, Sakai A. Cryopreservation of in vitro-grown axillary shoot-tip meristems of mint (Mentha spicata L.) by encapsulation vitrification. Plant Cell Reports, 1999a, 19(2): 150～155
64. Hirai D, Sakai A. Cryopreservation of in vitro-grown meristems of potato (Solanum tuberosum L.) by encapsulation-vitrification. Potato Research, 1999b, 42(2): 153～160
65. Hirai D, Shirai K, Shirai S, Sakai A. Cryopreservation of in vitro-grown meristems of strawberry (Fragaria x ananassa Duch.) by encapsulation-vitrification. Euphytica, 1998, 101(1): 109～115
66. Hornung R, Domas R, Lynch P T. Cryopreservation of plumular explants of coconut (Cocos nucifera L.) to support programmes for mass clonal propagation through somatic embryogenesis. Cryo-Letters, 2001, 22(4): 211～220
67. Hornung R, Holland A, Taylor H F, Lynch P T. Cryopreservation of auricula shoot tips using the encapsulation/dehydration technique. Cryo-Letters, 2001, 22(1): 27～34
68. IPGRI/CIAT. Establishment and operation of a pilot in vitro active genebank. Report
of a CIAT-IBPGR collaborative project using cassava (Manihot escu]enta Crantz) as a model. International Plant Genetic Resources Institute y Centro Internacional de Agricultura Tropical, Italia, 1994, 59p.
69. Kim S I, Choi H K, Son J S, Yun J H, Jang M S, Kim H R, Song J Y, Eim J H, Choi H J, Hong S S. Cryopreservation of Taxus Chinensis suspension cell cultures. Cryo-Letters, 2001, 22(1): 43～50
70. Joyce S M, Cassells A C. Variation in potato microplant morphology in vitro and DNA methylation. Plant Cell, Tissue and Organ Culture, 2002, 70(2): 125～137
71. Jung D W, Sung C K, Touno K, Yoshimatsu K, Shimomura K. Cryopreservation of Hyoscyamus niger adventitious roots by vitrification. Journal of Plant Physiology, 2001, 158(6): 801～805
72. Kartha K K, Mroginski L A, Pahl E K, Leung N L. Germplasm preservation of Coffea (Coffea spp L) by in vitro culture of shoot apical meristems. Plant Science Letters, 1981, 22: 301～307
73. Ko W H, Hwang S C, Ku F M. A new technique for storage of meristem-tip cultures of 'Cavendish' banana. Plant Cell, Tissue Organ culture, 1991, 25: 179～183
74. Kubota Chieri, Rajapakse N. C. Low-temperature storage of micropropagated plantlets under selected light environments. HortScience, 1996, 31(3): 449～452
75. Lambardi M, Fabbri A, Cacccavale A. Cryopreservation of white poplar (Populus alba L.) by vitrification of in vitro-grown shoot-tips. Plant Cell Reports, 2000, 19: 213～218
76. Larkin P J, Scowcroft W R. Somaclonal variation - a novel source of variability from cell cultures for plant improvement. Theor. Appl. Genet, 1981, 60: 197～214
77. Lyndsey A, Withers L A. in vitro approaches to conversation of plant genetic resources [A], in Plant Tissue Culture and it's Application. Lyndsey A. Withers and P. G. AldXson (eds). London, Butterworths, 1986, 261～275
78. Matsumoto T, Sakai A, Yamada K. Cryopreservation of in vitro grown apical meristems of Wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration. Plant Cell Report, 1994, 13: 442～446
79. Matsumoto T, Takahashi C, Sakai A, Nako Y. Cryopreservation of in vitro-grown apical meristems of hybrid statice by three different procedures. Scientia Horticulturae, 1998, 76(1-2): 105～114
80. Mix-Wagner G, Conner A J, Cross R J. Survival and recovery of asparagus shoottips after cyopreservation using the "droplet" method. New Zealand Journal of Crop and Horticultureal Science, 2000, 28(4): 283～287
81. Monette P L. Cold storage of kwifruit shoot tips in vitro[J]. Hortscience, 1986, 21(5): 1203～1205
82. Mullin R H, Schligel D E. Cold storage of strawberry meristem plantlets. Hortscience, 1976, 11(1): 100～102
83. Oka s, Niino T. Long term storage of Pear (Pyrus sup) shoot cultures in vitro by minimal growth method [J]. JARQ, 1997, 31(1): 1～7
84. Paulet F, Eksomtramage T, Lu Y H. Callus and apes cryopreservation for sugarcane germplasm management. In: Proceedings of ISSCT Sugar Breeding Workshop, CIRAD/ISSCT, Montpellier, France. 1994
85. Pence V C. Cryopreservation of bryophytes: The effects of abscisic acid and encapsulation dehydration. Bryologist, 1998, 101: 278～281
86. Pennycooke J C, Towill L E. Medium alterations improve regrowth of sweet potato [Ipomoea batatas (L.) Lam.] shoot tips cryopreservation by vitrification and encapsulation dehydration. Cryo-letters, 2001, 22(6): 381～389
87. Pennycooke J C, Towill L E. Cryopreservation of shoot tips from in vitro plants of sweet potato (Ipomoea batatas [L.] Lam.) by vitrification. Plant Cell Reports, 2000, 19: 733～737
88. Rajora O P, Mosseler A. Challenges and opportunities for conservation of forest genetic resources. Euphytica, 2001, 118(2): 197～212
89. Rall W F, Fahy G M. Ice-free cryopreservation of mouse embryos at -196℃ by vitrification. Nature, 1985, 313: 573～575
90. Ryynanen L A, Haggman H M. Recovery of cryopreserved silver birch shoot tips is affected by the pre-freezing age of the cultures and ammonium substitution. Plant Cell Reports, 2001, 20 (4): 354～360
91. Sarkar D, Chakrabarti S. K, Naik P. S. Slow-growth conservation of potato microplants: efficacy of ancymidol for long-termstorage in vitro. Euphytica, 2001, 117: 133～142
92. Shibli R A, Haagenson D M, Cunningham S M, Berg W K, Volenec J J. Cryopreservation of alfalfa (Medicago sativa L.) cells by encapsulation/dehydration. Plant Cell Reports, 2001, 20(5): 445～450
93. Sakai A, Kobayashi S, and Oiyama I. Cryopreservation of nuclear cells of navel orange by vitrification. Plant Cell Report, 1990, 9: 30～33
94. Staritsky A J. In vitro conservation of avoid germplasm at reduced temperature and under osmotie stress. In: Plant Tissue Culture and Its Agricultural Applications. London: Butterworths, 1986: 277～283
95. Takagi H ey al. Cryopreservation of in vitro-grown shoot tips of taro (Colocasia
esculenta (L.) Schott) by vitrification, 1. Investigation of basic conditions of the vitrification procedure. Plant Cell Reports. 1997, 16: 594～595
96. Tannoury M, Ralambosoa J, Kaminski M. cryopreservation by vitrification of coated shoot-tips of carnation (Dianthus caryophyllus L.) cultured in vitro. C. R. Acad. Sci (Paris), 1991, 313 Ser. Ⅲ: 633～638
97. Towill, L. E. and Jarret. R. L. Cryopreservation of shoot tips from in vitro plants of sweet potato [Ipomoea batatas(L.) Lam] by vitrification. Plant Cell Reports, 1992, 11: 175
98. Turner S R, Touchell D H, Senaratna T, Bunn E, Tan B, Dixon KW. Effects of plant growth regulators on survival and recovery growth following cryopreservation. Cryo-letters, 2001, 22(3): 163～174
99. Van den houwe I, De S K, Tezenas du M H, Swennen R. Variability in storage potential of banana shoot cultures under medium term storage conditions. Plant Cell, Tissue and Organ Culture. 1995, 42: 269～274
100. Vasil I K. Perspectives in Plant Cell and Tissue Culture, International Review of Cytoloty, Supplement 11B, New York/London/Sydney/San Francisco: Academic Press, 1980. 297～306
101. Vendrame W A, Kochert G, Wetzstein H Y. AFLP analysis of variation in pecan somatic embryos. Plant Cell Reports, 1999, 18: 853～857
102. Villalobos V M, Engelman F. Ex situ conservation of plant germplasm using biotechnology. Biotechol, 1995, 11(4): 375～382
103. Wang Q, Gafny R, Sahar N, Sela I, Mawassi M, Tanne E, Perl A. Cryopreservation of grapevine (Vitis vinifera L.) embryogenic cell suspensions by encapsulation/dehydration and subsequent plant regeneration. Plant Science, 2002, 162(4): 551～558
104. Watt M P, Thokoane N L, Mycock D, Blakeway F. In vitro storage of Eucalyptus grandis germplasm under minimal growth conditions. Plant Cell, Tissue and Organ Culture, 2000, 61: 161～164
2．陈品良。植物组织培养物的超低温保存。武汉植物学研究，1989，7(4)：390～398
3．陈箐英，兰贺胜。不同培养条件对菊花试管苗继代存活的影响。林业科技开发，2000，14(3)：18～20
4．陈世儒。蔬菜育种学。北京：农业出版社，1980
5．陈勇，王君晖，黄纯农。胡萝卜悬浮培养细胞和原生质体的玻璃化法超低温保存。浙江大学学报(理学版)，2002，29(1)：94～98
6．陈勇，王君晖，黄纯农。铁皮石斛种质资源的玻璃化法超低温保存。浙江大学学报(农业与生命科学版)，2001，27(4)：436～438
7．陈一华，陈正华。植物离体培养细胞的类减数分裂。遗传，1996，18(6)：32～36
8．陈学军，邢国明。大蒜组织培养进展与展望。长江蔬菜，1997(6)：1～3
9．邓才生。大蒜脱毒培养与试管快繁技术初探。江西农业科技，2002(4)：33～34
10．高山林，金雍安。大蒜分生组织培养脱病毒和快速繁殖技术。植物资源与环境学报，2000，9(3)，15～18
11．管正学，王建立，张学予。我国大蒜资源及其开发利用研究。自然资源，1994，(5)，54～58
12．郭延平，李嘉瑞。甘露醇对猕猴桃试管苗生长与保存的影响。落叶果树，1993(3)：24～27
13．郭延平，李嘉瑞，吉爱梅．ABA对猕猴桃种质离体保存的生理效应。西北农业学报，1994，3(4)：84～87
14．郝玉金。柑橘和苹果等果树种质资源的离体保存及其遗传变异。[博士学位论文]，武汉，华中农业大学，2000
15．黄菊辉。生姜种质资源的离体繁殖和保存。中国农业学，1995，28(2)：24～30
16．李昌华，李小川，赵美华，赵军良，罗春香。大蒜茎尖脱毒技术及组织培养研究。华北农学报，1995，10(3)：20～25
17．李锡香，戚春章，朱德蔚，王素，沈嫡。中国蔬菜种质资源保存和利用现状、问题与对策。高卫东主编，《中国植物遗传资源保存和利用》(中国植物遗传资源保存和利用战略研讨会论文集)，国际植物遗传资源研究所，2001。
18．刘高琼，李式军，张学军。大蒜试管鳞茎微繁技术研究。南京农业大学，1996，19(3)：31～36
19．刘高琼，李式军，张学军。温度和光照对大蒜试管鳞茎形成的影响(简报)。植物生理通讯，1997．33(2)：106～109
20．栾非时，陈典。大蒜茎尖愈伤组织诱导、植株分化及变异的研究。北方园艺，1993，(4)：23～25
21．马兵钢，崔辉梅，马连营．葡萄体细胞无性系变异中过氧化物酶同工酶的变化。石河子大学学报，2001，5(4)：299～301
22．彭民璋，邓九生，甘霖．油梨种质的离体低温保存。果树科学，1996，13(2)：96～98
23．沈嫡，漆小泉。大白菜超含水量种子保存研究。中国蔬菜，1996，(4)：19～22。
24．史永忠，潘瑞炽，王小箐，叶庆生，郭丽荣。铁皮石斛种质室温离体保存。华南师范大学学报(自然科学版)，1999(4)：73～77
25．史永忠，邓秀新，万蜀渊，薛光荣。苹果种质资源的离体保存。作物品种资源，1996(4)：42～44
26．王金龙。利用大豆硬实种子保存大豆种质的研究[J]。大豆科学，1999，18(4)：351～354。
27．王君晖，黄纯农。玻璃化法——园艺作物茎尖和分生组织超低温保存的新途径——文献综述。园艺学报，1994，21(3)：277～282
28．汪良驹，周夔。高等植物脱落酸的生物合成及调节。植物生理学通讯，1989，(2)：7～12
29．王善广，张华云。大蒜品种资源与分类。保鲜与加工，2002，2(3)：35
30．王子成。柑橘种质资源离体保存研究。[博士学位论文]，武汉，华中农业大学，2002
31．吴信民，陈秀兰，马鸿翔。草莓休眠芽离体培养与丛生芽低温保存实验。中国果树，1999(2)：19
32．辛淑英，谢欣。影响甘薯离体种质存活率的若干因素初探。作物品种资源，1996，(2)：36～37
33．臧新，梅兴国，龚伟，常俊丽．中国红豆杉悬浮培养细胞的超低温保存。生物技术，2002，12(2)：13～14
34．张金文，曹孜义，崔建垣。继代十七年的玉米花粉胚性细胞系的异常核和异常分裂现象。实验生物学报，1997，30(4)：407～415
35．张文海，李伯寿，黄红弟。莴苣种子贮藏条件研究。中国蔬菜，1999，(2)：18～20
36．张玉进，张兴国，刘佩瑛．魔芋花粉的低温和超低温保存。园艺学报，2000，27(2)：139～140
37．章志宏，胡中立。水稻单倍体不定芽的超低温保存和植株再生及其遗传稳定性。武汉植物学研究，2000，18(3)：169～173
38．赵树仁，武丽英，姚民昌，程爱华。番茄花粉超低温保存的研究。园艺学报，1993，20(1)：66～70
39．周小梅，王国英，敖光明，赵云云。玉米组培材料的玻璃化法超低温保存及冻后植株再生。农业生物技术学报，2001，9(4)：355～358
40．徐刚标。植物种质离体保存研究。中南林学院学报，2000，20(4)：81～87
41．徐刚标，李美娥，郑从义。银杏愈伤组织超低温保存的研究。林业科学，2001，37(3)：30～34
42．徐培文，曲士松，刘恒英。中国大蒜种质资源离体保存初步研究。中国农业科学，2002，35(3)：314～319
43．徐培文，孙慧生，介瑞杰，张培歧，张崇义，张春仁。大蒜茎尖培养脱毒及增产效果的研究。山东农业科学，1991，(6)：6～9
44. Angel, F, Barney V, Tohme J, Roca W. Stability of cassava plants at the DNA level after retrieval from 10 years of in vitro storage. Euphytica, 1996, 90(3): 307～313
45. Ashmore S E. Status reports on the development and application of in vitro techniques for the conservation and use of plant genetic resources. International Plant Genetic Resources Institute, Rome, 1997
46. Bernard F, Shaker-Bazarnow H, Kaviani B. Effects of salicylic acid on cold preservation and cryopreservation of encapsulated embryonic axes of Persian lilac (Melia azedarach L.). Euphytica, 2002, 123(1): 85～88
47. Bertrand—Desbrunais A, Noirot M, Charrier A. Minimal growth in vitro conservation of coffee (Coffea spp) 1. Influence of low concentrations of 6—benzyladenine. Plant Cell, Tissue and Organ Culture, 1991, 27: 333～339
48. Bertrand—Desbrunais A, Noirot M, Charrier A. Slow growth in vitro conservation of Coffea (Coffea Spp). Plant Cell, Tissue and Oragan Culture, 1992, 31: 105～110
49. Bessembinder J. J. E., Staritsky G, Zandvoort E A. Long term in vitro storage of Colocasia esculenta under minimal growth conditions. Plant Cell, Tissue and Organ Culture, 1993, 33: 121～127
50. Blakesley D, Kiernan R J. Cryopreservation of axillary buds of a Eucalyptus grandis × Eucalyptus camaldulensis hybrid. Cryo-Letters, 2001, 22(1): 13～18
51. Bouman H, De Klerk G J. Measurement of the extent of somaclonal variation in begonia plants regenerated under various conditions: Comparison of three assays. Theor Appl genet, 2001, 102: 111～117
52. Caplin S M. Mineral oil overlay for conservation of plant tissue cultures. Amer. Jour. Bot, 1959, 46(5): 324～329
53. Decruse S W, Seeni S. Ammonium nitrate in the culture medium influences regeneration potential of cryopreseved shoot-tips of Holostemma annulare. Cryo-letters, 2002, 23(1): 55～60
54. Desbrunais B A, Noirot M, Charrier A. Slow growth in vitro conservation of coffee (Cofffea spp.) 2: Influences of reduced concentrations of sucrose and low temperature. Plant Cell, Tissue and Organ Culture, 1992, 31: 105～110
55. Dolezel J, Lucretti S, Novak F J. The influence of 2,4-dichlorophenoxyacetic acid on cell cycle kinetics and sister-chromatid exchange frequency in garlic (Allium
sativa L.) meristem cells. Biolo plant, 1987, 29: 253～257
56. Eun G C, Yue LH, Haeng H K, Ramanatha Rao V, Englmann F. Cryopreservation of Citrus madurensis zygotic embryonic axes by vitrification: Importance of pregrowth and preculture conditions. Cryo-Letters, 2001, 22(6): 391～396
57. Gagliardi R F, Pacheco G P, Valls J F M, Mansur E. Cryopreservation of cultivated and wild Arachis species embryonic axes using desiccation and vitrification methods. Cryo-Letters, 2002, 23(1): 61～68
58. Gonzalez-Benito M E, Perez—Garcia F. Cryopreservation of lipid-rich seeds: Effect of moisture content and cooling rate on germination. Cryo-Letters, 2001, 22(2): 135～140
59. Harding K. Molecular stability of the ribosomal RNA gene in Solanum tuberosum plants recovered from slow growth and cryoconservation. Euphytica, 1991, 55: 141～146
60. Hashi G. Huettel R, Meyer R, Krusberg L, Hammerschlag F. RAPD analysis of somaclonal variants derived from embryo callus cultures of peach. Plant Cell Reports, 1997, 16: 624～627
61. Henshow G G. Tissue cultures and germplasm storage. Plant Cell Cultures: Results and Perspectives. Elsevier/North Holland Biomed, 1980. 227～282
62. Heo J W, Kubota C, Kozai T. Effects of CO_2 concentration, PPFD and sucrose concentration on Cymbidium plantlet growth in vitro. Acta Horticulturae, 1996, 440: 560～565
63. Hirai D, Sakai A. Cryopreservation of in vitro-grown axillary shoot-tip meristems of mint (Mentha spicata L.) by encapsulation vitrification. Plant Cell Reports, 1999a, 19(2): 150～155
64. Hirai D, Sakai A. Cryopreservation of in vitro-grown meristems of potato (Solanum tuberosum L.) by encapsulation-vitrification. Potato Research, 1999b, 42(2): 153～160
65. Hirai D, Shirai K, Shirai S, Sakai A. Cryopreservation of in vitro-grown meristems of strawberry (Fragaria x ananassa Duch.) by encapsulation-vitrification. Euphytica, 1998, 101(1): 109～115
66. Hornung R, Domas R, Lynch P T. Cryopreservation of plumular explants of coconut (Cocos nucifera L.) to support programmes for mass clonal propagation through somatic embryogenesis. Cryo-Letters, 2001, 22(4): 211～220
67. Hornung R, Holland A, Taylor H F, Lynch P T. Cryopreservation of auricula shoot tips using the encapsulation/dehydration technique. Cryo-Letters, 2001, 22(1): 27～34
68. IPGRI/CIAT. Establishment and operation of a pilot in vitro active genebank. Report
of a CIAT-IBPGR collaborative project using cassava (Manihot escu]enta Crantz) as a model. International Plant Genetic Resources Institute y Centro Internacional de Agricultura Tropical, Italia, 1994, 59p.
69. Kim S I, Choi H K, Son J S, Yun J H, Jang M S, Kim H R, Song J Y, Eim J H, Choi H J, Hong S S. Cryopreservation of Taxus Chinensis suspension cell cultures. Cryo-Letters, 2001, 22(1): 43～50
70. Joyce S M, Cassells A C. Variation in potato microplant morphology in vitro and DNA methylation. Plant Cell, Tissue and Organ Culture, 2002, 70(2): 125～137
71. Jung D W, Sung C K, Touno K, Yoshimatsu K, Shimomura K. Cryopreservation of Hyoscyamus niger adventitious roots by vitrification. Journal of Plant Physiology, 2001, 158(6): 801～805
72. Kartha K K, Mroginski L A, Pahl E K, Leung N L. Germplasm preservation of Coffea (Coffea spp L) by in vitro culture of shoot apical meristems. Plant Science Letters, 1981, 22: 301～307
73. Ko W H, Hwang S C, Ku F M. A new technique for storage of meristem-tip cultures of 'Cavendish' banana. Plant Cell, Tissue Organ culture, 1991, 25: 179～183
74. Kubota Chieri, Rajapakse N. C. Low-temperature storage of micropropagated plantlets under selected light environments. HortScience, 1996, 31(3): 449～452
75. Lambardi M, Fabbri A, Cacccavale A. Cryopreservation of white poplar (Populus alba L.) by vitrification of in vitro-grown shoot-tips. Plant Cell Reports, 2000, 19: 213～218
76. Larkin P J, Scowcroft W R. Somaclonal variation - a novel source of variability from cell cultures for plant improvement. Theor. Appl. Genet, 1981, 60: 197～214
77. Lyndsey A, Withers L A. in vitro approaches to conversation of plant genetic resources [A], in Plant Tissue Culture and it's Application. Lyndsey A. Withers and P. G. AldXson (eds). London, Butterworths, 1986, 261～275
78. Matsumoto T, Sakai A, Yamada K. Cryopreservation of in vitro grown apical meristems of Wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration. Plant Cell Report, 1994, 13: 442～446
79. Matsumoto T, Takahashi C, Sakai A, Nako Y. Cryopreservation of in vitro-grown apical meristems of hybrid statice by three different procedures. Scientia Horticulturae, 1998, 76(1-2): 105～114
80. Mix-Wagner G, Conner A J, Cross R J. Survival and recovery of asparagus shoottips after cyopreservation using the "droplet" method. New Zealand Journal of Crop and Horticultureal Science, 2000, 28(4): 283～287
81. Monette P L. Cold storage of kwifruit shoot tips in vitro[J]. Hortscience, 1986, 21(5): 1203～1205
82. Mullin R H, Schligel D E. Cold storage of strawberry meristem plantlets. Hortscience, 1976, 11(1): 100～102
83. Oka s, Niino T. Long term storage of Pear (Pyrus sup) shoot cultures in vitro by minimal growth method [J]. JARQ, 1997, 31(1): 1～7
84. Paulet F, Eksomtramage T, Lu Y H. Callus and apes cryopreservation for sugarcane germplasm management. In: Proceedings of ISSCT Sugar Breeding Workshop, CIRAD/ISSCT, Montpellier, France. 1994
85. Pence V C. Cryopreservation of bryophytes: The effects of abscisic acid and encapsulation dehydration. Bryologist, 1998, 101: 278～281
86. Pennycooke J C, Towill L E. Medium alterations improve regrowth of sweet potato [Ipomoea batatas (L.) Lam.] shoot tips cryopreservation by vitrification and encapsulation dehydration. Cryo-letters, 2001, 22(6): 381～389
87. Pennycooke J C, Towill L E. Cryopreservation of shoot tips from in vitro plants of sweet potato (Ipomoea batatas [L.] Lam.) by vitrification. Plant Cell Reports, 2000, 19: 733～737
88. Rajora O P, Mosseler A. Challenges and opportunities for conservation of forest genetic resources. Euphytica, 2001, 118(2): 197～212
89. Rall W F, Fahy G M. Ice-free cryopreservation of mouse embryos at -196℃ by vitrification. Nature, 1985, 313: 573～575
90. Ryynanen L A, Haggman H M. Recovery of cryopreserved silver birch shoot tips is affected by the pre-freezing age of the cultures and ammonium substitution. Plant Cell Reports, 2001, 20 (4): 354～360
91. Sarkar D, Chakrabarti S. K, Naik P. S. Slow-growth conservation of potato microplants: efficacy of ancymidol for long-termstorage in vitro. Euphytica, 2001, 117: 133～142
92. Shibli R A, Haagenson D M, Cunningham S M, Berg W K, Volenec J J. Cryopreservation of alfalfa (Medicago sativa L.) cells by encapsulation/dehydration. Plant Cell Reports, 2001, 20(5): 445～450
93. Sakai A, Kobayashi S, and Oiyama I. Cryopreservation of nuclear cells of navel orange by vitrification. Plant Cell Report, 1990, 9: 30～33
94. Staritsky A J. In vitro conservation of avoid germplasm at reduced temperature and under osmotie stress. In: Plant Tissue Culture and Its Agricultural Applications. London: Butterworths, 1986: 277～283
95. Takagi H ey al. Cryopreservation of in vitro-grown shoot tips of taro (Colocasia
esculenta (L.) Schott) by vitrification, 1. Investigation of basic conditions of the vitrification procedure. Plant Cell Reports. 1997, 16: 594～595
96. Tannoury M, Ralambosoa J, Kaminski M. cryopreservation by vitrification of coated shoot-tips of carnation (Dianthus caryophyllus L.) cultured in vitro. C. R. Acad. Sci (Paris), 1991, 313 Ser. Ⅲ: 633～638
97. Towill, L. E. and Jarret. R. L. Cryopreservation of shoot tips from in vitro plants of sweet potato [Ipomoea batatas(L.) Lam] by vitrification. Plant Cell Reports, 1992, 11: 175
98. Turner S R, Touchell D H, Senaratna T, Bunn E, Tan B, Dixon KW. Effects of plant growth regulators on survival and recovery growth following cryopreservation. Cryo-letters, 2001, 22(3): 163～174
99. Van den houwe I, De S K, Tezenas du M H, Swennen R. Variability in storage potential of banana shoot cultures under medium term storage conditions. Plant Cell, Tissue and Organ Culture. 1995, 42: 269～274
100. Vasil I K. Perspectives in Plant Cell and Tissue Culture, International Review of Cytoloty, Supplement 11B, New York/London/Sydney/San Francisco: Academic Press, 1980. 297～306
101. Vendrame W A, Kochert G, Wetzstein H Y. AFLP analysis of variation in pecan somatic embryos. Plant Cell Reports, 1999, 18: 853～857
102. Villalobos V M, Engelman F. Ex situ conservation of plant germplasm using biotechnology. Biotechol, 1995, 11(4): 375～382
103. Wang Q, Gafny R, Sahar N, Sela I, Mawassi M, Tanne E, Perl A. Cryopreservation of grapevine (Vitis vinifera L.) embryogenic cell suspensions by encapsulation/dehydration and subsequent plant regeneration. Plant Science, 2002, 162(4): 551～558
104. Watt M P, Thokoane N L, Mycock D, Blakeway F. In vitro storage of Eucalyptus grandis germplasm under minimal growth conditions. Plant Cell, Tissue and Organ Culture, 2000, 61: 161～164