紫花苜蓿再生体系的建立及农杆菌介导的BADH基因转化研究
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摘要
紫花苜蓿(Medicago sativa)为生长面积最大,利用价值最高的优质牧草。如何进一步提高其耐盐碱抗旱特性,扩大其种植范围,是当前苜蓿生产面临的重要研究课题,基因工程的发展和应用为苜蓿抗盐育种开辟了一条新的途径。本研究探讨了和田、阿尔冈金、甘农1号、甘农3号、甘农4号、陇中、德福、牧歌、润不勒、三得利10个苜蓿品种组织培养程序,确定出再生能力最强的品种及外植体。利用农杆菌介导法转化BADH(甜菜碱醛脱氢酶)基因,建立了一套农杆菌介导苜蓿基因转化的优化体系,主要研究结果如下:
1.再生体系的建立
试验表明,苜蓿不同品种间再生能力差异较大,在试验中所采用的10个品种中,再生能力的强弱依次为:甘农4号﹥牧歌﹥陇中﹥三得利﹥阿尔冈金﹥德福﹥润不勒﹥甘农3号﹥甘农1号﹥和田。胚轴是紫花苜蓿最佳的愈伤诱导外植体材料,最佳诱导培养基是MS+2mg/L2,4-D+0.25mg/LKT+30g/L蔗糖,愈伤组织诱导的最佳品种是甘农4号。10个品种最适的分化培养基为MS+0.5mg/LKT+0.1mg/LNAA+20g/L蔗糖。最佳诱导生根的培养基为1/2MS+0.5mg/LNAA+15g/L蔗糖。综合考虑各品种再生能力的强弱,最后确定以甘农4号苜蓿作为基因转化受体。
2. BADH基因转化体系的建立
以侵染后甘农4号号外植体的抗性愈伤组织出愈率、抗性芽形成率以及抗性苗的获得为指标建立了转化体系。
以培养7天的甘农4号的胚轴为受体材料,预培养3天,用于转化的侵染时间为10 min,脱菌中使用500mg/L的羧苄青霉素,菌液浓度为OD6000.3～0.5,共培养3天,50mg/L Kan的选择压下筛选90多天。通过上述转化体系获得了5株抗卡那霉素的再生植株,经PCR检测,均表现为阳性。
Abstract: Alfalfa (Medicago sativa) is the biggest for the growth area and the highest quality forage grass. Development and application of genetic engineering opened up a new way for the alfalfa in resisting salt breeding. Tissue culture technique of ten alfalfa breeds(Hetian,A’ergangjin,Gannong NO.1,Gannong NO.3,Gannong NO.4,Longzhong,Muge,Defu,Runbule,Sandeli) was researched and the BADH gene was transferred to alfalfa by Agrobacterium mediated transformation in this paper. The main achievements of our rearch are following:
1) The establishment of regeneration systems. The test indecated that to different breeds,the regeneration ability isn't same.From stronge to low,the order is:GannongNO.4>Muge>Longzhong>Sandeli>A’ergangjin>Defu>Runbule>Gannong NO.3>Gannong NO.1>Hetian.To different explant,hypocotyl has the best ability to induce callus. The effective media for inducing callus was on MS supplemented with 2,4-D 2.0 mg·L-1 and KT 0.25mg·L-1 and sucrose 30g·L-1, the best breeds for inducing callus was Gannong NO.4 . The effective media for bud inducing was MS containing KT 0.5mg·L-1and NAA 0.1mg/L and sucrose 20g·L-1.The effective root inducing meida was on 1/2MS supplementedwith NAA0.5 mg·L-1 and sucrose 15 g·L-1.
Because of the high or low of the regeneration ability,the test conform Gannong NO.4 Alfalfa as the gene transform ingredients.
2) Transformation system of BADH gene establishment: 500 mg·L-1 Carb could restrained growing of agrobacterium effectively.According to we established transformation system:Cultivated 7 days hypocotyls as explants as mediator, preculture 2 days, 10 minutes infection time and OD6000.3～0.5, 3 days coculture. After more than 90 days selection 3 regenerated Kan-resistant plantlets were abtained. The result of PCR analysis stated that the detected Kanamycin resistant plants transgenic BADH gene were all positive.
1.再生体系的建立
试验表明,苜蓿不同品种间再生能力差异较大,在试验中所采用的10个品种中,再生能力的强弱依次为:甘农4号﹥牧歌﹥陇中﹥三得利﹥阿尔冈金﹥德福﹥润不勒﹥甘农3号﹥甘农1号﹥和田。胚轴是紫花苜蓿最佳的愈伤诱导外植体材料,最佳诱导培养基是MS+2mg/L2,4-D+0.25mg/LKT+30g/L蔗糖,愈伤组织诱导的最佳品种是甘农4号。10个品种最适的分化培养基为MS+0.5mg/LKT+0.1mg/LNAA+20g/L蔗糖。最佳诱导生根的培养基为1/2MS+0.5mg/LNAA+15g/L蔗糖。综合考虑各品种再生能力的强弱,最后确定以甘农4号苜蓿作为基因转化受体。
2. BADH基因转化体系的建立
以侵染后甘农4号号外植体的抗性愈伤组织出愈率、抗性芽形成率以及抗性苗的获得为指标建立了转化体系。
以培养7天的甘农4号的胚轴为受体材料,预培养3天,用于转化的侵染时间为10 min,脱菌中使用500mg/L的羧苄青霉素,菌液浓度为OD6000.3～0.5,共培养3天,50mg/L Kan的选择压下筛选90多天。通过上述转化体系获得了5株抗卡那霉素的再生植株,经PCR检测,均表现为阳性。
Abstract: Alfalfa (Medicago sativa) is the biggest for the growth area and the highest quality forage grass. Development and application of genetic engineering opened up a new way for the alfalfa in resisting salt breeding. Tissue culture technique of ten alfalfa breeds(Hetian,A’ergangjin,Gannong NO.1,Gannong NO.3,Gannong NO.4,Longzhong,Muge,Defu,Runbule,Sandeli) was researched and the BADH gene was transferred to alfalfa by Agrobacterium mediated transformation in this paper. The main achievements of our rearch are following:
1) The establishment of regeneration systems. The test indecated that to different breeds,the regeneration ability isn't same.From stronge to low,the order is:GannongNO.4>Muge>Longzhong>Sandeli>A’ergangjin>Defu>Runbule>Gannong NO.3>Gannong NO.1>Hetian.To different explant,hypocotyl has the best ability to induce callus. The effective media for inducing callus was on MS supplemented with 2,4-D 2.0 mg·L-1 and KT 0.25mg·L-1 and sucrose 30g·L-1, the best breeds for inducing callus was Gannong NO.4 . The effective media for bud inducing was MS containing KT 0.5mg·L-1and NAA 0.1mg/L and sucrose 20g·L-1.The effective root inducing meida was on 1/2MS supplementedwith NAA0.5 mg·L-1 and sucrose 15 g·L-1.
Because of the high or low of the regeneration ability,the test conform Gannong NO.4 Alfalfa as the gene transform ingredients.
2) Transformation system of BADH gene establishment: 500 mg·L-1 Carb could restrained growing of agrobacterium effectively.According to we established transformation system:Cultivated 7 days hypocotyls as explants as mediator, preculture 2 days, 10 minutes infection time and OD6000.3～0.5, 3 days coculture. After more than 90 days selection 3 regenerated Kan-resistant plantlets were abtained. The result of PCR analysis stated that the detected Kanamycin resistant plants transgenic BADH gene were all positive.
引文
[1]张静妮.苜蓿不同品种植株高效再生体系的建立及根癌农杆菌介导的sacB基因转化因素的分析[A].甘肃农业大学硕士论文.2005.
[2]刘小琳,王继峰,胡晓艳,等.根癌农杆菌介导的紫花苜蓿遗传转化体系的建立与优化[J].中国草地学报.2007,29(2):102-106.
[3]骆爱玲,刘家尧,马德钦,等.转甜菜碱醛脱氢酶基因烟草叶片中抗氧化酶活性增高[J].科学通报.2000,45( 18):1953-1956.
[4] Csonka L N, Physiological and genetic responses of bacteria to osmotic stress. Microbiol Rev.1998 ,53:121-147.
[5] Rhodes D, hanson A D,Quaternary ammonium and tertiary sulfonium compounds in higher plants. Annu Rev Plant Physiol Plant Mol Biol.1993,44:357-384.
[6]许雯,孙梅好,朱亚芳,苏维埃.甘氨酸甜菜碱增强青菜抗盐的作用[J].植物学报.2001,43(8):809- 814.
[7] Eltjo G M Meijier, Daniel C W Brown.Role of exogenous reduced nitrogen and sucrosein rapid high frequencysomatic embryogenesis in Medicago sativa[J].Plant Cell, Tissue and Organ Culture.1987,(10):11-19.
[8]刘明志.大叶紫花苜蓿愈伤组织原生质体再生植株[J].武汉植物学研究.1996, 14(4):329-333.
[9]刘淑兰,罗松,王天原,等.苜蓿组织培养及小定根芽分化蛋白质研究[J].北京农业大学学报.1993,(7):35-39.
[10]时永杰,周丽霞.几种豆科牧草的组织培养[J].四川草原.1997,(3):27-29.
[11]舒文华,耿华株,阎伦兴.紫花苜蓿胚轴愈伤组织培养与植株再生[J].草业科学.1993,10(3): 65-67.
[12]杨志宏,李波,贾秀峰,李红,付园园.低温胁迫筛选苜蓿抗寒突变体细胞[J].现代化农业.2004,(7):13-15.
[13]高翠萍,石凤翎,孙静.苜蓿雄性不育系组织培养技术的研究[J].中国草地学报.2006,28(5):29-32.
[14]李聪,熊德邵,耿华株.苜蓿愈伤组织再生植株的研究[J].中国草地.1989,6(6):51-56.
[15] Tony H H Chen,Janet Marowitch,B G Thompson.Genotypic effects on somatic embryogenesis and plant regeneration from callus cultures of alfalfa[J].Plant Cell, Tissue and Organ Culture.1987,(8):73-81.
[16]邓百万,张兆清,刘世贵.几种豆科牧草植物组织培养体细胞胚诱导研究[J].汉中师范学院学报.2000,18(1) : 72-78.
[17] Navak F J,KonecnaD.Somatic embryogenesis in callus and cell suspension culture of alfalfa (Medicago sativa L.)[J].Z.P flanzenphysiol.Bd.1982,105:279-284.
[18]肖荷霞,王瑛,紫花苜蓿愈伤组织诱导和分化的研究[J].河北农业科学.2007,11(1):77-81.
[19]杨起简,周禾,孙彦,等.紫花苜蓿的愈伤组织诱导及组织培养[J].北京农学院学报.2004,19(2):28-30.
[20]梁慧敏,黄剑,夏阳,王太明,孙仲序.苜蓿外植体再生系统的建立研究[J].中国草地.2003, 25(4):8-14.
[21]黄远新,玉永雄,胡艳.南方紫花苜蓿不同外植体离体培养的研究[J].中国草地.2003, 25(3):42-47.
[22]危晓薇,蔡丽娟,李仁敬.紫花苜蓿组织培养及其再生植株[J].新疆农业科学.1999,(2):73-75.
[23]金淑梅,管清杰,罗秋香,等.苜蓿愈伤组织高频再生遗传和转化体系的建立[J].分子植物育种.2006,4(4):571-578.
[24]葛军,刘振虎,卢欣石.紫花苜蓿再生体系研究进展[J].中国草地.2004,26(2):63-67.
[25]刘进远,吴庆余译.植物分子生物学试验指南[M].北京:科学出版社.2000.
[26]卢泳全,吴为人.水稻遗传转化研究进展[J].福建农林大学学报(自然科学版.2003,32(1):28-29.
[27]王英国,张宏,丁群星,等.几种玉米基因转移技术的研究及转基因植株的获得[J].生物工程学报.1996,12 (1): 45-49.
[28]张晓东,李冬梅,徐文英.等.用基因枪法将除草剂Basta抗性基因与小麦HMW谷蛋白亚基基因导入小麦获得转基因植株[J].华北农学报.1997,(1 ): 133-136.
[29] Fromm M.E, Taylor L.P, Wallbot V. Expression of genes transferred into monocot and dicot plant cells by electroporation .Proc.Natl.Acad.Sci.USA. 1985,(82):5824-5828
[30]李宝健,吴敏.以改建的农杆菌Ti质粒转化外源基因于单了叶植物花叶芋的研究[J].科学通报.1989(8):615-617.
[31] Ohta Y. High-efficiency genetic transformation via electroporation of maize by a mixture of pollen and exogenous DNA. Proc.Natl.Acad.Sci.USA. 1986, (83):715-719.
[32]王才林,朱镇,赵凌,等.用花粉管通道法将慈姑蛋白酶抑制剂基因(caps)到入水稻获得转基因植株[J].江苏农业学报.2005,21(4):245-248.
[33] Reich, T, J. et al., Biotechnology. 1986,4:1001-1004.
[34]周兴龙,杨青川,王凭青,吴明生,谢伟伟.苜蓿转基因研究进展[J].重庆大学学报(自然科学版).2005,4,28(4),126-130.
[35]张新春,庄炳昌,李自超.植物耐盐性研究进展[J].玉米科学.2002,10 (1):50-56.
[36] Winisov, I, DR Bastola. Transgenic overexpression of the transcription factor Alfinl enhances expression of the endogenous MsPRP2 gene in alfalfa and improves salinity tolerance of the plants[J]. Plant Physiol.1999,120:473-480.
[37] GUO D J, CHEN F, NOUEK. Down regulation of Caffeciacid 3-O-methyltransferase and Caffeoy I CoA 3-O-methyltransferase inTransgenic Alfalfa Impacts on Lignin structure and In plications for the Biosynthesis of G and S Lignin[J].The Plan t Cell.2001, 13: 73-88.
[38] Liu Q, Kasuga M, Sakuma Y, etal. Two transcription factors DREB1 and DREB2 with an DREBP/AP2 DNA-binding domain separate two cellular signal transduction pathways in drought and low temperature-responsive gene expression in Arabidopsis[J].Plant Cell. 1998,(10):1391-1406.
[39]李新国,张建霞,孙中海.植物抗寒基因工程研究进展(综述)[J].亚热带植物科学.2004,33(4):69-73.
[40]杜秀敏,殷文璇,张慧,等.超氧化物歧化酶( SOD)研究进展[J].中国生物工程杂志.2003, 23(1): 49-51.
[41]由继红,杨文杰,李淑云.紫花苜蓿抗寒性突变体的筛选[J].东北师范大学学报(自然科学版).1996,(2):84-87.
[42]李波,白庆武,马兰,等.苜蓿抗性变异细胞系的筛选[J].草业科学.2003,20(4):5-9.
[43] Mckersie B D, et al. Manipulating freezing tolerance in transgenic plants[J].Acta Physiologiae Plantarum.1997,19(4):17-19.
[44]苏颖,王丕武,董英山,王永志,王中伟.苜蓿生物技术研究进展[J].分子植物育种.2004,2(5):733-739.
[45] Donn G,Tischer E,Smith J A,et al. Herbicide resist ant alfalfa cell an example of gene amplication in plants. Mol Appl Gent.1984,2:621-635.
[46] D'Halluin K, Boteerman J, de Greef W. Engineering of herbicide-resistant alfalfa and evaluation under field conditions [J].Crop Sci.1990,(30):866-871.
[47]黄绍兴,吕德扬,邵嘉红,紫花苜蓿原生质体转基因植株再生[J].科学通报.1991,36(17): 1345-1348.
[48]刘艳芝,王玉民,刘莉等.Bar基因转化草原1号苜蓿的研究[J].草地学报.2004,12 (4):273-280.
[49]高必达.植物抗病基因工程研究进展[J].湖南农学院学报.1994,20(6):587-596.
[50] Strizhov N, Keller M,Mathur J,Koncz-Kalman Z, Bosch D,Prudovsky E, Schell J, Sneh B, Koncz C and Zilberstein A,1996,A synthetic crylC gene, encoding a Bacillus thuringiensis delta-endotoxin, confers Spodoptera resistance in alfalfa and tobacco. Proc. Natl. Acad. Sci..1996, 93 (26):15012-15017.
[51] Oliver M.J., Deom C.M., De B. K., et al .In vitro transcription and translation of cloned cDNA soding the 30-kDa protein gene of TMV[J]. Virology.1986,177:277-283.
[52] Hill K.K., Jarvis-Eagan N., Halk E.L., et al. The development of virus-resistant alfalfa, Medicago satival[J]. Biol. Tech. 1991, 9:373-377.
[53] Mizukami Y,HoumuraI,Takamizo T,et al .Production of transgenic alfalfa with chintinase gene(RCC2)[A] Lorne and Hamitton,Victoria:Abstracts and Interna tional Symposium Molecular Breeding of Forage Crops[C] .2000:105.
[54] Hipshind J D, Paiva N L. Constitutive accumulation of a resveratrol-glucoside in transgenic alfalfa increase resistance to Phoma medicaginis[J].Mol Plant Microb Interact. 2000,(13):551-562.
[55] Tuboly T, Yo W, Baliey A. et al. Immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants [J].Vaccine. 2000. 18(19):2023-2028.
[56] Dus Samosa M J, Wigdorovitz A,Trono K. Mouth disease virus (FMDV) peptide-based vaccine in transgenic plants [J].Vaccine. 20(7-8):1141-1147.
[57] Miflin, B. 2000. Crop improvement in the 21st century[J]. Exp. Bot. 51(342):1-8.
[58]孙雷心译.美加联手开发转基因苜蓿[J].生物技术通报.2001,1: 53.
[59] Todorovska E,S arid P,Atanasov A.Inheritance of kanamycin and ethionine resistance introduced in alfalfa( Medicago sativa L.)by gene transfer and cell selection[J].Biotechnology and Biotechnological Equipncent.1995,9(1):45-51.
[60] Moftah A E, Michel B E.The effect of sodium cboloride on solute potential and proline accumulation in soybean leaves[J].Plant Physiol.1997, 83: 238-240.
[61] Clenn E P, Brown J J, Blumwald E. Salt tolerance and crop potential of halophytes [J]. Crit Rev Plant Sci.1999, 18:227- 255.
[62] Yeo A R. Molecular biology of salt tolerance in the context of whole-plant physiology [J]. J Exp Bot.1998, 49:915- 929.
[63] Shinozaki K, Yamaguchi-Shinozaki K. Molecular responses to drought and cold stress [J]. Curr Opin Biotechn.1996, 7:161-167.
[64] Shinozaki K, Yamaguchi-Shinozaki K. Gene expression and signal transduction in water-stress response [J]. Plant Physiol.1997, 115:327- 334.
[65]侯彩霞,汤章城.细胞相容性物质的生理功能及其作用机制[J].植物生理学通讯.1999, 35 (1):1.
[66] Hall JL,et al.Evidence for the cytoplasmic locating of betaine in leaf cells of Suaeda maririma[J].planta,1978,140:59-62.
[67] Zhao Ke-Fu,et al.Effects of salinity on the contents of osmotica of monocotyledenous halophtytes and their contribution to osmotic adjustment[J].Acta Botanica Sinica.1999,41(12):1287-1292.
[68] Waisel Y Biology of halophytes [M.New York: Academic Press.l972.
[69] KUIPER P J C. Auctal cytokinin concerntration in plant tissue as an indicator for salt resistance in cereal [J]. Plant and Soi1,1990,123:243-250.
[70]赵可夫,李法曾.中国盐生植物[M].北京科学出版社.1999.
[71]陆静梅,朱俊义,李建东,等.松嫩平原4种盐生植物根的结构研究[J].生态学报.1998,18(3):335-337.
[72] Apse MP, Blumwald E. Engineering salt tolerance in plants[J]. Current Opinion In Biotechnology. 2002,13:146-150.
[73] Willekens H, Chamnongpol S, Davey M; Catalase is a sink for H202 and is indispensable for stress defense in C3 plants[J].EMBO J, 1997, 16: 4806-4816.
[74] Yamada S, et al.A family of transcripts encoding water channel proteins tissue-specific expression in the common ice plant[J]. The Plant Cell,1995,(7):1129-1142.
[75]张新春,庄炳昌,李自超.植物耐盐性研究进展[J].玉米科学. 2002,10 (1) : 50-56.
[76]张建锋,李吉跃,宋玉民,邢尚军,郗金标,马丙尧.植物耐盐机理与耐盐植物选育研究进展[J].世界林业研究.2003,16(2):16-21.
[77] Story R, Wyn Jones RG.Quaternary ammoniun compounds in plants inrelation to salt resistance.Phytochemistry[J].1977, 16:447.
[78]梁峥,骆爱玲.甜菜碱和甜菜碱合成酶[J].植物生理学通讯.1995,35(1):1.
[79]张士诚,高吉寅.外源甜菜碱对盐胁迫下小麦幼苗体内几种与抗逆能力有关物质含量及钾钠吸收和运输的影响[J].植物生理学通讯.2000,36(1):23.
[80]钟国辉,王建林.外源甜菜碱对NaCl胁迫下白菜叶片的保护效应[J].植物生理学通讯.1997,33(5): 333.
[81]郭岩,张莉,肖岗,等.甜菜碱醛脱氢酶基因在水稻中的表达及转基因植株的耐盐性研究[J].中国科学.1997,427( 2) : 151-153.
[82]许仕珍.紫花苜蓿不同再生体系的比较及农杆菌介导的AtNHX1基因转化研究[A].甘肃农业大学硕士论文,2006.
[83]曹孜义,刘国民,王蒂等.实用植物组织培养技术教程[M].甘肃:甘肃科学技术出版社,1996.
[84]马晖玲,卢欣石,曹致中.紫花苜蓿不同栽培品种植株再生的研究[J].草业学报.2004,13(6):99-105.
[85]胡静.甘农2号和甘农3号苜蓿再生体系的建立及抗旱耐盐基因转化的研究[A].甘肃农业大学硕士论文.2007.
[86]王关林,方宏均.植物基因工程[M].北京:科学出版社.2002.
[87]崔凯荣,戴若兰.植物体细胞胚发生的分了生物学[M].北京:科学出版社.2000.
[88]王姝杰,闫淑珍,李世访.紫花苜蓿下胚轴愈伤组织诱导及再生植株的研究[J].植物保护.2005,30(3):50-52.
[89]吕冬霞,曲长福.植物生长调节剂对愈伤组织培养的影响[J].北方园艺.2004,5:68.
[90]刘艳芝,王玉民,刘莉等.Bar基因转化草原1号苜蓿的研究[J].草地学报.2004,12 (4):273-280.
[91]马菊兰,张博.培养基、蔗糖和激素对苜蓿花药愈伤组织诱导的影响[J].新疆农业科学.2007,44( 6):839-844.
[92] Y. BAI and R.Qu. Factors influencing tissue culture responses of mature seeds and immature embryos in turf- type tall fescue[J].Plant Breeding.2001,(120):239-242.
[93]崔凯荣,邢更生,周功克,等.植物激素对体细胞胚胎发生的诱导与调节[J].遗传.2000,22(5):349-354.
[94]王慧中,周晓云,赵培洁,等.影响籼稻愈伤组织分化频率的几个因素[J].浙江农业大学学报.2000,28(3):240-242.
[95]钱瑾,刘发央,谢小冬,贾彪.紫花苜蓿高频植株再生体系的建立[J].甘肃农业大学学报.2007,40(1):77-81.
[96]胡静,马晖玲,等.甘农2号杂花苜蓿愈伤组织诱导及体细胞胚和芽分化的研究[J].甘肃农业大学学报.2007,42(4):87-91.
[97] Dus Samosa M J, Wigdorovitz A, Trono K. Mouth disease virus (FMDV) peptide-based vaccine in transgenic plants[J]. Vaccine. 2002,(7-8):1141-1147.
[98]吕德扬,范云六,俞梅敏,等.苜蓿高含硫氨基酸蛋白转基因植株再生[J].遗传学报.2000,27(4): 331-337.
[99]吕德扬,曹学远,唐顺学.紫花苜蓿外源基因共转化植株的再生[J].中国科学.2000,30(4):342-348.
[100]耿华珠,苜蓿耐盐鉴定初报[J].中国草地.1990,(2): 67-69.
[101]郑进,康薇,洪华珠.抗菌素在农杆菌介导植物转基因中的应用[J].林业科技开发.2006,20(3):8-11.
[102]吴关庭,胡张华,等.抗生素对高羊茅胚性愈伤组织生长与分化的影响[J].核农学报.2005,19(2): 88-91.
[103]王紫萱,易自力.卡那霉素在植物转基因中的应用及其抗性基因的生物安全性评价[J].中国生物工程杂志.2003,23(6):9-13.
[104] Van ber Krol R.A, M.Leon, B.Beld.et al. Flavonoid genes in petunia addition of a limited maker of gene copies m ay leadt oa suppressiono f gene expression[J].Biotechnology.1992,3(12):667-674.
[105]贾小明,樊军锋.影响农杆菌介导的河北杨遗传转化的因素[J].西北林学院学报.2006,21(5):102-105.
[106]陈晨,曹致中,贺顺姬,等.农杆菌介导的紫花苜蓿遗传转化体系的建立与优化[J].甘肃农业大学学报.2004,39(4):516-519.
[107]王强龙,王锁民,张金林,等.紫花苜蓿体细胞胚高频再生体系的建立[J].草业科学.2006,23(11):21-27.
[2]刘小琳,王继峰,胡晓艳,等.根癌农杆菌介导的紫花苜蓿遗传转化体系的建立与优化[J].中国草地学报.2007,29(2):102-106.
[3]骆爱玲,刘家尧,马德钦,等.转甜菜碱醛脱氢酶基因烟草叶片中抗氧化酶活性增高[J].科学通报.2000,45( 18):1953-1956.
[4] Csonka L N, Physiological and genetic responses of bacteria to osmotic stress. Microbiol Rev.1998 ,53:121-147.
[5] Rhodes D, hanson A D,Quaternary ammonium and tertiary sulfonium compounds in higher plants. Annu Rev Plant Physiol Plant Mol Biol.1993,44:357-384.
[6]许雯,孙梅好,朱亚芳,苏维埃.甘氨酸甜菜碱增强青菜抗盐的作用[J].植物学报.2001,43(8):809- 814.
[7] Eltjo G M Meijier, Daniel C W Brown.Role of exogenous reduced nitrogen and sucrosein rapid high frequencysomatic embryogenesis in Medicago sativa[J].Plant Cell, Tissue and Organ Culture.1987,(10):11-19.
[8]刘明志.大叶紫花苜蓿愈伤组织原生质体再生植株[J].武汉植物学研究.1996, 14(4):329-333.
[9]刘淑兰,罗松,王天原,等.苜蓿组织培养及小定根芽分化蛋白质研究[J].北京农业大学学报.1993,(7):35-39.
[10]时永杰,周丽霞.几种豆科牧草的组织培养[J].四川草原.1997,(3):27-29.
[11]舒文华,耿华株,阎伦兴.紫花苜蓿胚轴愈伤组织培养与植株再生[J].草业科学.1993,10(3): 65-67.
[12]杨志宏,李波,贾秀峰,李红,付园园.低温胁迫筛选苜蓿抗寒突变体细胞[J].现代化农业.2004,(7):13-15.
[13]高翠萍,石凤翎,孙静.苜蓿雄性不育系组织培养技术的研究[J].中国草地学报.2006,28(5):29-32.
[14]李聪,熊德邵,耿华株.苜蓿愈伤组织再生植株的研究[J].中国草地.1989,6(6):51-56.
[15] Tony H H Chen,Janet Marowitch,B G Thompson.Genotypic effects on somatic embryogenesis and plant regeneration from callus cultures of alfalfa[J].Plant Cell, Tissue and Organ Culture.1987,(8):73-81.
[16]邓百万,张兆清,刘世贵.几种豆科牧草植物组织培养体细胞胚诱导研究[J].汉中师范学院学报.2000,18(1) : 72-78.
[17] Navak F J,KonecnaD.Somatic embryogenesis in callus and cell suspension culture of alfalfa (Medicago sativa L.)[J].Z.P flanzenphysiol.Bd.1982,105:279-284.
[18]肖荷霞,王瑛,紫花苜蓿愈伤组织诱导和分化的研究[J].河北农业科学.2007,11(1):77-81.
[19]杨起简,周禾,孙彦,等.紫花苜蓿的愈伤组织诱导及组织培养[J].北京农学院学报.2004,19(2):28-30.
[20]梁慧敏,黄剑,夏阳,王太明,孙仲序.苜蓿外植体再生系统的建立研究[J].中国草地.2003, 25(4):8-14.
[21]黄远新,玉永雄,胡艳.南方紫花苜蓿不同外植体离体培养的研究[J].中国草地.2003, 25(3):42-47.
[22]危晓薇,蔡丽娟,李仁敬.紫花苜蓿组织培养及其再生植株[J].新疆农业科学.1999,(2):73-75.
[23]金淑梅,管清杰,罗秋香,等.苜蓿愈伤组织高频再生遗传和转化体系的建立[J].分子植物育种.2006,4(4):571-578.
[24]葛军,刘振虎,卢欣石.紫花苜蓿再生体系研究进展[J].中国草地.2004,26(2):63-67.
[25]刘进远,吴庆余译.植物分子生物学试验指南[M].北京:科学出版社.2000.
[26]卢泳全,吴为人.水稻遗传转化研究进展[J].福建农林大学学报(自然科学版.2003,32(1):28-29.
[27]王英国,张宏,丁群星,等.几种玉米基因转移技术的研究及转基因植株的获得[J].生物工程学报.1996,12 (1): 45-49.
[28]张晓东,李冬梅,徐文英.等.用基因枪法将除草剂Basta抗性基因与小麦HMW谷蛋白亚基基因导入小麦获得转基因植株[J].华北农学报.1997,(1 ): 133-136.
[29] Fromm M.E, Taylor L.P, Wallbot V. Expression of genes transferred into monocot and dicot plant cells by electroporation .Proc.Natl.Acad.Sci.USA. 1985,(82):5824-5828
[30]李宝健,吴敏.以改建的农杆菌Ti质粒转化外源基因于单了叶植物花叶芋的研究[J].科学通报.1989(8):615-617.
[31] Ohta Y. High-efficiency genetic transformation via electroporation of maize by a mixture of pollen and exogenous DNA. Proc.Natl.Acad.Sci.USA. 1986, (83):715-719.
[32]王才林,朱镇,赵凌,等.用花粉管通道法将慈姑蛋白酶抑制剂基因(caps)到入水稻获得转基因植株[J].江苏农业学报.2005,21(4):245-248.
[33] Reich, T, J. et al., Biotechnology. 1986,4:1001-1004.
[34]周兴龙,杨青川,王凭青,吴明生,谢伟伟.苜蓿转基因研究进展[J].重庆大学学报(自然科学版).2005,4,28(4),126-130.
[35]张新春,庄炳昌,李自超.植物耐盐性研究进展[J].玉米科学.2002,10 (1):50-56.
[36] Winisov, I, DR Bastola. Transgenic overexpression of the transcription factor Alfinl enhances expression of the endogenous MsPRP2 gene in alfalfa and improves salinity tolerance of the plants[J]. Plant Physiol.1999,120:473-480.
[37] GUO D J, CHEN F, NOUEK. Down regulation of Caffeciacid 3-O-methyltransferase and Caffeoy I CoA 3-O-methyltransferase inTransgenic Alfalfa Impacts on Lignin structure and In plications for the Biosynthesis of G and S Lignin[J].The Plan t Cell.2001, 13: 73-88.
[38] Liu Q, Kasuga M, Sakuma Y, etal. Two transcription factors DREB1 and DREB2 with an DREBP/AP2 DNA-binding domain separate two cellular signal transduction pathways in drought and low temperature-responsive gene expression in Arabidopsis[J].Plant Cell. 1998,(10):1391-1406.
[39]李新国,张建霞,孙中海.植物抗寒基因工程研究进展(综述)[J].亚热带植物科学.2004,33(4):69-73.
[40]杜秀敏,殷文璇,张慧,等.超氧化物歧化酶( SOD)研究进展[J].中国生物工程杂志.2003, 23(1): 49-51.
[41]由继红,杨文杰,李淑云.紫花苜蓿抗寒性突变体的筛选[J].东北师范大学学报(自然科学版).1996,(2):84-87.
[42]李波,白庆武,马兰,等.苜蓿抗性变异细胞系的筛选[J].草业科学.2003,20(4):5-9.
[43] Mckersie B D, et al. Manipulating freezing tolerance in transgenic plants[J].Acta Physiologiae Plantarum.1997,19(4):17-19.
[44]苏颖,王丕武,董英山,王永志,王中伟.苜蓿生物技术研究进展[J].分子植物育种.2004,2(5):733-739.
[45] Donn G,Tischer E,Smith J A,et al. Herbicide resist ant alfalfa cell an example of gene amplication in plants. Mol Appl Gent.1984,2:621-635.
[46] D'Halluin K, Boteerman J, de Greef W. Engineering of herbicide-resistant alfalfa and evaluation under field conditions [J].Crop Sci.1990,(30):866-871.
[47]黄绍兴,吕德扬,邵嘉红,紫花苜蓿原生质体转基因植株再生[J].科学通报.1991,36(17): 1345-1348.
[48]刘艳芝,王玉民,刘莉等.Bar基因转化草原1号苜蓿的研究[J].草地学报.2004,12 (4):273-280.
[49]高必达.植物抗病基因工程研究进展[J].湖南农学院学报.1994,20(6):587-596.
[50] Strizhov N, Keller M,Mathur J,Koncz-Kalman Z, Bosch D,Prudovsky E, Schell J, Sneh B, Koncz C and Zilberstein A,1996,A synthetic crylC gene, encoding a Bacillus thuringiensis delta-endotoxin, confers Spodoptera resistance in alfalfa and tobacco. Proc. Natl. Acad. Sci..1996, 93 (26):15012-15017.
[51] Oliver M.J., Deom C.M., De B. K., et al .In vitro transcription and translation of cloned cDNA soding the 30-kDa protein gene of TMV[J]. Virology.1986,177:277-283.
[52] Hill K.K., Jarvis-Eagan N., Halk E.L., et al. The development of virus-resistant alfalfa, Medicago satival[J]. Biol. Tech. 1991, 9:373-377.
[53] Mizukami Y,HoumuraI,Takamizo T,et al .Production of transgenic alfalfa with chintinase gene(RCC2)[A] Lorne and Hamitton,Victoria:Abstracts and Interna tional Symposium Molecular Breeding of Forage Crops[C] .2000:105.
[54] Hipshind J D, Paiva N L. Constitutive accumulation of a resveratrol-glucoside in transgenic alfalfa increase resistance to Phoma medicaginis[J].Mol Plant Microb Interact. 2000,(13):551-562.
[55] Tuboly T, Yo W, Baliey A. et al. Immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants [J].Vaccine. 2000. 18(19):2023-2028.
[56] Dus Samosa M J, Wigdorovitz A,Trono K. Mouth disease virus (FMDV) peptide-based vaccine in transgenic plants [J].Vaccine. 20(7-8):1141-1147.
[57] Miflin, B. 2000. Crop improvement in the 21st century[J]. Exp. Bot. 51(342):1-8.
[58]孙雷心译.美加联手开发转基因苜蓿[J].生物技术通报.2001,1: 53.
[59] Todorovska E,S arid P,Atanasov A.Inheritance of kanamycin and ethionine resistance introduced in alfalfa( Medicago sativa L.)by gene transfer and cell selection[J].Biotechnology and Biotechnological Equipncent.1995,9(1):45-51.
[60] Moftah A E, Michel B E.The effect of sodium cboloride on solute potential and proline accumulation in soybean leaves[J].Plant Physiol.1997, 83: 238-240.
[61] Clenn E P, Brown J J, Blumwald E. Salt tolerance and crop potential of halophytes [J]. Crit Rev Plant Sci.1999, 18:227- 255.
[62] Yeo A R. Molecular biology of salt tolerance in the context of whole-plant physiology [J]. J Exp Bot.1998, 49:915- 929.
[63] Shinozaki K, Yamaguchi-Shinozaki K. Molecular responses to drought and cold stress [J]. Curr Opin Biotechn.1996, 7:161-167.
[64] Shinozaki K, Yamaguchi-Shinozaki K. Gene expression and signal transduction in water-stress response [J]. Plant Physiol.1997, 115:327- 334.
[65]侯彩霞,汤章城.细胞相容性物质的生理功能及其作用机制[J].植物生理学通讯.1999, 35 (1):1.
[66] Hall JL,et al.Evidence for the cytoplasmic locating of betaine in leaf cells of Suaeda maririma[J].planta,1978,140:59-62.
[67] Zhao Ke-Fu,et al.Effects of salinity on the contents of osmotica of monocotyledenous halophtytes and their contribution to osmotic adjustment[J].Acta Botanica Sinica.1999,41(12):1287-1292.
[68] Waisel Y Biology of halophytes [M.New York: Academic Press.l972.
[69] KUIPER P J C. Auctal cytokinin concerntration in plant tissue as an indicator for salt resistance in cereal [J]. Plant and Soi1,1990,123:243-250.
[70]赵可夫,李法曾.中国盐生植物[M].北京科学出版社.1999.
[71]陆静梅,朱俊义,李建东,等.松嫩平原4种盐生植物根的结构研究[J].生态学报.1998,18(3):335-337.
[72] Apse MP, Blumwald E. Engineering salt tolerance in plants[J]. Current Opinion In Biotechnology. 2002,13:146-150.
[73] Willekens H, Chamnongpol S, Davey M; Catalase is a sink for H202 and is indispensable for stress defense in C3 plants[J].EMBO J, 1997, 16: 4806-4816.
[74] Yamada S, et al.A family of transcripts encoding water channel proteins tissue-specific expression in the common ice plant[J]. The Plant Cell,1995,(7):1129-1142.
[75]张新春,庄炳昌,李自超.植物耐盐性研究进展[J].玉米科学. 2002,10 (1) : 50-56.
[76]张建锋,李吉跃,宋玉民,邢尚军,郗金标,马丙尧.植物耐盐机理与耐盐植物选育研究进展[J].世界林业研究.2003,16(2):16-21.
[77] Story R, Wyn Jones RG.Quaternary ammoniun compounds in plants inrelation to salt resistance.Phytochemistry[J].1977, 16:447.
[78]梁峥,骆爱玲.甜菜碱和甜菜碱合成酶[J].植物生理学通讯.1995,35(1):1.
[79]张士诚,高吉寅.外源甜菜碱对盐胁迫下小麦幼苗体内几种与抗逆能力有关物质含量及钾钠吸收和运输的影响[J].植物生理学通讯.2000,36(1):23.
[80]钟国辉,王建林.外源甜菜碱对NaCl胁迫下白菜叶片的保护效应[J].植物生理学通讯.1997,33(5): 333.
[81]郭岩,张莉,肖岗,等.甜菜碱醛脱氢酶基因在水稻中的表达及转基因植株的耐盐性研究[J].中国科学.1997,427( 2) : 151-153.
[82]许仕珍.紫花苜蓿不同再生体系的比较及农杆菌介导的AtNHX1基因转化研究[A].甘肃农业大学硕士论文,2006.
[83]曹孜义,刘国民,王蒂等.实用植物组织培养技术教程[M].甘肃:甘肃科学技术出版社,1996.
[84]马晖玲,卢欣石,曹致中.紫花苜蓿不同栽培品种植株再生的研究[J].草业学报.2004,13(6):99-105.
[85]胡静.甘农2号和甘农3号苜蓿再生体系的建立及抗旱耐盐基因转化的研究[A].甘肃农业大学硕士论文.2007.
[86]王关林,方宏均.植物基因工程[M].北京:科学出版社.2002.
[87]崔凯荣,戴若兰.植物体细胞胚发生的分了生物学[M].北京:科学出版社.2000.
[88]王姝杰,闫淑珍,李世访.紫花苜蓿下胚轴愈伤组织诱导及再生植株的研究[J].植物保护.2005,30(3):50-52.
[89]吕冬霞,曲长福.植物生长调节剂对愈伤组织培养的影响[J].北方园艺.2004,5:68.
[90]刘艳芝,王玉民,刘莉等.Bar基因转化草原1号苜蓿的研究[J].草地学报.2004,12 (4):273-280.
[91]马菊兰,张博.培养基、蔗糖和激素对苜蓿花药愈伤组织诱导的影响[J].新疆农业科学.2007,44( 6):839-844.
[92] Y. BAI and R.Qu. Factors influencing tissue culture responses of mature seeds and immature embryos in turf- type tall fescue[J].Plant Breeding.2001,(120):239-242.
[93]崔凯荣,邢更生,周功克,等.植物激素对体细胞胚胎发生的诱导与调节[J].遗传.2000,22(5):349-354.
[94]王慧中,周晓云,赵培洁,等.影响籼稻愈伤组织分化频率的几个因素[J].浙江农业大学学报.2000,28(3):240-242.
[95]钱瑾,刘发央,谢小冬,贾彪.紫花苜蓿高频植株再生体系的建立[J].甘肃农业大学学报.2007,40(1):77-81.
[96]胡静,马晖玲,等.甘农2号杂花苜蓿愈伤组织诱导及体细胞胚和芽分化的研究[J].甘肃农业大学学报.2007,42(4):87-91.
[97] Dus Samosa M J, Wigdorovitz A, Trono K. Mouth disease virus (FMDV) peptide-based vaccine in transgenic plants[J]. Vaccine. 2002,(7-8):1141-1147.
[98]吕德扬,范云六,俞梅敏,等.苜蓿高含硫氨基酸蛋白转基因植株再生[J].遗传学报.2000,27(4): 331-337.
[99]吕德扬,曹学远,唐顺学.紫花苜蓿外源基因共转化植株的再生[J].中国科学.2000,30(4):342-348.
[100]耿华珠,苜蓿耐盐鉴定初报[J].中国草地.1990,(2): 67-69.
[101]郑进,康薇,洪华珠.抗菌素在农杆菌介导植物转基因中的应用[J].林业科技开发.2006,20(3):8-11.
[102]吴关庭,胡张华,等.抗生素对高羊茅胚性愈伤组织生长与分化的影响[J].核农学报.2005,19(2): 88-91.
[103]王紫萱,易自力.卡那霉素在植物转基因中的应用及其抗性基因的生物安全性评价[J].中国生物工程杂志.2003,23(6):9-13.
[104] Van ber Krol R.A, M.Leon, B.Beld.et al. Flavonoid genes in petunia addition of a limited maker of gene copies m ay leadt oa suppressiono f gene expression[J].Biotechnology.1992,3(12):667-674.
[105]贾小明,樊军锋.影响农杆菌介导的河北杨遗传转化的因素[J].西北林学院学报.2006,21(5):102-105.
[106]陈晨,曹致中,贺顺姬,等.农杆菌介导的紫花苜蓿遗传转化体系的建立与优化[J].甘肃农业大学学报.2004,39(4):516-519.
[107]王强龙,王锁民,张金林,等.紫花苜蓿体细胞胚高频再生体系的建立[J].草业科学.2006,23(11):21-27.