摘要
21世纪是生命科学的世纪,谁占生物技术的优势地位,谁就会主导未来的生物经济时代。转基因技术是生物技术的重要内容之一,是改良品种的关键技术,在理论研究和实际应用中都具有极其重要的地位,但目前的转基因技术大都依赖于选择标记。本文利用分割法(即通过对转化处理的愈伤组织进行多次分割、培养和直接选择,富集转化细胞的方法)建立了不依赖选择标记的转基因技术,其目的在于解决伴随转基因作物越来越多而日益突出的选择标记问题,它可以消除由于应用选择标记可能在以下五个方面产生的不利影响,1)影响目标基因的表达,2)限制多个基因的转化,3)影响被改良植物的某些性状,4)危及环境安全,5)危害人身健康。近年来许多研究者利用共转化、位点特异性重组、转座子和同源重组等方法,开展了对转基因植物中的选择标记进行剔除的研究。但到目前为止还难以获得令人满意的结果。因此,建立不依赖选择标记的转基因技术比完成转基因后再剔除选择标记具有更重要的意义。
本文先以烟草(Nicotiana tabacum subsp.petit SR1)为受体,通过农杆菌介导转化,以GUS基因为追踪目标摸索了分割法的技术参数。当愈伤组织长至直径0.5cm时就进行均分,每次的分割块数(i)设4个处理,分别为64块、32块、16块和8块,分割次数(n)设4个处理,分别为2次、3次、4次和5次,为了便于将“子愈伤组织”的阳性结果与“母愈伤组织”的转化部位对应起来,均分后的小细胞团按其在“母愈伤组织”中的相邻关系依次摆放在培养基上。通过分割和培养可以削弱转化细胞与非转化细胞之间的竞争强度,给予转化细胞较充分的发展机会,提高有转化愈伤组织中转化细胞所占比例。自第二次分割起,每次分割前,都寻找“转化愈伤组织”集中出现的区域,并选择区域内最靠近中间的愈伤组织进行下次分割,如此重复使转化细胞得到富集,最终得到近似均一的转化愈伤组织,诱导出再生植株,并对再生植株的每个叶片进行检测确定其是否为纯合体(或嵌合体)。然后,通过用PCR代替X-gluc检测,以番茄为受体,以葡聚糖酶基因为供体基因,对该技术进行了验证。
本研究用分割法建立了不依赖选择标记的转基因技术,主要包括三方面内容:优化培养基,建立小细胞团培养技术;优化农杆菌介导的转化技术,提高“起始愈伤组织”中转化细胞的数目;确定分割法的可行性及其必要的参数。
本试验研究表明,较适合培养烟草小细胞团的培养基为:B_5+584mg/l谷氨酰胺+
董文琦中国农业科学院研究生硕士论文
0.6m留1 BA+0.03m留1 IAA十250m留ICarb。外植体为番茄时,将激素调整为BA
1 .2m酬+I AA 0.2 ms/l。较适合农杆菌侵染和共培养的培养基为:BS+谷氨酞胺584m幼十
甜菜碱117.2mg/1+脯氨酸0.12mg/1+肌醇400mg/卜309/1蔗糖,pH 5.5。较合适的侵染和
共培养条件为:将农杆菌稀释至OD(Optical Degree)600=0.05,加入smg/l乙酞T香酮,
侵染外体植30分钟,在附加10 mg/l Agar的培养基上,于避光条件下,共培养3天。
在烟草上,每当转化处理后的愈伤组织长至直径o.scm时就进行分割,前1一2次的
分割块数大于32块、分割递进度(分割块数X分割次数)大于128的处理效果比较理
想,获得的有效转化率都超过了50%。试验数据还表明,在一定的范围内,分割递进度
与有效转化率之间基本上呈现正比关系,因此,我们认为在利用分割法进行基因转化的
早期应尽量增加分割块数,以尽早降低非转化细胞的比率,早日获得转化体。但还应考
虑到不同受体小细胞团培养的难易程度及操作的难易程度,当小细胞团较难培养或转化
细胞在转化愈伤组织中的比率己较高时,应适当减少分割块数。
利用分割法,在不进行药物筛选的前提下,已将p一1,3一葡聚糖酶基因和几丁质酶成
功地转入了SRI烟草外植体,得到了纯合转化植株。并将p一l,3一葡聚糖酶基因转入了
番茄(却e叩ersscon esculentum)的栽培品种(毛T一5番茄)。
The 21th century is the century of life science, and one who keeps ahead in the biotechnology would occupy the predominant position in the bio-economy ages in future. Transgenic technique, an important part of biotechnology, has a great and significant prospective for developing new and improving the existing commercial cultivars. But nowadays the most transgenic techniques depend on the selection markers. We want to establish a new transgenic technique without selection marker by dividing technique in order to eliminate the potential disadvantages of selection markers, such as influencing the gene expression, limiting the further transformation, disturbing the plant characteristics, endangering the environment and jeopardizing the community health. In recent years many researchers have put a lot of efforts to eliminate the selection markers from transgenic plants, but up to now all the results are unsatisfactory. So it is more important to establish a new genetic transformation technique without selection m
arker than to eliminate the selection markers from transgenic plants.
In this study, the explants obtained from tobacco leaves were inoculated by Agrobacterium tumefaciens with GUS as reporter gene. The calli were grown and divided into 8, 16, 32 and 64 pieces for 2, 3, 4 and 5 times, respectively, and placed on media in original parental neighborhood sequence to provide an opportunity to transformed calli pieces to grow by weakening the competition from non-transformed cells. Every time the central calli were selected from the set of calli with positive GUS activity. Seedlings were regenerated from the finally obtained calli and their transformed status was confirmed through GUS or PCR activity.
This research primarily includes three parts: Improving the calli pieces culture technique, improving the DNA transfer technique by Agrobacterium and ascertaining the necessary parameters of dividing technique.
"B5+0.6 mg/L 2ip+0.03 mg/L IAA+584 mg/l Glutamine" was used for small piece of callus culture for tobacco. "B5+ 584 mg/1 Glutamine+117.2mg/l betaine+0.12mg/l proline+400mg/l inositol (pH5.5)" was used for bacterial infection and co-culture. The agro-bacteria was diluted to OD (Optical Degree) 600=0.5 with subsequent addition of
acetosyrimgone at concentration of 5 mg/l, and infected the explants for 30min, followed by 3days co-culture in the dark. When the diameter of callus reached about 0.5 cm, the next division was conducted. That the number of division and division degree(numbre of division X times) at first and second times were more than 32 and 128, respectively, was time saving and cost effective with more than 50% chances to get higher transformed plants. As a whole, the Dividing Degree and percentage of transformed plants are linearly correlated. How many pieces the callus should be divided relies on the difficulty of callus-culturing and operation too. We think, if possible, the more pieces we divide the callus, the better it will be, especially in the earlier stage, In this way we can decrease the ratio of non-transgenic cells and obtain the transgenic plants as early as possible. When there are enough transgenic cells in callus, we can decrease the number of pieces in the latter stage.
Using the transgenic technique by dividing treatment without selection marker, we have transferred chitinase and B-1,3-glucanase genes to SRI tobacco, and transferred B-1,3-glucanase gene to tomato cultivar (Mao T-5) successfully.
引文
1 陈峥,金红,程奕,杜胜利,魏爱民.提高黄瓜农杆菌遗传转化体系再生频率的研究.天津农业科学天津农业科学,2001,7(4):47-49
2 C.W.迪芬巴赫,G.S.德维克斯勒.PCR技术实验指南.科学出版社,1998
3 Clark MS,顾红雅,瞿礼嘉,陈章良.植物分子生物学.高等教育出版社,1998
4 F.奥斯伯,R.布伦特,R.E.金斯顿,D.D.穆尔,J.G.塞德曼,J.A.史密斯,K.斯特拉尔.精编分子生物学实验指南.科学出版社,1998
5 J.萨姆布鲁克,E.F.弗里奇,T.曼尼阿蒂斯.分子克隆实验指南.科学出版社,1995
6 蔡应繁,叶鹏盛,张利,赖家业.β-1,3-葡聚糖酶及其在植物抗真菌病基因工程中的应用.西南农业学报,2001,4(2):78-81
7 陈青,薛朝阳,周雪平.番茄花叶病毒移动蛋白基因转化烟草及在转基因烟草中的表达黄晓.南京农业大学学报,2001,24(4):5-8
8 程英豪.LacZ基因作为报道基因在植物基因工程中的应用.北京师范大学学报,1997,33(1):126-129
9 邓德旺,郭三堆.棉花花粉管通道法转基因的分子细胞学机理研究.中国农业科学,1999,32(6):113-114
10 董云洲,贾士荣.基因枪在植物遗传转化上的应用.生物工程进展,1993, 14(2):15-18
11 董志峰,马荣才,彭于发,管华诗.转基因植物中外源非目的基因片段的生物安全研究进展.植物学报,2001(7):661-672
12 傅荣昭,孙勇如,贾士荣主编.植物遗传转化技术手册.北京:中国科学技术出版社,1998
13 高必达.植物抗病基因工程研究进展.湖南农学报,1994,20(6):586-596
14 高川,王惠芳等.脂质体介导转染法的原理与应用.生命的化学,2002,22(1):73-75
15 郭勇,倪和民.显微注射法培育转基因哺乳动物的研究进展.生物工程进展,1997,17f1):42-47
16 候学文,姜悦,郭勇.转基因中的筛选标记.生物学通报,1997,32(10):19-21
17 候学文,曾庆平,郭勇.转基因植物的一个新型标记基因.生命的化学,1997,17(2):36
18 贾士荣.T-DNA转移机理.植物生理学通讯,1994,30(4):306-311
19 蓝海燕,田颖川,王长海,刘桂珍,张丽华,王兰岗,陈正华.表达β-1,3-葡聚糖酶及几丁质酶基因的转基因烟草及其抗真菌病的研究.遗传学报,2000:70-77
20 雷茂良,程金根.全球转基因植物发展现状.生物技术通报,1998,6:30-32
21 黎定军,赵开军,周清明,罗宽.转几丁质酶基因和β-1,3-葡聚糖酶基因烟草的研究.湖南农业大学学报,2002,6(28):211-213
22 陆晓春,薛庆中.转基因植物中标记基因的消除.生命科学,2001,13(2):78-81
23 莽克强.转基因植物的生物安全性的商榷.生物工程进展,1996,16(4):2-5
24 欧阳石文,谢丙炎,赵开军,冯兰香.转几丁质酶基因研究进展.生物技术通报,2001,3:28-31
25 佘建明等.氮源和激素对陆地棉原生质体体细胞胚发生和发育的影响.江苏农业学报,1991,7(4):20-24.
26 佘建明等.棉花原生质体培养体细胞胚胎发生及植株再生.江苏农业学报,1989,5(4):54-60.
27 孙效文,阎学春.用直接注射法生产转基因鱼.生物技术,1993, 3(3):12-14
28 田颖川,秦晓峰.表达苏云杆菌内毒素基因的转达基因烟草的抗虫性.生物工程学报1991,7(1):1-10
29 汪若海 李秀兰.我国转基因抗虫棉应用现状及建议.生物技术通报,2000(5):1-6
30 王关林,方宏筠.植物基因工程原理与技术.科学出版社,1998
31 王海波 赵和.转基因植物研究开发的国内外现状.河北农业科学,2001,5(11:67-69
32 王海波.植物组织细胞培养通用分析模式的探讨.中国农业科学院研究生院博士论文,1994。
33 王亮 游松.转基因植物的现状.沈阳药科大学学报,2001,18(3):217-222
34 王万军等.烟草叶外植体不定芽的诱导.西北植物学报,1993,13(4):308-311
35 王永胜,扈廷茂,刘明秋,李丽莉,孔威.马铃薯天冬氨酸蛋白酶抑制剂基因转化烟草研究.遗传,2000,22(3):129-132
36 王忠华,舒庆晓,叶庆富,崔海瑞,夏英武.转基因植物外源基因逃逸的途径.植物学通报,2001,18(2):137-142
37 吴明等.转基因农作物的研究.世界农业,1991,12:11-12
38 奚元龄,颜昌敬.植物细胞培养手册.农业出版社,1989
39 薛经卫,卫志明.通过PEG法转化甘蓝获得转基因植株.植物学报,1997,39(1):28-33
40 杨崇良 李长松.农业转基因工程及其产品安全性检测研究现状.山东农业科学,2000(5):48-51
41 杨崇良 张君亭.农业转基因生物研究现状及安全性管理.山东农业科学.2002(5):50-52
42 张丕方,倪德祥,包慈华.植物组织培养与繁殖上的应用.上海教育出版社,1985
43 赵开军.蔬菜转基因育种.中国蔬菜,1999(2):4-5
44 赵连元等.谷子原生质体高效成株体系建立研究.华北农学报1991,(增刊): 53-58.
45 周冀明,卫志明.PEG法介导转化诸葛菜下胚轴原生质体获得转基因植株.遗传学报,1996,3(1):69-76
46 朱生伟,徐仲.烟草叶组织培养再生系统的建立.吉林农大学报,1998,20(1):27-29
47 朱行.世界转基因作物发展现状和趋势.饲料博览,2002(3):19-20
48 Araki H., Jeampipatkul A., Tatsumi H., Sakurai T., Ushino K., Muta T., Oshima Y. Molecular and functional organization of yeast plasmid pSR1. J Mol Biol, 1987, 182:191-203
49 Arpad Pusztai. Report of Project Coordinator on data produced at the Rowett Research Institute (RRI). SOAEFD flexible Fund Project RO818, 22nd October, 1998
50 Ballas N., Citovsky V. Nuclear localization signal binding protein from arabidopsis mediates nuclear import of agrobacterium vird2 protein. Proc Natl Acad Sci (USA), 1997, 94(20): 10723-10728
51 Baron C., Llosa M., Zhou S., Zambryski P. C. Virbl, a component of the t-complex transfer machinery of agrobacterium tumefaciens, is processed to a C-terminal secreted product, virbl. J Bact, 1997, 179(4): 1203-1210
52 Bej A. K., Mahbubani M. H., Atlas R. M. Amplification of nucleic acids by polymerase chain reaction (PCR) and other methods and their applications. Critical Reviews in Biochemistry and Molecular Biology, 1991, 26:301-334
53 Belzile F., Lassner M. W., Tong Y., Khush R., Yoder J. I. Sexual transmission of transposed Activator elements in transgenic tomatoes. Genetics, 1989, 123: 181-189
54 Binns A. N., Thomashow M. F. Cell biology of Agrobacterium infection and transformation of plants. Ann Rev Microbiol, 1988, 42:575-606
55 Bollag R. J., Waldman A. S., Liskay R. M. Homologous recombination in mammalian cells. Annu Rev Genet, 1989, 23:199-225
56 Bravoangel A. M., Hohn B., Tinland B. The omega sequence of vird2 is important but not essential for efficient transfer of t-DNA by agrobacterium tumefaciens. Mol Plant- Microbe Interact, 1998, 11(1): 57-63
57 Clive James, Anatole F. K. Global Review of the Field Testing and Commercialization of Transgenic Plants 1986 to 1995. The International service for the Acquisition ofAgri-biotech Applications(ISAAA), 1996
58 Clive James, Anatole F. K. The Annual Global Review of Commercialized Transgenic (GM) Crops. ISAAA, 2003
59 Corneille S., Lutz K., Svab Z., Maliga P. Efficient elimination of selectable marker genes from the plastid gnome by the CRE/lox site-specific recombination system. Plant J, 2001, 27:171-178
60 Cregg J. M., Madden K. R. Use of site-specific recombination to generate selectable markers. Mol Gen Genet, 1989, 219:320-323
61 Cui M., Takayanagi K., Kamada H., Nishimura S., Handa T. Efficient shoot regeneration from hairy roots of Antirrhinummajus L. transformed by rol type MAT vector system. Plant Cell Rep, 2001, 20: 60-66
62 Dale E. C., Ow D. W. Gene transfer with subsequent removal of the selection gene from the host genome. Proc Natl Acad Sci USA, 1991, 88:10558-10562
63 Daley M., Knauf V. C., Summerfelt K. R., Turner J. C. Co-transformation with one Agrobacterium tumefaciens strain containing two binary plasmids as a method for producing marker free transgenic plants. Plant Cell Rep, 1998, 17:489-496
64 De Block M., Debrouwer D. Two T-DNAs co-transformed into Brassica napus by a double Agrobacterium tumefacien infection are mainly integrated at the same locus. Theor Appl Genet, 1991, 82:257-263
65 De Framond A. J., Back E. W., Chilton W.S., Kayes L., Chilton M.D. Two unlinked T-DNAs can transform the same tobacco plant cell and segregate in the F1 generation. Mol Gen Genet, 1986, 202: 125-131
66 De Neve M., De Buck S., Jacobs A., Van Montagu M., Depicker A. T-DNA integration patterns in co-transformed plant cells suggest that T-DNA repeats originate from co-integration of T-DNAs. Plant J, 1997, 11: 15-29
67 Depicker A., Herman L., Jacobs A., Schell J., Van Montagu M. Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium/plant cell interaction. Mol Gen Genet, 1985, 201:477-484
68 Dillen W., Declercq J., Kapila J., Zambre M., Vanmontagu M., Angenon G. The effect of temperature on agrobacterium tumefaciens-mediated gene transfer to plants. Plant J, 1997, 12(6): 1459-1463
69 Ebinuma H., Sugita K., Matsunaga E., Yamakado M. Selection of marker-free transgenic plants using the isopentenyl transferase gene as a selectable marker. Proc Natl Acad Sci USA, 1997, 94:2117-2121
70 FedoroffN. Maize transposable elements. American Society for Microbiology, 1989:375-411
71 Fillatti J. et al. Agrobacterium mediated transformation and regeneration of Populus. Mol Gen Genet, 1987, 206:192-199
72 Fischer N., Stampacchia O., Redding K., Rochaix J. D. Selectable marker recycling in the chloroplast. Mol Gen Genet, 1996, 251(3): 373-380
73 Fullner K. J. Role of agrobacterium Virb genes in transfer of T-complexes and rsfl010. J Bact, 1998, 180(2): 430-434
74 Gardner J. F., Nash H. A. Role of Escherichia coli IHF protein in lambda site-specific recombination. J Mol Biol, 1986, 191:181-189
75 Gatz C. Chemical control ofgene expression. Ann Rev Plant Physial Plant Mol Biol, 1997,48:89-108
76 Gautheret R. J. History of plant tissue and cell culture in Cell Culture and Somatic Cell Genetics of Plants. Academic Press Inc, 1985:1-59
77 Gebhand F. K. Monitoring field release of genetically modified sugar beets for persistence of transgenic DNA and horizontal gene transfer. FEMS Microbiol, 1999, 28:261-272
78 Gleave A. P., Mitra D. S., Mudge S. R., Morris B. A. Selectable marker-free transgenic plants without serual crossing transient expression of ere recombinase and use of a conditional lethal dominant gene. Plant Mol Biol, 1999, 40:223-325
79 Godwin I. et al. The effects of acetosyringone and pH on Agrobacterium-mediated transformation vary according to plant species. Plant Cell Rep, 1991, 9:671-675
80 Goldsbrough A. P., Lastrella C. N., Yoder J. I. Transposition mediated re-positioning and subsequent elimination of marker genes from transgenic tomato. Biotechnology, 1993, 11:1286-1292
81 Hehl R., Baker B. Properties of the maize transposable element of activator in transgenic tobacco plants A versatile inter-species genetic tool. Plant Cell, 1990, 2:709-721
82 Hong S. B., Hwang I., Dessaux Y., Guyon P., Kim K. S., Farrand S. K. A T-DNA gene required for agropine biosynthesis by transformed plants is functionally and evolutionarily related to a Ti plasmid gene required for catabolism of agropine by Agrobacterium strains. J Bact 1997, 179(15): 4831-4840
83 Hooykas P.J.J., Beijersbergen A.G.M. The virulence system of Agrobacterium tumefaciens. Annual Review of Phytopathology, 1994, 32:157-180
84 Jacq B. et al. Factors influencing T-DNA transfer in Agrobacterium-mediated transformation of sugar beet. Plant Cell Rep, 1993, 12:621-624
85 James C. Global review of commercialized transgenic crops[M]. ISAAA, 1998
86 James D. J. et al. Genetic transformation of apple Malus pumila Mill using a disarmed Ti-binary vector. Plant Cell Rep, 1989, 7:658-661
87 Jefferson R. A., Kavanagh T. A., Bevan M.W. GUS fusions,β-glucuronidase as a sensitive and versatile gene. EMBO J, 1987, 6:3901-3907
88 Jones J. D., Gilbert D. E., Grady K. L., Jorgensen R. A. T-DNA structure and gene expression in petunia plants transformed by Agrobacterium tumefaciens C58 derivatives. Mol Gen Genet, 1987, 207:478-485
89 Komari T., Hiei Y., Saito Y., Murai N., Kumashiro T. Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J, 1996, 10:165-174
90 Kumpatla S. P., Teng W., Buchholz W. G., Hall T. C. Epigenetic transcriptional silencing and 5-azacytidine-mediated reactivation of a complex transgene in rice. Plant Physiol, 1997, 115(2): 361-73
91 Kunkel T., Niu Q. W., Chan Y. S., Chua N. H. Inducible isopentenyl transferase as a high-efficiency marker for plant transformation. Nat Biotechnol 1999, 17:916-919
92 Lee Y. M. et al. Genetic evidence for direct sensing of phenolic compounds by the Vir A protein of Agrobacterium tumefaciens. Proc. Natl. Acad. Sci, 1995, 92: 12245-12249
93 Lessl M., Lanka E. Common mechanisms in bacterial conjugation and Ti-mediated T-DNA transfer to
plant cells. Cell, 1994, 77:321-324
94 Liu C. N., Steck T. R., Habeck L. L., Meyer J. A., Gelvin S. B. Multiple copies of virG allow induction of Agrobacterium tumefaciens Vir genes and T-DNA processing at alkaline pH. Mol. Plant-Microbe Interact, 1993, 6:144-156
95 Losey J. E., Rayor J. S., Carter M. E. Transgenic pollen harms monarch larvae. Nature(London), 1999: 214-399
96 Lyznik L. A., Mitchell J. C., Hirayama L., Hodges T. K. Activity of yeast FLP recombinase in maize and rice protoplasts. Nucleic acids research, 1993, 21:969-975
97 Maeser S., Kahmann R. The Gin recombinase of phage Mu can catalyse site-specific recombination in plant protoplasts. Mol Gen Genet, 1991, 230:170-176
98 Matsuzaki H., Nakajima R., Nishiyama J., Araki H., Oshima Y. Chromosome engineering in Saccharomyces cerevisiae by using a site-specific recombination system of a yeast plasmid. J Bacteriol, 1990, 172:610-618
99 McCormac A. C., Elliott M. C., Chen D. F. a novel plasmid series for the facile creation of complex binary vectors, which incorporates "clean-gene" facilities. Mol Gen Genet, 1999, 261:226-235
100 McKnight T. D., Lillis M. T., Simpson R. B. Segregation of genes transferred to one plant cell from two separate Agrobacterium strains. Plant Mol Biol, 1987, 8:439-445
101 Morten J., Okkeis F. A novel principle for selection of transgenic plant cells: positive selection. Plant Cell Rep, 1999, 16:219-221
102 Murashige T., Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant, 1962, 15:473-497
103 Negrotto D., Jolley M., Beer S., Wenck A. R., Hansen G. The use of phosphomannose-isomerase as a selectable marker to recover transgenic maize plants (Zea mays L.) via Agrobacterium transformation. Plant Cell Rep, 2000, 19:798-803
104 Nielsen K. M., Bones A. M., Smalla K. V., Elsas J. D. Horizontal gene transfer from transgenic plants to terrestrial bacteria. FEMS Microbiol, 1998, 22:79-103
105 Nielsen K. M., Elsas J. D., Smalla Ko Transformation ofAcinetobacrer sp. Strain BD413(_pFG4Dnpt Ⅱ) with transgenic plant DNA in soil microcosms and effects of kanamycin on selection of transformants. Appl Emiron Mirobiol, 2000, 66:1237-1242
106 Nordlee J. A., Taylor S. L., Townsel J. A. Investigations of the allergenicity of Brazil nut 2S seed storage protein in transgenic soybean. Paris OCED, 1996:151-155
107 Odell J., Caimi P., Sauer B., Russell S. Site-directed recombination in the genome oftransgenic tobacco. Mol Gen Genet, 1990, 223:369-378
108 Onouchi H., Nishihama R., Kudo M., Machida Y., Machida C. Visualization of site-specific recombination catalyzed by a recombinase from Zygosaccharomyces rouxii in Arabidopsisthaliana. Mol Gen Genet, 1995, 247:653-660
109 Pham C.T., Maclvor D. M., Hug B. A., Heusel J. W., Ley T. J. Long-range disruption ofgene expression by a selectable marker cassette. Pro Natl Acad Sci USA, 1996, 93 (23): 13090-13095
110 Pua E.C., Teo S.H., Loh C.S. Interactive role of ethylene and polyamines on shoot regenerability of Chinese kale (Brassica alboglabra Bailey) in vitro. Journal of Plant Physiology, 1996, 149(1): 138-148
111 Purnhauser L. P., Medgyesi M., Czako P. J., Marton L. et ai. Stimulation of shoot regeneration in Triticum aestivum and Nicotiana plumbaginifolia Viv. tissue cultures using an ethylene inhibitor AgNO3. Plant Cell Rep, 1987, 6:1-4
112 Qian Y. Q., Ma K. P., Sang W. G., Wei W. Termninator technology and biosafety, China Biodiv, 1999, 7: 151-155
113 Rempel H. C., Nelson L. M. Analysis of conditions for Agrobacterium-mediated transformation of tobaco cells in suspension. Transgen Res, 1995, 4:199-207
114 Reuhs B. L., Kim J. S., Matthysse A. G. Attachment ofAgrobacterium tumefaciens to carrot cells and Arabidopsis wound sites is correlated with the presence of a cell-associated, acidic polysaccharide. J Bact, 1997, 179(17): 5372-5379
115 Schluter K., Futterer J. Horizontal gene transfer from a transgenic potato line to a bacteria pathopen (Erninia chrysanthemi) occurs, if at all, at an extremely low frequency. Biotechnology, 1995,13:94-98
116 Sheikholeslam S. N., Weeks D. P. Acetosyringone promotes high efficiency of Arabidopsis thaliana explants by Agrobacterium tumefaciens. Plant Mol Biol, 1987, 8:291-298
117 Skoog F., Miller C. O. Chemical regulation of growth and organ formation. Symp Soc Exp Biol, 1959, 11:118-131
118 Skoog F., Tsui C. Chemical control of growth and bud formation in tobacco stem segments and callus
cultured in vitro. Am J Bot, 1948, 35:782-787
119 Song Y. N., Shibuya M., Ebizuka Yo, Sankawa U. Synergistic action of phenolic signal compounds and carbohydrates in the induction of virulence gene expression of Agrobacterium tumefaciens. Chem Pharm Bull, 1991, 39(10): 2613-2616
120 Song Y. N., Shibuya M., Ebizuka Y., Sankawa U. Identification of plant factors inducing virulence gene expression in Agrobacterium tumefaciens. Chem Pharm Bull, 1991, 39(9): 2347-2350
121 Sudupak M. A., Bennetzen J. L., Hulert S. H. Unequal exchange and meiotic instability of disease-resistance genes in the Rpl region of maize. Genetics, 1993, 133:119-125
122 Taylor S. L. Evaluation of the allergenicity of foods developed through biotechnoiogy. The University of California, 1994:185-198
123 Vasill V. et al. Regeneration of plants from embryogenic suspension culture protoplats of Wheat. Biotechnology, 1990, 8:429-434
124 Wabiko H., Kagaya M., Kodama I., Masuda K., Kodama Y., Yamamoto H., Shibano Y., Sano H. Isolation and characterization of diverse nopaline type Ti plasmids ofAgrobacterium tumefaciens from Japan. Arch Microbiol, 1989, 152:119-124
125 Wang H. B. et al. Plantlet regeneration from protoplasts of Wheat. Genetic Manipulation in Crops Newsletter, 1988, 4 (2): 11-16
126 Weber, T. E., Richert B. T. Grower-finisher growth performance and carcass characteristics including attempts to detect transgenic plant DNA and protein in muscle from pigs fed genetically modified "Bt" com. J Anim Sci, 2001, 79(Suppl. 2): 67-69
127 Widmer F., Seidler R. J., Donegan K. K., Reed G. L. Quantification of transgenic plant marker gene persistence in the field. Mol Ecol, 1997, 6:1-7
128 Yikang S., Simon R., Titen W., Heng B. Targted mutagenesis by homologous recombination in D. melanogaster. Gene & development, 2002(16): 1568-1581
129 Zhou X. R., Christie P. J. Suppression of mutant phenotypes of the agrobacterium tumefaciens virbl 1 ATPase by overproduction of virb proteins. J Bact, 1997, 179(18): 5835-5842
130 Zubko E., Scutt C., Meyer P. Intrachromosomal recombination between attP regions as a tool to remove selectable marker genes from tobacco transgenes. Nal Biotechnol, 2000, 18:442-445
131 Zuo J., Niu Q. W., Chua N. H. An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J, 2000, 24:265-273
132 Zuo J., Niu Q. W., Mφller S. G., Chua N. H. Chemical-regulated, site-specific DNA excision in transgenic plants. Nat Biotechnal, 2001, 19:157-161
133 Zupan J., Zambryski P. The agrobacterium DNA transfer complex. Crit Rev Plant Sci, 1997, 16(3): 279-295.