橡胶农杆菌转化体系及抗寒转基因种质的研究
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摘要
巴西橡胶(Hevea brasiliensis)是重要的热带经济作物,是具有不可替代性的重要工业原料和军事物资,但我国橡胶自给严重不足,低温寒害是限制我国橡胶发展的主要原因。常规育种只能部分解决抗寒问题,不能从根本上解决橡胶寒害的瓶颈。基因工程育种成为橡胶树生物技术研究、品种改良的核心内容。巴西橡胶树体外再生技术在20世纪70年代已经成功,但目前其组织培养技术仍然比较落后,至今只有少数无性系能够实现体外形态发生,且大多数品系体外植株再生的效率非常低下,这已经成为建立高效遗传转化体系,从分子水平进行橡胶树遗传改造的重要障碍。相比于模式植物和粮食等经济作物来说,橡胶分子生物学研究严重滞后。
项目以海南目前大规模推广种植的橡胶树新品系热研7-33-97为植物材料,通过农杆菌(Agribacterium tumefaciens)介导法,分别选择花药和内珠被脱分化愈伤,以及长期继代增殖的胚性愈伤为转化受体进行转化,采取导入抗寒调控基因和关键性抗寒基因相结合的策略,将新疆小拟南芥抗冻调节基因——CBF基因及其下游功能基因
——cor15a转入橡胶中,以期完善组织培养技术,获得抗寒转基因植株,探索提高橡胶抗寒性的基因工程策略,建立稳定、高效的遗传转化体系。研究主要解决了以下问题:
1、优化了橡胶热研7-33-97品种花药组织培养配方。
2、成功建立了橡胶胚性愈伤长期继代保存和增殖技术(已申请专利,申请号200710084622.7,申请日2007年2月12日),国内外还未见相关报道。为提高橡胶树组培效率,建立新的高效的转化技术路线,以及为橡胶花药悬浮培养和橡胶原生质体培养长期提供大量的好材料提供了技术基础。
3、在橡胶花药组培和胚性愈伤长期继代增殖技术的基础上,利用花药脱分化愈伤和经长期继代增殖的胚性愈伤拟行农杆菌转化,实验表明胚性愈伤是更适宜转化的受体,在转化的侵染、共培养、抑菌、筛选、成胚等各环节均表现出显著的优越性。胚性愈伤经农杆菌转化后形成的抗性胚,经PCR检测已有部分转基因体胚存在。开拓了橡胶转化技术新的技术路线。
4、探索和优化了橡胶农杆菌转化技术各环节的技术指标。
5、为研究和开发利用通过向橡胶中导入抗寒基因来提高橡胶的抗寒性提供了一条新的途径和方法,具有一定的理论和应用前景。
Rubber tree (Hevea brasiliensis) is an important economic crop in tropics. It has very important value especially in the fields of industry and military. At present, the consumption of natural rubber in China increases annually, and the shortage increases annually. Cold injury is the key factor that restricts the growth and distribution of rubber. Routine breeding of rubber tree can only partially, but not radically settle the problem. Gene engineering breeding becomes the core content of rubber tree biotechnological research and variety improvement.The technique of somatic embryogenesis in Hevea brasiliensis was succeeded in 1970s. But now the technique is dropped behind because that so far, only a few the clones can produce somatic embryogenesis through this technique, and the rate of the in vitro plant regeneration in most of the clones is very low. This becomes the main obstacle in the way to found the high genetic transformation system for reconstructing the inheritance of Hevea in molecule level. Compare of studies on model plants and economic crops, there are lags in advances in molecular biology of rubber tree.
In this research item,we chosed the rubber variety of RY 7-33-97 widely generalized in Hainan, China. CBF gene, its downstream COR15a gene and regulating element CRT/DRE in promoter of COR15a gene, were transfered to firsthand calli from anther or inner integument culture and the long-term proliferated embryogenic calli via somatic embryogenesis by Agrobacterium tumefaciens to develop cold endurance rubber trees and to establish of genetic transformation system in Hevea including efficient culture via somatic embryogenesis. The results were shown as follow.
1. The anther culture media of RY 7-33-97 was optimized.
2. The long-term proliferation technique of Hevea embryonic calli was successfully established. The technique could increase the efficient of Hevea tissue culture, carve out a new way to found the high Hevea genetic transformation system and chronically afford plentiful good material for Hevea embryogenic cell suspension culture and protoplast culture.
3. On the basis of Hevea anther culture and long-term proliferation technique of Hevea embryonic calli, we used the firsthand calli of Hevea anther and long-term proliferated embryonic calli to transfer by Agrobacterium tumefaciens. The result showed that embryonic calli were consumedly superior to the firsthand calli in transformation. And we have gained transformed embryoids via long-term proliferated embryonic calli. The study caved out a new way to found the high Hevea genetic transformation system.
4. The study explored or optimized the index of every step of transformation mediated by Agrobacterium tumefaciens.
5.This research laid a foundation for researching on fouction of CBF1、CBF3、ApCOR15a and provided a new method for cold endurance, presenting theorical significance and applicative prospect.
项目以海南目前大规模推广种植的橡胶树新品系热研7-33-97为植物材料,通过农杆菌(Agribacterium tumefaciens)介导法,分别选择花药和内珠被脱分化愈伤,以及长期继代增殖的胚性愈伤为转化受体进行转化,采取导入抗寒调控基因和关键性抗寒基因相结合的策略,将新疆小拟南芥抗冻调节基因——CBF基因及其下游功能基因
——cor15a转入橡胶中,以期完善组织培养技术,获得抗寒转基因植株,探索提高橡胶抗寒性的基因工程策略,建立稳定、高效的遗传转化体系。研究主要解决了以下问题:
1、优化了橡胶热研7-33-97品种花药组织培养配方。
2、成功建立了橡胶胚性愈伤长期继代保存和增殖技术(已申请专利,申请号200710084622.7,申请日2007年2月12日),国内外还未见相关报道。为提高橡胶树组培效率,建立新的高效的转化技术路线,以及为橡胶花药悬浮培养和橡胶原生质体培养长期提供大量的好材料提供了技术基础。
3、在橡胶花药组培和胚性愈伤长期继代增殖技术的基础上,利用花药脱分化愈伤和经长期继代增殖的胚性愈伤拟行农杆菌转化,实验表明胚性愈伤是更适宜转化的受体,在转化的侵染、共培养、抑菌、筛选、成胚等各环节均表现出显著的优越性。胚性愈伤经农杆菌转化后形成的抗性胚,经PCR检测已有部分转基因体胚存在。开拓了橡胶转化技术新的技术路线。
4、探索和优化了橡胶农杆菌转化技术各环节的技术指标。
5、为研究和开发利用通过向橡胶中导入抗寒基因来提高橡胶的抗寒性提供了一条新的途径和方法,具有一定的理论和应用前景。
Rubber tree (Hevea brasiliensis) is an important economic crop in tropics. It has very important value especially in the fields of industry and military. At present, the consumption of natural rubber in China increases annually, and the shortage increases annually. Cold injury is the key factor that restricts the growth and distribution of rubber. Routine breeding of rubber tree can only partially, but not radically settle the problem. Gene engineering breeding becomes the core content of rubber tree biotechnological research and variety improvement.The technique of somatic embryogenesis in Hevea brasiliensis was succeeded in 1970s. But now the technique is dropped behind because that so far, only a few the clones can produce somatic embryogenesis through this technique, and the rate of the in vitro plant regeneration in most of the clones is very low. This becomes the main obstacle in the way to found the high genetic transformation system for reconstructing the inheritance of Hevea in molecule level. Compare of studies on model plants and economic crops, there are lags in advances in molecular biology of rubber tree.
In this research item,we chosed the rubber variety of RY 7-33-97 widely generalized in Hainan, China. CBF gene, its downstream COR15a gene and regulating element CRT/DRE in promoter of COR15a gene, were transfered to firsthand calli from anther or inner integument culture and the long-term proliferated embryogenic calli via somatic embryogenesis by Agrobacterium tumefaciens to develop cold endurance rubber trees and to establish of genetic transformation system in Hevea including efficient culture via somatic embryogenesis. The results were shown as follow.
1. The anther culture media of RY 7-33-97 was optimized.
2. The long-term proliferation technique of Hevea embryonic calli was successfully established. The technique could increase the efficient of Hevea tissue culture, carve out a new way to found the high Hevea genetic transformation system and chronically afford plentiful good material for Hevea embryogenic cell suspension culture and protoplast culture.
3. On the basis of Hevea anther culture and long-term proliferation technique of Hevea embryonic calli, we used the firsthand calli of Hevea anther and long-term proliferated embryonic calli to transfer by Agrobacterium tumefaciens. The result showed that embryonic calli were consumedly superior to the firsthand calli in transformation. And we have gained transformed embryoids via long-term proliferated embryonic calli. The study caved out a new way to found the high Hevea genetic transformation system.
4. The study explored or optimized the index of every step of transformation mediated by Agrobacterium tumefaciens.
5.This research laid a foundation for researching on fouction of CBF1、CBF3、ApCOR15a and provided a new method for cold endurance, presenting theorical significance and applicative prospect.
引文
1. 陈雄庭, 王泽云, 吴胡蝶. 加快橡胶花药植株推广应用的几种方法探讨. 热带作物学报, 1993, 14(2): 17-20
2. 陈雄庭. 橡胶速生高产新型种植材料的培育与试种. 热带作物产业带建设规划研讨会—天然橡胶产业发展论文集. 2006: 104-107
3. 陈正华, 许绪恩, 庞任声等. 橡胶树属的花药培养及花粉植株的研究. 木本植物组织培养及其应用. 北京:高等教育出版社, 1986.481-500
4. 广东省农垦总局、海南省农垦总局编著.橡胶树良种选育与推广.广州:广东科技出版社: 1994
5. 简令成,王红. 钙(Ca2+)在植物抗寒中的作用. 细胞生物学杂志,2002,24(3):166-171.
6. 简令成, 孙龙华, 孙德兰. 几种植物细胞表面糖蛋白的电镜细胞化学及其与植物抗逆性的关系.实验生物学报, 1986, 19:261-271
7. 李卫, 孙中海,章文才等. 钙与钙调素对柑橘原生质体抗冻性的影响. 植物生理学报, 1997, 23(3):262-266
8. 刘炜, 孙德兰, 王红等. 2℃低温下抗寒冬小麦与冷敏感春小麦幼苗细胞质膜a2+-ATpase 活性比较. 作物学报, 2002, 28(2):227-29
9. 刘祖棋, 张石城主编. 植物抗性生理学.北京.中国农业出版社.1994:369-386
10. 罗雯, 刘阳. 根癌农杆菌转化条件优化的研究. 生物技术, 2006, 16(1): 41-43
11. 马建忠. 植物的冷诱导基因. 农业生物技术学报, 1996,4(1):8-13
12. 祁忠占, 彭永康, 宋久雪. 汞对蔬菜幼苗生长及过氧化酶同工酶的影响.环境科学学报, 1991,11(3):370-374
13. 衰鹰, 李启云, 孔祥梅等. 农杆菌介导玉米遗传转化影响因子的研究. 分子植物育种, 2006, 4(2): 228-232
14. 孙龙华. 抗寒剂 CR-4 提高玉米幼苗抗寒力及质膜 5'-核苷酸酶冷稳定性的研究. 植物学通报, 1994,S2: 24-28
15. 谭德寇, 孙雪飘, 张家明. 巴西橡胶树的组织培养技术. 植物生理学通讯, 2005, 41 (5):674-678
16. 王景雪, 孙毅, 杜建中等. 处理时间对农杆菌介导甘蓝转化中抗性芽分化率的影响. 山东农业科学, 2006, 2: 27-29
17. 王凭青, 吴明生; 王远亮等. 植物抗寒基因工程研究最新进展. 重庆大学学报, 2003,26(7) : 35-40
18. 王瑞云, 贺润喜; 岳文斌等. 植物抗寒性基因工程研究进展. 中国生态农业学报, 2004,12(1) : 14-18
19. 王孝宣, 李树德, 东惠茹等. 齐茄品种耐寒性与 ABA 和可溶性糖含量的关系. 园艺学报, 1998, 25(l):56-60
20. 王亚丽. 巴西橡胶树体外体细胞胚发育的细胞学和组织学研究. 广州:华南热带农业大学, 2004
21. 王颖, 陈雄庭, 张秀娟等.基因枪法将GAI基因导入巴西橡胶的研究. 热带亚热带植物学报, 2006, 14(3):179-182
22. 王泽云, 陈雄庭, 吴胡蝶. 橡胶树新型种植材料——体胚植株. 热带农业科学, 2001, 94(6):11-15
23. 王泽云, 曾宪松, 陈传琴等.从离体花药诱导巴西橡胶植株. 热带作物科技通讯, 1978, (4):1-7
24. 王泽云, 曾宪松, 陈伟琴等. 用离体花药诱导巴西橡的研究.热带作物学报, 1980, 1(1):16-25
25. 吴胡蝶, 王泽云, 陈雄庭. 6-BA、ABA 对橡胶花药体细胞胚形成及植株再生的影响.热带作物研究, 1994, (3):1-3
26. 吴胡蝶, 王泽云, 陈雄庭等. 影响橡胶体细胞胚萌发成植株的几个因素. 热带作物研究, 1997, (2):5-8
27. 押辉远, 秦广雍, 霍裕平. Prd29A 及 DREB1A 的克隆和干旱诱导型植物表达载体的构建与鉴定.植物生理学通讯, 2005, 41(3):371-375
28. 颜子颖、王海林. 精编分子生物学实验指南(第四版)北京:科学出版社. 1998.37-38
29. 张红宇, 汪秀志, 赵秀云等. 农杆菌介导的共转化体系的研究进展. 农业生物技术科学, 2004, 20(2): 26-28
30. 赵军,王以柔,李美茹等. 低温锻炼对水稻幼苗叶片中 Rubisco 的影响. 植物生理学报, 1997,23(2):123-129
31. 赵可夫.植物抗盐生理. 北京:中国科学技术出版社, 1993, 187-191
32. 周碧燕, 陈杰忠, 季作梁等. 香蕉越冬期间 SOD 活性和可溶性蛋白质含量的变化.果树科学.1999, 16(3):192-196
33. 朱广廉, 张爱琴. 植物生理学实验指导. 北京大学出版社,1990
34. Arokiaraj P, Jones H, Cheong K F, et al. Gene insertion into Hevea brasiliensis. Plant Cell Rep, 1994, 13: 425- 431
35. Arokiaraj P, Jones H, Cheong K F, et al. Gene insertion intoHevea brasiliensis.Plant Cell Re-ports , 1994, 13: 425-431
36. Arokiaraj P, Jones H, Hafsah Jaafar, et al. A-grobacterium-mediated transformation ofHevea anther calli and their regeneration into plantlets. Journal of Natural Rubber Research, 1996, 11(2): 77-87
37. Arokiaraj P, Yeang H Y, Cheong K F, et al.CaMV 35S promoter directsβ- glucuronidase expression in the laticiferous system of transgenicHevea brasiliensis(rubber tree). Plant Cell Re-ports, 1998, 17: 621-625
38. Arokiaraj P. Agrobacterium-mediated transformation ofHeveacells de-rived fromin vitroandin vivoseeding cultures. J Nat Rubber Res,1991, 6(1):551
39. Blanc G, Baptiste C, Oliver G, Martin F, Montoro P. Efficient Agrobacterium tumefaciens-mediated transformation of embryogenic calli and regeneration of Hevea brasiliensis Mull¨ Arg. plants. Plant Cell Rep, 2006, 24: 724–733
40. Carron M P, Laredt L, Dea B G. Hevea microprogation by somatic embryogensis, Plantations Res Dev, 1998, (5):187-192
41. Cazaux E, Auzac J D. Isolation and culture of Hevea brasillensis protoplasts. Physiol Plant, 1991,82,(1): 1-14
42. Chinnusamy V, Ohta M, Kanrar S, Lee BH, Hong X, Agarwal M, Zhu JK. ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Genes Dev, 2003,17(8):1043-54
43. Claudia Kaye,Lisa Neven,Andrea Flofig. Characterization of a gene for sPinach CAP160 and exPression of two sPinach cold-acclimation Protein in tobacco. Plant Physiol, 1998, 116:1307-1377
44. Cook D, Fowler S, Fiehn O, Thomashow M F. A prominent role for the CBF cold response pathway in configuring the low-temperature metabolome of Arabidopsis. ProcNatl Acad Sci USA, 2004, 101(42):15243-15248
45. Dejardin A, Sokolov L N, Kleczkowski L A. Sugar/osmoticum levels modulate differential abscisic acid-independent expression of two stress-responsive sucrose synthase genes in Arabidopsis. Biochem Jour, 1999,344(2):503-509
46. Fowler S, Thomashow M F. Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. Plant Cell, 2002,14, 1675-1690
47. Gao M J, Allard G, Byass L, Flanagan A M. Singh J. Regulation and characterization of four CBF transcription factors from Brassica napus. Plant Mol Biol, 2002, 49:459-471
48. Blanc G, Baptiste C, Oliver G, et al.Efficient Agrobacterium tumefaciens-mediated transformation of embrygenic calli and regeneration of Hevea brasiliensis. Plant Cell Rep, 2006, 24:724- 733
49. Gilmour S J, Sebolt A M, Salazar M P, et al. Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiol, 2000,124(4):1854-65
50. Gilmour S J, Zarka D G, Stockinger E J, et al. Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression. Plant Jour, 1998, 16(4):433-42
51. Gong Z, Dong C H, Lee H, et al. A DEAD Box RNA Helicase Is Essential for mRNA Export and Important for Development and Stress Responses in Arabidopsis. The Plant Cell, 2005,17:256-267
52. Guy C L. Cold acclimation and freezing stress tolerance role of Protein metabolism·A nnuRev Plant Mol Biol, 1990,41:187-223
53. Haake V, Cook D, Riechmann J L, et al. Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis. Plant Physiol, 2002,130(2):639-48
54. Haris N, Darussamin A, Dodd W A. Isolation of rubber tree (Hevea brasiliensis Muell-Arg) protoplast from callus and cell suspension. Menara Perkebunan (Indonesia), 1993, 61: 25-31
55. Jaglo K R, Kleff S, Amundsen KL, et al. Components of the ArabidopsisC-repeat/dehydration-responsive element binding factor cold-response pathway are conserved in Brassica napus and other plant species. Plant Physiol, 2001,127(3):910-917
56. Jayashree R, Pekha K, Venkatachalam P, et al. Genetic transformation and regeneration of rubber tree Hevea brasiliensis Muell. Arg) transgenic plants with a constitutive version of an anti-oxidative stress uperoxide dismutase gene. Plant Cell Rep, 2003, 22:201-209
57. Kasuga M, Liu Q, Miura S, et al. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biotechnol, 1999, 17:287-291
58. Kreps J A, Wu Y, Chang H S. Transcriptome Changes for Arabidopsis in Response to Salt, Osmotic, and Cold Stress. Plant Physiol, 2002, 130:2129-2141
59. Liu Q, Kasuga M, Sakuma Y et al. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell, 1998,10:1391-1406
60. MonroyA F, Dhindsa R S. Temperature signal transduction:induction fcold acclimation-specific genes of lfalfe by calcium at 25℃. Plantcell, 1995, 7: 321-331
61. Montoro P, Teinseree N, Rattana W, et al. Effect of exogenous calcium on Agrobacterium tumefaciens-mediated gene transfer in Hevea brasiliensis (rubber tree) friable calli. Plant Cell Rep, 2000, 19:851-855
62. Stockinger E J, Gilmour S J, Thomashow M F. Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc Natl Acad Sci USA, 1997, 94(3):1035-1040
63. Stockinger E J, Gilmour S J, Thomashow M F. Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc Natl Acad Sci USA, 1997, 94(3):1035-40
64. Suraninpong P, Te-chato S. Effect of cytokines on cell suspension culture, isolation andculture of protoplast of rubber, Songklanakarin J Sci Technol, 1999, 21(2): 169-177
65. Sushamakumar S, Asokan M P, Anthony P, et al. Plant regeneration from embryogenic cell suspension-derived protoplasts of rubber. Plant Cell Tiss Org Cult, 2000, 61(1): 81-85
66. Thomashow M R. Plant cold acclimation: freezing tolerance genes and regulatory mechanisms, Annu Rev Plant Mol Biol, 1999, 50:571-599
67. Thomashow M F. Plant Cold Acclimattion: Freezing Tolerance Genes and Regulatory Mechanisms. Annu Rev Plant Physiol Plant Mol Biol, 1999, 50:571-599
68. Wanner L A. Cold-induced fteezing tolerance in AlabidoPsis.PlantPhysiol, 1999, 120:391-400
69. Wanner L A.Cold-induced fteezing tolerance inAlabidoPsis. PlantPhysiol.1999 ,120:391-400
70. Wrren G J. The molecular biological aPProach to understanding fteezing-tolerance in the model Plant AlabidoPsis thaliana.Environmental Stressors and Gene Responses, 2000, l:245-258
71. Yokio S. Introduction of the cDNA for ArabidoPsis glycerol-3, PhosPhate acyltransferase(GPAT)confers unsaturation of fatty acids and chilling tolerance of Phorosynthesis on rice. Mol Breed, 1998, 4:269-275
72. Zhang X, Fowler S, Cheng H, et al. Freezing-sensitive tomato has a functional CBF cold response pathway, but a CBF regulon that differs from that of freezing-tolerant Arabidopsis. Plant Jour, 2004, 39 (6):905-919
2. 陈雄庭. 橡胶速生高产新型种植材料的培育与试种. 热带作物产业带建设规划研讨会—天然橡胶产业发展论文集. 2006: 104-107
3. 陈正华, 许绪恩, 庞任声等. 橡胶树属的花药培养及花粉植株的研究. 木本植物组织培养及其应用. 北京:高等教育出版社, 1986.481-500
4. 广东省农垦总局、海南省农垦总局编著.橡胶树良种选育与推广.广州:广东科技出版社: 1994
5. 简令成,王红. 钙(Ca2+)在植物抗寒中的作用. 细胞生物学杂志,2002,24(3):166-171.
6. 简令成, 孙龙华, 孙德兰. 几种植物细胞表面糖蛋白的电镜细胞化学及其与植物抗逆性的关系.实验生物学报, 1986, 19:261-271
7. 李卫, 孙中海,章文才等. 钙与钙调素对柑橘原生质体抗冻性的影响. 植物生理学报, 1997, 23(3):262-266
8. 刘炜, 孙德兰, 王红等. 2℃低温下抗寒冬小麦与冷敏感春小麦幼苗细胞质膜a2+-ATpase 活性比较. 作物学报, 2002, 28(2):227-29
9. 刘祖棋, 张石城主编. 植物抗性生理学.北京.中国农业出版社.1994:369-386
10. 罗雯, 刘阳. 根癌农杆菌转化条件优化的研究. 生物技术, 2006, 16(1): 41-43
11. 马建忠. 植物的冷诱导基因. 农业生物技术学报, 1996,4(1):8-13
12. 祁忠占, 彭永康, 宋久雪. 汞对蔬菜幼苗生长及过氧化酶同工酶的影响.环境科学学报, 1991,11(3):370-374
13. 衰鹰, 李启云, 孔祥梅等. 农杆菌介导玉米遗传转化影响因子的研究. 分子植物育种, 2006, 4(2): 228-232
14. 孙龙华. 抗寒剂 CR-4 提高玉米幼苗抗寒力及质膜 5'-核苷酸酶冷稳定性的研究. 植物学通报, 1994,S2: 24-28
15. 谭德寇, 孙雪飘, 张家明. 巴西橡胶树的组织培养技术. 植物生理学通讯, 2005, 41 (5):674-678
16. 王景雪, 孙毅, 杜建中等. 处理时间对农杆菌介导甘蓝转化中抗性芽分化率的影响. 山东农业科学, 2006, 2: 27-29
17. 王凭青, 吴明生; 王远亮等. 植物抗寒基因工程研究最新进展. 重庆大学学报, 2003,26(7) : 35-40
18. 王瑞云, 贺润喜; 岳文斌等. 植物抗寒性基因工程研究进展. 中国生态农业学报, 2004,12(1) : 14-18
19. 王孝宣, 李树德, 东惠茹等. 齐茄品种耐寒性与 ABA 和可溶性糖含量的关系. 园艺学报, 1998, 25(l):56-60
20. 王亚丽. 巴西橡胶树体外体细胞胚发育的细胞学和组织学研究. 广州:华南热带农业大学, 2004
21. 王颖, 陈雄庭, 张秀娟等.基因枪法将GAI基因导入巴西橡胶的研究. 热带亚热带植物学报, 2006, 14(3):179-182
22. 王泽云, 陈雄庭, 吴胡蝶. 橡胶树新型种植材料——体胚植株. 热带农业科学, 2001, 94(6):11-15
23. 王泽云, 曾宪松, 陈传琴等.从离体花药诱导巴西橡胶植株. 热带作物科技通讯, 1978, (4):1-7
24. 王泽云, 曾宪松, 陈伟琴等. 用离体花药诱导巴西橡的研究.热带作物学报, 1980, 1(1):16-25
25. 吴胡蝶, 王泽云, 陈雄庭. 6-BA、ABA 对橡胶花药体细胞胚形成及植株再生的影响.热带作物研究, 1994, (3):1-3
26. 吴胡蝶, 王泽云, 陈雄庭等. 影响橡胶体细胞胚萌发成植株的几个因素. 热带作物研究, 1997, (2):5-8
27. 押辉远, 秦广雍, 霍裕平. Prd29A 及 DREB1A 的克隆和干旱诱导型植物表达载体的构建与鉴定.植物生理学通讯, 2005, 41(3):371-375
28. 颜子颖、王海林. 精编分子生物学实验指南(第四版)北京:科学出版社. 1998.37-38
29. 张红宇, 汪秀志, 赵秀云等. 农杆菌介导的共转化体系的研究进展. 农业生物技术科学, 2004, 20(2): 26-28
30. 赵军,王以柔,李美茹等. 低温锻炼对水稻幼苗叶片中 Rubisco 的影响. 植物生理学报, 1997,23(2):123-129
31. 赵可夫.植物抗盐生理. 北京:中国科学技术出版社, 1993, 187-191
32. 周碧燕, 陈杰忠, 季作梁等. 香蕉越冬期间 SOD 活性和可溶性蛋白质含量的变化.果树科学.1999, 16(3):192-196
33. 朱广廉, 张爱琴. 植物生理学实验指导. 北京大学出版社,1990
34. Arokiaraj P, Jones H, Cheong K F, et al. Gene insertion into Hevea brasiliensis. Plant Cell Rep, 1994, 13: 425- 431
35. Arokiaraj P, Jones H, Cheong K F, et al. Gene insertion intoHevea brasiliensis.Plant Cell Re-ports , 1994, 13: 425-431
36. Arokiaraj P, Jones H, Hafsah Jaafar, et al. A-grobacterium-mediated transformation ofHevea anther calli and their regeneration into plantlets. Journal of Natural Rubber Research, 1996, 11(2): 77-87
37. Arokiaraj P, Yeang H Y, Cheong K F, et al.CaMV 35S promoter directsβ- glucuronidase expression in the laticiferous system of transgenicHevea brasiliensis(rubber tree). Plant Cell Re-ports, 1998, 17: 621-625
38. Arokiaraj P. Agrobacterium-mediated transformation ofHeveacells de-rived fromin vitroandin vivoseeding cultures. J Nat Rubber Res,1991, 6(1):551
39. Blanc G, Baptiste C, Oliver G, Martin F, Montoro P. Efficient Agrobacterium tumefaciens-mediated transformation of embryogenic calli and regeneration of Hevea brasiliensis Mull¨ Arg. plants. Plant Cell Rep, 2006, 24: 724–733
40. Carron M P, Laredt L, Dea B G. Hevea microprogation by somatic embryogensis, Plantations Res Dev, 1998, (5):187-192
41. Cazaux E, Auzac J D. Isolation and culture of Hevea brasillensis protoplasts. Physiol Plant, 1991,82,(1): 1-14
42. Chinnusamy V, Ohta M, Kanrar S, Lee BH, Hong X, Agarwal M, Zhu JK. ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Genes Dev, 2003,17(8):1043-54
43. Claudia Kaye,Lisa Neven,Andrea Flofig. Characterization of a gene for sPinach CAP160 and exPression of two sPinach cold-acclimation Protein in tobacco. Plant Physiol, 1998, 116:1307-1377
44. Cook D, Fowler S, Fiehn O, Thomashow M F. A prominent role for the CBF cold response pathway in configuring the low-temperature metabolome of Arabidopsis. ProcNatl Acad Sci USA, 2004, 101(42):15243-15248
45. Dejardin A, Sokolov L N, Kleczkowski L A. Sugar/osmoticum levels modulate differential abscisic acid-independent expression of two stress-responsive sucrose synthase genes in Arabidopsis. Biochem Jour, 1999,344(2):503-509
46. Fowler S, Thomashow M F. Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. Plant Cell, 2002,14, 1675-1690
47. Gao M J, Allard G, Byass L, Flanagan A M. Singh J. Regulation and characterization of four CBF transcription factors from Brassica napus. Plant Mol Biol, 2002, 49:459-471
48. Blanc G, Baptiste C, Oliver G, et al.Efficient Agrobacterium tumefaciens-mediated transformation of embrygenic calli and regeneration of Hevea brasiliensis. Plant Cell Rep, 2006, 24:724- 733
49. Gilmour S J, Sebolt A M, Salazar M P, et al. Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiol, 2000,124(4):1854-65
50. Gilmour S J, Zarka D G, Stockinger E J, et al. Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression. Plant Jour, 1998, 16(4):433-42
51. Gong Z, Dong C H, Lee H, et al. A DEAD Box RNA Helicase Is Essential for mRNA Export and Important for Development and Stress Responses in Arabidopsis. The Plant Cell, 2005,17:256-267
52. Guy C L. Cold acclimation and freezing stress tolerance role of Protein metabolism·A nnuRev Plant Mol Biol, 1990,41:187-223
53. Haake V, Cook D, Riechmann J L, et al. Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis. Plant Physiol, 2002,130(2):639-48
54. Haris N, Darussamin A, Dodd W A. Isolation of rubber tree (Hevea brasiliensis Muell-Arg) protoplast from callus and cell suspension. Menara Perkebunan (Indonesia), 1993, 61: 25-31
55. Jaglo K R, Kleff S, Amundsen KL, et al. Components of the ArabidopsisC-repeat/dehydration-responsive element binding factor cold-response pathway are conserved in Brassica napus and other plant species. Plant Physiol, 2001,127(3):910-917
56. Jayashree R, Pekha K, Venkatachalam P, et al. Genetic transformation and regeneration of rubber tree Hevea brasiliensis Muell. Arg) transgenic plants with a constitutive version of an anti-oxidative stress uperoxide dismutase gene. Plant Cell Rep, 2003, 22:201-209
57. Kasuga M, Liu Q, Miura S, et al. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biotechnol, 1999, 17:287-291
58. Kreps J A, Wu Y, Chang H S. Transcriptome Changes for Arabidopsis in Response to Salt, Osmotic, and Cold Stress. Plant Physiol, 2002, 130:2129-2141
59. Liu Q, Kasuga M, Sakuma Y et al. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell, 1998,10:1391-1406
60. MonroyA F, Dhindsa R S. Temperature signal transduction:induction fcold acclimation-specific genes of lfalfe by calcium at 25℃. Plantcell, 1995, 7: 321-331
61. Montoro P, Teinseree N, Rattana W, et al. Effect of exogenous calcium on Agrobacterium tumefaciens-mediated gene transfer in Hevea brasiliensis (rubber tree) friable calli. Plant Cell Rep, 2000, 19:851-855
62. Stockinger E J, Gilmour S J, Thomashow M F. Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc Natl Acad Sci USA, 1997, 94(3):1035-1040
63. Stockinger E J, Gilmour S J, Thomashow M F. Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc Natl Acad Sci USA, 1997, 94(3):1035-40
64. Suraninpong P, Te-chato S. Effect of cytokines on cell suspension culture, isolation andculture of protoplast of rubber, Songklanakarin J Sci Technol, 1999, 21(2): 169-177
65. Sushamakumar S, Asokan M P, Anthony P, et al. Plant regeneration from embryogenic cell suspension-derived protoplasts of rubber. Plant Cell Tiss Org Cult, 2000, 61(1): 81-85
66. Thomashow M R. Plant cold acclimation: freezing tolerance genes and regulatory mechanisms, Annu Rev Plant Mol Biol, 1999, 50:571-599
67. Thomashow M F. Plant Cold Acclimattion: Freezing Tolerance Genes and Regulatory Mechanisms. Annu Rev Plant Physiol Plant Mol Biol, 1999, 50:571-599
68. Wanner L A. Cold-induced fteezing tolerance in AlabidoPsis.PlantPhysiol, 1999, 120:391-400
69. Wanner L A.Cold-induced fteezing tolerance inAlabidoPsis. PlantPhysiol.1999 ,120:391-400
70. Wrren G J. The molecular biological aPProach to understanding fteezing-tolerance in the model Plant AlabidoPsis thaliana.Environmental Stressors and Gene Responses, 2000, l:245-258
71. Yokio S. Introduction of the cDNA for ArabidoPsis glycerol-3, PhosPhate acyltransferase(GPAT)confers unsaturation of fatty acids and chilling tolerance of Phorosynthesis on rice. Mol Breed, 1998, 4:269-275
72. Zhang X, Fowler S, Cheng H, et al. Freezing-sensitive tomato has a functional CBF cold response pathway, but a CBF regulon that differs from that of freezing-tolerant Arabidopsis. Plant Jour, 2004, 39 (6):905-919