摘要
植物基因工程技术为培育抗病植物品种开辟了一条全新而有效的途径。近年来，在植物抗病虫害方面取得了很大的进展，已经克隆和鉴定了不少与抗真菌病害有关的基因，并获得了许多能稳定遗传的抗病转化植株。
由于酿酒葡萄品种主要是欧洲葡萄（Vitis vinifera L.），其丰产、优质，但抗病性差。尤其是我国大多数酿酒葡萄种植区域在葡萄生长期降雨偏多，导致葡萄霜霉病、白粉病等真菌病害严重，对酿酒葡萄的产量和质量影响很大。故培育优质抗病的酿酒葡萄品种对我国酿酒葡萄的生产和葡萄酒质量的提高具有重要意义。
本研究利用基因工程技术构建了含有几丁质酶（Chi.）、β-1,3-葡聚糖酶（Glu.）、萝卜抗真菌蛋白（Rs-AFP2）的三价植物表达载体，并通过农杆菌直接转化法将三价植物表达载体转入农杆菌EHA105。以酿酒葡萄“梅尔诺”叶柄为材料，通过控制激素水平，光照和温度等，对建立再生体系的器官发生途径和体胚发生途径进行了研究，获得以下实验结果：
通过控制激素水平，光照和温度等，对建立再生体系的器官发生途径和体胚发生途径进行了研究。试验结果表明，获得梅尔诺单芽增殖生根的培养基配方为1/2MS＋IBA0.1mg/L，获得了梅尔诺离体叶柄的再生不定芽，最佳培养基配方为MS+TDZ2.0mg/L，再生率为62.42%。同时获得了体胚萌发芽的再生不定芽最佳培养基配方为MS+TDZ4.0mg/L，再生率为52.25%，在此培养基上可直接诱导出绿色不定芽。二者的再生不定芽增殖的最佳培养基配方均为MS+BA0.5mg/L。
试验结果表明，细胞分裂素TDZ对梅尔诺离体叶柄再生不定芽效果显著，较嫩的叶柄容易再生出不定芽，并且不同的暗培养时间对不定芽再生有很大的影响，暗培养时间30d时效果较好。
本研究利用基因工程技术成功地构建了两种不同组合的含潮霉素（Hyg.）的几丁质酶、β-1，3-葡聚糖酶与萝卜抗真菌蛋白基因三价植物表达载体。用直接导入法将两种三价表达载体转化农杆菌EHA105，获得了农杆菌工程菌株，为下一步转化酿酒葡萄，获得对真菌有更广泛抗病性的新品系奠定基础。
本研究为下一步转化酿酒葡萄，提高抗生素对酿酒葡萄转化细胞的有效筛选，获得优质、对真菌有更强抗病性和更广泛抑菌范围的酿酒葡萄新品系奠定了基础。
Plant gene engineering techniques offer new and efficient methods for breeding cultivars resistant to disease. In recent years, great achievements have been obtained in genetically engineering plants resistant to disease and pests. Several genes for resistance to fungal disease have been cloned and demonstrated, leading to many disease resistant plants with stable heredity.
Most cultivars used in viticulture are Vitis vinifera L., productive and high-quality but with poor disease resistance. In China especially, most areas suitable for cultivation have high rainfall, which results in fungal diseases such as powdery mildew and downy mildew affecting both the productivity and quality of wine grapes. So, it is very important to grow wine grape cultivars of high-quality and resistance to disease.
This paper describes construction of trivalent-expression vectors containing Chitinase (Chi.), β-1,3-Glucanase (Glu.) and Raphnus sativus - antifungal protein (Rs-AFPs) by gene engineering techniques. By manipulating hormone levels, light intensities and temperature, we have developed two successful regeneration systems for the winegrape varieties Merlot Noir.
The results were as follows:
By manipulating hormone levels, light intensities and temperatures, we have developed two successful regeneration systems for the winegrape varieties Merlot Noir, For Merlot Poir, The best rooting culture medium was: ?MS + IBA (0.1mg/L). The optimum medium for adventitious bud regeneration from petioles was MS+TDZ2.0mg/L, and the germination rate was 62.42%. The optimum medium for adventitious buds regeneration from germination of somatic embryos of Merlot Noir was MS+TDZ(4.0mg/L), and the germination rate was 52.25%. The best adventitious bud propagation culture medium was MS + BA (0.5mg/L).
In this study, TDZ was best for inducing adventitious bud from petioles in vitro, and it was easy to regenerate adventitious bud from the younger petiole. Maintaining cultures in dark had an important effect on regenerated adventitious bud, 30 days is best.
The trivalent-expression vectors containing Chitinase(Chi.)/β-1, 3-Glucanase (Glu.)/Raphnus sativus-antifingal protein (Rs-AFPs), were constructed and transferred into Agrobacterium tumefaciens EHA105 through direct transformation by gene engineering techniques.
This work will help us to: research genetic transformation of grapevines; select transgenic cells using antibiotics; develop new wine grape cultivars of high quality and with resistance to disease.
引文
蔡应繁，裴 炎，叶鹏盛等．抗真菌基因导入棉花创造高抗黄萎病材料研究[J]．西南农业学报，2000，13(4)：45- 49
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[J]． 西南农业学报，2001，14（2）：78-82
陈崇顺，朱雪峰，郁志芳．豇豆几丁质酶的诱导与纯化[J]．园艺学报，2000，27（5）：351-355
陈三凤，刘德虎，李季伦．植物几丁质酶的结构、基因及其表达[J]．生物工程进展，1998, 18（2）：32-37
陈肖英，叶庆生，刘伟．TDZ研究进展[J]．亚热带植物科学，2003，32（3）：59-63
崔 欣，杨庆凯．植物几丁质酶在抗真菌病害基因工程中的应用[J]．植物保护，2002，28（1）：39-43
崔百明，祝建波，肖璐等．转β- 1,3 -葡聚糖酶基因和几丁质酶基因棉花[J]．生物技术，2002，12（4）：1-2
崔洪志，郭三堆．双价杀虫基因植物表达载体的构建及其作用机理[J]．农业生物技术学报，1998，1（6）：7-13
崔萍，蒋红蕾．几丁质酶在植物抗病工程中的应用[J]．山东轻工业学院学报，2000，14（3）：65-69
邓晓东，费小雯，胡新文．萝卜抗真菌蛋白基因Rs-AFPs在大肠杆菌中的表达及其转化番茄的研究[J]．园艺学报，2001,28(4):3 61-363
冯道荣，许新萍，邱国华，李宝健．多个抗病抗虫基因在水稻中的遗传和表达[J]．科学通报，2000，45（15）：1593-1599
高武军，孙毅，王景雪，李润植．植物几丁质酶及其基因工程研究进展[J]．世界农业，1999，3：21-24
韩放，李景鹏．植物几丁质酶的研究进展[J]．生物技术，2001，11（5）：25-29
郝林，李茂辉．质粒对受体细胞的转化及注意问题[J]．生物技术，1995：5（1）：42-43
何迎春，贺思学，高必达．几丁质酶及其在植物遗传转化中的应用[J]．生物学通报，2003，38（7）：9-14
胡新文，郭建春，符少萍，郑学勤．抗真菌蛋白 Rs- AFPs基因在大肠杆菌中的表达[J]．生命科学研究，1998，2（1）：17-24
胡新文，郭建春，郑学勤．萝卜抗真菌蛋白Rs-AFPs基因克隆与序列分析[J]．热带作物学报，1998，19（3）：62-69
黄玉杰，杨合同，丁爱云． 几丁质酶和葡聚糖酶生物学特性及其编码基因的克隆和转化[J]． 山东科学, 2002,15(1): 28-35
蒋昌顺，邹冬梅，张义正．几丁质酶基因及其在作物抗病育种中的应用[J]．华南热带农业大学学报，2002，8(2)：11-18
蒋昌顺，邹冬梅．几丁质酶基因在大肠杆菌中的表达及其酶对柱花草胶孢炭疽病原菌的抑菌效果[J]．草地学报，2000，8（2）：107-115
蒋红彬，张瀛，蒋千里，李树品．几丁质酶的研究概况[J]．山东科学，2000，13（4）：41-46
蒋选利，李振歧，康振生，闫海林．几丁质酶与植物的抗病性[J]．西北农业学报, 2002，11(3)：71-75
蓝海燕，陈正华．几丁质酶及其研究进展[J]．生命科学研究，1998，2（3）：163-173
蓝海燕, 陈正华．葡聚糖酶及其在植物中的发育调节和防卫反应[J]．生物技术通报,1998，4：10-17
蓝海燕，田颖川，王长海等，β-1，3-葡聚糖酶基因及几丁质酶基因的转基因烟草及其抗真菌病的研究[J]．遗传学报，2000，27(1)：70-77
蓝海燕，田颖川，张丽华等. 烟草β-1, 3葡聚糖酶cDNA克隆、大肠杆菌表达及植物表达载体的构建[J]．生物工程学报，1998，14(4)：412-419
蓝海燕，王长海，陈正华，田颖川．农杆菌介导法将β-1，3 -葡聚糖酶基因导入油菜的研究初报[J]．中国油料作物学报，2000，22（1）：6-11
蓝海燕，王长海，张丽华等.导入β-1 , 3-葡聚糖酶及几丁质酶基因的转基因可育油菜及其抗菌核病的研究[J]． 生物工程学报，2000，16（2）：141-146
黎定军，赵开军，周清明等．转几丁质酶基因和β-1，3-葡聚糖酶基因烟草的研究[J]．湖南农业大学学报 (自然科学版 )，2002，28（3）：211-214
李继红，邵寒霜，郑学勤． 烟草 I类几丁质酶、β-1，3 -葡聚糖酶c DNA的克隆和序列分析[J]．农业生物技术学报，1997，5（2）：125-131
李继红，邵寒霜，郑学勤. 烟草I类几丁质酶和β-1,3葡聚糖酶cDNA在大肠杆菌中的表达[J] .生物工程学报，1998，14（3）：309-313
李继红等．烟草I类几丁质酶和β-1，3葡聚糖酶cDNA植物双价表达载体的构建及转化番茄的研究[J]．热带作物学报，1999，20（1）：29-33
李云, 冯慧, 田砚亭. “红地球”葡萄叶片、叶柄不定芽再生系统的建立[J]．园艺学报，2002, 29(1): 60-62
刘 柱，胡新文，刘志昕，郭建春．抗真菌蛋白分子生物学研究进展[J]．莱阳农学院学报，2001，18(2):197-201
刘 志，袁小玲，张天真．获得多价转基因作物的策略[J]．遗传，2001，23（2）：182-186
刘 柱，胡新文，朱建清，杨志荣．萝卜抗真菌蛋白Rs-AFP1基因PCR定点突变及其在大肠杆菌中的表达[J]．西南农业学报，2002，15（3）：85-90
刘爱新，董汉松，梁元存等．烟草几丁酶β-1，3-葡聚糖酶的抑菌作用[J]．微生物学通报，1999，26（1）：15-18
刘焕利，王金生等．苦草杆菌B3抗植物病原真菌蛋白的纯化及其性质的研究[J]．农业生物技术
学报，1995，3（3）：32-38
欧阳波，季汉霞，叶志彪．植物β-1，3-葡聚糖酶及其基因[J]．中国生物工程杂志，2002，12:18-24
欧阳石文，谢丙炎，赵开军，冯兰香．转几丁质酶基因研究进展[J]．生物技术通报，2001，3：28-32
欧阳石文，赵开军，冯兰香，谢丙炎．植物几丁质酶的研究进展[J]．生物工程进展，2000，1（21）：30-35
潘学军．无核抗病葡萄胚挽救育种的研究．西北农林科技大学硕士论文. 2002
彭仁旺．壳多糖酶研究的概况及最新进展[J] ．生物化学与生物物理进展，1996，23（2）：102-106
彭秀玲等编著．基因工程实验技术．湖南科学技术出版社，1987：75-82
秦玲．苹果遗传转化体系的建立及ipt基因的导入．西北农林科技大学硕士论文，2002
谭文斌．两种质粒转化方法的比较[J]．生物技术．1996，6（5）：42-44
王 伟，朱 祯，高越峰．等双价抗虫基因陆地棉转化植株的获得[J]. 植物学报，1994，41：84-388
王关林、方宏筠主编．植物基因工程原理与技术（第一版）[Ｍ]．北京：科学出版社，1998
王华，周鹏，郭安平．几丁质酶基因与抗真菌蛋白、葡聚糖酶基因和萝卜双价表达载体的构建及农杆菌工程菌株的重组[J]．西北植物学报，2002，22（2）：250-256
王新力，索桂英，彭学贤．植物果实成熟基因的转录调控[J]．生物技术通报，2001，4:33-38
王毓美，徐云远，贾敬芬．亚麻遗传转化体系的建立及几丁质酶基因导入的研究[J]．西北植物学报，2000，20（3）：346-351
温进坤，韩 梅. 医学分子生物学理论与研究技术[M] ．北京：科学出版社，2002：41-51
吴家和，张献龙，罗晓丽等．转几丁质酶和葡聚糖酶基因棉花的获得及其对黄萎病的抗性[J]．遗传学报，2004，31（2）：183-188
邢全华，王 斌．植物葡聚糖酶基因抗病作用的研究进展[J]．遗传，2002，24（6）：715-720
邢全华，王广金，石金锋等．β-1，3 -葡聚糖酶基因高效表达载体的构建及对小麦的转化[J]． 遗传学报，2003, 30 (8):717-722
徐凌飞，马锋旺，王之等．梨叶片离体培养和植株再生[J]．园艺学报，2002，29（4）：367-368
徐香玲，李集临，马兴红．几丁质酶基因表达载体构建及转化烟草的研究[J]．植物研究，2000，22（7）：318-324
许明辉，李成云，李进斌．转几丁质酶、聚糖酶基因水稻稻瘟病抗谱分析[J]．中国水稻科学 ，2003 , 1 7(4):307～310
曾 艳，赵南明，刘进元．几丁质酶与植物防卫反应[J]．生物工程进展，1 997，17（4）：31-36
张小红，张红燕，武军等．TDZ对香椿愈伤组织诱导及芽增殖生长等的影响［J］．西北农林科技大学学报，2002，30（5）：35－39
赵晓琴，李 冠． 几丁质酶研究进展[J]．新疆大学学报 (自然科学版 )，2003，20（4）：422-426
郑秀芳，缪为等，2002．抗菌肽及在植物抗病基因工程中的应用[J]．生物学通报，2002，37（10）：
9-10
郑用琏，G.M.Balance．小麦与褐斑病之间的互作I.受病原菌诱导的寄主PR基因的表达[J]．华中农业大学学报，1994，13（3）：213-218
周淼平，陆维忠．植物几丁质酶及其在抗真菌基因工程中的应用[J]．江苏农业学报, 1998, 1 4(3 ):183～187
周鹏，郭安平，王跃进等．2个葡萄品系外植体愈伤组织诱导和植株再生[J]．热带作物学报，2002，23（3）：52-56
周思军，李希臣，刘昭军等．通过农杆菌介导将菜豆几丁质酶基因导入马铃薯[J]．中国马铃薯，2000，14（2）：70-73
周向军，王仑山，林芝萍等．萝卜抗真菌蛋白1 (Rｓ-AFP1 )在大肠杆菌中的融合表达及其对大丽轮枝菌抑制活性的研究[J]．植物学报，2000,42(7):703-707
朱雪峰，陈崇顺，郁志芳．植物几丁质酶的生物功能[J]．生命的化学，2000，20（1）：36-37
Ajith Anand, Tian Zhou, Harold N. Trick, Bikram S. Gill et al..Greenhouse and field testing of transgenic wheat plants stably expressing gens for thaumatin-like protein, chitinase and glucanase against Fusarium graminearum [J]. Journal of Experimental Botany, 2003, 54(384): 1101-1111
Ana Beatriz Moreno, Alvaro Martinez del Pozo, Marise Borjia et al., Activity of the antifungal protein from Aspergillus giganteus against Botrytis cinerea [J]. Phytopathology, 2003, 93(11): 1344-1350
Beintema J J. Structure feature of plant chitinase and chititin-binding proteins [J]. FEBS Lett, 1994,350(2-3): 159-163
Berres R., Otten L., Thnland B., Malgarini-Clog E. and Walter B..Transfomation of vitis by different srains of Agrobacterium tumefacient containing the T-6b gene [J]. Plant Cell Reports, 1992, 11:192-195
Botha A.M., M.A.C. Nagel, A.J. Van der Westhuizen1, F.C. Botha. Chitinase isoenzymes in near-isogenic wheat lines challenged with Russian wheat aphid, exogenous ethylene, and mechanical wounding [J]. Bot. Bull. Acad. Sin, 1998, 39: 99-106
Brameld K A, Goddard W A. The role of enzyme distortion in the single displacement mechanism of family chitinase [J]. Proc Natl Acad Sci USA, 1999,8:4276-4281
Broglie K E, Chet I, Holliday M et al., Transgenic plant with enhanced resistance to the fungal pathogen Rhizotonia solani [J]. Science,1991,254:1194-1197
Broglie, K, Gaynor, J, and Broglie, R. Ethylene-Regulated Gene Expression: Molecular Cloning of the Genes Encoding an Endochitinase from Phaseolus vulgaris [J]. Proceedings of the National Academy of Sciences, 1986,83: 6820-6824
Bruno Mezzetti, Tiziana Pandolfini, Oriano Navacchi and Lucia Landi. Genetic transformation of Vitis vinifera via organogenesis [J]. BMC Biotechnology, 2002, 2:1-10
Caius M Rommens, Ganesh M Kishore. Exploiting the full potential of disease resistance genes for agricultural use [J]. Current Opinion in Biotechnology 2000, 11:120–125
Carol Robacker. Somatic embryogenesis and plant regeneration from Muscadine grape leaf explants [J]. Hortscience, 1993, 28(1): 53-55
Choonkyun Jung, Seoung Hyun Lyou, Yeon Jong Koo et al., Constitutive Expression of Defense Genes in Transgenic Arabidopsis Overproducing Methyl Jasmonate. Agric [J]. Chem. Biotechnol, 2003, 46(2): 52-57
Chun-Ta Wu, Kent J Bradford. Class I chitinase and [beta]-1, 3-glucanase are differentially regulated by wounding, methyl jasmonate, ethylene, and gibberellin tomato seeds and leaves1 [J]. Plant Physiology, 2003,133(1): 263-268
Colby S.M., Juncosa A.M., Meredith C.P., Cellular Differences in Agrobacterium Susceptibility and Regenerative Capacity Restrict the Development of Transgenic Grapevines J [J]. Amer. Soc. Hort. Sci. 1991, 116(2): 356-361
Das D.K. Reddy M.K., Upadyaya K.C., Sopory S.K. An efficient leaf-disc culture method for the regeneration via somatic embryogenesis and transformation of grape (Vitis vinifera L.) [J]. Plant Cell Rep, 2002,20:999-1005
Dusgupta S et al. Cordinated expression of multiple enzymes in different sub-celluar compartments in plants [J]. Plant J., 1998,16:107-116
El-Katatny M. H., W. Somitsch1, K.H. Robra1. Production of chitinase andβ- 1,3-glucanase by Trichoderma harzianum for control of the phytopathogenic fungus sclerotium rolfsii. Food technol [J]. Biotechnol, 2000, 38 (3) 173–180
Frank Siefert, Klaus Grossmann. Induction of chitinase andβ-1,3-glucanase activity in sunflower suspension cells in response to an elicitor from Phytophthora megasperma f. sp. glycinea (Pmg). Evidence for regulation by ethylene and 1-aminocyclopropane-1-carboxylic acid (ACC) [J]. Journal of Experimental Botany, 1997, 48(317): 2023–2029
Georgy P. G., Ivan J. T., Violeta M. C et al., Genetically Engineered Grapevines Carrying GFLV Coat Protein and Antifreeze Genes [J]. Agriculturae Conspectus Scientificus, 2001,66(1): 71-76
Hamilton C M. Stable transfer of intact high molecular weight DNA into chromosome [J]. Proc. Natl.Acad.Sci.USA, 1996,93:9975-9979
Horst Vierheilig, Monika Alt, Ju¨RG Lange et al., Colonization of Transgenic Tobacco Constitutively Expressing Pathogenesis-Related Proteins by the Vesicular-Arbuscular Mycorrhizal Fungus Glomus mosseae [J]. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1995, 61(8): 3031–3034
Iocco P., Franks T. Thomas M.R. Genetic transformation of major wine grape cultivars of Vitis vinifera L [J]. Transgenic Reseach, 2001,10:105-112
J．萨姆布录克 等著．植物基因工程原理和技术[M]．科学技术出版社，1998：469-490
Jayasankar S., Gray D. J., Litz. R. E. High-efficiency somatic embryogenesis and plant regeneration from suspension cultures of grapevine [J]. Plant Cell Reports, 1999, 18:533-537
Peros J.P., Laurent Torregrosa and Gilles Berger. Variability among Vitis vinifera cultivars in micropropagation, organogenesis and antibiotic sensitivity [J]. Journal of Experimental Botany, 1998, 49(319): 171-179
Kalliopi A. Roubelakis-Angelakis, Molecular Biology & Biotechnology of the Grapevine [M]. Kluwer Academic Publishers, c2001: 50-70
Kikkert JR, Ali GS, Wallace PG, Reisch BI, Reustle GM. Expression of a fungal chitinase in Vitis vinifera L. 'Merlot' and 'Chardonnay' plants produced by biolistic transformation [J]. Acta Hort., 2000, 528, 297-303
Kikkert Julie R.Hebert-Soule Dominique, Wallace Patricia G.etc. Transgenic plantlets of ‘Chancellor’grapevine (Vitis sp.) from boilistic transformation of embryogenic cell suspendsions [J]. Plant Cell Reports, 1996,15:311-316
Klarzynski O, Plesse B, Joubert J M et al., Linear β-1，3-glucanase are elicitors of defense response in tobacco [J]. Plant Physiol, 2000,124:1027-1037
Leo S Melchers, Maarten H Stuiver. Novel genes for disease-resistance breeding [J]. Current Opinion in Plant Biology 2000, 3:147–152
LI Ji, LIU Jin-Yuan. A Novel Cotton Gene Encoding a New Class of Chitinase [J]. Acta Botanica Sinica 2003, 45 (12): 1489-1496
LI Ying-Zhang, ZHENG Xiao-Hua, TANG Hai-Lin, ZHU Jian-Wei, Increase of β-1, 3-Glucanase and Chitinase Activities in Cotton Callus Cells Treated by Salicylic Acid and Toxin of Verticillium dahliae[J]. Acta Botanica Sinica, 2003, 45 (7): 802-808
Li ZJ, Jayasankar S., Gray DJ. Expression of a bifunctional green fluorescent protein (GFP) fusion marker under the control of three constitutive promoters and enhanced derivatives in transgenic grape (Vitis vinifera) [J]. Plant Science, 2001, 160:877-887
LIU Mei (刘梅), SUN Zong-xiu (孙宗修), ZHU Jie (朱洁), et al., Enhancing rice resistance to fungal pathogens by transformation with cell wall degrading enzyme genes from Trichoderma atroviride [J]. Journal of Zhejiang University SCIENCE, 2004,5(2): 133-136
Luciana Petruzzelli, Christian Kunz, Rosa Waldvoge et al., Distinct ethylene- and tissue-specific regulation of β-1, 3-glucanases and chitinases during pea seed germination [J]. Planta, 1999, 209: 195-201
Martinelli L, Mandolino G. Genetic transformation and regeneration of transgenic plants in grapevine (Vitis rupestris S.) [J]. Theor Appl Genet, 1994, 88: 621-628 (R4)
Mauch F, Mani B, Boiler T. Antifungal hydrolases in pea tissue. II. Inhibition of fungal growth by combinations of chitinase and beta-1, 3-glucanase [J]. Plant. Physiol. 1988, 88:936-942
Mauch Felix, L. Andrew Staehelin. Functional lmplications of the Subcellular Localization of Ethylene-induced Chitinase and β-1,3-Glucanase in Bean Leaves [J]. The Plant Cell, 1989,1:447-457
Mauro M C,Toutain S,Walter B,et al. High efficiency regeneration of grapevine plants transformed with the GFLV coat protein gene [J]. Plant Science, 1995,112:97-106
Mojtaba Mohammadi, Ramin Roohparvar, Mohammad Torabi. Induced chitinase activity in resistance wheat leaves inoculated incompatible race of Puccinia striiforms f.sp.tritici, the cause yellow rust disease [J]. Mycopathologia, 2001, 154:119-126
Mullins Michael G., Archie Tang F.C., Facciotti Daniel. Agrobacterium-mediated genetic transformation of grapevines: transgenic plants of Vitis rupeestris scheele and nuds of Vitis vinifera L. [J]. Bio/Technology, 1990,8:1041-1045
Nakano M., Hoshino Y., Mii M. Regeneration of transgenic plants of grapevine (Vitis vinifera L.) via Agtobacterium rhizogenes- mediated transformation of embryogenic calli [J]. Journal of Experimental Botany, 1994, 45(274): 649-656
Perl. A., Saad, S., Sahar, N., and D. Holland. Establishment of long-term embryogenic cultures of seedless V. vinifera cultivars - a synergistic effect of auxins and the role of abscisic acid [J]. Plant Sci., 1995, 104:193- 200
Porat R., A. Lers, S. Dori et al., Induction of Chitinase andβ-1, 3-endoglucanase Proteins by UV Irradiation and Wounding in Grapefruit Peel Tissue [J]. Phytoparasitica, 1999, 27(3): 1-6
Pozo M.J., C. Azcon-Aguilar, E. Dumas-Gaudot et al., Chitosanase and chitinase activities in tomato roots during interactions with arbuscular mycorrhizal fungi or Phytophthore parastica [J]. Journal Experimental Botany, 1998,49(327): 1729-1739
Rozi Mohamed, Richard Meilan, Michael E Ostry et al., Bacterio-opsin gene overexpression fails to elevate fungal disease resistance in transgenic poplar (Populus) [J]. Canadian Journal of Forest Research, 2001, 31(2): 268-276
Salunkhe, C.K., Rao, P.S., and M. Mhatre. Induction of somatic embryogenesis and plantlets in tendrils of V. vinifera L [J]. Plant Cell Rep. 1997,17: 65-70
Scorza R., Cordts J. M., Producing transgenic ‘Thompson seedless’ grape(Vitis vinifera L.) plants [J]. J. Amer. Soc.Hort. Sci., 1996,12 (4): 616-619
Scorza R., Cordts J.M., Ramming D. W., Emershad R.L., Transformation of grape (Vitis vinifera L.) zygotic-derived somatic embryos and regeneration of transgenic plants [J]. Plant Cell Reports, 1995,14:589-592
Selitrennikoff C P. Antifungal proteins [J]. Appl Environ Microbiol, 2001, 67:2883-2894
Seung-Heui Kim, Seon-Kyu Kim. Effects of Auxins and Cytokinins on Callus Induction from Leaf Blade, Petiole, and Stem Segments of In Vitro-grown ‘Sheridan’Grape Shoots [J]. J. Plant Biotechnology, 2002, 4(1): 17~21
Stamp J. A., C. P. Meredith. Proliferative Somatic embryogenesis from Zygotic Embryos of grapevine [J]. Scientia Horticulture, 1988,113(6): 941-945
Stamp JA, Colby SM, Meredith CP. Improved shoot organogenesis from leaves of grape [J]. J. Amer Soc Hort Sci, 1990, 115(6): 1038–1042
Terras, F.R.G., Eggermont, K. et al., Small Cysteine-rich antifungal proteins from radish: their role in host defense [J]. Plant Cell,1995,7:753-758
Terras, F.R.G., Schoofs, H.M.E. et al., Analysis of two novel classes of antifungal proteins from radish (Raphanus sativus) seeds [J]. J. Biol. Chem, 1992b, 267:15301-15309
Terras, F.R.G., Torrekens, S., van Leuven, F., Osborn, R.W., Vanderleyden J., Cammue, B.P.A., Broekaert W.F, A new family of of basic cysteine-rich antifungal proteins from Brassicaceae-species [J]. FEBS Lett, 1993,316:233-240
Thomas Hollis, Yuji Honda, Tamo Fukamizoet al., Kinetic Analysis of Barley Chitinase [J]. Archives of Biochemistry and biophysics, 1997, 344(2): 335–342
Tsvetkov I.J., Atanassov A.I., Tsolova V.M., Gene transfer for stress resistance in Grapes [J]. Acta Horticulturae, 2000, 528:389-394
Velazhahan R., R. Samiyappan and P. Vidhyasekaran1, Purification of an elicitor- inducible antifungal chitinase from suspension-cultured rice cells [J]. Phytoparasitica, 2000, 28(2):1-9
Vera V. Lozovaya, Aree Waranyuwat, and Jack M. Widholm. β-1,3-Glucanase and Resistance to Aspergillus flavus Infection in Maize [J]. Published in Crop Sci. 1998, 38:1255–1260
Vierheilig Horst, Monika Alt, JU¨RG Lange et al., Colonization of Transgenic Tobacco Constitutively Expressing Pathogenesis-Related Proteins by the Vesicular-Arbuscular Mycorrhizal Fungus Glomus mosseae [J]. Applied and Nvironmental Microbiology, 1995, 61(8) : 3031–3034
Von Bodman SB，Domier LL, Farrand SK. Expression of multiple eukaryotic genes from a single promoter in Nicotiana [J]. Bio/Technology, 1995, 13:587-591
Wen-Chi Hou, Ying-chou Chen, Yaw-Huei Lin. Chitinase activity of sweet potato (Ipomoea batatas (L.) Lam var Tainong 57) [J]. Bot. Bull. Acad Sin, 1998,39:93-97
Yamamoto T, Iketani H, Ieki H, Nishizawa Y, Notsuka K, Hibi T, Hayashi T, Matsuta N. Transgenic grapevine plants expressing a rice chitinase with enhanced resistance to fungal pathogens [J]. Plant Cell Reports, 2000, 19:639-646
Zamir K. Punja. Genetic engineering of plants to enhance resistance to fungal pathogens- review of progress and future prospects. Can. J [J]. Plant Pathol, 2001, 23: 216–235