转基因水稻转育杂交稻亲本及其快速检测技术的研究
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摘要
稻瘟病是一种严重危害水稻的真菌病害。D2-1-2(中花9号ZH9(R))是中国科学院遗传与发育生物学研究所以溶菌酶目的基因和npt-Ⅱ选择标记基因作外源基因，用基因枪法转化、经过连续5年田间选育和抗稻瘟病实验获得的T7代转基因水稻纯合品系，抗谱分析表明，该材料对所有参试的37个稻瘟病生理小种均表现出免疫级抗性。针对部分杂交稻在生产上不抗病的问题，本研究以ZH9(R)为溶菌酶基因供体，利用几个目前生产上有广泛应用价值的杂交水稻组合两优培九、汕优63、培两优288和培两优E32的恢复系9311、明恢63(MH63)、288、E32和两优培九、培两优288的不育系培矮64s(PA64s)、进行回交转育，选育出优良的抗病恢复系和不育系，进而配制新的杂交稻组合，以期获得既抗稻瘟病又农艺性状优良的超级杂交稻，以克服杂交稻稻瘟病抗性差的弱点。
(一)带npt-Ⅱ选择标记基因转基因水稻快速检测技术的研究
本研究部分以转基因水稻纯系材料ZH9(R)及受体品种中花9号(ZH9(CK))为材料。以ZH9(R)中携带的npt-Ⅱ基因作为辅助筛选标记，利用抗生素对其进行处理，建立了一套快速检测转基因水稻的技术体系。
通过使用不同浓度的卡那霉素和G418溶液对ZH9(R)和ZH9(CK)成株离体叶片进行处理，当卡那霉素浓度为700mg/l时，到第六天，ZH9(R)和ZH9(CK)二者的叶片都没有受到明显伤害，当G418浓度为80mg/l时，到第四天，ZH9(CK)叶片黄化死亡，而ZH9(R)叶片仍为正常绿色。所以，我们将G418作为筛选转基因水稻的抗生素；将G418(溶液)80mg/l浓度作为筛选转基因水稻成株叶片的临界浓度。同时，我们进一步用G418对ZH9(R)和ZH9(CK)种子、幼胚和幼苗进行了处理，将：①G418(溶液)300mg/l浓度(处理7天)作为筛选转基因水稻种子的临界浓度。②G418(培养基)200mg/l浓度(处理10天)作为筛选转基因水稻幼胚的临界浓度。③G418(培养基)150mg/l浓度(处理12天)作为筛选转基因水稻幼苗的临界浓度。
根据npt-Ⅱ基因和溶菌酶基因序列设计两对引物，对上述筛选方法进行了PCR验证，发现G418阳性株均扩增出了与npt-Ⅱ基因和溶菌酶基因大小相同的带，这验证了G418检测的正确性，同时也说明了npt-Ⅱ基因和溶菌酶基因是紧密连锁的。应用以上确定的临界浓度对转育后代的叶片、种子、幼胚和幼苗进行了筛选，效果
都非常明显，经xZ测验，转育回交后代呈1:1分离规律，回交自交后代呈3:1
分离规律，符合孟德尔遗传规律，说明外源溶菌酶基因是呈单拷贝、显性、稳定遗
传的。
(二)zH，卫)转育杂交稻亲本的研究
将双9(R)分别与恢复系9311、MH63、288、E32和不育系PA64s杂交获得
Fl代，然后以各亲本9311、MH63、288、E32、PA64s为轮回亲本，进行溶菌酶抗
病基因的转育。通过研究得到了如下结论:
(1) G418筛选与PcR、PCR一Southem检测的结果是一致的。通过对转育后代
进行G418筛选和PCR检测，结果表明以18筛选的阳性株有99%以上扩增出了与
溶菌醉基因大小相同的带。为进一步验证目的基因已整合到水稻基因组DNA中，
用辣根过氧化物酶标记的目的基因作探针，对部分PcR结果进行了southem杂交，
发现PcR阴性株没有杂交信号出现，而阳性株都有明显杂交信号，证实了PCR扩
增带确实是溶菌酶基因的特异性片段。(2)将轮回亲本93n、MH63、288、E3么
.
PA64s;转育后代93n/zHg(R)、MH63八王9(R)的B几Fl和Bq凡代;288左升9冈、
E32龙升9(R)、PA64s/ZHg(R)的BqFI和BCI凡代;杂交组合两优培九、汕优63
和培两优288;测交组合BZRA64s旧293n，珍汕97A/B2MH63，扩RA从s旧1288种植
于病圈中，以培两优邓8为诱发品种。采取培养稻瘟病菌人工接种和自然诱发相结
合，整体发病状况较好。通过发病率和病情指数调查，发现转育后代抗病能力比亲
本大大增强，各测交组合的抗病能力与对应的杂交组合相比也有很大的提高。
(3)通过对亲本及各转育后代的株商、穗长、结实率、千粒重和单株产童筹
进行农艺性状分析，表明随着回交世代的增加，各转育后代的农艺性状越来越接近
于亲本。
(4)为了考查转育后代的杂种优势，本研究对各侧交组合和对照杂交组合进
行了农艺性状分析，各测交组合的大部分农艺性状都比对照组合要差一点，这主要
是由于低世代回交代(BqFI代或BCIF;代)还不是很纯合，与亲本差异太大的缘
故。但部分测交组合有些性状与对照组合还是没有构成显著性差异。
Blast is a fungus disease of rice. Using lysozme and npt-II gene as the external genes, transforming by bombardment, by continuous five-year selection and breeding and blast-resistance experiments, Institute of genetics and developmental Biology, Chinese Academy of Sciences obtained Transgenic rice D 2-1-2 (Zhonghua 9 ZH9 (R) ). The analysis showed that it was immune to 37 physiological races of blast. Using the transgenic ZH9 (R) as donor, restorer lines 9311, MH63, 288, E32 and male sterile line PA64s as receptor , restorer lines and male sterile line with lysozme will be developed. Resistant restorer lines crossing male sterile line will obtain good super-hybrid rice that are resistant to blast.
(一) Research on quick testing-technology of transgenic rice with npt -II screen marker gene
By using npt- II in ZH9 (R) as auxiliary selection-marker, conducting ZH9 (R) and ZH9 (CK) with antibiotic, we built a system of quickly testing transgenic rice offspring.
Detached leaves of ZH9 (R) and ZH9 (CK) were treated by Kanamycin and G418 with different concentration. When Kanamycin was 700 mg/1, on the sixth day, the leaves of ZH9(R)and ZH9(CK)were not damaged. When G418 was 80mg/l, on the fourth day, the leaves of ZH9 (CK) had been yellow and dead, but the leaves of ZH9 (R) remained green. Therefore, G418 was selected as the optimal antibiotic and 80mg/l was treated as the optimal critical concentration to test detached leaves of transgenic rice. Further study indicated: 300mg/l was the best critical concentration to test seeds of transgenic rice; 200mg/L was the best critical concentration to test young embryo of transgenic rice;
150mg/Lwas the best critical concentration to test seedlings of transgenic rice.
Two primers were designed based on npt- II and iysozme gene sequences. PCR was done to confirm the above detection system. Results showed the wpt-II was closely linked with lysozme gene. Above confirmed critical concentrations were adopted to select detached leaves, seeds, young embryos and seedlings of transferred offspring. Hie effect was very significant. By x testing, the separation law of backcrossed offspring was 1:1 and one of self-crossed offspring was 3:1, which was coincident with Mendelian genetic law. So external lysozme gene was single-copied, dominant and stably genetic.
(二) Research on ZH9(R) transferring different hybrid rice parents
ZH9(R ) crossed with excellent restorer lines 9311, MH63, 288, E32 and sterile line PA64s, which produced FI generation. Then 9311 , MH63, 288 , E32, PA64s as rotation parents, disease-resistance gene of Lysozme was transferred. The main conclusions are described as follows:
(1) G418 testing is consistent with PCR, PCR-Southern testing. Transferring offspring were tested with G418 and PCR. Positive plants of G418 had the same amplifications as one of lysozme gene. Furthermore, PCR conclusion was proved correctly through PCR-Southern. G418 testing is consistent with blast-resistance. 99% of G418 positive plants wete resistant through resistance evaluations.
(2) PeiLiangYou 288 as induction variety, rotation parents, transferring generations, hybrid rice and testcrosses were evaluated in rice blast nursery. Combined with inoculation and induction, the effect is satisfactory. The disease-resistance ability of transferring generations was stronger than one of rotation parents. The disease-resistance ability of testcrosses also had been improved greatly compared with corresponding hybrid rice.
(3) Investigation on agronomic characters of transferring progenies and rotation parents were made. With the generations increasing, the agronomic characters of backcross progeny are closer with parents. That is to say, all agronomic characters of
BC1F1 have significant differences with rotation parents; some agronomic characters of have significant differences with parents; none of BC3F1 has significant differences with them.
(4) Domination of transferring generations was examined. The agronomic characters of testcrosses were worse than one of hybrid rice,
(一)带npt-Ⅱ选择标记基因转基因水稻快速检测技术的研究
本研究部分以转基因水稻纯系材料ZH9(R)及受体品种中花9号(ZH9(CK))为材料。以ZH9(R)中携带的npt-Ⅱ基因作为辅助筛选标记，利用抗生素对其进行处理，建立了一套快速检测转基因水稻的技术体系。
通过使用不同浓度的卡那霉素和G418溶液对ZH9(R)和ZH9(CK)成株离体叶片进行处理，当卡那霉素浓度为700mg/l时，到第六天，ZH9(R)和ZH9(CK)二者的叶片都没有受到明显伤害，当G418浓度为80mg/l时，到第四天，ZH9(CK)叶片黄化死亡，而ZH9(R)叶片仍为正常绿色。所以，我们将G418作为筛选转基因水稻的抗生素；将G418(溶液)80mg/l浓度作为筛选转基因水稻成株叶片的临界浓度。同时，我们进一步用G418对ZH9(R)和ZH9(CK)种子、幼胚和幼苗进行了处理，将：①G418(溶液)300mg/l浓度(处理7天)作为筛选转基因水稻种子的临界浓度。②G418(培养基)200mg/l浓度(处理10天)作为筛选转基因水稻幼胚的临界浓度。③G418(培养基)150mg/l浓度(处理12天)作为筛选转基因水稻幼苗的临界浓度。
根据npt-Ⅱ基因和溶菌酶基因序列设计两对引物，对上述筛选方法进行了PCR验证，发现G418阳性株均扩增出了与npt-Ⅱ基因和溶菌酶基因大小相同的带，这验证了G418检测的正确性，同时也说明了npt-Ⅱ基因和溶菌酶基因是紧密连锁的。应用以上确定的临界浓度对转育后代的叶片、种子、幼胚和幼苗进行了筛选，效果
都非常明显，经xZ测验，转育回交后代呈1:1分离规律，回交自交后代呈3:1
分离规律，符合孟德尔遗传规律，说明外源溶菌酶基因是呈单拷贝、显性、稳定遗
传的。
(二)zH，卫)转育杂交稻亲本的研究
将双9(R)分别与恢复系9311、MH63、288、E32和不育系PA64s杂交获得
Fl代，然后以各亲本9311、MH63、288、E32、PA64s为轮回亲本，进行溶菌酶抗
病基因的转育。通过研究得到了如下结论:
(1) G418筛选与PcR、PCR一Southem检测的结果是一致的。通过对转育后代
进行G418筛选和PCR检测，结果表明以18筛选的阳性株有99%以上扩增出了与
溶菌醉基因大小相同的带。为进一步验证目的基因已整合到水稻基因组DNA中，
用辣根过氧化物酶标记的目的基因作探针，对部分PcR结果进行了southem杂交，
发现PcR阴性株没有杂交信号出现，而阳性株都有明显杂交信号，证实了PCR扩
增带确实是溶菌酶基因的特异性片段。(2)将轮回亲本93n、MH63、288、E3么
.
PA64s;转育后代93n/zHg(R)、MH63八王9(R)的B几Fl和Bq凡代;288左升9冈、
E32龙升9(R)、PA64s/ZHg(R)的BqFI和BCI凡代;杂交组合两优培九、汕优63
和培两优288;测交组合BZRA64s旧293n，珍汕97A/B2MH63，扩RA从s旧1288种植
于病圈中，以培两优邓8为诱发品种。采取培养稻瘟病菌人工接种和自然诱发相结
合，整体发病状况较好。通过发病率和病情指数调查，发现转育后代抗病能力比亲
本大大增强，各测交组合的抗病能力与对应的杂交组合相比也有很大的提高。
(3)通过对亲本及各转育后代的株商、穗长、结实率、千粒重和单株产童筹
进行农艺性状分析，表明随着回交世代的增加，各转育后代的农艺性状越来越接近
于亲本。
(4)为了考查转育后代的杂种优势，本研究对各侧交组合和对照杂交组合进
行了农艺性状分析，各测交组合的大部分农艺性状都比对照组合要差一点，这主要
是由于低世代回交代(BqFI代或BCIF;代)还不是很纯合，与亲本差异太大的缘
故。但部分测交组合有些性状与对照组合还是没有构成显著性差异。
Blast is a fungus disease of rice. Using lysozme and npt-II gene as the external genes, transforming by bombardment, by continuous five-year selection and breeding and blast-resistance experiments, Institute of genetics and developmental Biology, Chinese Academy of Sciences obtained Transgenic rice D 2-1-2 (Zhonghua 9 ZH9 (R) ). The analysis showed that it was immune to 37 physiological races of blast. Using the transgenic ZH9 (R) as donor, restorer lines 9311, MH63, 288, E32 and male sterile line PA64s as receptor , restorer lines and male sterile line with lysozme will be developed. Resistant restorer lines crossing male sterile line will obtain good super-hybrid rice that are resistant to blast.
(一) Research on quick testing-technology of transgenic rice with npt -II screen marker gene
By using npt- II in ZH9 (R) as auxiliary selection-marker, conducting ZH9 (R) and ZH9 (CK) with antibiotic, we built a system of quickly testing transgenic rice offspring.
Detached leaves of ZH9 (R) and ZH9 (CK) were treated by Kanamycin and G418 with different concentration. When Kanamycin was 700 mg/1, on the sixth day, the leaves of ZH9(R)and ZH9(CK)were not damaged. When G418 was 80mg/l, on the fourth day, the leaves of ZH9 (CK) had been yellow and dead, but the leaves of ZH9 (R) remained green. Therefore, G418 was selected as the optimal antibiotic and 80mg/l was treated as the optimal critical concentration to test detached leaves of transgenic rice. Further study indicated: 300mg/l was the best critical concentration to test seeds of transgenic rice; 200mg/L was the best critical concentration to test young embryo of transgenic rice;
150mg/Lwas the best critical concentration to test seedlings of transgenic rice.
Two primers were designed based on npt- II and iysozme gene sequences. PCR was done to confirm the above detection system. Results showed the wpt-II was closely linked with lysozme gene. Above confirmed critical concentrations were adopted to select detached leaves, seeds, young embryos and seedlings of transferred offspring. Hie effect was very significant. By x testing, the separation law of backcrossed offspring was 1:1 and one of self-crossed offspring was 3:1, which was coincident with Mendelian genetic law. So external lysozme gene was single-copied, dominant and stably genetic.
(二) Research on ZH9(R) transferring different hybrid rice parents
ZH9(R ) crossed with excellent restorer lines 9311, MH63, 288, E32 and sterile line PA64s, which produced FI generation. Then 9311 , MH63, 288 , E32, PA64s as rotation parents, disease-resistance gene of Lysozme was transferred. The main conclusions are described as follows:
(1) G418 testing is consistent with PCR, PCR-Southern testing. Transferring offspring were tested with G418 and PCR. Positive plants of G418 had the same amplifications as one of lysozme gene. Furthermore, PCR conclusion was proved correctly through PCR-Southern. G418 testing is consistent with blast-resistance. 99% of G418 positive plants wete resistant through resistance evaluations.
(2) PeiLiangYou 288 as induction variety, rotation parents, transferring generations, hybrid rice and testcrosses were evaluated in rice blast nursery. Combined with inoculation and induction, the effect is satisfactory. The disease-resistance ability of transferring generations was stronger than one of rotation parents. The disease-resistance ability of testcrosses also had been improved greatly compared with corresponding hybrid rice.
(3) Investigation on agronomic characters of transferring progenies and rotation parents were made. With the generations increasing, the agronomic characters of backcross progeny are closer with parents. That is to say, all agronomic characters of
BC1F1 have significant differences with rotation parents; some agronomic characters of have significant differences with parents; none of BC3F1 has significant differences with them.
(4) Domination of transferring generations was examined. The agronomic characters of testcrosses were worse than one of hybrid rice,
引文
[1] 陈素生，刘生祥，宋晓华，1995．回交育种在春小麦育种上的应用．宁夏农学院学报，16(2)：79-82．
[2] 陈先彰，1996．水稻航天育种获突破性进展．农业科技要闻，3：4-5．
[3] 陈小荣，钱海丰，薛庆中，2002．转Xa21基因水稻培矮64s回交后代白叶枯病抗性与育性研究．中国水稻科学，16(3)：270-272．
[4] 陈璋，陈启峰等，1993．运用α-吡啶羧酸筛选抗稻瘟病细胞变异体研究．植物学报，35(3)：165-170．
[5] 程振东，卫志明，许智宏，1994．根癌农杆菌对甘蓝型油菜的转化及转基因植株的再生．植物学报，36(9)：657-663．
[6] 崔海瑞，舒庆尧，项友斌等，1998．转crylAb基因水稻的田间表现[A]．见：朱睦元，李亚南．生命科学探索与进展．杭州：杭州大学出版社，810-816．
[7] 方宣均，贾士荣，1999．中国转基因抗虫棉产业化进展．生物技术通报，139(2)：39-40．
[8] 付凤玲．张莉萍，朱祯，2000．玉米优良自交系转基因受体系统建立及转化后的筛选与再生．四川农业大学学报，18(2)：97-99．
[9] 傅荣昭，孙勇如，贾士荣．植物遗传转化技术手册．北京：中国科学出版社，1994，4．
[10] 郭风琴，马慧英，郭庆瑞等，2002．硬粒小麦温光敏雄性不育系DT—94的转育及利用研究简报．麦类作物学报，22(3)：87—88．
[11] 何月秋，Hei Leung，Robert S等，2003．水稻抗稻瘟病鉴定中几个相关问题的探讨．云南农业大学学报，18(3)：234-238．
[12] 黄大年，1993．转基因技术在水稻上的应用．科学出版社，12-26．
[13] 黄大年，李敬阳，章善庆等，1998．用抗除草剂基因快速检测和提高杂交稻纯度的新技术．43(1)：67-70．
[14] 贾士荣，1997．转基因植物食品中标记基因的安全性评价．中国农业科学．30(2)：1-15．
[15] 金万梅，巩振辉，宋正旭等，2003．通过植株原位真空渗入遗传转化获得转基因芥菜．西北农林科技大学学报(自然科学版)，31(5)：39-42．
[16] 李金国，李源祥，华育坚，2001．利用搭载卫星水稻干种子选育出“赣早籼47号”的研究．航天医学与医学工程，14(4)：286-290．
[17] 李晚忱，荣廷昭，雷本鸣等．3个玉米细胞质雄性不育系的选育及分组鉴定作物学报，2001，27(2)：308-312
[18] 李燕娥，焦改丽，李淑君等，1998．转基因棉花纯合系的获得和遗传分析研究．中国农业科学，31(5)：44-47．
[19] 林久生，王根轩，雒梅，2001．谷胱甘肽还原酶基因的表达载体构建及对马铃薯的转化．中国农业科学，34(2)：223-226．
[20] 林丽明，张春媚，谢荔岩，2003．农杆菌介导的水稻草矮病毒NS6基因的转化．福建农林大学学报(自然科学版)，32(3)：288-291．．
[21] 刘梅，覃宏涛，孙宗修等，2003．转基因水稻中ThEn-42基因的稳定遗传及其抗病性的提高．
农业生物技术学报，11(6)：444-449．
[22] 刘巧泉，陈秀花，王兴稳等，2001．一种快速检测转基因水稻中潮霉素抗性的简易方法．农业生物技术学报，9(3)，264，268．
[23] 刘伟华，李文雄，胡尚连等，2002．小麦组织培养和基因枪轰击影响因素探讨．西北植物学报，22(3)：602-610．
[24] 刘宗华，汤继华，李宝健等，2003．玉米转Bt基因自交系的抗玉米螟特性鉴定初报．作物学报，29(4)：621-625．
[25] 毛慧珠，唐惕，曹湘玲等，1996．抗虫基因甘蓝及后代的研究．中国科学(C辑)，26(4)：339-347．
[26] 毛建辉，卢代华，何明等，1999．持续低温对水稻稻瘟病抗性的影响．植物保护学报，26(2)：1-6．
[27] 闵绍楷，申宗坦，熊振民等，1996．水稻育种学．北京：中国农业出版社，269～275．
[28] 全国稻病防泊科研协作组，1993．全国稻瘟病防治协作组20年工作成就回顾．浙江农业学报，(5)：1-4．
[29] 全国稻瘟病菌生理小种联合试验组，1980．我国稻瘟病菌生理小种研究．植物病理学报，10(2)：71-82．
[30] 孙国昌，孙漱沅，1996．中国部分水稻主栽品种稻瘟病的抗性分析和利用评价．中国农业科学，29(6)：55-59．
[31] 孙敬，唐灿明，袁小玲等，2000．利用卡那霉素间接鉴定法进行大規模的棉花转基因育种技术．棉花学报，12(5)：270-276．
[32] 孙漱沅等，1996．我国稻瘟病研究现状和展望．植保技术与推广，16(3)：39～40．
[33] 孙学永，林国平，殷凤生等，2003．烟草体细胞杂交优质抗病株系的选育．烟草科技／栽培与调制．7：36-40．
[34] 唐柞舜，王象坤，李良才等，2000．基因枪法转基因水稻中 hpt 基因稳定遗传．遗传学报，27(1)：26-33．
[35] 陶家凤，1995．稻瘟病菌致病性变异研究现状(评述)．四川农业大学学报，13(4)：518-521．
[36] 田文忠，丁力，曹守云等，1998．植物抗毒素转化水稻和转基因檀株的生物鉴定．植物学报，40(9)：803-808
[37] 王关林，方宏筠，2003．植物基因工程(第二版)．北京：科学出版杜，134～178．
[38] 奚亚军，范学科，侯文胜等，2003．小麦遗传转化中潮霉素适宜筛选浓度的研究．西北农林科技大学学报(自然科学版)，31(1)：39-42．
[39] 许明辉，李成云，李进斌．等．转溶菌酶基因水稻稻瘟病抗圃分析．中国农业科学，2003，36(4)：387-392．
[40] 许明辉，唐祚舜，谭亚玲等，2003．几丁质酶-葡聚糖酶双价基因导入滇型杂交稻恢复系提高稻瘟病抗性的研究．遗传学报，30(4)：330-334．
[41] 许明辉，唐祚舜，赵丰萍等，2003．抗生素G418胁迫条件下转基因水稻种子发芽特性及应用．遗传，25(1)：45-48．
[42] 薛石玉，项寿南，谢加华等，1999．应用聚合回交法选育抗稻瘟病恢复系新组合．浙江农业大学学报，25(1)：5-9．
[43] 薛玉石，刘建慧，吴跃，1999．水稻转bar基因恢复系选育初报．杂交水稻，14(3)：11-12．
[44] 杨长登，唐克轩，吴连斌．1998．农杆菌介导将雪莲凝集素(GNA)基因转入籼稻单倍体微芽的初步研究．中国水稻科学，12(3)：129-133．
[45] 杨广东，朱祯，李燕娥等，2002．几种抗生素对大白菜种子发芽及离体子叶再生的影响．华北农学报，17(1)：55-59．
[46] 袁隆平，辛业芸，2001．希望之光—超级杂交稻．杂交水稻，(10)：46．
[47] 岳建雄，张慧军，张炼辉．2002．以对潮霉素抗性为筛选标记的棉花遗传转化．棉花学报，14(4)：195-199．
[48] 张凯，李富强，2002．转Bt基因玉米回交二代抗虫性鉴定．信阳农业高等专科学校学报．12(4)：7-9．
[49] 张连平，张丽娟，1999．提高水稻花培综合效益的研究．见：白新盛等主编．生物技术在水稻育种中的应用研究．北京：中国农业科技出版社，51-54．
[50] 张铭铣，骆荣挺，施德等，1997．抗稻瘟病新种质R917的抗瘟基因遗传分析及其转育研究．核农学报，11(4)：193-198．
[51] 张天真，郭三堆等，2001．转基因抗虫杂交棉南抗3号的选育及应用．中国棉花，28(10)：25-26．
[52] 章冰，卫志明，1999．根癌农杆菌介导的水稻转化及转基因R_1代植株表型特征．植物生理学报，25(4)：313-320．
[53] 郑祖玲，褚启人，张承姝等，1984．利用水道花药培养筛选稻瘟病抗性突变体．植物学通报，2(4)：41-43．
[54] 周光召，1999．面向21世纪的科技进步与社会经济发展．中国科技出版社，522．
[55] 朱启升，王安东，杨前进等，2002．水稻除草剂敏感基因导入恢复系的研究．杂交水稻，15(3)：5-6．
[56] Altman D W, 1991. Quantitative trait variation in phenotypically normal regenerants of cotton. In Vitro Cell Dev Biol, 27: 132-38.
[57] Ayres N M, 1994. Genetic transformation of rice. Critical Rev, Plant Sci, 13: 219-239.
[58] Baker B, Zambryski P, Staskewicz B, et al, 1997. Science, 276: 726-733.
[59] Benedict J H, Sachs E S, Altman D W, et al, 1996. Field performance of cottons expressing transgenic CrylA insecticidal proteins for resistance to Heliothis virescens and Helicoverpa zea. J Econ Entomal, 89: 230-238.
[60] Bevan M W, Flavell R B, Chilton N D, 1990. A chimaetic antibiotic resistance gene as a selectable marker for plant cell transformation. Nature, 304:184-187.
[61] Bolar J P, Norelli J L, Harman G E, et al, 2001. Synergistic activity of endochitinase and exochitinase from Trichoderma atroviride (T. harzianum) against the pathogenic fungus (Venturia inaequalis)in transgenic apple plants. Transg Res., 1067:1-11.
[62] Bolar J P, Norelli J L, Wong K W, et al, 2000. Expression of endochitinase from Trichoderma harzianumin in transgenic apple increases resistance to apple scab and reduces vigor. Phytopathology, 90: 72-77.
[63] Bryant J., Leather S., 1992. Removal of selectable marker genes from transgenic plants: needless sophistication or social necessity? Trends in Biotechnology, 10: 274-275.
[64] Cao J, Duan X, McElory D, et al, 1992. Regeneration of herbicide resistant transgenic rice plants following microprojectile-mediated transformation of suspension culture cells. Plant Cell Rep. 11: 586-591.
[65] Cho H. J, Farrand S. K, Noel G. R, et al, 2000. High-effeciency induction of soybean hairy roots and propagation of the soybean cyst nematode. Planta, 210:195-204.
[66] Dale P J, Irwin J A, Schefler J A, 1993. The experimental and commercial release of transgenic crop planta. Plant breeding, 111: 1-22.
[67] Datta S K, 1992. Herbicide-resistant Indica rice plants from IRRI line IR72 after PEG-mediated transformation of protoplasts. Plant Mol. Biol, 20: 619-627.
[68] Davey M R, Kothari S L, Zhang H, et al, 1991. Transgenic rice: characterization of protoplast-derived plants and their seed progeny. J. Of Experimental Botany, 42(242): 1159-1169.
[69] De greef W, R. Delon, M. De Block, et al, 1989. Evaluation of herbicide resistance in transgenic crops under field conditions. Biotechnology, 7: 61-64.
[70] Dekeyaer R, Claes B, Marichal M, et al, 1989. Evalution of selectable markers for rice transformation.Plant Physiol, 90: 217-233.
[71] Dung J, 1996. Agrobacterium-mediated transformation of Javnica rice. Molecular Breeding, 2: 267-276.
[72] D. ring K, Porsch P, Fladung M, et al, 1993. Transgenic potato plants resistant to the phytopathogenic bacterium Erwinia carotovora. Plant Journal, 4(3): 587-598.
[73] Esposito S, Coluoci M G, Lorito M, et al, 2000. Antifungal transgnes expression in Petunia hybrida. Proc. 19th Int'1 Symposium Improvement Ornamental Plants, Acta Hort. 508, ISHS, 7860-7865.
[74] Feng D R, Xu X P, Wei J W, et al, 1999. Enhancement of rice disease resistance by two antifungal protein genes. Acta Botanica Sinica, 41(11): 1187-1191.
[75] Flor H H, 1971. Ann Rev Phyfopathol, 9:275-296.
[76] Fromm M. E., Morrish E, Armstrong C., et al, 1990. Inheritance and expression of chimerical genes on the progeny of transgenic maize plants. Biotechnology, 8: 833-839.
[77] Fu X D, Duraialagaraja Sudhakar, Jinrong Peng, et al, 2001. Expression of Arabidopsis GAI in Transgenic Rice Represses Multiple Gibberellin Responses. Plant Cell, 13: 1791-1802.
[78] Hamid R. Shuuji Y, Kinya T, et al, 1996. Transgenic plant production mediated by Arobacterium in Indica rice. Plant Cell Reports, 15: 727-730.
[79] Hayashimoto A, Jian L, Mural N, et al, 1990. A polyethylene glycolmediated protoplast transformation system for the production of fertile transgenic rice plants. Plant Physiol, 93: 857-863.
[80] Hayashimoto A, 1990. A polyethylene glycol-mediated protoplast transformation system for production of fertile transgenic rice plant. Plantphysiol, 83: 857-863.
[81] Hayes C K, Klemsdal S, Lorito M, et al, 1994. Isolatiom and sepuence of an endochitinase-encoding gene from a cDNA library of Trichodermaharzianum. Gene, 138: 143-148.
[82] Hiei Y, Shozo O. Toshihiko K, et al, 1994. Effcient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. The Plant Journal, 6(2): 271-282.
[83] Igasaki T, Mohri T, Ichikawa T, et al, 2000. Agrobacterium tumefaciens mediated transformation of Robinia pseudoacacia. Plant Cell Rep., 19(5): 448-453.
[84] Kanzaki H, Nirasawa S, Saitoh H, 2002. Overexpression of the wasabi defensin gene confers enhanced resistance to blast fungus (Magnaporthe grisea) in tranagenic rice. Theoretical and Applied Genetics, 105: 809-814.
[85] Koenraad EM, Tierens J., Bart P. H. J., et al, 2001. Study of the Role of Antimicrobial Glucosinolate-Derived Isothiocyanstes in Resistance of Arabidopsis to Microbial Pathogens. Plant Physiology, 125: 1688-1699.
[86] Latterell F M, Tullis E C, Otten R J L, et al, 1954. Physiologic races of Piricularia oryzae. Phytopathology, 44: 495-496.
[87] Leuhrsen K R, 1990. Insertion of Mul elements in the first intron of the Adh 1-S gene of maize results in novel RNA processing evems. Plant Cell, 2:1225-1238.
[88] Lorito M, Woo S L, Fernandez I G, et al, 1998. Genes from mycoparasitic fungi as a source for improving plant resistance to fungal pathogens. Proc Natl Acad Sci USA, 95: 7860-7865.
[89] Lynch P T, Jones J, Blackball N W, et al, 1995. The phenotypic characterization of R_2 generation transgenic rice plants under field and glasshouse conditions. Euphytica, 85:395-401.
[90] Ming X T, Wang L J, An C C, et al, 2000. Resistance to rice blast (Pyricularia oryzae) caused by the expression of trichosanthin gene in transgenic rice plants transferred through Agrobcterium method. Chinese Science Bulletin, 19: 1774-1778.
[91] Nakajima H, Muranaka T, Ishige F, et al, 1997. Fungel and bacterial disease resistance in transgenic plants expressing human lysozyme. Plant Cell Reports, 16: 674-679.
[92] Nishizawa Y, Nishio Z, Nakazono K, et al, 1999. Enhanced resistance to blast (Magnaporthe grisea) in transgenic Japonica rice by constitutive expression of rice chitiuase. Theor Appl Genet. 99: 383-390.
[93] Oard J H, Cock K A, Gomez J H, et al, 1996. Development, field evaluation, and agoronomic performance of transgenic herbicide resistance rice. Molecular Breeding, 2:359-368.
[94] Okada Y, Nishiguchi M, 2002. Plant breeding, 121(3): 249-253.
[95] Ou S H, 1985. Rice disease. Farnham Royal, UK; Commonwealth Agricultural Bureaux.
[96] Owen J E, Schultz D W, Taylor A, et al, 1983. Nucleotide sequence of the lysozyme gene of bacteriophage T4. Analysis of mutation involving repeated sequence. J Mol Biol, 165: 229-248.
[97] Peng J, Kononowicz H, Hodges T K, 1992. Transgenic indica rice plants. Theor Appl Genet, 83: 855-863.
[98] Rashid H, Yokoi S, Toriyama K et al, 1996. Transgenic plant production mediated by Agrobacterium in Indica rice. Plant Cell Rep., 15: 727-730.
[99] Sachs E S, 1998. Expression and segregation of genes encoding crylA insecticidal proteins in cotton. Crop Science, 38(1):1-11.
[100] Schaffrath U, Mauch F, Freydl E, et al, 2000. Constitutive expression of the defense-related Rirlb gene in transgenic rice plants confers enhanced resistance to the rice blast fungus Magnaporthe grisea. Plant Mol.Bi-ol, 43:59-66.
[101] Silue D, Notteghem J L, Tharreau D, 1992. Evidence of a gene-for-gene relationship in the oryza sativa-Magnaporthe grisea pathosystem. Phytopathology, 82(5): 577-580.
[102] Stark-lorenzen P, Nelke B, H(?)nBler G, et al, 1997. Transfer of a grapevine stilbene synthase gene to rice (Oryza sativa L.). Plant Cell Rep, 16: 668-673.
[103] Stelly D M, Altman D W, Kohel R J, et al, 1989. Cytogenetic abnormalities of cotton somaclones from callus cultures. Genome, 32: 762-770.
[104] Tang H R, Wallbraun M, Ren Z L, et al, 2001. Genetic transformatiom of the Trichoderma endochitinase gene ThEn-42 to somatic embryos of English walnut. Acta Horticul Sin 28(1): 12-18.
[105] Toriyama K, Arimoto Y, Uchimiya H, et al, 1988. Transgenic rice plants after direct gene tranafer into protoplasts. Bio/Technology, 6: 1072-1074.
[106] Trudel J, Potvin C, Asselin A, 1995. Secreted hen lysozyme in transgenic tobacco: recovery of bound enzyme and in vitro growth inhibition of plant pathogens. Plant Science, 106: 55-62.
[107] Valent B, Chemley F G, 1991. Molecular genetic analysis of the rice blast fungus, Magnaporthe grisea.Annu Rev Phytopathol, 29: 443-467.
[108] Van Lijsebetens, Vanderhaeghen V, Montagu Van M, 1991. Insertional mutagenesis in Arbidopsis thaliana: isolation of a T-DNA linked mutation that alters leaf morphology. Theor Appl Genet, 81:277-284.
[109] Wang Z. H, 2000. Genetic analysis of transgenes in the progenies of Bt rice cross to conventional rice varieties. Hereditas, 22(5): 309-312.
[110] Wang Z X, Yano M, Yamanouchi U et al, 1999. The Pib gene for rice blast resistance belonge to the nucleotide binding and leucine-rice, repeat class of plant disease resistance genes. The Plant Journal, 19(1): 55-64.
[111] Zhang H M, Yang H, Rech E L, et al, 1988. Transgenic rice plant produced by electroporation-mediated plasmid uptake into protoplasts. Plarit Cell Rep, 7: 379-384.
[112] Zhang S, Warkentin D, Sun B, et al, 1996. Variation in the inheritance of expression among subclones for unselected (uid A) and selected (bar) transgenes in maize (Zea mays L,). Theor Appl Genet, 92: 752-761.
[113] Dale P. J., Irwin J. A., Schefler J. A., et al, 1993. The experimental and commercial release of transgenic crop plant. Plant breeding, 111: 1-22.
[2] 陈先彰，1996．水稻航天育种获突破性进展．农业科技要闻，3：4-5．
[3] 陈小荣，钱海丰，薛庆中，2002．转Xa21基因水稻培矮64s回交后代白叶枯病抗性与育性研究．中国水稻科学，16(3)：270-272．
[4] 陈璋，陈启峰等，1993．运用α-吡啶羧酸筛选抗稻瘟病细胞变异体研究．植物学报，35(3)：165-170．
[5] 程振东，卫志明，许智宏，1994．根癌农杆菌对甘蓝型油菜的转化及转基因植株的再生．植物学报，36(9)：657-663．
[6] 崔海瑞，舒庆尧，项友斌等，1998．转crylAb基因水稻的田间表现[A]．见：朱睦元，李亚南．生命科学探索与进展．杭州：杭州大学出版社，810-816．
[7] 方宣均，贾士荣，1999．中国转基因抗虫棉产业化进展．生物技术通报，139(2)：39-40．
[8] 付凤玲．张莉萍，朱祯，2000．玉米优良自交系转基因受体系统建立及转化后的筛选与再生．四川农业大学学报，18(2)：97-99．
[9] 傅荣昭，孙勇如，贾士荣．植物遗传转化技术手册．北京：中国科学出版社，1994，4．
[10] 郭风琴，马慧英，郭庆瑞等，2002．硬粒小麦温光敏雄性不育系DT—94的转育及利用研究简报．麦类作物学报，22(3)：87—88．
[11] 何月秋，Hei Leung，Robert S等，2003．水稻抗稻瘟病鉴定中几个相关问题的探讨．云南农业大学学报，18(3)：234-238．
[12] 黄大年，1993．转基因技术在水稻上的应用．科学出版社，12-26．
[13] 黄大年，李敬阳，章善庆等，1998．用抗除草剂基因快速检测和提高杂交稻纯度的新技术．43(1)：67-70．
[14] 贾士荣，1997．转基因植物食品中标记基因的安全性评价．中国农业科学．30(2)：1-15．
[15] 金万梅，巩振辉，宋正旭等，2003．通过植株原位真空渗入遗传转化获得转基因芥菜．西北农林科技大学学报(自然科学版)，31(5)：39-42．
[16] 李金国，李源祥，华育坚，2001．利用搭载卫星水稻干种子选育出“赣早籼47号”的研究．航天医学与医学工程，14(4)：286-290．
[17] 李晚忱，荣廷昭，雷本鸣等．3个玉米细胞质雄性不育系的选育及分组鉴定作物学报，2001，27(2)：308-312
[18] 李燕娥，焦改丽，李淑君等，1998．转基因棉花纯合系的获得和遗传分析研究．中国农业科学，31(5)：44-47．
[19] 林久生，王根轩，雒梅，2001．谷胱甘肽还原酶基因的表达载体构建及对马铃薯的转化．中国农业科学，34(2)：223-226．
[20] 林丽明，张春媚，谢荔岩，2003．农杆菌介导的水稻草矮病毒NS6基因的转化．福建农林大学学报(自然科学版)，32(3)：288-291．．
[21] 刘梅，覃宏涛，孙宗修等，2003．转基因水稻中ThEn-42基因的稳定遗传及其抗病性的提高．
农业生物技术学报，11(6)：444-449．
[22] 刘巧泉，陈秀花，王兴稳等，2001．一种快速检测转基因水稻中潮霉素抗性的简易方法．农业生物技术学报，9(3)，264，268．
[23] 刘伟华，李文雄，胡尚连等，2002．小麦组织培养和基因枪轰击影响因素探讨．西北植物学报，22(3)：602-610．
[24] 刘宗华，汤继华，李宝健等，2003．玉米转Bt基因自交系的抗玉米螟特性鉴定初报．作物学报，29(4)：621-625．
[25] 毛慧珠，唐惕，曹湘玲等，1996．抗虫基因甘蓝及后代的研究．中国科学(C辑)，26(4)：339-347．
[26] 毛建辉，卢代华，何明等，1999．持续低温对水稻稻瘟病抗性的影响．植物保护学报，26(2)：1-6．
[27] 闵绍楷，申宗坦，熊振民等，1996．水稻育种学．北京：中国农业出版社，269～275．
[28] 全国稻病防泊科研协作组，1993．全国稻瘟病防治协作组20年工作成就回顾．浙江农业学报，(5)：1-4．
[29] 全国稻瘟病菌生理小种联合试验组，1980．我国稻瘟病菌生理小种研究．植物病理学报，10(2)：71-82．
[30] 孙国昌，孙漱沅，1996．中国部分水稻主栽品种稻瘟病的抗性分析和利用评价．中国农业科学，29(6)：55-59．
[31] 孙敬，唐灿明，袁小玲等，2000．利用卡那霉素间接鉴定法进行大規模的棉花转基因育种技术．棉花学报，12(5)：270-276．
[32] 孙漱沅等，1996．我国稻瘟病研究现状和展望．植保技术与推广，16(3)：39～40．
[33] 孙学永，林国平，殷凤生等，2003．烟草体细胞杂交优质抗病株系的选育．烟草科技／栽培与调制．7：36-40．
[34] 唐柞舜，王象坤，李良才等，2000．基因枪法转基因水稻中 hpt 基因稳定遗传．遗传学报，27(1)：26-33．
[35] 陶家凤，1995．稻瘟病菌致病性变异研究现状(评述)．四川农业大学学报，13(4)：518-521．
[36] 田文忠，丁力，曹守云等，1998．植物抗毒素转化水稻和转基因檀株的生物鉴定．植物学报，40(9)：803-808
[37] 王关林，方宏筠，2003．植物基因工程(第二版)．北京：科学出版杜，134～178．
[38] 奚亚军，范学科，侯文胜等，2003．小麦遗传转化中潮霉素适宜筛选浓度的研究．西北农林科技大学学报(自然科学版)，31(1)：39-42．
[39] 许明辉，李成云，李进斌．等．转溶菌酶基因水稻稻瘟病抗圃分析．中国农业科学，2003，36(4)：387-392．
[40] 许明辉，唐祚舜，谭亚玲等，2003．几丁质酶-葡聚糖酶双价基因导入滇型杂交稻恢复系提高稻瘟病抗性的研究．遗传学报，30(4)：330-334．
[41] 许明辉，唐祚舜，赵丰萍等，2003．抗生素G418胁迫条件下转基因水稻种子发芽特性及应用．遗传，25(1)：45-48．
[42] 薛石玉，项寿南，谢加华等，1999．应用聚合回交法选育抗稻瘟病恢复系新组合．浙江农业大学学报，25(1)：5-9．
[43] 薛玉石，刘建慧，吴跃，1999．水稻转bar基因恢复系选育初报．杂交水稻，14(3)：11-12．
[44] 杨长登，唐克轩，吴连斌．1998．农杆菌介导将雪莲凝集素(GNA)基因转入籼稻单倍体微芽的初步研究．中国水稻科学，12(3)：129-133．
[45] 杨广东，朱祯，李燕娥等，2002．几种抗生素对大白菜种子发芽及离体子叶再生的影响．华北农学报，17(1)：55-59．
[46] 袁隆平，辛业芸，2001．希望之光—超级杂交稻．杂交水稻，(10)：46．
[47] 岳建雄，张慧军，张炼辉．2002．以对潮霉素抗性为筛选标记的棉花遗传转化．棉花学报，14(4)：195-199．
[48] 张凯，李富强，2002．转Bt基因玉米回交二代抗虫性鉴定．信阳农业高等专科学校学报．12(4)：7-9．
[49] 张连平，张丽娟，1999．提高水稻花培综合效益的研究．见：白新盛等主编．生物技术在水稻育种中的应用研究．北京：中国农业科技出版社，51-54．
[50] 张铭铣，骆荣挺，施德等，1997．抗稻瘟病新种质R917的抗瘟基因遗传分析及其转育研究．核农学报，11(4)：193-198．
[51] 张天真，郭三堆等，2001．转基因抗虫杂交棉南抗3号的选育及应用．中国棉花，28(10)：25-26．
[52] 章冰，卫志明，1999．根癌农杆菌介导的水稻转化及转基因R_1代植株表型特征．植物生理学报，25(4)：313-320．
[53] 郑祖玲，褚启人，张承姝等，1984．利用水道花药培养筛选稻瘟病抗性突变体．植物学通报，2(4)：41-43．
[54] 周光召，1999．面向21世纪的科技进步与社会经济发展．中国科技出版社，522．
[55] 朱启升，王安东，杨前进等，2002．水稻除草剂敏感基因导入恢复系的研究．杂交水稻，15(3)：5-6．
[56] Altman D W, 1991. Quantitative trait variation in phenotypically normal regenerants of cotton. In Vitro Cell Dev Biol, 27: 132-38.
[57] Ayres N M, 1994. Genetic transformation of rice. Critical Rev, Plant Sci, 13: 219-239.
[58] Baker B, Zambryski P, Staskewicz B, et al, 1997. Science, 276: 726-733.
[59] Benedict J H, Sachs E S, Altman D W, et al, 1996. Field performance of cottons expressing transgenic CrylA insecticidal proteins for resistance to Heliothis virescens and Helicoverpa zea. J Econ Entomal, 89: 230-238.
[60] Bevan M W, Flavell R B, Chilton N D, 1990. A chimaetic antibiotic resistance gene as a selectable marker for plant cell transformation. Nature, 304:184-187.
[61] Bolar J P, Norelli J L, Harman G E, et al, 2001. Synergistic activity of endochitinase and exochitinase from Trichoderma atroviride (T. harzianum) against the pathogenic fungus (Venturia inaequalis)in transgenic apple plants. Transg Res., 1067:1-11.
[62] Bolar J P, Norelli J L, Wong K W, et al, 2000. Expression of endochitinase from Trichoderma harzianumin in transgenic apple increases resistance to apple scab and reduces vigor. Phytopathology, 90: 72-77.
[63] Bryant J., Leather S., 1992. Removal of selectable marker genes from transgenic plants: needless sophistication or social necessity? Trends in Biotechnology, 10: 274-275.
[64] Cao J, Duan X, McElory D, et al, 1992. Regeneration of herbicide resistant transgenic rice plants following microprojectile-mediated transformation of suspension culture cells. Plant Cell Rep. 11: 586-591.
[65] Cho H. J, Farrand S. K, Noel G. R, et al, 2000. High-effeciency induction of soybean hairy roots and propagation of the soybean cyst nematode. Planta, 210:195-204.
[66] Dale P J, Irwin J A, Schefler J A, 1993. The experimental and commercial release of transgenic crop planta. Plant breeding, 111: 1-22.
[67] Datta S K, 1992. Herbicide-resistant Indica rice plants from IRRI line IR72 after PEG-mediated transformation of protoplasts. Plant Mol. Biol, 20: 619-627.
[68] Davey M R, Kothari S L, Zhang H, et al, 1991. Transgenic rice: characterization of protoplast-derived plants and their seed progeny. J. Of Experimental Botany, 42(242): 1159-1169.
[69] De greef W, R. Delon, M. De Block, et al, 1989. Evaluation of herbicide resistance in transgenic crops under field conditions. Biotechnology, 7: 61-64.
[70] Dekeyaer R, Claes B, Marichal M, et al, 1989. Evalution of selectable markers for rice transformation.Plant Physiol, 90: 217-233.
[71] Dung J, 1996. Agrobacterium-mediated transformation of Javnica rice. Molecular Breeding, 2: 267-276.
[72] D. ring K, Porsch P, Fladung M, et al, 1993. Transgenic potato plants resistant to the phytopathogenic bacterium Erwinia carotovora. Plant Journal, 4(3): 587-598.
[73] Esposito S, Coluoci M G, Lorito M, et al, 2000. Antifungal transgnes expression in Petunia hybrida. Proc. 19th Int'1 Symposium Improvement Ornamental Plants, Acta Hort. 508, ISHS, 7860-7865.
[74] Feng D R, Xu X P, Wei J W, et al, 1999. Enhancement of rice disease resistance by two antifungal protein genes. Acta Botanica Sinica, 41(11): 1187-1191.
[75] Flor H H, 1971. Ann Rev Phyfopathol, 9:275-296.
[76] Fromm M. E., Morrish E, Armstrong C., et al, 1990. Inheritance and expression of chimerical genes on the progeny of transgenic maize plants. Biotechnology, 8: 833-839.
[77] Fu X D, Duraialagaraja Sudhakar, Jinrong Peng, et al, 2001. Expression of Arabidopsis GAI in Transgenic Rice Represses Multiple Gibberellin Responses. Plant Cell, 13: 1791-1802.
[78] Hamid R. Shuuji Y, Kinya T, et al, 1996. Transgenic plant production mediated by Arobacterium in Indica rice. Plant Cell Reports, 15: 727-730.
[79] Hayashimoto A, Jian L, Mural N, et al, 1990. A polyethylene glycolmediated protoplast transformation system for the production of fertile transgenic rice plants. Plant Physiol, 93: 857-863.
[80] Hayashimoto A, 1990. A polyethylene glycol-mediated protoplast transformation system for production of fertile transgenic rice plant. Plantphysiol, 83: 857-863.
[81] Hayes C K, Klemsdal S, Lorito M, et al, 1994. Isolatiom and sepuence of an endochitinase-encoding gene from a cDNA library of Trichodermaharzianum. Gene, 138: 143-148.
[82] Hiei Y, Shozo O. Toshihiko K, et al, 1994. Effcient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. The Plant Journal, 6(2): 271-282.
[83] Igasaki T, Mohri T, Ichikawa T, et al, 2000. Agrobacterium tumefaciens mediated transformation of Robinia pseudoacacia. Plant Cell Rep., 19(5): 448-453.
[84] Kanzaki H, Nirasawa S, Saitoh H, 2002. Overexpression of the wasabi defensin gene confers enhanced resistance to blast fungus (Magnaporthe grisea) in tranagenic rice. Theoretical and Applied Genetics, 105: 809-814.
[85] Koenraad EM, Tierens J., Bart P. H. J., et al, 2001. Study of the Role of Antimicrobial Glucosinolate-Derived Isothiocyanstes in Resistance of Arabidopsis to Microbial Pathogens. Plant Physiology, 125: 1688-1699.
[86] Latterell F M, Tullis E C, Otten R J L, et al, 1954. Physiologic races of Piricularia oryzae. Phytopathology, 44: 495-496.
[87] Leuhrsen K R, 1990. Insertion of Mul elements in the first intron of the Adh 1-S gene of maize results in novel RNA processing evems. Plant Cell, 2:1225-1238.
[88] Lorito M, Woo S L, Fernandez I G, et al, 1998. Genes from mycoparasitic fungi as a source for improving plant resistance to fungal pathogens. Proc Natl Acad Sci USA, 95: 7860-7865.
[89] Lynch P T, Jones J, Blackball N W, et al, 1995. The phenotypic characterization of R_2 generation transgenic rice plants under field and glasshouse conditions. Euphytica, 85:395-401.
[90] Ming X T, Wang L J, An C C, et al, 2000. Resistance to rice blast (Pyricularia oryzae) caused by the expression of trichosanthin gene in transgenic rice plants transferred through Agrobcterium method. Chinese Science Bulletin, 19: 1774-1778.
[91] Nakajima H, Muranaka T, Ishige F, et al, 1997. Fungel and bacterial disease resistance in transgenic plants expressing human lysozyme. Plant Cell Reports, 16: 674-679.
[92] Nishizawa Y, Nishio Z, Nakazono K, et al, 1999. Enhanced resistance to blast (Magnaporthe grisea) in transgenic Japonica rice by constitutive expression of rice chitiuase. Theor Appl Genet. 99: 383-390.
[93] Oard J H, Cock K A, Gomez J H, et al, 1996. Development, field evaluation, and agoronomic performance of transgenic herbicide resistance rice. Molecular Breeding, 2:359-368.
[94] Okada Y, Nishiguchi M, 2002. Plant breeding, 121(3): 249-253.
[95] Ou S H, 1985. Rice disease. Farnham Royal, UK; Commonwealth Agricultural Bureaux.
[96] Owen J E, Schultz D W, Taylor A, et al, 1983. Nucleotide sequence of the lysozyme gene of bacteriophage T4. Analysis of mutation involving repeated sequence. J Mol Biol, 165: 229-248.
[97] Peng J, Kononowicz H, Hodges T K, 1992. Transgenic indica rice plants. Theor Appl Genet, 83: 855-863.
[98] Rashid H, Yokoi S, Toriyama K et al, 1996. Transgenic plant production mediated by Agrobacterium in Indica rice. Plant Cell Rep., 15: 727-730.
[99] Sachs E S, 1998. Expression and segregation of genes encoding crylA insecticidal proteins in cotton. Crop Science, 38(1):1-11.
[100] Schaffrath U, Mauch F, Freydl E, et al, 2000. Constitutive expression of the defense-related Rirlb gene in transgenic rice plants confers enhanced resistance to the rice blast fungus Magnaporthe grisea. Plant Mol.Bi-ol, 43:59-66.
[101] Silue D, Notteghem J L, Tharreau D, 1992. Evidence of a gene-for-gene relationship in the oryza sativa-Magnaporthe grisea pathosystem. Phytopathology, 82(5): 577-580.
[102] Stark-lorenzen P, Nelke B, H(?)nBler G, et al, 1997. Transfer of a grapevine stilbene synthase gene to rice (Oryza sativa L.). Plant Cell Rep, 16: 668-673.
[103] Stelly D M, Altman D W, Kohel R J, et al, 1989. Cytogenetic abnormalities of cotton somaclones from callus cultures. Genome, 32: 762-770.
[104] Tang H R, Wallbraun M, Ren Z L, et al, 2001. Genetic transformatiom of the Trichoderma endochitinase gene ThEn-42 to somatic embryos of English walnut. Acta Horticul Sin 28(1): 12-18.
[105] Toriyama K, Arimoto Y, Uchimiya H, et al, 1988. Transgenic rice plants after direct gene tranafer into protoplasts. Bio/Technology, 6: 1072-1074.
[106] Trudel J, Potvin C, Asselin A, 1995. Secreted hen lysozyme in transgenic tobacco: recovery of bound enzyme and in vitro growth inhibition of plant pathogens. Plant Science, 106: 55-62.
[107] Valent B, Chemley F G, 1991. Molecular genetic analysis of the rice blast fungus, Magnaporthe grisea.Annu Rev Phytopathol, 29: 443-467.
[108] Van Lijsebetens, Vanderhaeghen V, Montagu Van M, 1991. Insertional mutagenesis in Arbidopsis thaliana: isolation of a T-DNA linked mutation that alters leaf morphology. Theor Appl Genet, 81:277-284.
[109] Wang Z. H, 2000. Genetic analysis of transgenes in the progenies of Bt rice cross to conventional rice varieties. Hereditas, 22(5): 309-312.
[110] Wang Z X, Yano M, Yamanouchi U et al, 1999. The Pib gene for rice blast resistance belonge to the nucleotide binding and leucine-rice, repeat class of plant disease resistance genes. The Plant Journal, 19(1): 55-64.
[111] Zhang H M, Yang H, Rech E L, et al, 1988. Transgenic rice plant produced by electroporation-mediated plasmid uptake into protoplasts. Plarit Cell Rep, 7: 379-384.
[112] Zhang S, Warkentin D, Sun B, et al, 1996. Variation in the inheritance of expression among subclones for unselected (uid A) and selected (bar) transgenes in maize (Zea mays L,). Theor Appl Genet, 92: 752-761.
[113] Dale P. J., Irwin J. A., Schefler J. A., et al, 1993. The experimental and commercial release of transgenic crop plant. Plant breeding, 111: 1-22.