唐菖蒲再生体系建立及NPR1基因的遗传转化
|
摘要
唐菖蒲(Gladiolus hybridus)是鸢尾科唐菖蒲属多年生单子叶球根花卉，是世界著名的四大切花之一。唐菖蒲的切花生产是以无性繁殖的方式生产球茎作为栽培材料，因此附着在球茎上的致病菌会不断传染给后代种球，经过多年积累导致种球种植过程中病害严重发生。
应用抗病品种是病害防治最直接有效的方法，但唐菖蒲抗病种质资源匮乏加之常规育种周期长，使得抗病品种远远不能满足生产上的需求。随着生物技术的迅猛发展，运用基因工程手段将外源抗病基因导入植物中，为唐菖蒲抗病育种带来了新的希望。
本研究在由唐菖蒲球茎芽诱导出的胚性愈伤组织基础上，建立了胚性愈伤组织经器官发生途径和经体细胞胚发生途径两套再生体系，并对器官发生途径中不定芽的壮苗培养和体细胞胚发生途径中胚状体的发育过程进行了较深入的研究。在此基础上以唐菖蒲愈伤组织为基因转化受体，将植物广谱抗病基因(NPR1基因)通过农杆菌介导转化唐菖蒲，建立了稳定的遗传转化体系，得到了经PCR检测为阳性的植株。本研究的主要结果如下：
1．唐菖蒲愈伤组织经器官发生途径再生体系：以球茎芽切片为外植体，胚性愈伤组织诱导最佳培养基为MS+2，4-D4mg／L+6-BA0.5mg／L，诱导率达95％；不定芽诱导最佳培养基为MS+6-BA0.5mg／L+NAA0.1mg／L，诱导率达92.7％；壮苗培养基为1／4MS+PP_(333)1mg／L+NAA1mg／L+蔗糖50g／L，壮苗率达100％，生根培养基为MS+IBA1.5mg／L，生根率达100％，移栽成活率达100％。
2．唐菖蒲愈伤组织经体细胞胚发生途径再生体系：以球茎芽切片为外植体，胚性愈伤组织诱导培养基为MS+2，4-D4.0mg／L+6-BA0.5 mg门L；胚状体诱导培养基为MS+2，4-D1.0mg／L+TDZ0.2mg／L+蔗糖30g／L+甘露醇30g／L，诱导率为68.3％；将产生的胚状体首先接种于MS培养基使其充分发育，之后转入MS+6-BA2.0mg／L培养基中诱导发芽，在转入MS培养基中使其形成完整植株。通过石蜡切片法和临时压片法对胚状体的发育过程观察发现，首先外植体表层薄壁细胞经脱分化恢复分生能力，形成愈伤组织，随后在愈伤组织表面形成许多瘤状突起即胚性细胞团，胚性细胞团继续发育成球形胚、盾形胚，最后发育成熟形成完整植株。
3．唐菖蒲遗传转化体系：以胚性愈伤组织为基因转化受体，将胚性愈伤组织预培养14天后用浓度为OD_(600)=0.3～0.5的农杆菌菌液侵染10～15min，侵染的愈伤组织与农杆菌共培养3天后，用250mg／L的头孢霉素进行去菌培养，20天后用2.0mg／L的草甘磷(PPT)进行筛选培养直至抗性愈伤组织的产生。将产生的抗性愈伤组织分别经器官发生途径和体细胞胚发生途径进行再生。
4．转化植株的PCR检测：经愈伤组织器官再生途径得到的69株抗性植株中有3株呈阳性，阳性率为4.3％，转化率为0.3％。经愈伤组织体细胞胚再生途径没有得到抗性植株。
Gladiolus (Gladiolus hybridus) is one of perennial and monocotyledonous bulbous flowers,which belongs to the gladiolus of iridaceous and is one of the four world-famous cut flowers. Theproduction of corm by asexual reproduction way during cut flower production of gladiolus,so thepathogen on the old corm would infection offspring. The accumulation of pathogen by a few yearscan lead to serious disease in the production of cut flower.
Using the disease-resistant varieties is the best way for preventing and curing the plantdiseases. But the disease-resistant resource of gladiolus is so little and the cycle of the generalbreeding is so long that the disease-resistant variety is far from meeting the demand of the gladiolusproduction. With the rapid development of biotechnology, transferring new disease-resistant geneof other sources into the gladiolus by using genetic engineering means brings new hope in thedisease-resistant breeding of gladiolus.
This study include that we get embryogenic callus from gladiolus'bud,using it setup upregeneration system by organogenesis and somatic embryogenesis and study the robust culture ofbuds in organogenesis way and the formation of somatic embryo in the embryogenesis way. We usethe callus of gladiolus as the explants of genetic transformation, The broad-spectrum diseaseresistance gene (NPR1 gene) transferred into gladiolus mediated by the agrobacterium, weestablish genetic transformation system of gladiolus. Finally, we get PCR positive plants. The maincontent and result of experiment are as follows:
1. The regeneration system by organogenesis: Taking the slice of gladious'corms as explant,MS basal medium supplemented with 2,4-D4mg/L and 6-BA0.5mg/L was advantageous toembryogenic callus induction,The frequency of callus induction was 95%;The combination of6-BA0.5mg/L and NAA 0.1mg/L was the best for callus differentiation with the rate of 86%;100%of the robust and regenerated shoot treated with 1/4MS and PP_(333)1mg/L and NAA1mg/L andsucrose50g/L; MS basal medium supplemented with IBA1.5 mg/L could develop their roots,therate of robust plantlets was 100%.The survival ratio of transplanting reach 100%.
2. The regeneration system by somatic embryogenesis: Taking the slice of gladious'corms asexplants, MS basal medium supplemented with 2,4-D4mg/L and 6-BA0.5mg/L was advantageousto callus induction,The frequency of callus induction was 95%; MS+2,4-D1.0mg/L+TDZ0.2 rag/L+sucrose30g/L+30g/L was the best culture medium that the induction of somatic embryo,the rate was 57%. After that there was three steps to complete the culture: First, the regenerated somatic embryowere cultured in the MS medium to growth enough.; Second,the growful somatic embryo wascultured in the MS+6-BA2.0mg/L to induce shoots;Third,the regenerated plants were obtained inthe MS medium.The appearance and growth of somatic embryo were observed through paraffinslices and press slices. Histological observations showed that parenchyma cells first experienceddedifferentiation and followed with callus-forming, ensued with embryogenic cell masses which likea tubercular structure.and these embryogenic cell masses experienced globular, scutellate stages anddeveloped into whole plantlets.
3. The establishment of genetic transformation system: Taking the slice of gladious'corms asexplants, pre-culture of callus for 14d;the agrobacterium tumefaciens solution wasOD_(600)=0.3～0.5,the time of infect was 10～15min, the time of co-culture was 3d,the delayedscreening on regeneration for 20d, during this time,the proliferation of Agrobacterium tumefacienswas selected Cef, the suit concentration was 250mg/L. After 20d, we start to select resistant callus,the select pressure of PPT was 2.0mg/L. The regeneration of resistant callus by organogenesis andsomatic embryogenesis way.
4. Detection of the transformed plant: For callus' regeneration by organic regeneration, wegained 69 resistant plants. Three of them is PCR positive plant, positive percentage is 4.3% andtransformation percentage is 0.3%. For callus' regeneration by somatic embryo regeneration,therewas no resistant plants obtioned from it..
应用抗病品种是病害防治最直接有效的方法，但唐菖蒲抗病种质资源匮乏加之常规育种周期长，使得抗病品种远远不能满足生产上的需求。随着生物技术的迅猛发展，运用基因工程手段将外源抗病基因导入植物中，为唐菖蒲抗病育种带来了新的希望。
本研究在由唐菖蒲球茎芽诱导出的胚性愈伤组织基础上，建立了胚性愈伤组织经器官发生途径和经体细胞胚发生途径两套再生体系，并对器官发生途径中不定芽的壮苗培养和体细胞胚发生途径中胚状体的发育过程进行了较深入的研究。在此基础上以唐菖蒲愈伤组织为基因转化受体，将植物广谱抗病基因(NPR1基因)通过农杆菌介导转化唐菖蒲，建立了稳定的遗传转化体系，得到了经PCR检测为阳性的植株。本研究的主要结果如下：
1．唐菖蒲愈伤组织经器官发生途径再生体系：以球茎芽切片为外植体，胚性愈伤组织诱导最佳培养基为MS+2，4-D4mg／L+6-BA0.5mg／L，诱导率达95％；不定芽诱导最佳培养基为MS+6-BA0.5mg／L+NAA0.1mg／L，诱导率达92.7％；壮苗培养基为1／4MS+PP_(333)1mg／L+NAA1mg／L+蔗糖50g／L，壮苗率达100％，生根培养基为MS+IBA1.5mg／L，生根率达100％，移栽成活率达100％。
2．唐菖蒲愈伤组织经体细胞胚发生途径再生体系：以球茎芽切片为外植体，胚性愈伤组织诱导培养基为MS+2，4-D4.0mg／L+6-BA0.5 mg门L；胚状体诱导培养基为MS+2，4-D1.0mg／L+TDZ0.2mg／L+蔗糖30g／L+甘露醇30g／L，诱导率为68.3％；将产生的胚状体首先接种于MS培养基使其充分发育，之后转入MS+6-BA2.0mg／L培养基中诱导发芽，在转入MS培养基中使其形成完整植株。通过石蜡切片法和临时压片法对胚状体的发育过程观察发现，首先外植体表层薄壁细胞经脱分化恢复分生能力，形成愈伤组织，随后在愈伤组织表面形成许多瘤状突起即胚性细胞团，胚性细胞团继续发育成球形胚、盾形胚，最后发育成熟形成完整植株。
3．唐菖蒲遗传转化体系：以胚性愈伤组织为基因转化受体，将胚性愈伤组织预培养14天后用浓度为OD_(600)=0.3～0.5的农杆菌菌液侵染10～15min，侵染的愈伤组织与农杆菌共培养3天后，用250mg／L的头孢霉素进行去菌培养，20天后用2.0mg／L的草甘磷(PPT)进行筛选培养直至抗性愈伤组织的产生。将产生的抗性愈伤组织分别经器官发生途径和体细胞胚发生途径进行再生。
4．转化植株的PCR检测：经愈伤组织器官再生途径得到的69株抗性植株中有3株呈阳性，阳性率为4.3％，转化率为0.3％。经愈伤组织体细胞胚再生途径没有得到抗性植株。
Gladiolus (Gladiolus hybridus) is one of perennial and monocotyledonous bulbous flowers,which belongs to the gladiolus of iridaceous and is one of the four world-famous cut flowers. Theproduction of corm by asexual reproduction way during cut flower production of gladiolus,so thepathogen on the old corm would infection offspring. The accumulation of pathogen by a few yearscan lead to serious disease in the production of cut flower.
Using the disease-resistant varieties is the best way for preventing and curing the plantdiseases. But the disease-resistant resource of gladiolus is so little and the cycle of the generalbreeding is so long that the disease-resistant variety is far from meeting the demand of the gladiolusproduction. With the rapid development of biotechnology, transferring new disease-resistant geneof other sources into the gladiolus by using genetic engineering means brings new hope in thedisease-resistant breeding of gladiolus.
This study include that we get embryogenic callus from gladiolus'bud,using it setup upregeneration system by organogenesis and somatic embryogenesis and study the robust culture ofbuds in organogenesis way and the formation of somatic embryo in the embryogenesis way. We usethe callus of gladiolus as the explants of genetic transformation, The broad-spectrum diseaseresistance gene (NPR1 gene) transferred into gladiolus mediated by the agrobacterium, weestablish genetic transformation system of gladiolus. Finally, we get PCR positive plants. The maincontent and result of experiment are as follows:
1. The regeneration system by organogenesis: Taking the slice of gladious'corms as explant,MS basal medium supplemented with 2,4-D4mg/L and 6-BA0.5mg/L was advantageous toembryogenic callus induction,The frequency of callus induction was 95%;The combination of6-BA0.5mg/L and NAA 0.1mg/L was the best for callus differentiation with the rate of 86%;100%of the robust and regenerated shoot treated with 1/4MS and PP_(333)1mg/L and NAA1mg/L andsucrose50g/L; MS basal medium supplemented with IBA1.5 mg/L could develop their roots,therate of robust plantlets was 100%.The survival ratio of transplanting reach 100%.
2. The regeneration system by somatic embryogenesis: Taking the slice of gladious'corms asexplants, MS basal medium supplemented with 2,4-D4mg/L and 6-BA0.5mg/L was advantageousto callus induction,The frequency of callus induction was 95%; MS+2,4-D1.0mg/L+TDZ0.2 rag/L+sucrose30g/L+30g/L was the best culture medium that the induction of somatic embryo,the rate was 57%. After that there was three steps to complete the culture: First, the regenerated somatic embryowere cultured in the MS medium to growth enough.; Second,the growful somatic embryo wascultured in the MS+6-BA2.0mg/L to induce shoots;Third,the regenerated plants were obtained inthe MS medium.The appearance and growth of somatic embryo were observed through paraffinslices and press slices. Histological observations showed that parenchyma cells first experienceddedifferentiation and followed with callus-forming, ensued with embryogenic cell masses which likea tubercular structure.and these embryogenic cell masses experienced globular, scutellate stages anddeveloped into whole plantlets.
3. The establishment of genetic transformation system: Taking the slice of gladious'corms asexplants, pre-culture of callus for 14d;the agrobacterium tumefaciens solution wasOD_(600)=0.3～0.5,the time of infect was 10～15min, the time of co-culture was 3d,the delayedscreening on regeneration for 20d, during this time,the proliferation of Agrobacterium tumefacienswas selected Cef, the suit concentration was 250mg/L. After 20d, we start to select resistant callus,the select pressure of PPT was 2.0mg/L. The regeneration of resistant callus by organogenesis andsomatic embryogenesis way.
4. Detection of the transformed plant: For callus' regeneration by organic regeneration, wegained 69 resistant plants. Three of them is PCR positive plant, positive percentage is 4.3% andtransformation percentage is 0.3%. For callus' regeneration by somatic embryo regeneration,therewas no resistant plants obtioned from it..
引文
蔡文启，董光军，徐绍华等．1995．根癌农杆菌ATC15和438T与唐菖蒲细胞结合的扫描电镜研究．微生物学报．35(3)．186-189
车代弟，刘志洋．2004．唐菖蒲子球茎直接分化再生体系的建立．分子植物育种．2(4)．531-534
陈典，徐启江．2001．分蘖洋葱茎尖愈伤组织诱导及植株再生．园艺学报．28(4)．359-360
陈俊愉．1990．中国花经．上海文化出版社(上海)．610-611
程金水．2000．园林植物遗传育种学．中国林业出版社(北京)．340-350
崔凯荣，戴若兰．植物体细胞胚胎发生的分子生物学．北京：科学出版社．2000．59-62。
金淑梅，管清杰，罗秋香等．2006．苜蓿愈伤组织高频再生遗传和转化体系的建立．分子植物育种．4(4)．571-578。
韩继成，陈宁，陈霜莹．2006．不同处理对无病毒葡萄离体叶片遗传转化的影响． 分子植物育种．4(5)．670-674
郝建平，金竹萍，王永康等．2006．枣合子胚和体细胞胚发育过程的观察与比较．分子细胞生物学报．39(5)．423-426
雷玉明．2002．唐菖蒲腐烂病侵染规律和发病因子研究．植物保护．28(3)．36-38
李俊明主编．2000．植物组织培养教程．中国农业大学出版社．
李哲，黄霞，李筱菊．2005．大蕉花序愈伤组织的诱导及其体细胞胚发生．植物生理学通讯．41(5)．616-619
刘春朝，欧阳藩，郭仲琛．1997．唐菖蒲原球茎培养的研究．应用与环境生物学报．3(3)．208-212
刘非燕，郭达初，高卓．1997．唐菖蒲组织培养研究．浙江农业学报，9(1)．51-53
刘建利，张占路，吴燕民．2006．百脉根农杆菌快速高效遗传转化体系的建立．草业学报．15(3)．228-234
刘志洋，车代弟．2004．唐菖蒲愈伤组织的诱导及植株再生．分子植物育种．2(5)．695-698
马晖玲，卢欣石，曹致中．2006．紫花苜蓿基因转化的影响因素分析．草业学报．15(5)．95-99
王萍，王罡，季静等．2004．大豆体细胞胚胎发生与农杆菌介导的遗传转化遗传．26(5)．694-699
王关林，方宏筠主编．2002．植物基因工程．科学出版社(北京)．347-350
王俐，龙春林，赵燕．2001．唐菖蒲快速繁殖条件的优化．西南林学院学报． 21(3)：152-155
王晓春，刘尚前，季静．2004．影响大豆体细胞胚萌发率的因素．大豆科学．23(2)：151-153
王晓春，刘尚前，王罡．2006．大豆体细胞胚再生植株的研究．大豆科学．25(2)．149-153
吴小芹，吴少华．2000．鲜切花病虫害防治技术．科学技术文献出版社(北京)．112-118
谢海燕，毛碧增，单兰兰等．2004．狗牙根颖果胚性愈伤组织的诱导和胚性细胞的超微
结构及植株再生．植物生理与分子生物学学报．30(2)．209-215
明慧．2002．花卉病虫害防治．金盾出版社(北京)．95-97
徐启江，李玉花，陈典．2004．PP_(333)对分蘖洋葱试管苗增殖和生根的影响．植物生理学通讯．40(3)．256-260
义鸣放，王玉国，缪珊．2000．唐菖蒲．中国农业出版社(北京)．1-66
詹园凤，王广东．2006．大蒜体细胞胚胎发生的组织学研究．农业生物技术科学．22(1)．46-49
张秦英，陈俊愉．2004．不同激素对‘美人’梅叶片离体培养的影响及其细胞学观察．北京林业大学．26(5)．42-45
赵宏．1997，庸菖蒲球茎腐烂病原鉴定及其致病力．甘肃农业大学学报．6：163-166
赵云鹏，郭维明，王广东．2005．花烛不同愈伤组织解剖结构的观察．园艺学报．32(1)．60-67
周盈，林拥军，柴鑫莉．2006．根癌农杆菌介导的花魔芋遗传转化体系的研究．分子植物育种．4(4)．559-564
Attree SM, Pomeroy MK, Fowke LC. 1991. Enhanced maturation and desiccation tolerance of whitespruce (Picea glaucaMoench Voss) somatic embryos. Amm Bot. 57(8). 519-525
Babu-P, Chawla-HS. 2000. In vitro regeneration and Agrobacterium mediated transformation in gladiolus. Journal of Horticultural Science and Biotechnology. 75(4). 400-404
Bi YM, Cammue BPA, Goodw in PH, et al. 1999. Resistance of Botrytis cinerea in cented geranium transformed with a gene encoding the antimicrobial protein A ce-AmP1. Plant Cell Rep. 18(4). 835-840
Biahoua A, Bormeau L 1999. Control of in vitro somatic embryogenesis of the spindle tree by the sugar type and the osmotic potential of the culturemedium. Plant Cell Reports. 35(4). 185-190
Bliffeld M. 1999. Genetic engineering of wheat for increased resistance to powdery mildew disease. Theoretical andApplied Genetics. 98 (67). 1079-1086
Cao H, Bow ling S A, Gordon A S, et al.. 1994. Characterization of anArabidopsis thaliana sis mutant that is non responsive to inducers of systemic acquired resistance. Plant Cell. 6(5). 1583-1592
Cao H, Glazebrook J, Clarke JD et al. 1997. The Arabidopsis NPRI gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell. 88(7). 57-63
Chern M S, Fitzgerald H A, Yadav R C, et al. 2001. A disease resistance pathway in rice similar to the mediated pathway in Arabidopsis Plant. 27 (2). 101-113.
Dantu-PK, Acconada M.C. and Mele E.. 1995. Adventitious shoot regeneration in gladiolus. Plant Cell Tissue and Organ Culture. 31(2). 123-128
Dantu-PK, Bhojwani-SS. 1992. In vitro corm formation and field evaluation of corm-derived plants of Gladiolus. Scientia Horticulturae. 61(12). 115-129
De Block M.. 1995. Factors influencing the culture and the Agrobacterium tumef aciens-mediated transformation of hybrid aspenand and poplar clones. Plant Cell Tissue Organ Culture. 43(23). 1110-1116
Diqiu Yu, Chunhong Chen and Zhixiang Chen. 2001. Evidence for an Important Role of WRKY DNA Binding Proteins in the Regulation of NPR1 Gene Expression . Plant Cell. 13(3). 1527-1540
Diqiu Yu, Yidong Liu, Baofang Fan, Daniel F.. 1997. Is the high basal level of salicylic acid important for disease resistance in potato? Plant Physiology. 115(5). 343-349
Dong X. 1998. Generation of broad-spectrum disease resistance by over expression of an essential regulatory gene in systemic acquired resistance. Pro Nat Acad Sci USA. 95(6). 6531-6536
Graves, ACF, Goldman, SL. 1987. Agrobacterium tumefaciens-mediated transformation of the monocot genus Gladiolus: detection of expression of T-DNA-encoded genes. Journal of Bacteriology. 169(6). 1745-1746
Gupta PK, Pullman GS, Timmis R. 1993. Forestry in the 21 st Century ～ The biotechnology of somatic embrygenesis. Bio Technology. 11(2). 454-459
Gupta PK, Pullman GS. 1991. Method for reproducing coniferous plants by somatic embryo genesisusing adscisic acid and somatic potential varition. U S Patent. 35(7). 5 -36
Hsia Blowers. 1996, 23(10). The factor of vitamins and inorganic micronutrients on callus growth from different explant of gladious. Journal of Plant Nutrition. 1407-1420
Hsia ChiNi, KorbanSS. 1996. Organogenesis and somatic embryogenesis in callus cultures of gladiolus hybrid. Plant Cell Tissue and Organ Culture. 44(6).
Iseli B. 1993. The N-terminal cysteine-rich domain of Tobacco Class I chitinase is essential for chitin binding but not for catalytic or activity. Plant Physiol. 103(7). 221-226
Jach G, Grnhardt B, Mundy J, et al. 1995. Enhanced quantitative resistance against fungal diseaseby combination expression of different barley antifungal proteins in transgenic tobacco. Plant Journal. 8(6). 97-109
Jayanti Sen, Koehorst H and Jong J. 1992. Adventitious shoot formation from in vitro leaf ex-plants of gladiolus Sci Hortic. 51(5). 223-235
Johanna Wagner. 1996. Changes in dormancy levels of Fraxinus excelsiorL embryos at different stages of morphological and physiological maturity . 10(7). 177-182
Kamada H, Tachikawa Y, Saitou T, et al. 1994. Heat stresses induction of carrot somatic embryo genesis. Plant Tissue Cult Lett. 11(3). 229-232
Kamo K and, Van Eck J. 1997. Effect of bialaphos and phosphinothricin on plant regeneration from long- and short-term callus cultures of Gladiolus. In Vitro Cell. Biol.-Plant 33(7). 180-183.
Kamo K, Blowers A, Smith F , Van Eck J. 1996. Genetic transformation of Gladiolus. Biotechnology in Agriculture and Forestry. Springer-Verlag. 38(7). 222-232
Kamo K, Blowers A, McElory D. 2000. Effect of the cauliflower mosaic 35S, actin, and ubiquitin promoters on uidA expression from a bar-uidA fusion gene in transgenic Gladiolus plants. In Vitro Cell Dev Biol Plant. 36(9). 13-20
Kamo K, Blowers A- Smith F. 1995. Stable transformation of Gladiolus, by particle gun bombardment of cormels. Plant Sci. 110(10). 105-111
Kamo K, Blowers A, Smith F. 1995. Stable transformation of Gladiolus using suspension cells and callus. J Am Soc Hort Sci. 120(4). 347-352
Kamo K. Blowers A. 1999. Tissue specificity and expression level of gusA under rolD, mannopine synthase and translation elongation factor 1 subunit a promoters in transgenic Gladiolus plants. Plant Cell Reports. 18(5). 809-815
Kamo K, Hammond J and Roh M. 1997. Transformation of Gladiolus for disease resistance. Journal of the Korean Society for Horticultural Science. 38(7). 188-193
Kamo K, McElroy D and Chamberlain D. 2000. Transforming embryogenic cells lines of Gladiolus with either a bar-uidA fusion gene or cobombardment. In Vitro Cell. Biol Plant. 36(4). 182-187
Kamo K., Gera A, Cohen J, Hammond J. 2004. Transgenic Gladiolus plants transformed with the Bean yellow mosaic virus coat protein gene in either sense or antisense orientations. Plant Cell. 23(5). 654-663
Kamo K. 1994. Effect of phytohormones on plant regeneration from callus of Gladiolus cultivar 'Jenny Lee.'. In Vitro Dev. Biol. 30(6). 26-31
Kamo K. 1995. A cultivar comparison of plant regeneration from suspension cells, callus and cormel slices of Gladuolus. In Vitro Cell Dev Biol Plant. 31(7). 113-115
Kamo K. 1996. Bean yellow mosaic virus coat protein and gusA gene expression in transgenic Gladiolus plants. Acta Hort. 447(8). 393-401.
Kamo K. 1997. Factors affecting Agrobacterium mediated gusA expression and opine synthesis in Gladuolus. Plant Cell Rep. 16(4). 389-392
Kamo K. 2001. Expression of the bar and uidA genes by Gladiolus following three seasons of dormancy. ActaHort. 560(3). 165-167
Kamo K. 2003. Long-term expression of the uidA gene in Gladiolus plants under control of either the ubiquitin, rolD, mannopine synthase, or cauliflower mosaic virus promoters following three seasons of dormancy. Plant Cell . 21(7). 797-803
Kamo, B.Jones, J.Castillon, J.Bolar, F.Smith, 2004. Dispersal and size fractionation of embryogenic callus increases the frequency of embryo maturation and conversion in hybrid tea roses. Plant Cell Rep. 22(6). 787-792
Kintzios S, Manos C, Makri O. 1999. Somatic embryogenesis from mature leaves of rose (Rosa spp.). Plant Cell Rep. 18(5). 467-472
Kintzios SE, Hiureas G, Shortsianitis. 1998. The effect of genotype on the induction, development and maturation of somatic embryos from various horticultural and ornamental species. Acta Horticulturae. 461(7). 427～432
Kong L, Yeung EC. 1994. Effects of ethylene and ethylene inhibitors on white spruce somatic embryo maturation. Plant Sci. 104(6). 71-80
Li XY, Huang FH, Gbur EE. 1998. Effect of basal medium , growth regulators and Phytagel concentration on initia tion of embryogenic cultures from immature zygotic embryos of loblolly pine (Pinus taeda L). Plant Cell Reports. 17(5). 298-301
LouH, KakoS. 1995. Role of high sugar concentration in inducing somatic embryogenesis from cucumber cotyledons. SciHortic, 6(8). 411-20
Ryals J, Weymann K, Lawton K et al. 1997. The Arabidopsis NINE protein shows homology to the mammalian transcription factor inhibitor LB. Plant Cell. 9(9). 425-439.
Shah J, Tsui F, Klessig DF. 1997. Characterization of a salicylic acid-insensitive mutant(sail) of Arabidopsis thaliana, identified in a selective screen utilizing the S.4inducible expression of the tms2 gene. At Plant-microbe Interact. 10(3). 69-78
Sumitra Sen. 1995. Two-step bud culture technique for a high frequency regeneration of Gladiolus corms. Scientia Horticulturae. 24 (4). 133-138
Takaichi M, Oeda K. 2000. Transgenic carrots with enhanced resistance against two major pathogens, Erysiphe heraclei and Alternaria dauci. Plant Sci. 153(6). 135-144
Takatsu Y, Nishizaw Y, Hibi T et al. 1999. Transgenic chrysanthemum expressing a rice chitinase gene shows enhanced resistance to gray mold ( Botrytis cinerea ) . Scientia Horticulturae. 82(7). 113-123
Vierheilig H, Alt M, Neuhaus JM. 1993. Colonization of transgenic Nicotiana sylvestris plants, expressing different forms of Nicotiana tabacum chitinase by the root pathogen Rhizoctonia solani and by the mycorrhizal symbiont Glomus mosseae . Molecular Plant-Microbe Interactions. 6(8). 261-264
Wan-Chi Lin, Ching-Fang Lu, Jia-Wei WU,. et al. 2004. Transgenic tomato plants expressing the Arabidopsis NPR1 gene display enhanced resistance to a spectrum of fungal and bacterial diseases. Transgenic Research. 13(5). 567-581
ZHANG Hong Zhi and CAI Xin Zhong. 2005. Nonexpressor of Pathogenesis related Genes 1(NPR1):A Key Node of Plant Disease Resistance Signaling . Chinese Journal Biotechnology. 4(21). 511-515.
Zhang Y, Ooritschnig S, Dong X et al. 2003. Again-of-function mutation in a plant disease resistance gene leads to constitutive activation of downstream signal transduction pathways in suppressor of nprl-1. Plant Cell. 15(5). 2636-2646
Zhang Y, Ooritschnig S, Dong X et al. 2003. Again-of-function mutation in a plant disease resistance gene leads to constitutive activation of downstream signal transduction pathways in suppressor of nprl-1. Plant Cell. 15(5). 2636-2646
车代弟，刘志洋．2004．唐菖蒲子球茎直接分化再生体系的建立．分子植物育种．2(4)．531-534
陈典，徐启江．2001．分蘖洋葱茎尖愈伤组织诱导及植株再生．园艺学报．28(4)．359-360
陈俊愉．1990．中国花经．上海文化出版社(上海)．610-611
程金水．2000．园林植物遗传育种学．中国林业出版社(北京)．340-350
崔凯荣，戴若兰．植物体细胞胚胎发生的分子生物学．北京：科学出版社．2000．59-62。
金淑梅，管清杰，罗秋香等．2006．苜蓿愈伤组织高频再生遗传和转化体系的建立．分子植物育种．4(4)．571-578。
韩继成，陈宁，陈霜莹．2006．不同处理对无病毒葡萄离体叶片遗传转化的影响． 分子植物育种．4(5)．670-674
郝建平，金竹萍，王永康等．2006．枣合子胚和体细胞胚发育过程的观察与比较．分子细胞生物学报．39(5)．423-426
雷玉明．2002．唐菖蒲腐烂病侵染规律和发病因子研究．植物保护．28(3)．36-38
李俊明主编．2000．植物组织培养教程．中国农业大学出版社．
李哲，黄霞，李筱菊．2005．大蕉花序愈伤组织的诱导及其体细胞胚发生．植物生理学通讯．41(5)．616-619
刘春朝，欧阳藩，郭仲琛．1997．唐菖蒲原球茎培养的研究．应用与环境生物学报．3(3)．208-212
刘非燕，郭达初，高卓．1997．唐菖蒲组织培养研究．浙江农业学报，9(1)．51-53
刘建利，张占路，吴燕民．2006．百脉根农杆菌快速高效遗传转化体系的建立．草业学报．15(3)．228-234
刘志洋，车代弟．2004．唐菖蒲愈伤组织的诱导及植株再生．分子植物育种．2(5)．695-698
马晖玲，卢欣石，曹致中．2006．紫花苜蓿基因转化的影响因素分析．草业学报．15(5)．95-99
王萍，王罡，季静等．2004．大豆体细胞胚胎发生与农杆菌介导的遗传转化遗传．26(5)．694-699
王关林，方宏筠主编．2002．植物基因工程．科学出版社(北京)．347-350
王俐，龙春林，赵燕．2001．唐菖蒲快速繁殖条件的优化．西南林学院学报． 21(3)：152-155
王晓春，刘尚前，季静．2004．影响大豆体细胞胚萌发率的因素．大豆科学．23(2)：151-153
王晓春，刘尚前，王罡．2006．大豆体细胞胚再生植株的研究．大豆科学．25(2)．149-153
吴小芹，吴少华．2000．鲜切花病虫害防治技术．科学技术文献出版社(北京)．112-118
谢海燕，毛碧增，单兰兰等．2004．狗牙根颖果胚性愈伤组织的诱导和胚性细胞的超微
结构及植株再生．植物生理与分子生物学学报．30(2)．209-215
明慧．2002．花卉病虫害防治．金盾出版社(北京)．95-97
徐启江，李玉花，陈典．2004．PP_(333)对分蘖洋葱试管苗增殖和生根的影响．植物生理学通讯．40(3)．256-260
义鸣放，王玉国，缪珊．2000．唐菖蒲．中国农业出版社(北京)．1-66
詹园凤，王广东．2006．大蒜体细胞胚胎发生的组织学研究．农业生物技术科学．22(1)．46-49
张秦英，陈俊愉．2004．不同激素对‘美人’梅叶片离体培养的影响及其细胞学观察．北京林业大学．26(5)．42-45
赵宏．1997，庸菖蒲球茎腐烂病原鉴定及其致病力．甘肃农业大学学报．6：163-166
赵云鹏，郭维明，王广东．2005．花烛不同愈伤组织解剖结构的观察．园艺学报．32(1)．60-67
周盈，林拥军，柴鑫莉．2006．根癌农杆菌介导的花魔芋遗传转化体系的研究．分子植物育种．4(4)．559-564
Attree SM, Pomeroy MK, Fowke LC. 1991. Enhanced maturation and desiccation tolerance of whitespruce (Picea glaucaMoench Voss) somatic embryos. Amm Bot. 57(8). 519-525
Babu-P, Chawla-HS. 2000. In vitro regeneration and Agrobacterium mediated transformation in gladiolus. Journal of Horticultural Science and Biotechnology. 75(4). 400-404
Bi YM, Cammue BPA, Goodw in PH, et al. 1999. Resistance of Botrytis cinerea in cented geranium transformed with a gene encoding the antimicrobial protein A ce-AmP1. Plant Cell Rep. 18(4). 835-840
Biahoua A, Bormeau L 1999. Control of in vitro somatic embryogenesis of the spindle tree by the sugar type and the osmotic potential of the culturemedium. Plant Cell Reports. 35(4). 185-190
Bliffeld M. 1999. Genetic engineering of wheat for increased resistance to powdery mildew disease. Theoretical andApplied Genetics. 98 (67). 1079-1086
Cao H, Bow ling S A, Gordon A S, et al.. 1994. Characterization of anArabidopsis thaliana sis mutant that is non responsive to inducers of systemic acquired resistance. Plant Cell. 6(5). 1583-1592
Cao H, Glazebrook J, Clarke JD et al. 1997. The Arabidopsis NPRI gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell. 88(7). 57-63
Chern M S, Fitzgerald H A, Yadav R C, et al. 2001. A disease resistance pathway in rice similar to the mediated pathway in Arabidopsis Plant. 27 (2). 101-113.
Dantu-PK, Acconada M.C. and Mele E.. 1995. Adventitious shoot regeneration in gladiolus. Plant Cell Tissue and Organ Culture. 31(2). 123-128
Dantu-PK, Bhojwani-SS. 1992. In vitro corm formation and field evaluation of corm-derived plants of Gladiolus. Scientia Horticulturae. 61(12). 115-129
De Block M.. 1995. Factors influencing the culture and the Agrobacterium tumef aciens-mediated transformation of hybrid aspenand and poplar clones. Plant Cell Tissue Organ Culture. 43(23). 1110-1116
Diqiu Yu, Chunhong Chen and Zhixiang Chen. 2001. Evidence for an Important Role of WRKY DNA Binding Proteins in the Regulation of NPR1 Gene Expression . Plant Cell. 13(3). 1527-1540
Diqiu Yu, Yidong Liu, Baofang Fan, Daniel F.. 1997. Is the high basal level of salicylic acid important for disease resistance in potato? Plant Physiology. 115(5). 343-349
Dong X. 1998. Generation of broad-spectrum disease resistance by over expression of an essential regulatory gene in systemic acquired resistance. Pro Nat Acad Sci USA. 95(6). 6531-6536
Graves, ACF, Goldman, SL. 1987. Agrobacterium tumefaciens-mediated transformation of the monocot genus Gladiolus: detection of expression of T-DNA-encoded genes. Journal of Bacteriology. 169(6). 1745-1746
Gupta PK, Pullman GS, Timmis R. 1993. Forestry in the 21 st Century ～ The biotechnology of somatic embrygenesis. Bio Technology. 11(2). 454-459
Gupta PK, Pullman GS. 1991. Method for reproducing coniferous plants by somatic embryo genesisusing adscisic acid and somatic potential varition. U S Patent. 35(7). 5 -36
Hsia Blowers. 1996, 23(10). The factor of vitamins and inorganic micronutrients on callus growth from different explant of gladious. Journal of Plant Nutrition. 1407-1420
Hsia ChiNi, KorbanSS. 1996. Organogenesis and somatic embryogenesis in callus cultures of gladiolus hybrid. Plant Cell Tissue and Organ Culture. 44(6).
Iseli B. 1993. The N-terminal cysteine-rich domain of Tobacco Class I chitinase is essential for chitin binding but not for catalytic or activity. Plant Physiol. 103(7). 221-226
Jach G, Grnhardt B, Mundy J, et al. 1995. Enhanced quantitative resistance against fungal diseaseby combination expression of different barley antifungal proteins in transgenic tobacco. Plant Journal. 8(6). 97-109
Jayanti Sen, Koehorst H and Jong J. 1992. Adventitious shoot formation from in vitro leaf ex-plants of gladiolus Sci Hortic. 51(5). 223-235
Johanna Wagner. 1996. Changes in dormancy levels of Fraxinus excelsiorL embryos at different stages of morphological and physiological maturity . 10(7). 177-182
Kamada H, Tachikawa Y, Saitou T, et al. 1994. Heat stresses induction of carrot somatic embryo genesis. Plant Tissue Cult Lett. 11(3). 229-232
Kamo K and, Van Eck J. 1997. Effect of bialaphos and phosphinothricin on plant regeneration from long- and short-term callus cultures of Gladiolus. In Vitro Cell. Biol.-Plant 33(7). 180-183.
Kamo K, Blowers A, Smith F , Van Eck J. 1996. Genetic transformation of Gladiolus. Biotechnology in Agriculture and Forestry. Springer-Verlag. 38(7). 222-232
Kamo K, Blowers A, McElory D. 2000. Effect of the cauliflower mosaic 35S, actin, and ubiquitin promoters on uidA expression from a bar-uidA fusion gene in transgenic Gladiolus plants. In Vitro Cell Dev Biol Plant. 36(9). 13-20
Kamo K, Blowers A- Smith F. 1995. Stable transformation of Gladiolus, by particle gun bombardment of cormels. Plant Sci. 110(10). 105-111
Kamo K, Blowers A, Smith F. 1995. Stable transformation of Gladiolus using suspension cells and callus. J Am Soc Hort Sci. 120(4). 347-352
Kamo K. Blowers A. 1999. Tissue specificity and expression level of gusA under rolD, mannopine synthase and translation elongation factor 1 subunit a promoters in transgenic Gladiolus plants. Plant Cell Reports. 18(5). 809-815
Kamo K, Hammond J and Roh M. 1997. Transformation of Gladiolus for disease resistance. Journal of the Korean Society for Horticultural Science. 38(7). 188-193
Kamo K, McElroy D and Chamberlain D. 2000. Transforming embryogenic cells lines of Gladiolus with either a bar-uidA fusion gene or cobombardment. In Vitro Cell. Biol Plant. 36(4). 182-187
Kamo K., Gera A, Cohen J, Hammond J. 2004. Transgenic Gladiolus plants transformed with the Bean yellow mosaic virus coat protein gene in either sense or antisense orientations. Plant Cell. 23(5). 654-663
Kamo K. 1994. Effect of phytohormones on plant regeneration from callus of Gladiolus cultivar 'Jenny Lee.'. In Vitro Dev. Biol. 30(6). 26-31
Kamo K. 1995. A cultivar comparison of plant regeneration from suspension cells, callus and cormel slices of Gladuolus. In Vitro Cell Dev Biol Plant. 31(7). 113-115
Kamo K. 1996. Bean yellow mosaic virus coat protein and gusA gene expression in transgenic Gladiolus plants. Acta Hort. 447(8). 393-401.
Kamo K. 1997. Factors affecting Agrobacterium mediated gusA expression and opine synthesis in Gladuolus. Plant Cell Rep. 16(4). 389-392
Kamo K. 2001. Expression of the bar and uidA genes by Gladiolus following three seasons of dormancy. ActaHort. 560(3). 165-167
Kamo K. 2003. Long-term expression of the uidA gene in Gladiolus plants under control of either the ubiquitin, rolD, mannopine synthase, or cauliflower mosaic virus promoters following three seasons of dormancy. Plant Cell . 21(7). 797-803
Kamo, B.Jones, J.Castillon, J.Bolar, F.Smith, 2004. Dispersal and size fractionation of embryogenic callus increases the frequency of embryo maturation and conversion in hybrid tea roses. Plant Cell Rep. 22(6). 787-792
Kintzios S, Manos C, Makri O. 1999. Somatic embryogenesis from mature leaves of rose (Rosa spp.). Plant Cell Rep. 18(5). 467-472
Kintzios SE, Hiureas G, Shortsianitis. 1998. The effect of genotype on the induction, development and maturation of somatic embryos from various horticultural and ornamental species. Acta Horticulturae. 461(7). 427～432
Kong L, Yeung EC. 1994. Effects of ethylene and ethylene inhibitors on white spruce somatic embryo maturation. Plant Sci. 104(6). 71-80
Li XY, Huang FH, Gbur EE. 1998. Effect of basal medium , growth regulators and Phytagel concentration on initia tion of embryogenic cultures from immature zygotic embryos of loblolly pine (Pinus taeda L). Plant Cell Reports. 17(5). 298-301
LouH, KakoS. 1995. Role of high sugar concentration in inducing somatic embryogenesis from cucumber cotyledons. SciHortic, 6(8). 411-20
Ryals J, Weymann K, Lawton K et al. 1997. The Arabidopsis NINE protein shows homology to the mammalian transcription factor inhibitor LB. Plant Cell. 9(9). 425-439.
Shah J, Tsui F, Klessig DF. 1997. Characterization of a salicylic acid-insensitive mutant(sail) of Arabidopsis thaliana, identified in a selective screen utilizing the S.4inducible expression of the tms2 gene. At Plant-microbe Interact. 10(3). 69-78
Sumitra Sen. 1995. Two-step bud culture technique for a high frequency regeneration of Gladiolus corms. Scientia Horticulturae. 24 (4). 133-138
Takaichi M, Oeda K. 2000. Transgenic carrots with enhanced resistance against two major pathogens, Erysiphe heraclei and Alternaria dauci. Plant Sci. 153(6). 135-144
Takatsu Y, Nishizaw Y, Hibi T et al. 1999. Transgenic chrysanthemum expressing a rice chitinase gene shows enhanced resistance to gray mold ( Botrytis cinerea ) . Scientia Horticulturae. 82(7). 113-123
Vierheilig H, Alt M, Neuhaus JM. 1993. Colonization of transgenic Nicotiana sylvestris plants, expressing different forms of Nicotiana tabacum chitinase by the root pathogen Rhizoctonia solani and by the mycorrhizal symbiont Glomus mosseae . Molecular Plant-Microbe Interactions. 6(8). 261-264
Wan-Chi Lin, Ching-Fang Lu, Jia-Wei WU,. et al. 2004. Transgenic tomato plants expressing the Arabidopsis NPR1 gene display enhanced resistance to a spectrum of fungal and bacterial diseases. Transgenic Research. 13(5). 567-581
ZHANG Hong Zhi and CAI Xin Zhong. 2005. Nonexpressor of Pathogenesis related Genes 1(NPR1):A Key Node of Plant Disease Resistance Signaling . Chinese Journal Biotechnology. 4(21). 511-515.
Zhang Y, Ooritschnig S, Dong X et al. 2003. Again-of-function mutation in a plant disease resistance gene leads to constitutive activation of downstream signal transduction pathways in suppressor of nprl-1. Plant Cell. 15(5). 2636-2646
Zhang Y, Ooritschnig S, Dong X et al. 2003. Again-of-function mutation in a plant disease resistance gene leads to constitutive activation of downstream signal transduction pathways in suppressor of nprl-1. Plant Cell. 15(5). 2636-2646