农杆菌介导的甘蓝遗传转化体系的研究
|
摘要
本研究以三个甘蓝品种为试材，在两个农杆菌菌株LBA4404(携带nptⅡ基因和gus基因)和GV3101(携带Bari-1基因和hpt基因)的介导下，重点研究了植株原位真空渗入遗传转化体系，包括适宜苗态建成方法研究、转化影响因素研究、转化体筛选及鉴定三部分。另外，初步对甘蓝高频芽再生技术进行了研究。实验结论如下：
1．鸡心、黄苗甘蓝品种于8月5日、8月10日露地播种，鸡心甘蓝品种叶数超过30片，株高23.03cm，茎粗达1.758cm；黄苗品种叶数超过37片，株高18.40cm，茎粗达1.739cm，均易形成甘蓝植株原位真空渗入遗传转化的适宜苗态；
2．影响甘蓝植株原位真空渗入遗传转化诸因素的研究表明：(1)鸡心品种外源基因的转化频率明显高于黄苗品种；(2)菌株LBA4404介导的转化频率较GV3101高；(3)渗入培养基中添加乙酰丁香酮以及pH为6时，其转化率较对照有明显提高，而蔗糖浓度的高低对转化率没有明显影响；(4)黄苗品种的最佳真空渗入时间为8min／间歇2min／30s，鸡心为6min／间歇2min／30s，转化率不因增加真空渗入间歇次数而有明显的提高；(5)在蕾期对花蕾进行真空渗入比对根、茎、叶进行真空渗入转化率高。
3．建立了培养基筛选甘蓝转基因植株的卡那霉素最佳浓度和筛选最佳时期。鸡心品种卡那霉素筛选的最佳浓度为30mg／L，黄苗品种卡那霉素筛选的最佳浓度为40mg／L；筛选时间以20日龄开始。鉴别转化体的标准：在卡那霉素筛选培养基上，20日龄以上的甘蓝植株生长正常，叶片显绿，有侧根发出或真叶长出或既有侧根发出又有真叶出现。
4．建立了生长点点滴法筛选甘蓝转基因植株的潮霉素最佳浓度和筛选最佳时期。鸡心、黄苗品种潮霉素筛选的最佳浓度为800mg／L，筛选时间自接种潮霉素后10d开始。筛选转化体的标准：真叶长出，叶色显绿，生长正常。
5．本研究共得到卡那霉素抗性植株150株，潮霉素抗性植株55株。从潮霉素抗性植株中随机抽取7株进行PCR检测，均有目标条带扩增出来，说明外源基因已整合到植物基因组中。
6．以牛心品种为试材，研究了外植体类型、激素配比、AgNO_3、苗龄、预培养等对甘蓝离体培养不定芽诱导的影响。研究表明，6d苗龄的甘蓝下胚轴外植体在附加4.0mg/L 6-BA的MS培养基上培养，芽分化率最高，达64.28％，平均每个外植体产芽数2.28个；AgNO_3对下胚轴不定芽的分化有促进作用；预培养2d再转入分化培养基中芽再生率提高28.57％。
In this study, the system for in-planta Agrobacterium-mediated transformation with vacuum infiltration in cabbage was investigated with three cabbage varieties (Brassica Oleracea Var. capitata), mediated by two Agrobacterium strains-LBA4404(carrying npt II gene and gus gene) and GV3101 (carrying Bari-1 gene and hpt gene), which includes methods of building the suitable plant shape > transformation influencing factors, transformant screening. In addition, high frequency of shoot regeneration in cabbage was studied. The conclusions are as followed:
l.Jinxin and Huangmiao varieties are easy to build the optimum plant shape for vacuum infiltration on August 5th and 10th in autumn under natural conditions. The optimum plant shape of two varieties:No. of leaves of Jinxin variety are over 30,the plant height is over 23.03cm, and diameter of root is 1.758cm ;and No.of leaves of Huangmiao variety are over 37,the plant height is over 18.40cm,and diameter of root is 1.739cm.
2. The study on the influencing factor of transformation shows that, (1) the transformation frequency of Jinxin is distinctly higher than Huangmiao;(2) LBA4404 is more efficient than GV3101 for transformation;(3) the culture medium including AS and the pH being 6 have obvious effect on the transformation, but the higher or lower of sue. concentration has no evident effect on the transformation;(4) the best infiltration tune for Huangmiao is 8min+intermission 2min+30S and for Jixin is 6min+intermission 2min+30S,and increasing the times of intermission has no evident effect on the transformation;(5) in bud stage, the buds being treated by the vacuum infiltration have higher transformation than roots, leaves and stems.
3. On screening Jixin and Huangmiao seeds through medium with Kan. Optimum concentration is 30mg/Lfor Jinxin and for Huangmiao is 40mg/L, and optimum time of screening is 20 days. The identified standard of the transformant is that the plant keeps normal in the medium including Kan, has green leaves or lateral roots or both.
4.On screening Jixin and Huangmiao seeds through growth point selection with Hyg. Optimum concentration is 800mg/L for Jinxin and Huangmiao, and optimum time of screening is 10 days after inoculated. The Hyg resistant seedlings: leaf buds appear, become green and grow normal.
5.150 Kan resistant seedlings and 55 Hyg resistant seedlings were achieved. 7 Hyg resistant seedlings were at random selected for PCR and all got the target strip. It proved that the foreign gene had been integrated into the plant genome.
6.Using Niuxin variety, the type of explants, hormone compositions, AgNO3, age of seedlings and preculture affecting shoot regeneration were also studied. The results showed the highest frequency of shoot regeneration is hypocotyls explants from 6-d-old in vitro grown seedling was 64.28% and No. of per explant with shoots was 2.28,using the MS media containing 4.0mg/L6-BA.It was also found AgN03(8mg/L) was advantageous to the shoot formation; via a preculture procedure of 2 days, the frequency of shoot regeneration increased 28.57%.
1．鸡心、黄苗甘蓝品种于8月5日、8月10日露地播种，鸡心甘蓝品种叶数超过30片，株高23.03cm，茎粗达1.758cm；黄苗品种叶数超过37片，株高18.40cm，茎粗达1.739cm，均易形成甘蓝植株原位真空渗入遗传转化的适宜苗态；
2．影响甘蓝植株原位真空渗入遗传转化诸因素的研究表明：(1)鸡心品种外源基因的转化频率明显高于黄苗品种；(2)菌株LBA4404介导的转化频率较GV3101高；(3)渗入培养基中添加乙酰丁香酮以及pH为6时，其转化率较对照有明显提高，而蔗糖浓度的高低对转化率没有明显影响；(4)黄苗品种的最佳真空渗入时间为8min／间歇2min／30s，鸡心为6min／间歇2min／30s，转化率不因增加真空渗入间歇次数而有明显的提高；(5)在蕾期对花蕾进行真空渗入比对根、茎、叶进行真空渗入转化率高。
3．建立了培养基筛选甘蓝转基因植株的卡那霉素最佳浓度和筛选最佳时期。鸡心品种卡那霉素筛选的最佳浓度为30mg／L，黄苗品种卡那霉素筛选的最佳浓度为40mg／L；筛选时间以20日龄开始。鉴别转化体的标准：在卡那霉素筛选培养基上，20日龄以上的甘蓝植株生长正常，叶片显绿，有侧根发出或真叶长出或既有侧根发出又有真叶出现。
4．建立了生长点点滴法筛选甘蓝转基因植株的潮霉素最佳浓度和筛选最佳时期。鸡心、黄苗品种潮霉素筛选的最佳浓度为800mg／L，筛选时间自接种潮霉素后10d开始。筛选转化体的标准：真叶长出，叶色显绿，生长正常。
5．本研究共得到卡那霉素抗性植株150株，潮霉素抗性植株55株。从潮霉素抗性植株中随机抽取7株进行PCR检测，均有目标条带扩增出来，说明外源基因已整合到植物基因组中。
6．以牛心品种为试材，研究了外植体类型、激素配比、AgNO_3、苗龄、预培养等对甘蓝离体培养不定芽诱导的影响。研究表明，6d苗龄的甘蓝下胚轴外植体在附加4.0mg/L 6-BA的MS培养基上培养，芽分化率最高，达64.28％，平均每个外植体产芽数2.28个；AgNO_3对下胚轴不定芽的分化有促进作用；预培养2d再转入分化培养基中芽再生率提高28.57％。
In this study, the system for in-planta Agrobacterium-mediated transformation with vacuum infiltration in cabbage was investigated with three cabbage varieties (Brassica Oleracea Var. capitata), mediated by two Agrobacterium strains-LBA4404(carrying npt II gene and gus gene) and GV3101 (carrying Bari-1 gene and hpt gene), which includes methods of building the suitable plant shape > transformation influencing factors, transformant screening. In addition, high frequency of shoot regeneration in cabbage was studied. The conclusions are as followed:
l.Jinxin and Huangmiao varieties are easy to build the optimum plant shape for vacuum infiltration on August 5th and 10th in autumn under natural conditions. The optimum plant shape of two varieties:No. of leaves of Jinxin variety are over 30,the plant height is over 23.03cm, and diameter of root is 1.758cm ;and No.of leaves of Huangmiao variety are over 37,the plant height is over 18.40cm,and diameter of root is 1.739cm.
2. The study on the influencing factor of transformation shows that, (1) the transformation frequency of Jinxin is distinctly higher than Huangmiao;(2) LBA4404 is more efficient than GV3101 for transformation;(3) the culture medium including AS and the pH being 6 have obvious effect on the transformation, but the higher or lower of sue. concentration has no evident effect on the transformation;(4) the best infiltration tune for Huangmiao is 8min+intermission 2min+30S and for Jixin is 6min+intermission 2min+30S,and increasing the times of intermission has no evident effect on the transformation;(5) in bud stage, the buds being treated by the vacuum infiltration have higher transformation than roots, leaves and stems.
3. On screening Jixin and Huangmiao seeds through medium with Kan. Optimum concentration is 30mg/Lfor Jinxin and for Huangmiao is 40mg/L, and optimum time of screening is 20 days. The identified standard of the transformant is that the plant keeps normal in the medium including Kan, has green leaves or lateral roots or both.
4.On screening Jixin and Huangmiao seeds through growth point selection with Hyg. Optimum concentration is 800mg/L for Jinxin and Huangmiao, and optimum time of screening is 10 days after inoculated. The Hyg resistant seedlings: leaf buds appear, become green and grow normal.
5.150 Kan resistant seedlings and 55 Hyg resistant seedlings were achieved. 7 Hyg resistant seedlings were at random selected for PCR and all got the target strip. It proved that the foreign gene had been integrated into the plant genome.
6.Using Niuxin variety, the type of explants, hormone compositions, AgNO3, age of seedlings and preculture affecting shoot regeneration were also studied. The results showed the highest frequency of shoot regeneration is hypocotyls explants from 6-d-old in vitro grown seedling was 64.28% and No. of per explant with shoots was 2.28,using the MS media containing 4.0mg/L6-BA.It was also found AgN03(8mg/L) was advantageous to the shoot formation; via a preculture procedure of 2 days, the frequency of shoot regeneration increased 28.57%.
引文
[1] Bird C.R,et al.番茄多聚半乳糖醛酸酶基因及其转基因植物中的熟化特性．植物分子生物学，1998，11(5)：651-661．
[2] Clark M S主编，顾红雅，瞿礼嘉主译，陈章良主校．植物分子生学实验手册．高等教育出版社，施普林格出版社出版．1998，北京，3-11．
[3] 蔡小宁，余建明，朱祯等．建立青菜(Barssica chinese)农杆菌介导基因转化体系．江苏农业学报，1997，13(2)：110-114．
[4] 沉宗英，张智良．花椰菜幼苗下胚轴诱导再生植株[J]．植物生理学通讯，1984,vol(1)：37．
[5] 成卓敏．应用基因工程创造黄锈病转基因小麦新种质．植物保护，1996，22(3)：18-20
[6] 成卓敏．应用基因工程创造小麦研究进展．生物技术通报，1997(4)：11-13．
[7] 程振东，卫志明，许智宏．芸薹属作物的遗传转化．植物生理学通讯，1992，28(3)：161-164．
[8] 邓万银，邵启全．酚类化合物能促进致瘤农杆菌对单子叶植物鸭跖草的转化．科学通报，1989：776-779．
[9] 傅忠昭等．植物遗传转化技术手册，北京：中国科学出版社，1994，5：97-123．
[10] 龚蓁蓁，沈慰芳，周光宇等．授粉后外源DNA导入植物——DNA通过花粉管通道进入胚囊，中国农业科学(B)，1988，6：661-664．
[11] 巩振辉，Cecchini E，Millner JJ.以PCR鉴定转基因植株的微量DNA提取方法．西北农业大学学报，1997，25(1)：45-48．
[12] 巩振辉，Millner J J，何玉科．拟南芥基因转移新方法—真空渗入法的研究，西北植物学报，1996，16(3)：277-283．
[13] 巩振辉．芸薹属作物基因转移新方法——植株原位真空渗入遗传转化方法，见冯延辉主编，新世纪的思索——中国当代改革发展文集．新疆人民出版社，2000，1712-1713．
[14] 官春云，李旬．转基因油菜应用研究．细胞生物学杂志，1997，19(1)：18—33[106]
[15] 郭启兴，范国强等．植物抗病毒基因工程育种策略及其进展．生命科学研究，2000，4(2)：113-117．
[16] 郭三堆，崔洪亮，倪万潮等．双价抗虫转基因棉花研究．中国农业科学，1999，32(3)：1-7．
[17] 郭三堆，植物Bt抗虫基因工程研究进展，中国农业科学，1993，28(5)：8-13．
[18] 郭元林，向平，飞速发展的生物技术．作物研究，1994，8(4)：4-6．
[19] 郭元林，向平．转基因技术在作物育种上的应用．西南农业学报，1997，10(4)：109-113．
[20] 华志华，黄大年．转基因植物中外源基因的遗传学行为．植物学报，1999，41(1)：1-5．
[21] 贾士荣．转基因作物的安全性争论及对策．生物技术通报，1999，(6)：1-7．
[22] 金万梅，巩振辉．芥菜真空渗入遗传转化体系适宜苗态的建成，西北农业学报，2000，9(4)：22-25．
[23] 金万梅，巩振辉等，植物遗传转化方法和转基因植株的鉴定．陕西农业科学，2000，1：24-29．
[24] 金万梅．芥菜植株原位真空渗入遗传转化体系的研究．西北农林科技大学硕士论文，2001．
[25] 荆家海主编，植物生理学．陕西科学技术出版社,1994年(第二版)，167-179．
[26] 朗春秀，胡张华，刘智宏等，油菜农杆菌转基因体系的建立及转PEP反义基因油菜的获得．浙江农业学报，1999，11(2)：55-58．
[27] 李宝健，曾庆平．植物生物技术原理与方法．科学技术出版社，1990，190-191．
[28] 李恩义．多聚阳离子基因导入法．上海农业科技，1999，1：8-10．
[29] 李洪清，李美茹．影响农杆菌介导植物基因转化的因素问题．植物生理学通讯，1999，35(2)：145-151．
[30] 李天然，张治中等．番茄ACC合酶反义基因对河套蜜瓜的转化．植物学报，1999，41(2)：142-145．
[31] 凌定厚，陶利珍，马镇荣等．以基因枪介导获转Psl-barnase基因的工程雄性不育水稻植株．遗传学报，1998，25(5)：433-442．
[32] 刘进元等．植物抗病基因工程的进展．生物工程进展，1994，14(2)：31-34．
[33] 卢爱兰，陈正华，孔令洁等，抗芜菁花叶病毒转基因甘蓝型油菜的研究．遗传学报，1996，23(1)：77-83．
[34] 陆璐，甜瓜ACC氧化酶cDNA克隆及其反义序列转化栽培品种．西北农业大学博士论文，1999，1-12．
[35] 毛慧琳，唐惕．抗虫转基因甘蓝及后代的研究[J]．中国科学(C辑)，1996，26(4)：339-349．
[36] 彭仁旺，周雪莱，王峻岭等．表达barstar基因及bar基因的转基因油菜的研究．遗传学报，1998，25(1)：74-79．
[37] 钱迎倩．生物多样性的几个问题．植物学通报，1998，15(5)．
[38] 宋鸣凤等．植物抗真菌和细菌病害基因工程的策略及其进展．植物生理学通讯，1996，32(1)：64-70．
[39] 宋正旭．小白菜植株原位真空渗入遗传转化体系的研究．西北农林科技大学硕士论文，2002，17-19．
[40] 苏军，段榕琦等．农杆菌介导小白菜的GUS基因瞬间表达．福建果树。2000，2：3-5．
[41]孙雷心译．开发转基因新作物．生物技术通报，2000，4：44．
[42]孙月芳，黄剑华，陆瑞菊等．叶用莴苣高频再生体系建立[J]．上海农业学报，1998，14(3)：17-20．
[43]王瓞，林其淮．阳离子脂质体介导基因转染的最优化条件．生命的化学，1997，17(1)：32-34．
[44]王关林，方宏筠．植物基因工程原理与技术．北京：科学出版社，1998．
[45]王景雪，孙毅．农杆菌介导的植物基因转化研究进展．生物技术通报，1999，(1)：7-13．
[46]王小军，刘玉乐，Huang Yong等．可育和抗除草剂溴苯睛转基因小麦．植物学报，1996，38(12)：942-948．
[47]王忠华，舒庆尧，崔海瑞等．Bt杀虫基因与Bt转基因抗虫植物研究进展．植物学通报，1999，16(1)：51-58．
[48]王忠华，夏英武．转基因植物中报告基因GUS的表达及其安全性评价．生命科学，2000，12(5)：207-209．
[49]卫志明，黄健秋等．甘蓝下胚轴的高效再生体系和农杆菌介导Bt基因转化甘蓝．上海农业学报，1998，14(2)：11-18．
[50]周音，张智奇，王少欧等．十字花科植物转基因技术研究进展及存在问题．吉林农业大学学报，1996，18(1)：96-102．
[51]徐茂军．转基因植物中卡那霉素(Kan~r)抗性标记基因的生物安全性．生物学通讯，2001，18-19．
[52]徐明良，杨金水，葛扣麟．植物转基因的整合机制．植物生理学通讯，1996，32(3)：234-240．
[53]许耀，王艇，李宝健．根癌农杆菌介导的外源基因转化植物萌动种胚的研究．实验生物学报，1991，24(2)：109-117．
[54]旋和平，李玲等，发根农杆菌对黄瓜的遗传转化，植物学报，1998，40(5)：470-473．
[55]薛红卫，卫志明，许智宏．通过PEG法转化甘蓝获得转基因植株[J]．植物学报，1997，39(1)：28-33．
[56]薛万新，巩振辉等．油菜基因转移适宜苗态建成方法的研究，西北农业大学学报，1999，27(3)：6-10．
[57]薛万新．白菜类蔬菜叶球发育相关基因的分子克隆及其转基因的研究．西北农林科技大学博士论文，2001．
[58]阎隆飞，刘国琴，肖兴国．从花粉肌动蛋白到作用雄性不育．科学通报，1999，44(23)：2471-2475．
[59]杨剑波，许智宏等．影响根癌农杆菌附着禾谷类作物培养细胞的因素．实验生物学报，1993,26(1)：1-7．
[60]叶志彪等，两个反义基因在番茄工程植株中的生理抑制效应分析．植物生理学报。1996，22(2)：2280-2283．
[61]于志章，程智慧，郭奠盈编著．蔬菜种子生产原理与技术．陕西天则出版社，1992．
[62]余文贵．现代生物技术在蔬菜品种改良中的应用，(上，下)．长江蔬菜，1996，9、10：1-3．
[63]张广辉，巩振辉等．大白菜和油菜真空渗入遗传转化法初报．西北农业大学学报，1998，26(4)：1-4．
[64]张桂华，巩振辉等．农杆菌介导的芸薹属作物遗传转化研究进展．西北农业大学学报，2000，28(2)：80-83．
[65]张宏宇，李中奎，喻子牛．转苏云金芽孢杆菌杀虫晶体蛋白基因抗虫植物的研究进展和商品化．生物技术通报，1997，(3)：13-15．
[66]张利平，曹孜义，李唯．高等植物基因转移技术研究进展，甘肃农业大学学报，1991，26(3)：270-275．
[67]张锐，郭三堆．植物抗虫基因工程研究进展，生物技术通报，2001，(2)：8-12．
[68]张钰，Adrian J，et a1．真空渗透法转化植物的研究．生物技术，1999，9(2)：35-36．
[69]张志宏，方宏筠等．苹果主栽品种高效遗传转化系统的建立及其影响因子的研究．遗传学报，1998，25(2)：160-165．
[70]中棉所．中棉所转基因抗虫棉选育及试种示范．生物技术通报，1997(1)：37-38．
[71]钟蓉，刘玉乐，罗鹏等．植物杂种优势利用的基因工程研究进展．生物技术通报，1996，(1)：1-5．
[72]周光宇．授粉后外源DNA导入植物技术．中国农业科学，1988，21(3)：1-4．
[73]周冀明，卫志明等．根癌农杆菌介导转化诸葛菜获得转基因植株．植物生理学报，1997，23(1)：21-28．
[74]周延清．转基因油菜研究进展，生物学通报，1994，29(5)：3-5．
[75]周奕华，韩历玲．利用激光微束穿刺法将GUS基因导入百脉根并获得转基因植株的研究，激光生物学报，1998，7(1)：57．
[76]朱玉贤，李毅．现代分子生物学．北京，高等教育出版社，1998，435-438．
[77]朱作言，曾志强．转基因鱼离市场还有多远．生物技术通报，2000，(1)：1-6．
[78]Ahl GP. Field release of genetically modified plants worldwide. Plant Industrial Plant from Newsletter, 1993,3:18-20.
[79]Alejandro P V.Plant Cell Report, 1995,14:482-487.
[80]Bechtold N, Ellis J, et al. In-planta Agrobacterium-mcdiated gent transfer by infiltration of adult Arabidopsis thaliana plants.C.T.Acad.Sci.Paris. Life Science,316: 1194-1199.
[81]Berthomiou P, Jouanin L.Transformation of rapid cycling cabbage(Brassica Oleracea
var. capitata) with Agrobacterium rhizogenes[J]. Plant Cell Rep., 1992,vol(11) :334-338．
[82] Block M D, Browwer D D, Tenning P. transformation of Brassica napus and Brassica Oleralea using Agrobacterirm tumefaciens and the expression of the bar and neo-genes in the transgenic plants[J]. Plant Physiol, 1989,vol(91) :694-701[150]
[83] Bouchez D, Camilleri C, Caboche M.A binary vector based on Basta resistance for in planta transformation of Arabidopsis thaliana. C.R. Acad. Sci. Paris. Life Science, 316: 1188-1193．
[84] Broekaert W F, et al. Antimicrobial peptides from Amaranthus caudatus seed with sequence homology to the cysteine/glycine-rich domain of chitin-binding proteins. Biochemistry, 1 992, 3 1 :4308-43 1 4．
[85] Broglia K. Transgenic plant with enhanced resistance to the fugal pathogen Rhizoctonia Solani.Science,1991, 254:1194-1197．
[86] Cao Ming Qing, Liu Fan, Yao Lei, et al. Transformation of pakchoi (Brassica rapa L. ssp. Chinese) by Agrobacterium infiltration. Molecular Breeding, 2000(6) :67-72．
[87] Chaleff R S, Ray T B. Herbicide-resistant mutants from tobacco cell culture . Sci.,1984, 223:1148-1151．
[88] Chan M T, Chang H H, Ho S L, et al. Agrobacterium-mediated production of transgenic rice plants expressing glucuronidase gene. Plant Mol Biol., 1993,22: 491-506．
[89] Charest P J, Hattori J, Demoor J. In vitro study of transgenic tobacco expressing Arabidopsis wild type and mutant ace to hydroxyacid synthase genes. Plant Cell Rep., 1990, 8:634-646．
[90] Chee P P, Slighton J L. Transformation of cucumber tissues by micro-projectile bombardment identification of plants containing functional and non-functional transferred genes. Gene, 1992, 118:255-260．
[91] Chraibi K M, Latche A, Roustan J P, et al. stimulation of shoot regeneration from cotyledons of Helianthus annuus by the ethylene inhibitors silver and cobalt (J). Plant Cell Rep., 1991,10:204-207．
[92] Chris Bright. Life out of Bounds-Bioinvasion hi a Borders less World. The World Watch Environmental Alert Series. World-Watch Institute,1998, W.W Norto & Commpany, New York,London.
[93] Clive James. Global Review of Commercialized Transgenic Crops: 1999． ISAAA Briefs.
[94] Comai L D, Facciotti D, Hiatt W R. Expression in plants of a mutant aroA gene from salmonella typhimurium confers tolerance to glyphosafe. Nature,
1985,317:741-744．
[95] Corughan T P. Application of tissue culture techniques to the development of herbicide resistant rice. Louisiana Agri., 1994,3 7(3) :25-26．
[96] Damgaard O, Hollund J L, kasmussen O S. Agrobacterium tumefaciens-mediated transformation of Brassica napus winter cultivars [J]. Transgenic Research, 1997, 6(4) : 279-288．
[97] Della-cippa G, Bauer S C,Taylor M L.Targetting a herbicide-resistant enzyme from E.coli to chloroplasts of higher plants.Bio. Technology, 1987, 5:579-584．
[98] Delzer B W, Somer D A, et al. Agrobacterium tumefaciens susceptibility and plant regeneration of 10 soybean genotypes in maturity groups oo to Ⅱ. Crop Sci, 1990,30: 320-322．
[99] Donn G, Tischer E, Smith J A. Herbicide-resistant alfalfa cell: an example of gene amplification in plants. Journal Mol. Appl. Genet, 1984,2:621-635．
[100] Enriguez OGA, Vazque Z P R I, Prieto SDL, et al. Herbicide-resistant sugarcane (Saccharum Officinarum L.) plants by Agrobacterium-mediated transformation. Planta, 1998,206(1) ; 20-27．
[101] FAO/WHO.Transfer gene food safety. FAO, 1996．
[102] Fillatti J J, et al. Efficient transfer of glyphosafe tolerance gene into tomato using a brinary Agrobacterium tumefaciens vector. Bio/Technology, 1987,5:726-730．
[103] Fromm M E, et al. Inheritance and expression of chamameric genes in the progeny of transgenic maize plants.Bio.Technology, 1990,8:833-839．
[104] Gelvin S B. Agrobacterium VirE2 protein can form a complex T strands in the plant cytoplasm. J. Bacteriol, 1998,180(16) : 4300-4302．
[105] Gong Zhenhui, Zhang Guanghui, et al. Studies on vacuum infiltration transformation method in Brassica Campestris Var.Purpurea and B. campestris Var. Utilis. International symposium on biotechnology application in horticultural crops, Sept,4-8, 2000,Beijing,China PP70．
[106] Gritz L, Davis J. Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Sac charomyces cerevisiae. Gene, 1983,25:179-188．
[107] Haughn R S, Somerville C. Sunfonylurea-resistant mutations of Arabidopsis thaliana. Mol .Gen. Genet, 1986, 204:430-434．
[108] Hirhoberg J, Mcintosh L.Molecular basis of herbicide resistance in Amaranthus hybridus.Sci.,1983,222:1346-1349．
[109] Jain S M. Recent advances in plant genetic engineering. Curr. Sci., 1993,64:705-714．
[110] James C. Global status of the field testing and commercialization of transgenic plants: 1986 to 1995--The first decade in crop biotechnology. ISAAA BriefJSAAA, USA.1996, 1:35-38．
[111] James D J, Passey A J, et al. Agrobacterium mediated transformation of the cultivated strawberry (Fragaria Anannassa Duch) using disarmed binary vectors.
[112] Jefferson R A, Kavanagh T A, Bevan M W. GUS fusion:p-glucaronidase as a sensitive and versatile gene fusion marker in high plants. EMBO J, 1987,6:3901-3907．
[113] Kenneth A, Feldman K A, et al. Agrobacterium-mediated transformation of germination seeds of Arabidopsis thaliana non-tissue culture approach. Mol. Gen. Gent., 1987,208:1-9．
[114] Klein T M , et al. Factors influencing gene delivery into zea mays cell by high-velocity microjectiles.Bio.Technology, 1988,6:559-563．
[115] Klein T M, et al. High-velocity microprojectiles for delivery of nucleic acid into living cells. Nature, 1987,327:70-73．
[116] Krens P, Oancia T, et al. Effects of seed-specific expression of a cytokinin gene on tabacco. Transgenic Research, 1997,6(2) : 133-141．
[117] Lecchine E, Gong Z H, et al. Transgenic Arabidopsis lines expressing genes Ⅵ from cauliflower mosaic virus variants exhibit a range of symptom-like phenotype accumulate inclusion bodies. Molecular plant Microbe Interactions, 1997, 10(9) : 1094-1101．
[118] Li Z, Hayashimoto A, Murai N A. Sulfonylurea herbicide resistance gene from Arabidopsis thaliana as a new selectable marker for production of fertile transgenic rice. Plant Physiol., 1992, 100:662-668．
[119] Lodge J K, Kaniewski W K, et al. Broad-spectrum virus resistance in transgenic plants expressing poke weed antiviral protein. Proc. Nat. Acad. Sci, USA,1993,8(1) : 90．
[120] Logermamm J. Expression of a barley ribosome-inactivating protein leads to increased fungal protection in transgenic tobacoo plants. Biotechnology, 1992,10: 305-308．
[121] Losey J E, Rayor L S, Cater M E. Transgenic pollen harms monarch larvae. Nature, 1999,399:224．
[122] Mariani C, Gossele V, Beuckeleer M D, et al. A chimaeric ribonuclease-inhibitor gene restores fertility to male sterile plants. Nature, 1992,357: 384-387．
[123] Mathews H, et al. Regeneration of shoots from Brasica juncea(L)Caern and Coss cell transformation by Agrobacterium tumefaciens and expression of napaline
dehydr-ogenat genes. Plant Sci., 1985,39(1) :49-54．
[124] Mazur B J Carl Falco S. The development of herbicide-resistant crops. Annu. Plant Physiol&Plant Mol. Biol., 1989,40:441-470．
[125] Mei-zhu Yang, Shi-Rong Jia, Eng-chong Pua. High frequency of plant regeneration from hypocotyls explants of Brassica Carinata A-Br plant cell[J]. Tissue and Organ Culture, 199 1,24:79-82．
[126] Miki B L, Labbe H, Hattori J. Transformation of B.napus canola cultivars with Arabidopis Thaliana to hydroxyacid synthase genes and analysis of herbicide resistance. TAG, 1990, 80:449-458．
[127] Nordlee J A, Tayler S L, Townsend J A, Thomas L A, Bush R K. Identification of a Brazil nut allergen in transgenic soybeans. New England Journal of Medicine, 1996, (334) :688-692．
[128] O'Neill C. Polyethylene glycol(PEG) treatment of protoplast is an alternative to biolistic delievery systems.The Plant Journal, 1993,3:729-738．
[129] Penarrubia L,Kim R.Bio/Techonlogy, 1992,10:561-564．
[130] Purahauser L, Medgyesy M, Czeko P J, et al. stimulation of shoot regeneration in Triticum aestivum and nicotiana plumbagmifolia vitro tissue culture using the ethylene inhibitors of AgNO3[J]. Plant Cell Rep., 1987, Vol(6) :1-4．
[131] Radke S E, Turner J C, Facciotti D. Transformation and regeneration of Brassica rapa using Agrobacterium tumefaciens. Plant Cell Rep., 1992,11 :499-505．
[132] Review of date on possible toxicity of GM potatoes. The Royal Society Report, 1999,5．
[133] Sanford J C, et al. Particulate Sci. Technol., 1987,5:27-37．
[134] Sen Gupta A, Heinen J L, Holaday A S. Increased resistance to oxidative stress in transgenic plants that overexpress chloroplastic Cu/Zn superoxide dismutase.Proc.Natl.Acad.Sci.USA, 1993,90: 1629-1633．
[135] Shan D M, Horsch R B, Klee H J. Engineering herbicide tolerance in transgenic plants. Sci., 1986, 233:478-481．
[136] Spencer T M, et al. Segregation of transgenes hi maize. Plant Molec.Biol., 1992,18: 201-210．
[137] Stark D M, Beachy R N. protection against polyvirus in transgenic plants: evidence for broad spectrum resistance. Bio/Technology, 1989,7:1257-1262．
[138] Steven J, Clough S J, et al. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J., 1998,16(6) : 735-743．
[139] Stoger E, William S, Christon P, et al. Molecular Breeding,1999,5(1) :65-73．
[140] Stromvik M V, Sundaraman V P, et al. A novel promoter from soybean that is active
in a complex developmental pattern with and without its proximal 650 base paris. Plant Mol.Biol., 1999,9:41．
[141] Tian Wenzhong, et al. Rice transformation with a phytoalexin gene and bioassay of the transgenic plants. Acta Botanica Sinica, 1998,40(9) : 803-808．
[142] Tjokrokusumo D, Heinrich T, et al. Vacuum infiltration of petunia hybrida pollen with Agrobacterium tumefaciens to achieve plant transformation. Plant Cell Reports, 2000, 19(8) :792-797．
[143] Van Roekel Jsc, Pamm B, et al. Factors influencing transformation frequency of tomato(Lycopersicon esculentum). Plant Cell Rep., 1993,12:644-647．
[144] Wang Y L, et al. Transient expression of foreign gene in rice, wheat and soybean cells following particle bombardment. Plant Molec. Bio., 1998,11 :433-439．
[145] Wenck A, Czako M, et al. Frequent collinear long transfer of DNA inclusive of the whole binary vector during Agrobacterium-mediated transformation. Plant Mol. Biol., 1997,34(6) :913-922．
[146] WHO.Health aspects of marker gene in genetically modified plants. Report of a WHO work shop. WHO Food Safety Unit.WHO/FUN/FOS/93,6,1993．
[147] Wilknson J E, Twell D, et al. Methanol does not specifically inhibit endogenous β-glucuronidase(Gus)activity. Plant Sci., 1994,97:61-67．
[148] Willams J, pink A C, Bidding N L. Effect of silver nitrate on long term culture and regeneration of callus from Brassica Oleracea Var.gemmifera plant cell[J]. Tissue and Organ Culture, 1990,21:61-66．
[149] Witty M. Nucleic Acids Research, 1989,17:3312．
[150] Wuk K, Butgust W, Sorrells M E, et al. The detection and estimation of linkage in polyploids using single-dose restriction fragments. Theory Appl Genent, 1992,83, 294-300．
[151] Yang M.S. Expression of a synthetic gene for improved protein quality in transformed potato plants. Plant Sci., 1989,64:99-111．
[152] Zupan J R, Zambryski P. Transfer of I-DNA from Agrobacterium to the plant cell. Plant Physiol.,1995, 107:1041-1047．
[153] Zupan J R, Zarnbruski P C. Transfer of T-DNA from Agrobacterium to the plant cell. Plant Physiol., 1995,107:1041-1047．
[2] Clark M S主编，顾红雅，瞿礼嘉主译，陈章良主校．植物分子生学实验手册．高等教育出版社，施普林格出版社出版．1998，北京，3-11．
[3] 蔡小宁，余建明，朱祯等．建立青菜(Barssica chinese)农杆菌介导基因转化体系．江苏农业学报，1997，13(2)：110-114．
[4] 沉宗英，张智良．花椰菜幼苗下胚轴诱导再生植株[J]．植物生理学通讯，1984,vol(1)：37．
[5] 成卓敏．应用基因工程创造黄锈病转基因小麦新种质．植物保护，1996，22(3)：18-20
[6] 成卓敏．应用基因工程创造小麦研究进展．生物技术通报，1997(4)：11-13．
[7] 程振东，卫志明，许智宏．芸薹属作物的遗传转化．植物生理学通讯，1992，28(3)：161-164．
[8] 邓万银，邵启全．酚类化合物能促进致瘤农杆菌对单子叶植物鸭跖草的转化．科学通报，1989：776-779．
[9] 傅忠昭等．植物遗传转化技术手册，北京：中国科学出版社，1994，5：97-123．
[10] 龚蓁蓁，沈慰芳，周光宇等．授粉后外源DNA导入植物——DNA通过花粉管通道进入胚囊，中国农业科学(B)，1988，6：661-664．
[11] 巩振辉，Cecchini E，Millner JJ.以PCR鉴定转基因植株的微量DNA提取方法．西北农业大学学报，1997，25(1)：45-48．
[12] 巩振辉，Millner J J，何玉科．拟南芥基因转移新方法—真空渗入法的研究，西北植物学报，1996，16(3)：277-283．
[13] 巩振辉．芸薹属作物基因转移新方法——植株原位真空渗入遗传转化方法，见冯延辉主编，新世纪的思索——中国当代改革发展文集．新疆人民出版社，2000，1712-1713．
[14] 官春云，李旬．转基因油菜应用研究．细胞生物学杂志，1997，19(1)：18—33[106]
[15] 郭启兴，范国强等．植物抗病毒基因工程育种策略及其进展．生命科学研究，2000，4(2)：113-117．
[16] 郭三堆，崔洪亮，倪万潮等．双价抗虫转基因棉花研究．中国农业科学，1999，32(3)：1-7．
[17] 郭三堆，植物Bt抗虫基因工程研究进展，中国农业科学，1993，28(5)：8-13．
[18] 郭元林，向平，飞速发展的生物技术．作物研究，1994，8(4)：4-6．
[19] 郭元林，向平．转基因技术在作物育种上的应用．西南农业学报，1997，10(4)：109-113．
[20] 华志华，黄大年．转基因植物中外源基因的遗传学行为．植物学报，1999，41(1)：1-5．
[21] 贾士荣．转基因作物的安全性争论及对策．生物技术通报，1999，(6)：1-7．
[22] 金万梅，巩振辉．芥菜真空渗入遗传转化体系适宜苗态的建成，西北农业学报，2000，9(4)：22-25．
[23] 金万梅，巩振辉等，植物遗传转化方法和转基因植株的鉴定．陕西农业科学，2000，1：24-29．
[24] 金万梅．芥菜植株原位真空渗入遗传转化体系的研究．西北农林科技大学硕士论文，2001．
[25] 荆家海主编，植物生理学．陕西科学技术出版社,1994年(第二版)，167-179．
[26] 朗春秀，胡张华，刘智宏等，油菜农杆菌转基因体系的建立及转PEP反义基因油菜的获得．浙江农业学报，1999，11(2)：55-58．
[27] 李宝健，曾庆平．植物生物技术原理与方法．科学技术出版社，1990，190-191．
[28] 李恩义．多聚阳离子基因导入法．上海农业科技，1999，1：8-10．
[29] 李洪清，李美茹．影响农杆菌介导植物基因转化的因素问题．植物生理学通讯，1999，35(2)：145-151．
[30] 李天然，张治中等．番茄ACC合酶反义基因对河套蜜瓜的转化．植物学报，1999，41(2)：142-145．
[31] 凌定厚，陶利珍，马镇荣等．以基因枪介导获转Psl-barnase基因的工程雄性不育水稻植株．遗传学报，1998，25(5)：433-442．
[32] 刘进元等．植物抗病基因工程的进展．生物工程进展，1994，14(2)：31-34．
[33] 卢爱兰，陈正华，孔令洁等，抗芜菁花叶病毒转基因甘蓝型油菜的研究．遗传学报，1996，23(1)：77-83．
[34] 陆璐，甜瓜ACC氧化酶cDNA克隆及其反义序列转化栽培品种．西北农业大学博士论文，1999，1-12．
[35] 毛慧琳，唐惕．抗虫转基因甘蓝及后代的研究[J]．中国科学(C辑)，1996，26(4)：339-349．
[36] 彭仁旺，周雪莱，王峻岭等．表达barstar基因及bar基因的转基因油菜的研究．遗传学报，1998，25(1)：74-79．
[37] 钱迎倩．生物多样性的几个问题．植物学通报，1998，15(5)．
[38] 宋鸣凤等．植物抗真菌和细菌病害基因工程的策略及其进展．植物生理学通讯，1996，32(1)：64-70．
[39] 宋正旭．小白菜植株原位真空渗入遗传转化体系的研究．西北农林科技大学硕士论文，2002，17-19．
[40] 苏军，段榕琦等．农杆菌介导小白菜的GUS基因瞬间表达．福建果树。2000，2：3-5．
[41]孙雷心译．开发转基因新作物．生物技术通报，2000，4：44．
[42]孙月芳，黄剑华，陆瑞菊等．叶用莴苣高频再生体系建立[J]．上海农业学报，1998，14(3)：17-20．
[43]王瓞，林其淮．阳离子脂质体介导基因转染的最优化条件．生命的化学，1997，17(1)：32-34．
[44]王关林，方宏筠．植物基因工程原理与技术．北京：科学出版社，1998．
[45]王景雪，孙毅．农杆菌介导的植物基因转化研究进展．生物技术通报，1999，(1)：7-13．
[46]王小军，刘玉乐，Huang Yong等．可育和抗除草剂溴苯睛转基因小麦．植物学报，1996，38(12)：942-948．
[47]王忠华，舒庆尧，崔海瑞等．Bt杀虫基因与Bt转基因抗虫植物研究进展．植物学通报，1999，16(1)：51-58．
[48]王忠华，夏英武．转基因植物中报告基因GUS的表达及其安全性评价．生命科学，2000，12(5)：207-209．
[49]卫志明，黄健秋等．甘蓝下胚轴的高效再生体系和农杆菌介导Bt基因转化甘蓝．上海农业学报，1998，14(2)：11-18．
[50]周音，张智奇，王少欧等．十字花科植物转基因技术研究进展及存在问题．吉林农业大学学报，1996，18(1)：96-102．
[51]徐茂军．转基因植物中卡那霉素(Kan~r)抗性标记基因的生物安全性．生物学通讯，2001，18-19．
[52]徐明良，杨金水，葛扣麟．植物转基因的整合机制．植物生理学通讯，1996，32(3)：234-240．
[53]许耀，王艇，李宝健．根癌农杆菌介导的外源基因转化植物萌动种胚的研究．实验生物学报，1991，24(2)：109-117．
[54]旋和平，李玲等，发根农杆菌对黄瓜的遗传转化，植物学报，1998，40(5)：470-473．
[55]薛红卫，卫志明，许智宏．通过PEG法转化甘蓝获得转基因植株[J]．植物学报，1997，39(1)：28-33．
[56]薛万新，巩振辉等．油菜基因转移适宜苗态建成方法的研究，西北农业大学学报，1999，27(3)：6-10．
[57]薛万新．白菜类蔬菜叶球发育相关基因的分子克隆及其转基因的研究．西北农林科技大学博士论文，2001．
[58]阎隆飞，刘国琴，肖兴国．从花粉肌动蛋白到作用雄性不育．科学通报，1999，44(23)：2471-2475．
[59]杨剑波，许智宏等．影响根癌农杆菌附着禾谷类作物培养细胞的因素．实验生物学报，1993,26(1)：1-7．
[60]叶志彪等，两个反义基因在番茄工程植株中的生理抑制效应分析．植物生理学报。1996，22(2)：2280-2283．
[61]于志章，程智慧，郭奠盈编著．蔬菜种子生产原理与技术．陕西天则出版社，1992．
[62]余文贵．现代生物技术在蔬菜品种改良中的应用，(上，下)．长江蔬菜，1996，9、10：1-3．
[63]张广辉，巩振辉等．大白菜和油菜真空渗入遗传转化法初报．西北农业大学学报，1998，26(4)：1-4．
[64]张桂华，巩振辉等．农杆菌介导的芸薹属作物遗传转化研究进展．西北农业大学学报，2000，28(2)：80-83．
[65]张宏宇，李中奎，喻子牛．转苏云金芽孢杆菌杀虫晶体蛋白基因抗虫植物的研究进展和商品化．生物技术通报，1997，(3)：13-15．
[66]张利平，曹孜义，李唯．高等植物基因转移技术研究进展，甘肃农业大学学报，1991，26(3)：270-275．
[67]张锐，郭三堆．植物抗虫基因工程研究进展，生物技术通报，2001，(2)：8-12．
[68]张钰，Adrian J，et a1．真空渗透法转化植物的研究．生物技术，1999，9(2)：35-36．
[69]张志宏，方宏筠等．苹果主栽品种高效遗传转化系统的建立及其影响因子的研究．遗传学报，1998，25(2)：160-165．
[70]中棉所．中棉所转基因抗虫棉选育及试种示范．生物技术通报，1997(1)：37-38．
[71]钟蓉，刘玉乐，罗鹏等．植物杂种优势利用的基因工程研究进展．生物技术通报，1996，(1)：1-5．
[72]周光宇．授粉后外源DNA导入植物技术．中国农业科学，1988，21(3)：1-4．
[73]周冀明，卫志明等．根癌农杆菌介导转化诸葛菜获得转基因植株．植物生理学报，1997，23(1)：21-28．
[74]周延清．转基因油菜研究进展，生物学通报，1994，29(5)：3-5．
[75]周奕华，韩历玲．利用激光微束穿刺法将GUS基因导入百脉根并获得转基因植株的研究，激光生物学报，1998，7(1)：57．
[76]朱玉贤，李毅．现代分子生物学．北京，高等教育出版社，1998，435-438．
[77]朱作言，曾志强．转基因鱼离市场还有多远．生物技术通报，2000，(1)：1-6．
[78]Ahl GP. Field release of genetically modified plants worldwide. Plant Industrial Plant from Newsletter, 1993,3:18-20.
[79]Alejandro P V.Plant Cell Report, 1995,14:482-487.
[80]Bechtold N, Ellis J, et al. In-planta Agrobacterium-mcdiated gent transfer by infiltration of adult Arabidopsis thaliana plants.C.T.Acad.Sci.Paris. Life Science,316: 1194-1199.
[81]Berthomiou P, Jouanin L.Transformation of rapid cycling cabbage(Brassica Oleracea
var. capitata) with Agrobacterium rhizogenes[J]. Plant Cell Rep., 1992,vol(11) :334-338．
[82] Block M D, Browwer D D, Tenning P. transformation of Brassica napus and Brassica Oleralea using Agrobacterirm tumefaciens and the expression of the bar and neo-genes in the transgenic plants[J]. Plant Physiol, 1989,vol(91) :694-701[150]
[83] Bouchez D, Camilleri C, Caboche M.A binary vector based on Basta resistance for in planta transformation of Arabidopsis thaliana. C.R. Acad. Sci. Paris. Life Science, 316: 1188-1193．
[84] Broekaert W F, et al. Antimicrobial peptides from Amaranthus caudatus seed with sequence homology to the cysteine/glycine-rich domain of chitin-binding proteins. Biochemistry, 1 992, 3 1 :4308-43 1 4．
[85] Broglia K. Transgenic plant with enhanced resistance to the fugal pathogen Rhizoctonia Solani.Science,1991, 254:1194-1197．
[86] Cao Ming Qing, Liu Fan, Yao Lei, et al. Transformation of pakchoi (Brassica rapa L. ssp. Chinese) by Agrobacterium infiltration. Molecular Breeding, 2000(6) :67-72．
[87] Chaleff R S, Ray T B. Herbicide-resistant mutants from tobacco cell culture . Sci.,1984, 223:1148-1151．
[88] Chan M T, Chang H H, Ho S L, et al. Agrobacterium-mediated production of transgenic rice plants expressing glucuronidase gene. Plant Mol Biol., 1993,22: 491-506．
[89] Charest P J, Hattori J, Demoor J. In vitro study of transgenic tobacco expressing Arabidopsis wild type and mutant ace to hydroxyacid synthase genes. Plant Cell Rep., 1990, 8:634-646．
[90] Chee P P, Slighton J L. Transformation of cucumber tissues by micro-projectile bombardment identification of plants containing functional and non-functional transferred genes. Gene, 1992, 118:255-260．
[91] Chraibi K M, Latche A, Roustan J P, et al. stimulation of shoot regeneration from cotyledons of Helianthus annuus by the ethylene inhibitors silver and cobalt (J). Plant Cell Rep., 1991,10:204-207．
[92] Chris Bright. Life out of Bounds-Bioinvasion hi a Borders less World. The World Watch Environmental Alert Series. World-Watch Institute,1998, W.W Norto & Commpany, New York,London.
[93] Clive James. Global Review of Commercialized Transgenic Crops: 1999． ISAAA Briefs.
[94] Comai L D, Facciotti D, Hiatt W R. Expression in plants of a mutant aroA gene from salmonella typhimurium confers tolerance to glyphosafe. Nature,
1985,317:741-744．
[95] Corughan T P. Application of tissue culture techniques to the development of herbicide resistant rice. Louisiana Agri., 1994,3 7(3) :25-26．
[96] Damgaard O, Hollund J L, kasmussen O S. Agrobacterium tumefaciens-mediated transformation of Brassica napus winter cultivars [J]. Transgenic Research, 1997, 6(4) : 279-288．
[97] Della-cippa G, Bauer S C,Taylor M L.Targetting a herbicide-resistant enzyme from E.coli to chloroplasts of higher plants.Bio. Technology, 1987, 5:579-584．
[98] Delzer B W, Somer D A, et al. Agrobacterium tumefaciens susceptibility and plant regeneration of 10 soybean genotypes in maturity groups oo to Ⅱ. Crop Sci, 1990,30: 320-322．
[99] Donn G, Tischer E, Smith J A. Herbicide-resistant alfalfa cell: an example of gene amplification in plants. Journal Mol. Appl. Genet, 1984,2:621-635．
[100] Enriguez OGA, Vazque Z P R I, Prieto SDL, et al. Herbicide-resistant sugarcane (Saccharum Officinarum L.) plants by Agrobacterium-mediated transformation. Planta, 1998,206(1) ; 20-27．
[101] FAO/WHO.Transfer gene food safety. FAO, 1996．
[102] Fillatti J J, et al. Efficient transfer of glyphosafe tolerance gene into tomato using a brinary Agrobacterium tumefaciens vector. Bio/Technology, 1987,5:726-730．
[103] Fromm M E, et al. Inheritance and expression of chamameric genes in the progeny of transgenic maize plants.Bio.Technology, 1990,8:833-839．
[104] Gelvin S B. Agrobacterium VirE2 protein can form a complex T strands in the plant cytoplasm. J. Bacteriol, 1998,180(16) : 4300-4302．
[105] Gong Zhenhui, Zhang Guanghui, et al. Studies on vacuum infiltration transformation method in Brassica Campestris Var.Purpurea and B. campestris Var. Utilis. International symposium on biotechnology application in horticultural crops, Sept,4-8, 2000,Beijing,China PP70．
[106] Gritz L, Davis J. Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Sac charomyces cerevisiae. Gene, 1983,25:179-188．
[107] Haughn R S, Somerville C. Sunfonylurea-resistant mutations of Arabidopsis thaliana. Mol .Gen. Genet, 1986, 204:430-434．
[108] Hirhoberg J, Mcintosh L.Molecular basis of herbicide resistance in Amaranthus hybridus.Sci.,1983,222:1346-1349．
[109] Jain S M. Recent advances in plant genetic engineering. Curr. Sci., 1993,64:705-714．
[110] James C. Global status of the field testing and commercialization of transgenic plants: 1986 to 1995--The first decade in crop biotechnology. ISAAA BriefJSAAA, USA.1996, 1:35-38．
[111] James D J, Passey A J, et al. Agrobacterium mediated transformation of the cultivated strawberry (Fragaria Anannassa Duch) using disarmed binary vectors.
[112] Jefferson R A, Kavanagh T A, Bevan M W. GUS fusion:p-glucaronidase as a sensitive and versatile gene fusion marker in high plants. EMBO J, 1987,6:3901-3907．
[113] Kenneth A, Feldman K A, et al. Agrobacterium-mediated transformation of germination seeds of Arabidopsis thaliana non-tissue culture approach. Mol. Gen. Gent., 1987,208:1-9．
[114] Klein T M , et al. Factors influencing gene delivery into zea mays cell by high-velocity microjectiles.Bio.Technology, 1988,6:559-563．
[115] Klein T M, et al. High-velocity microprojectiles for delivery of nucleic acid into living cells. Nature, 1987,327:70-73．
[116] Krens P, Oancia T, et al. Effects of seed-specific expression of a cytokinin gene on tabacco. Transgenic Research, 1997,6(2) : 133-141．
[117] Lecchine E, Gong Z H, et al. Transgenic Arabidopsis lines expressing genes Ⅵ from cauliflower mosaic virus variants exhibit a range of symptom-like phenotype accumulate inclusion bodies. Molecular plant Microbe Interactions, 1997, 10(9) : 1094-1101．
[118] Li Z, Hayashimoto A, Murai N A. Sulfonylurea herbicide resistance gene from Arabidopsis thaliana as a new selectable marker for production of fertile transgenic rice. Plant Physiol., 1992, 100:662-668．
[119] Lodge J K, Kaniewski W K, et al. Broad-spectrum virus resistance in transgenic plants expressing poke weed antiviral protein. Proc. Nat. Acad. Sci, USA,1993,8(1) : 90．
[120] Logermamm J. Expression of a barley ribosome-inactivating protein leads to increased fungal protection in transgenic tobacoo plants. Biotechnology, 1992,10: 305-308．
[121] Losey J E, Rayor L S, Cater M E. Transgenic pollen harms monarch larvae. Nature, 1999,399:224．
[122] Mariani C, Gossele V, Beuckeleer M D, et al. A chimaeric ribonuclease-inhibitor gene restores fertility to male sterile plants. Nature, 1992,357: 384-387．
[123] Mathews H, et al. Regeneration of shoots from Brasica juncea(L)Caern and Coss cell transformation by Agrobacterium tumefaciens and expression of napaline
dehydr-ogenat genes. Plant Sci., 1985,39(1) :49-54．
[124] Mazur B J Carl Falco S. The development of herbicide-resistant crops. Annu. Plant Physiol&Plant Mol. Biol., 1989,40:441-470．
[125] Mei-zhu Yang, Shi-Rong Jia, Eng-chong Pua. High frequency of plant regeneration from hypocotyls explants of Brassica Carinata A-Br plant cell[J]. Tissue and Organ Culture, 199 1,24:79-82．
[126] Miki B L, Labbe H, Hattori J. Transformation of B.napus canola cultivars with Arabidopis Thaliana to hydroxyacid synthase genes and analysis of herbicide resistance. TAG, 1990, 80:449-458．
[127] Nordlee J A, Tayler S L, Townsend J A, Thomas L A, Bush R K. Identification of a Brazil nut allergen in transgenic soybeans. New England Journal of Medicine, 1996, (334) :688-692．
[128] O'Neill C. Polyethylene glycol(PEG) treatment of protoplast is an alternative to biolistic delievery systems.The Plant Journal, 1993,3:729-738．
[129] Penarrubia L,Kim R.Bio/Techonlogy, 1992,10:561-564．
[130] Purahauser L, Medgyesy M, Czeko P J, et al. stimulation of shoot regeneration in Triticum aestivum and nicotiana plumbagmifolia vitro tissue culture using the ethylene inhibitors of AgNO3[J]. Plant Cell Rep., 1987, Vol(6) :1-4．
[131] Radke S E, Turner J C, Facciotti D. Transformation and regeneration of Brassica rapa using Agrobacterium tumefaciens. Plant Cell Rep., 1992,11 :499-505．
[132] Review of date on possible toxicity of GM potatoes. The Royal Society Report, 1999,5．
[133] Sanford J C, et al. Particulate Sci. Technol., 1987,5:27-37．
[134] Sen Gupta A, Heinen J L, Holaday A S. Increased resistance to oxidative stress in transgenic plants that overexpress chloroplastic Cu/Zn superoxide dismutase.Proc.Natl.Acad.Sci.USA, 1993,90: 1629-1633．
[135] Shan D M, Horsch R B, Klee H J. Engineering herbicide tolerance in transgenic plants. Sci., 1986, 233:478-481．
[136] Spencer T M, et al. Segregation of transgenes hi maize. Plant Molec.Biol., 1992,18: 201-210．
[137] Stark D M, Beachy R N. protection against polyvirus in transgenic plants: evidence for broad spectrum resistance. Bio/Technology, 1989,7:1257-1262．
[138] Steven J, Clough S J, et al. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J., 1998,16(6) : 735-743．
[139] Stoger E, William S, Christon P, et al. Molecular Breeding,1999,5(1) :65-73．
[140] Stromvik M V, Sundaraman V P, et al. A novel promoter from soybean that is active
in a complex developmental pattern with and without its proximal 650 base paris. Plant Mol.Biol., 1999,9:41．
[141] Tian Wenzhong, et al. Rice transformation with a phytoalexin gene and bioassay of the transgenic plants. Acta Botanica Sinica, 1998,40(9) : 803-808．
[142] Tjokrokusumo D, Heinrich T, et al. Vacuum infiltration of petunia hybrida pollen with Agrobacterium tumefaciens to achieve plant transformation. Plant Cell Reports, 2000, 19(8) :792-797．
[143] Van Roekel Jsc, Pamm B, et al. Factors influencing transformation frequency of tomato(Lycopersicon esculentum). Plant Cell Rep., 1993,12:644-647．
[144] Wang Y L, et al. Transient expression of foreign gene in rice, wheat and soybean cells following particle bombardment. Plant Molec. Bio., 1998,11 :433-439．
[145] Wenck A, Czako M, et al. Frequent collinear long transfer of DNA inclusive of the whole binary vector during Agrobacterium-mediated transformation. Plant Mol. Biol., 1997,34(6) :913-922．
[146] WHO.Health aspects of marker gene in genetically modified plants. Report of a WHO work shop. WHO Food Safety Unit.WHO/FUN/FOS/93,6,1993．
[147] Wilknson J E, Twell D, et al. Methanol does not specifically inhibit endogenous β-glucuronidase(Gus)activity. Plant Sci., 1994,97:61-67．
[148] Willams J, pink A C, Bidding N L. Effect of silver nitrate on long term culture and regeneration of callus from Brassica Oleracea Var.gemmifera plant cell[J]. Tissue and Organ Culture, 1990,21:61-66．
[149] Witty M. Nucleic Acids Research, 1989,17:3312．
[150] Wuk K, Butgust W, Sorrells M E, et al. The detection and estimation of linkage in polyploids using single-dose restriction fragments. Theory Appl Genent, 1992,83, 294-300．
[151] Yang M.S. Expression of a synthetic gene for improved protein quality in transformed potato plants. Plant Sci., 1989,64:99-111．
[152] Zupan J R, Zambryski P. Transfer of I-DNA from Agrobacterium to the plant cell. Plant Physiol.,1995, 107:1041-1047．
[153] Zupan J R, Zarnbruski P C. Transfer of T-DNA from Agrobacterium to the plant cell. Plant Physiol., 1995,107:1041-1047．