摘要
苜蓿(Medicago Sativa L.)是世界上分布最广泛的一种豆科多年生牧草,素有“牧草之王”的美誉。随着分子生物学的发展,传统农业正在向分子农业转变。植物生物反应器作为一种大规模重组蛋白的生产系统,已广泛运用于工业、农业尤其是生命科学以及医学制造领域。
与微生物发酵、动物细胞和转基因动物等生产系统相比,苜蓿作为一种生物反应器,为人类提供了一个更加安全和廉价的生产体系。它具有操作方便、便于大面积推广等许多潜在的优势。利用转基因苜蓿作为植物生物反应器生产工业酶制剂、疫苗、药用蛋白等的操作路线与现在的苜蓿转基因技术路线相同,即目的基因的克隆→载体构建→苜蓿外植体的转化→转基因植株再生;只需在得到转基因植株以后,再从转基因植株中进行疫苗、酶制剂、药用蛋白等的下一步提取工作。论文针对外源蛋白在转基因苜蓿植物组织中表达量低、转基因苜蓿的种质保存困难的问题进行研究。
本论文选用农杆菌株系GV3101,含gus/npt II的双相载体pCAMBIA2301和pBin9,以N4.4.2作为转化受体进行转化,在卡那霉素筛选压下,经过一系列的诱导培养,形成转基因植株。然后,通过GUS组织化学定位分析转基因植株中的GUS表达,并进一步通过PCR分子检测。经分析检测确定为阳性植株后,对转基因植株进行体细胞胚诱导,通过GUS荧光定量分析不同组织中的GUS表达,评价外源GUS在苜蓿体细胞胚中的积累。同时,对获得的7个转基因苜蓿株系进行体细胞胚的诱导,随后利用外源ABA和6种干燥剂处理来获得转基因苜蓿人工种子,考察不同株系转基因苜蓿人工种子的萌发率和遗传特性。以上研究为苜蓿体细胞胚作为植物反应器生产外源有用蛋白和转基因人工种质保存提供理论依据。主要结论如下:
1.进一步完善了苜蓿转化体系。以N4.4.2为转化受体,感染的子叶期苜蓿体细胞胚在75mg/L卡那霉素筛选压下,经过一系列诱导培养,最终获得转基因植株。然后,通过GUS组织化学定位分析检测转基因植株不同组织器官中的GUS表达,并进一步通过PCR和Southern杂交确定转基因的整合和转化率。结果证实:苜蓿次级体细胞胚的遗传转化法相对于叶柄、叶子和上胚轴等为外植体的常规的转化方法,缩短了苜蓿转化的时间;次级体细胞胚作为外植体的遗传转化方法大大提高了次级体细胞胚的诱导频率;它还提供了一个导入多基因到苜蓿植株中的方法。同时,这种方法也适用于别的转化效率低的一些苜蓿品种,拓宽了苜蓿遗传转化方法。
2.证明了苜蓿体细胞胚是最佳的外源蛋白表达材料,为苜蓿体细胞胚作为生物反应器生产外源蛋白提供理论依据。以N4.4.2紫花苜蓿品种叶柄为材料,进行农杆菌GV3101介导的苜蓿遗传转化。农杆菌株系GV3101包括双向载体pBin9。这个载体含有2个CaMV35s启动子调控下的gus/npt II融合基因。感染的叶柄外植体置于不同的转化培养基中培养,形成了适合于农杆菌介导的转基因体系。获得的转基因植株,采用组织化学定位分析GUS表达,进一步的PCR方法检测其中11个抗性株系,并对表现为阳性的转基因植株的不同组织器官进行GUS荧光定量分析。结果表明:GUS外源蛋白表达量在体细胞胚中明显高于根、茎和叶,是根、茎和叶的3~6倍;而在根、茎和叶中GUS表达量相似,没有明显的差异。
3.本试验建立了使用干燥的转基因苜蓿体细胞胚作为种质保藏的模式体系。将转基因苜蓿植株进行体细胞胚的诱导,随后,子叶期的体细胞胚在含有10mM ABA的BOi2Y培养基中培养14d,随后,体细胞胚依次放在相对湿度恒定的饱和溶液中:硫酸钾饱和溶液(87%)、碳酸钠饱和溶液(87%)、氯化钠饱和溶液(75%)、硝酸铵饱和溶液(63%)、四水硝酸钙饱和溶液(50%)、二水碳酸钾饱和溶液(43%)6种干燥剂中缓慢干燥,获得干化的转基因体细胞胚。干化的体细胞在MSO培养基中萌发,获得转基因苜蓿植株。结果表明:干化的体细胞胚和未干化的体细胞胚具有相似的萌发率,恢复的植株在形态上相似。经过Southern杂交分析遗传变异特性,源自干化的和未干燥的苜蓿体细胞胚恢复的植株具有相同的整合位点,研究证实了干燥的体细胞胚可以用于转基因苜蓿种质保藏的可行性。
4.35s CaMV是一种强启动子,通常用于转基因的组成型表达调控。在苜蓿转化试验中还发现,使用2个35s CaMV启动子调控GUS表达时,明显高于单个35s CaMV启动子调控的GUS表达。为了获得高水平的外源蛋白表达,可以使用双强35s CaMV启动子的载体。
Alfalfa (Medicago sativa L.) is important, perennial and leguminous forage and is widelydistributed in the world with the reputation of “King Forage”. With the development ofmolecular biology, the traditional farming is gradually transiting to the molecular farming. Theplant bioreactor has been extensively used as the scale production system of recombinantprotein in field of industry and agriculture, especially in life science and medicinalmanufacture.
Alfalfa as a bioreactor provided a more safe and cheap production system of recombinantprotein comparing with which used the microbe fermentation, animal cell or transgenicanimal as bioreactor. Alfalfa production system has more potential superiority such as easyoperation and easy spread to large area. The application of transgenic alfalfa as a plantbioreactor has the similar technical route with the operation process of current transgenicalfalfa in the production of vaccine, industry enzyme preparation and pharmaceutical protein.That is to say: Clone of interest gene→construction of vector→alfalfa transformation in vitro→Regeneration of transgenic alfalfa; Then we may extract the vaccine, enzyme preparation,pharmaceutical protein from these transgenic plants. This paper focused on the issue of lowexogenous protein expression in transgenic plants and difficult storage of transgenic alfalfagermplasm.
The Agrobacterium strain GV3101contained binary vector pCAMBIA2301and pBin19which had gus gene as reporter and nptⅡ gene as selectable marker. N4.4.2genotype(Medicago sativa) as donor plants was induced through series of medium under75mg/Lkanamycin selection. And we obtained the transgenic alfalfa plants. Then, GUS expression indifferent tissue of transgenic alfalfa was tested by GUS histochemical analysis. Further, thestable integration and transformation efficiency were tested by polymerase chain reaction. Toevaluate the accumulation of the exogenous GUS in somatic embryos, the somatic embryoswere induced from transgenic plantlets after the positive transgenic alfalfa plantlets wereconfirmed by GUS stain and polymerase chain reaction. And we compare the expression ofGUS in different transgenic alfalfa tissue (roots, stems, leaves and somatic embryos) by GUSfluorescence assay. At the same time, somatic embryos were induced from3transgenic alfalfa lines. Subsequently, the artificial transgenic alfalfa seeds were obtained by exogenousABA and6series of desiccators. The germination rate and genetic characteristics oftransgenic artificial alfalfa seeds was investigated. These studies provide the theoretical basisfor alfalfa somatic embryos used as bioreactor to produce exogenous pharmaceutical proteinand for artificial transgenic alfalfa used as germplasm storage. The followings are the majorconclusions:
1. The alfalfa transformation system was further improved. N4.4.2alfalfa genotype wasused as donor plants. The infected cotyledonary-stage embryos were induced through seriesof medium under75mg/L kanamycin selection. And we obtained the transgenic alfalfa plants.Then, GUS expression in different tissue of transgenic alfalfa was tested by GUShistochemical analysis. Further, the stable integration and transformation efficiency weretested by polymerase chain reaction and Southern blot hybridization. The result showed thatthe protocol of genetic transformation via secondary somatic embryos can shorten alfalfatransformation time than the protocol of genetic transformation via explants of petiole, leavesand hypocotyls and so on. The protocol significantly improved the induce frequency ofsecondary somatic embryos and provide a method of transferring multigene to alfalfa plants.At the same time, the protocol is also suited to those alfalfa genotypes which have lowtransformation efficiency to extend the method of genetic alfalfa transformation.
2. Alfalfa somatic embryos are the optimal materials of exogenous protein expressionwhich provides theoretical basis for alfalfa somatic embryos used as plant bioreactor toproduce recombinant protein. In this paper, petiole explants from N4.4.2alfalfa genotype astransformation materials were induced by Agrobacterium tumefaciens strain GV3101whichharbors the plasmid pBin9. pBin9vector contains gus/npt II fusion gene Both genes weredriven by double35s CaMV35S promoter. Infected petiole explants were cultured in differentmedia and the alfalfa tissue culture and transgenic system was developed. We tested11resistant Kanamycin alfalfa lines selected from the recovered transgenic plantlets by GUSstain and PCR. Moreover, we tested GUS activity expression in different tissue in transgenicalfalfa by GUS fluorometric measure in positive plantlets. The result showed that the amountof exogenous protein GUS expression in somatic embryos is significantly higher than in othertissue (roots, stems and leaves). The amount in somatic embryos is3~6folds than in roots,stems and leaves. However, the amount of GUS expression was no significant difference inroots, stems and leaves. Alfalfa somatic embryos can accumulate higher exogenous protein.
3. In this paper, we set up a germplasm preservation model system from desiccationsomatic embryos of transgenic alfalfa. The somatic embryos were induced original from thetransgenic pants and the cotyledonary-stage somatic embryos were cultured on a maturation medium supplemented with10mM abscisic acid for14d. Then somatic embryos weredesiccated in a series of desiccators K2SO4(RH87%), Na2CO3(RH87%), NaCl (RH75%), NH4NO3(RH63%), Ca (NO3)2.4H2O (RH51%), and K2CO3.2H2O (RH43%) over1week to slowly dry.Finally, the desiccation somatic embryos treated by the MSO medium were germinated totransgenic plantlets. Our result showed that the germination rate of transgenic somaticembryos is74.3%after desiccation. The recovery transgenic alfalfa posterity from desiccationsomatic embryos could express stable GUS. In addition, the plants produced were similarmorphology. Moreover, southern hybridization has confirmed that integration of the gene inrecovery plant was consistent in three different alfalfa lines. So these findings have proved thefeasibility of using desiccation somatic embryos as germplasm preservation method fortransgenic plants.
4.35s CaMV is a constitutive, strong promoter and is used for induce of constitutiveexpression in the transgenic plants. In this paper, the result show that GUS expression ishigher by double35s CaMV promoter than single one. To obtain higher exogenous proteinexpression, The vector with double35s CaMV promoter can be possible.
引文
陈德富,陈喜文,程炳嵩.1995.人工种子几个问题的探讨[J].山东农业大学学报,26(2):249-256
陈世勇.1993.外源基因导入作物的分子育种研究进展[J].山东农业科学,2:4-9
程霞英,杨宗岐.2005.转基因植物生产药用蛋白研究进展[J].生物技术,15(2):86-88
陈学平,朱显灵.1997.分子生物技术在烟草改良中的应用[J].安徽农学通报,31(1):50-52
迟君道,崔艳莉,卢华.2002.转基因作物种植及生产概况[J].黑龙江农业科学,2:30-31
丁绍欢,张明生,史梦娜.2011,植物人工种子技术研究进展[J].种子.30(3):59-66
范钦,邱国华,许新萍.2000.植物表达载体pKC-3的构建及大分子DNA连接策略[J].生物技术,10(4):1-3
范钦,许新萍,李宝健.2002.多个抗虫基因转化水稻两用系培矮-64S [J].实验生物学报,35(1):42-46
范亚军,陈朴,倪秀珍.2008.植物生物反应器瞬时表达外源蛋白的研究进展[J].长春师范学院学报,27(6):59-62
冯道荣,邱国华,许新萍.2001a.多基因植物表达载体的构建[J].西北植物学报,21(4):609-614
冯道荣,许新萍,李宝健.2001b.转多基因水稻植株的获得[J].植物生理学报,27(4):331-336
冯道荣,许新萍,邱国华.2000.多个抗病抗虫基因在水稻中的遗传和表达[J].科学通报,45(15):1593-1599
冯道荣,卫剑文,许新萍.1999.转多个抗真菌蛋白基因水稻植株的获得及其抗稻瘟病菌的初步研究[J].中山大学学报(自然科学版),38(4):62-66
傅荣昭,孙勇如,贾士荣主编.1994.植物遗传转化技术手册[M].北京:中国科学技术出版社
傅学政,朱薇,管天球.2006.我国红薯生产燃料乙醇的综合效益分析[J].湖南科技学院学报,27(11):183-185
耿德贵,孙勇如.2001.利用转基因植物生产口服疫苗的研究进展.生物技术[J].11(6):33-35
关荣伟,郭长虹,司亮.2006.叶绿体转基因植物---种新型生物反应器[J].植物学通报,23(3):294-301
顾垒,毕玉芬.2004.转基因苜蓿研究的现状和前景[J].草原和草坪,1:17-21
黄绍兴,吕德扬,邵嘉红,邵嘉红,李良材,陈英.1991.紫花苜蓿原生质体转基因植株再生[J].科学通报,36(17):13451347
黄文惠,刘自学.1995.概论苜蓿的分布和发展[J].中国苜蓿,2-7
黄治玲.2002.燃料乙醇产业的全球发展概况[J].化工管理,(3):34-35
焦瑞身.2001.展望即将到来的“分子农业”[J].生物工程学报,17:4
康杰芳,王喆之.2006.转基因植物生产药用蛋白的研究进展[J].现代生物医学进展,6(9):73-76
柯善强,桂耀林.1990.植物体细胞胚胎发生和人工种子,北京:科学出版社,35-42
李明,邱国华,许新萍.1999.含多种抗真菌病基因植物表达载体的构建[J].中山大学学报(自然科学版),38(5):67-71
李修庆.1990.植物人工种子研究[M].北京:北京大学出版社,1-12
黎万奎,陈幼竹,周宇.2003.肝片吸虫抗原基因转基因苜蓿再生的研究[J].四川大学学报(自然科学版),2:144-147
刘常金,吴定.2000.植物基因工程生产药用蛋白[J].安徽农业技术示范学院学报,14(1):51-53
刘晶莹,扬丽萍,范亚军.2008.植物生物反应器瞬时表达外源蛋白的新方法[J].中国遗传学第八次代表大会暨学术讨论会论文摘要汇编
刘铁男.2002.中国燃料乙醇产业发展[J].中国能源,3:6-10
刘翔,何国庆.2003.利用木素纤维素生产燃料乙醇的微生物代谢工程[J].粮油加工与食品机械,8:67-69
刘艳芝,王玉民,黄学林.2005.苜蓿组织培养体细胞胚发生体系的建立[J].草业科学,23(1):34-36.
卢雄斌,龚祖埙.1998.植物转基因方法及进展[J].生命科学,10(3):125-131
倪端祥,邓志龙,岑益群,蒋如敏,杨光孝,于静.1995.旱芹人工种子内胚状体的优化[J].上海农业学报,11(1):9-14.
倪娜娜,祝建波,陈创夫.2009.转基因植物疫苗的开发及其在动物传染病防治中的应用[J].畜牧与兽医,41(6):92-95
牛颜冰,史正文,王德富.2009.重组马铃薯X病毒注射番茄果实高效表达胸腺素α1[J].生物工程学报,25(4):537-541
秦小波,高继海,徐莺,张金平,邵彩霞,林莎,张淑文,江璐玎,李月琴,陈放.2008.麻疯树curcin启动子的分离及其在转基因烟草中的功能分析[J].植物学通报,25:407-414
任迎虹.2001.人工种子的若干理论问题与应用研究[J].西昌农业高等专科学校学报,1:33-36
沈黎明.1996.林生山黧豆抗旱生理指标的比较研究[J].中国草地,18(3):53-56
谭甜,季勤,窦秉德.2009.禽流感病毒H5HA基因在马铃薯中的表达[J].南京师大学报(自然科学版),32(3):104-108
汤绍虎,孙敏.1994.蔬菜人工种子研究[J].园艺学报,21(1):71-75
唐巍,欧阳藩.1996.马铃薯人工种子的研究[J].生物技术,6(5):11-13
童继平,孙林静,马忠友.1998.浅谈水稻超高产育种[J].生物学杂志,15(6):45-46
王志华,夏英武.1998.水稻农杆菌介导转化关键因子研究进展[J].生物技术,8(3):5-8
王国英,杜天兵,张宏.1995.用基因枪法将Bt毒蛋白基因转入玉米及转基因植物再生[J].中国科学,25(1):71-76
王文国,王胜华,陈放.2006.植物人工种子包被与储藏技术研究进展[J].种子,2:51-57
魏玉清,许兴.2003.植物转基因技术及其应用[J].宁夏农林科技,4:41-44
温志良.1999.重金属污染生物技术研究[J].环境科学动态,3:15-18
吴立东,桂耀林.1993.植物体细胞胚干化处理研究述评[J].植物学通报,10(1):22-27
许新萍,剑文,范云六.1999.用基因枪法转化籼稻胚性组织获得可育的转基因植株[J].植物学报,26(3):219-227
徐宜为,步志高.2001.转基因植物可饲(食)疫苗[J].中国兽医学报,6:14-17
薛红卫,卫志明.1997.通过PEG法转化甘蓝获得转基因植株[J].植物学报,39(1):28-33
谢鑫星,路扬,梁晶,马翠,何奕昆.高效农杆菌介导的紫花苜蓿遗传转化体系的建立[J].中国农业科技导报,2010,12(1):128134
杨蓉,朱睦元,周晴.2000.转人γIFN基因烟草的初步研究[J].浙江大学学报(理学版),27(4):464-468
杨振泉,刘巧泉,于恒秀.2004.转新城疫病毒融合蛋白基因水稻植株的获得[J].生物技术,14(1):10-12
张万军,王涛.2002.紫花苜蓿愈伤成苗高频再生体系的建立及其影响因子的研究[J].中国农业科学,35(12):1579-1583.
张小明,鲍根良,叶胜海,石春海.2002.作物人工种子的研究进展[J].种子,2:41-43
赵桂琴, Kaila R.2003. AMV和WCMV在转基因红三叶中的基因累集[J].草业学报,12(2):106-111
赵恢武.2000.干旱诱导性启动子驱动的海藻糖-6-磷酸合酶基因载体的构建及转基因烟草的耐旱性[J].植物学报,42(6):616-619
赵建聪,江婷,李维.2009.利用植物生物反应器表达胸腺素α1的研究[J].四川师范大学学报(自然科学版),32(1):112-117.
萨姆布鲁克等著,分子克隆实验指南(第二版)北京:科学出版社,1996
Akhtar N, Kumari N, Pandey S, Ara H, Singh M, aiswal U, Jaiswal V S.2000. Somatic embryogenesisin tropical fruit trees. Dordrecht: Kluwer,93-144
Antonietta G, Ahmad H I, Maurizio M, Alvaro S.2007. Preliminary research on conversion ofencapsulated somatic embryos of Citrus reticulata Blanco, cv. Mandarino Tardivo di Ciaculli [J]. Plant CellTiss Organ Cult,88(1):117–120
Angoshtari R, Tayakkol Afshari R, kalantari S, Omidi M.2009. Effects of abscisic acid on somaticembryogenesis and induction of desiccation telerance in Brassica napus [J]. Asian J Plant Sciences,8(4):276-284
Aquea F,Poupin M J, Matus J T, Gebauer M, Medina C, Arce-Johnson P.2008. Synthetic seedproduction from somatic embryos of Pinus radiata [J]. Biotechnol Lett,30:1847–1852
Arakawa T, Chong D K, Langridge W H.1998. Mucosal vaccines: nontoxic derivatives of LT and CTas mucosal adjuvants[J]. Nat Biotechnol,16(3):292-297
Austin S, Bingham E, Mathews D, Shahan M, Will J, Burgess R.1995. Production and fieldperformance of transgenic alfalfa (Medicago sativa L.) expressing alpha-amylase andmanganese-dependent lignin peroxidase [J]. Euphytica,85(1):381-393
Austin S, Bingham E T.1997.The potential use of transgenic alfalfa as a bioreactor for the p roductionof industrial enzymes. In: Biotechnology and the Improvement of Forage Legumes, UK: CAB International,409-424
Barton K A, Whitely H R, Yang N S.1987. Bacillus thuringiensis endotoxin expressed in transgenicNicotiana tabacum provides resistance to Lepidopteran insects [J]. Plant physiol,85:1103-1109
Bavage A D, Buck E, Dale P, Moyes C, Senior A.2002. Analysisof a backcross population from amulti-copy transgenic Brassica napus line [J]. Euphytica,124:333-340
Bevan, M.1984. Binary Agrobacterium vectors for plant transformation[J].Nucleic Acids Res,12:8711-8721
Bizily S P.1999. Phytoremediation of methylmercury pollution: merB expression in Arbidopsisthaliana confers resistance to organomercurials [J]. Proe Natl Acad Sci USA,96:6808-6813
Blaydes DF.1966. Interaction of kinetin and various inhibitors in the growth of soybean tissue [J].Physio Planta,19:753-758
Bouton J.2007. The economic benefits of forage improvement in the United States [J]. Euphytica,154(3):263-270
Carrillo C, Wigdorovitz A. Trono K.2001. Induction of a virus specific antibody response to food andmonth disease virus using the structural protein VP1expressed in transgenic potato [J]. Viral Immunol,14(1):49-57
Castanon S, Martin M S, Martin J M.1999. Immunization with potato expressing VP60proteinprotects against rabbit hemorrhagic disease virus [J]. J Virol,73(5):4452-4455
Castillo B.1998. Plant regeneration from encapsulated somatic embryos of Carica papaya L [J]. PlantCell Reports,17:172-176
Cervera M, Pin J A, Juarez J, Navarro L, Pena L.2000. A broadexploration of a transgenic populationof citrus: stability of gene expression and phenotype [J]. Theor appl Genet,100:670-677
Chabaud M, Larsonneau C, Marmouget C, Huguet T.1996.Transformation of barrel medic (Medicagotruncatula Gaertn.) by Agrobacterium tumfaciens and regeneration via somatic embryogenesis oftransgenic plants with the M tENOD1-nodulin promoter fused to the gus reporter gene [J]. Plant Cell Rep,15:305-310.
Chand S,Singh A K2004. Plant regeneration from encapsulated nodal segments of DalbergiasissooRoxb., a timber-yielding leguminous tree species [J]. Journal of Plant Physiology,161(2):237–243
Chong D K X.1997. Expression of the human milk protein b-casein in transgenic potato plants [J].Transgenic Res,6:289-296
Chong D K X, Langridge WHR.2000. Expression of fulllength bioactive antimicrobial humanlactoferrin in potato plants [J]. Transgenic Res,9:71-78
Chris R S, Dario B.2001. Plants as factories for technical materials [J]. Plant Physiology,125:168-171
Commandeur U, Twyman R M, Fisher R.2003.The biosafety of molecular farming in plant [J].Biotech Net,5:110-111
Dai Z Y, Hooker B S, Anderson D B, Thomas S R..2000. Improved plant-based production of E1endoglucanase using potato: expression optimization and tissue targeting [J]. Mol Breed,6:277-285
D’Aoust, M A.2004.Perennial plants as a production system for pharmaceuticals. In: Handbook ofPlant Biotechnology (Christou Pand Klee H, eds), Weinheim, Wiley-VCH
Dalta S K, Datta K, Soltanifar N.1992. Herbicide-resistant Indiarice plants from IRR1breeding lineIR72after PEG-mediated transformation of protoplasts [J]. Plant Mol Biol,20:619-629
Danso K E, Ford Llyod B V.2003. Encapsulation of nadal cuttings and shoot tips for storage andexchange of cassava germplasm [J]. Plant Cell Rep,21:718-725
Danie H, Streatfield S J, Wycoff K.2001. Medical molecular farming: production of antibodies,biopharmaceuticals and edible vaccines in plants [J]. Trends Plant Sci,6:219-226
Datta S K, Potrykus I.1989. Artificial seeds in barley: encapsulation of microspore-derived embryos[J]. Ther Appl Genet,77:820-824
De Buck S, J acobs A, Van Montagu M.1998. Agrobacterium tumefaciens transformation andcotransformation frequencies of Arabidopsis thaliana root explants and tobacoo protoplasts [J]. MP MI,11:449-457
De Marchis F, Bellucci M, and Arcioni S.2008. Agrobacterium-mediated genetic transformation ofalfalfa. In Handbook of New Technologies for Genetic Improvement of Legumes. CRC Press.29-43
Denbow D M, Grabau E, Lacy G, Kornegay E T, Russell D R, Umbeck P1998. Soybeans transformedwith fungal phytase gene improve phosphorus availability for broilers [J]. Poult Sci,77:878-881
De Neve M, De Buck S, J acobs A.1997.T-DNA integration patterns in co-transformed plant cellssuggest that T-DNA repeats originateform co-integration of separate T-DNAs[J]. The Plant J,11:15-29
Desai N S, Suprasannu P, Bapat V A.2004. Partial desiccation of embryogenous calli improves plantregeneratio in sugarcane (Saccharum spp.)[J]. J Plant Biotechnol,5:1-5
Desgagnes R, Laberge S, Allard G, Khoudi H, Castonguay Y, Lapointe J, Michaud R, and Vezina L P.1995. Genetic transformation of commercial breeding lines of alfalfa (Medicago sativa)[J]. Plant CellTissue Organ Cult,42(2):129-140
De Wilde C, Peters K, Jacobs A.2002. Expression of antibodies and Fab fragments in transgenicpotato plants: a case study for bulk production in crop plants [J]. Mol. Breed,9:2871-2882
Dus Santos M J, Wigdor A, Trono K.2002. A novel met hodology to develop a foot and mouthdisease virus (FMDV) peptide-based vaccine in transgenic plants [J].Vaccine,20(8):1141-1147
Duval Y, Engelmann F, Durand G T.1995. Somatic Embryogenesis and Synthetic Seed I:Biotechnology in Agriculture and Forestry. Berlin: Springer Verlag,335352
Finer J J, Vain P, Jones M W.1992. Development of the particle inflow gun for DNA delivery to plantcells[J]. Plant Cell Rep,11:323-328
Fiore M C, Trabace T and Sunseri F.1997. High frequency of plant regeneration in sunflower fromcotyledons via somatic embryogenesis [J]. Plant Cell Rep,16(5):295-298
Fischer R, Emans N.2000. Molecular farming of pharmaceutical proteins [J]. Transgenic Res,9:279-299
Fischhoff D A, Bowdish K S, Perlak F J.1987. Insect tolerant transgenic tomato plants [J].Biotechnology,5:807-813
Fitch M M, Manshardt R M, Gonsalves D.1992. Virus resistant papaya derived from tissuesbombarded with the coat protein gene of papaya ringspot virus [J]. Bio/Technology,10:1466-1472
Forreiter C, Kirschner M, Nover L.1997. Stable transformation of an Arabidopsis cell suspensionculture with firefly luciferase providing a cellular system for analysis of chaperone activity in vivo [J].Plant Cell,9:2171-2181
Frigerio Lorenzo, Vine N D. Pedrazzini E, Hein M B,Wang F, Ma J K C, Vitale A.2000. Assembly,Secretion, and Vacuolar Delivery of a Hybrid Immunoglobulin in Plants [J]. Plant Physiology,123:1483–1494
Furmanowa M, Sowińska D, Pietrosiuk A.1991. XI Carum carvi L.(caraway): in vitro culture,embryogenesis, and the production of aromatic compounds. In: Bajaj YPS (ed) Biotechnology inagriculture and forestry15, medicinal and aromatic plants III. Springer-Verlag, Berlin, Heidelberg,176–192
Gamborg O L, Miller R A, and Ojima K.1968. Nutrient requirements of suspension cultures ofsoybean root cells [J]. Exp Cell Res,50(1):151-158
Germana M, Micheli M, Chiancone B, Macaluso L, Standardi A.2011. Organogenesis andencapsulation of in vitro-derived propagules of Carrizo citrange. Citrus sinensis (L.); Poncirius trifoliata(L.)[J]. Plant Cell Tiss Organ Cult,78:1–9
Goto F.1999. Iron fortification of riceseed by the soybean ferritin gene [J]. Nature Biotechnology,17:282-286
Hasegawa I.1997. Genetic improvement of heavy metal tolerance in plants by transfer of the yeastmetallothionein gene (CUP)[J]. Plant and Soil,196:277-281
Hilder V A, Gatehouse A M R, Sheerman SE.1987. A novel mechanism of insect resistanceengineered into tobacco [J]. Nature,300:160-163
Hood E E, itcher D R, Maddock S, Meyer T, Baszczynski C, Bailey M, Flynn P, Register J,Marshall L, Bond D.1997. Commercial production of avidin from transgenic maize: characterization oftransformant, production, processing, extraction and purification [J]. Mol Breed,3:291-306
Hood E E.2002. From green plants to industrial enzymes [J]. Enzyme&Microbial Technol,30:279-283
Hood E E, Woodard S L, Horn M E.2002. Monoclonal antibody manufacturing in transgenic plants–myths and realities [J]. Curr Opin Biotechnol,13:630-635
Hood E E, Gelvin S B, Melchers L S, and Hoekema A.1993. New Agrobacterium helper plasmids forgene transfer to plants [J]. Transgenic Res,2(4):208-218
Horsch R B, Fry J E, Hofimann N L.1985. A simple and general method for transferring genes intoplants [J]. Science,227:1229-1231
Idrees L Z, Nasir A, Riazuddin S.2007. Indigernous production of synthetic seed in Daucus Carota [J].Pak J Bot,39(3):849-855
Ipekci Z, Gozukirmizi N.2011. Direct somatic embryogenesis and synthetic seed production fromPaulownia elongate [J]. Plant Cell Rep,22:16-24
Jaiswal H A U, Jaiswal V S.1999. Germination and plantlet regeneration from encapsulated somaticembryos of mango (Mangifera indica L.)[J]. Plant Cell Reports,19:166-170
Jefferson R A, Kavanagh T A, Bevan M W.1987.GUS fusion: β-glucuronidaseas a sensitive andversatile gene fusion marker in higher plants [J]. EMBO J,6:3901-3907
Kapusta J, Modelska A, Figlerowiez M.1999. A plant-derived edible vaccine against hepatitis Bvirus[J]. Faseb J,13:1796-1799
Karenlampi S.2000.Genetic engineering in the improvement of plants for phytoremediation of metalpolluted soil [J]. Environmental Pollution,107:225-231
Kevin N R.1998.Clean air and renewable fuels: the market for fuel ethanol in the US from1984to1993[J]. Energy Economics,20:325-345
Khan A G.2000. Role of plants, mycorrhizae and phytochelators in heavy metal contaminated landremediation [J]. Chemosphere,41:197-207
Khoudi H, Laberge S, Ferullo J M.1999. Production of a diagnostic monoclonal antibody in perennialalfalfa plants [J]. Biotechnology and Bioengineering,64(2):135-143
Kisung K, Yoram T, Pauline M.2003. Function and glycosylation of plant-derived antiviralmonoclonal antibody [J]. PNAS,100(13):8013-8018
Kitto S L, Janick J.1985. A citrus embryo assay to screen water-soluble resins as synthetic seed coats[J]. Hort Science,20(1):98-100
Klein T M, Wolf E D, Sandford J C.1987. Highly-velocity microprojectiles for delivering nucleicacidsinto living cells [J]. Nature,327:70-73
Konan E E, Durand-Gasselin T, Kouadio J Y.2006. A modeling approach of the in vitro conversion ofoil palm (Elaeis guineensis) somatic embryos. Plant Cell Tissue Organ Cult,84:99–112
Koncz C, Schell J.1986. The promoter of TL-DNA gene5controls the tissue-specific expression ofchimeric genes carried by a novel type of Agobacterium binary vector [J]. Mol Gen Genet,204:383-396
Korban S S, Krasnyanski S F, Buetow D E.2002. Foods as production and delivery vehicles forhuman vaccines [J]. Am Coll Nutr,21:212-217
Krens F H.1982. In vitro transformation of plant protoplast swith Ti-plasmid [J]. Nature,296:72.
Kumagai M H, Donson J D, Cioppa G.2000. Rapid high-level expression of glycosylated ricealphaamylase in transfected plants by an RNA viral vector [J]. Gene,245:169-174
Leelavathi S, Sunnichan V G, Kumria R, Vijaykanth G P, Bhatnagar R K, and Reddy V S.2004. Asimple and rapid Agrobacterium-mediated transformation protocol for cotton (Gossypium hirsutum L.):Embryogenic calli as a source to generate large numbers of transgenic plants [J]. Plant Cell Rep,22(7):465-470
Lee R W H, Strommer J, Hodgins D, Shewen P E, Niu Y, Lo R Y C.2001. Towards Development ofan Edible Vaccine against Bovine Pneumonic Pasteurellosis Using Transgenic White Clover Expressing aMannheimia haemolytica A1Leukotoxin50Fusion Protein [J]. Infection and Immunity,69(9):5786-5790
Levee V, Garin E, Klimaszewska K, and Seguin A.1999. Stable genetic transformation of white pine(Pinus strobus L.) after cocultivation of embryogenic tissues with Agrobacterium tumefaciens[J]. MolBreed,5:429-440
Lico C, Chen Q, Santi L.2008. Viral vectors for production of recombinant proteins in plants [J]. CellPhysiol,216(2):366-377
Lin L, Liu Y G, Xu X P.2003. Efficient linking andtransfer of multiple genes by a multigeneassembly and transformation vector system [J]. PNAS,100(10):5962-5967
Lodhi, M A, Ye G N, Weeden NF, Reisch BI.1994. A simple and efficient method for DNA extractionfrom grapevine cultivars and Vitis species [J]. Plant Mol Biol Reporter,12:6-13
Lopez S J, Kumar R R, Pius R K.2004. Agrobacterium tumefaciens-Mediated Genetic transformationin Tea (Camellia sinensis [L.] O. Kuntze)[J]. Plant Mol Biol Reporter,22:201-202
Loss A, Eib C, Harloff H J.2003. Polyester synthesis in transplastomic tobacco (Nicotiana tobacumL.): Significant contents of polyhydroxybutyrate are associated with growth reduction [J]. Plant Cell Rep,21,891-899
Malabadi RB, van Staden J.2005. Storability and germination of sodium alginate encapsulated somaticembryos derived from the vegetative shoot apices of mature Pinus patula trees. Plant Cell Tissue OrganCult,82:259–265
Mandal J, Pattnaik S, Chand P.2000. Alginate encapsulation of axillary buds of Ocimum americanumL.(hoary basil), Basilicum L.(sweet basil), Gratissimum L.(shrubby basil), and Sanctum L [J]. In VitroCell Dev Biol Plant,36(4):287–292
Mason H S, Haq T A, Clements J D.1998. Edible vaccine protects mice against Escherichia coliheat-labile enterotoxin (LT): potatoes expressing a synthetic LT2B gene [J]. Vaccine,16(13):1336-1343
Mason H S, Warzencha H, Mor T.2002. Edible plant vaccines: applications for prophylactic andtherapeuticmolecular medicine [J]. Trends in Molecular Medicine,8:324-329
Ma S W, Zhao D L.1997.Transgenic plants expessing autoatigens fed tomice to induce oral immunetolerance [J]. Nature Med,3:793-796
Mc Cabe D E, Swain W F, Martinell B J.1988. Starle transformation of soybean (Glycine max) byparticle acceleration [J]. Biotechnology,6:923-926
McGarvey P B, Hammond J, Diene M.1995. Expression of the rabies virus glycoprotein in transgenictomatoes [J]. Biotechnology,13:1484-1487
McKersie B D and Brown D C W.1996. Somatic embryogenesis and artificial seeds in forage legumes[J]. Seed Sci Res,6(3):109-126
Mckersie B D, Murnaghan J, Jones K S.2000. Iron-superoxide dismutase expression in transgenicalfalfa increase winter survivial without a detectable increase in photosynthetic oxidative stress tolerance[J]. Plant Physil,122:1427-1437
Merton L, Wullenms G L, Molendijk L.1979. In vitro transformation of cultured cells from Nicotianatobacum by Agrobacterium tumefaciens [J]. Nature,277:129-131
Meza T J, Kamfjord D, H kelien A M, Evans I, Godager L H, Mandal A, Jakobsen K S, Aalen R B.2001. The frequency of silencing in Arabidopsis thaliana varies highly between progeny of siblings andcan be influenced by environmental factors [J]. Transgenic Res,10(1):53-67
Michelle B, Francesca D M, and Sergio A.2007. Zeolin is a recombinant storage protein that can beused to produce value-added proteins in alfalfa (Medicago sativa L.)[J]. Plant Cell Tissue Organ Cult,90:85-91
Micheli M, Hafiz I A, Standardi A.2007. Encapsulation of in vitroderived explants of olive (Oleaeuropaea L. cv. Moraiolo): II. Effects of storage on capsule and derived shoots performance [J]. Sci Hortic,113(3):286–292
Moloney M.1995. The future of molecular farming. Canada: Plant Biotechnology Institute (PB I).National Research Council,36
Montague A, Ziauddin A, Lee R, Ainley W, and Strommer J.2007. High efficiencyphosphinothricin-based selection for alfalfa transformation [J]. Plant Cell Tissue Organ Cult,91(1):29-36
Montalbán A, De Diego N, Moncaleán P.2012. Enhancing initiation and proliferation in radiata pine
(Pinus radiata D. Don) somatic embryogenesis through seed family screening, zygotic embryo stagingand media adjustments [J]. Acta Physiol Plant,34:451–460
Moreira J R. and Goldemberg J.1999. Alcohol program [J]. Energy Policy,27(4):229-245
Mujib A, Samaj J.2006. Somatic embryogenesis. Berlin, Heidelberg
Murashige T, and Skoog F.1962. A revised medium for rapid growth and bioassays with tobacco tissuecultures [J]. Physio Planta,15(3):473-497
Murashige T,1978. The impact of plant tissue culture on agriculture [J]. Canada: University ofCalgary Alberta,15-26
Murphy M.1995. A Comparison of metallothionein gene expression and nonprotein thiols in tenArabidopsis ecotypes [J]. Plant Physiology,109:945-954
Naik S K, Chand P K.2006. Nutrient-alginate encapsulation of in vitro nodal segments ofpomegranate (Punica granatum L.) for germplasm distribution and exchange [J]. Sci Hortic,108(3):247–252
Nakashimade Y.1996. Efficient culture method for production of plantlets frim mechanically cuthorseradish hairy roots [J]. Journal of Fementation and Engineering,81:87-89
Nieves N, Zambrano Y, Tapia R, Cid M, Pina D, Castillo R.2003. Field performance of artificial
seed derived sugarcane plants [J]. Plant Cell Tiss Organ Cult,75:279-282
Nitzche W.1978. Immunochemical evidence that Abscisic Acid is produced by several species ofanthocerotae and marchantiales [J]. The bryologist,90(4):393-400
Ninkovi S, Milju-DuKic J, Vinterhalter B, Ne kovi M.2004. Improved transformation of alfalfasomatic embryos using a superbinary vector [J]. Acta Biol Cracoviensia Ser Bot,46:139-143
Nor Asmah H, Nor Hasnida H, Nashatul Zaimah N A, Noraliza A, Nadiah Salmi N.2010. Syntheticseed technology for encapsulation and regrowth of in vitro-derived Acacia hyrid shoot and axillary buds [J].African Journal of Biotechnology,40(10):7820-7824
Novotry M J.1984. Purification and properties of D-mannitol-phosphate dehydrogenase andD-glucitol-6-phosphate dehydrogenase from Escherichia coli [J]. Journal of Bacteriology,159:986-990
Peferoen M.1997. Progress and prospects for field use of bt genes in crops [J]. Trends inbiotechnology,15:173-177
Pintos B, Bueno M, Cuenca B, Manzanera J A.2008. Synthetic seed production from encapsulatedsomatic embryos of cork oak (Quercus suber L.) and automated growth monitoring [J]. Plant Cell TissOrgan Cult,95(2):217–225
Potera C.1999. Eplcyte produces antibodies in plants [J]. Genetic Engineering News,2:15
Preeti S, Pandey S, Bhattacharya A, Nagar P K, Ahuja P S.2004. ABA associatedbiochemical changes during somatic embryo development in Camellia sinensis (L.) Kuntze O [J]. PlantPhysiol,161:1269-1276
Radovi J, Sokolovi D, Markovi J.2009. Alfafa most importamt perennial forgage legume inanimal husbandry [J]. Biotechnology in Animal Husbandry,25:465-475
Rai M K, Jaiswal V S, Jaiswal U.2008. Encapsulation of shoot tips of guava (Psidium guajava L.) forshort-term storage and germplasm exchange [J]. Sci Hortic,118(1):33–38
Raymond W, Lee H, Strommer J.2001. Towards development of an edible vaccine against bovinepneumonic pasteurellosis using transgenic white clover expressing a mannheimia haemolytica A1leukotoxin50fusion protein [J]. Infection and Immunity,69(9):5786-5790
Redenbaugh K, Paasch B, Nichol J, Kossler M, Viss P, Walker K.1986. Somatic seeds: encapsulationof asexual plant embryos [J]. Bio/Technology,4:797-801
Redenbaugh K, Slade D, Viss P, Fujii J.1987. Encapsulation of somatic embryos in synthetic seedcoats [J]. Hort Science,20:803-809
Richter L J, Thanavala Y, Arntzen C J, Mason H S.2000. Production of hepatitis B surface antigen intransgenic plants for oral immunization [J]. Nat Biotechnol.18:1167-1171
Robinson C L.1999. A ferric-chelate reductase for iron uptake from soils [J]. Nature,397:694-697
Rugh C L.1998. Development of transgenic yellow for murcury phytoremediation [J]. NatureBiotechnology,23:925-928
Sala F, Rigano M M, Barbante.2003. Vaccine antigen production in transgenic plants: strateges, geneconstructs and perspectives [J]. Vaccine,21:803-808
Salt D E.1995. Phytoremediation: a novel strategy for removal of toxic metals from the environmentusing plants [J]. Biotechnology,13:468-478
Samac D A.1995. Strain specificity in transformation of alfalfa by Agrobacterium tumefaciens [J].Plant Cell Tissue Organ Cult,43(3):271-277
Sambrook J, Fritsch E E, Maniatis T.1989. Molecular Cloning: A Laboratory Manual,2nd ed., ColdSpring Harbor: Cold Spring Harbor Laboratory Press,126-138
Saruul P, Srienc F, David A.2002. Production of a biodegra dable plastic polymer, poly-B-hydroxybutyrate, in transgenic alfalfa [J]. Crop Sci,42:919-927
Saunders J W and Bingham E T.1972. Production of alfalfa plants from callus tissue [J]. Plant Sci,12(6):804-808
Schenk R U, and Hildebrandt A C.1972. Medium and techniques for induction and growth ofmonocotyledonous and dicotyledonous plant cell cultures [J]. Can J Bot,50(1):199-204
Schillberg S.2002. Antibody molecular farming in plants and plant cells [J]. Phytochem Rev,10:45-54
Schlessinger J.2000. Cell signaling byreceptor tyrosine kinases [J]. Cell,123:211-225
Senaratna T, McKersie B D,Bowley S R.1990. Artificial seeds of alfalfa (Medicago sativa L.).Induction of desiccation tolerance in somatic embryos [J]. In Vitro Cell Dev Biol Plant,26,85-90
Sghaier B, Kriaa W, Bahloul M, Jorrń Novo J, Drira N.2009. Effect of ABA, arginine and sucrose onprotein content of date palm somatic embryos [J]. Scientia Horticulturae,120:379-385
Sharma A.1992. Regeneration Dendrobium wardcamum warner (orchidaceae) from synthetic seeds[J]. Indian Journal of Experimental Biology,30(8):747-748
Shetty K, and McKersie B D.1993. Proline, thioproline and potassium mediated stimulation ofsomatic embryogenesis in alfalfa (Medicago sativa L.)[J]. Plant Sci,88(2):185-193
Singh A K, Chand S.2010. Plant regeneration from alginate encapulated somatic embryos ofDelbergia sissoo Roxb [J].Indian Journal of biotechnology,9:319-324
Singh S, Rai M, Asthana P, Pandey S, Jaiswal V S,Jaiswal U.2009. Plant regeneration fromalginate-encapsulated shoot tips of Spilanthes acmella L. Murr., a medicinally important and herbalpesticidal plant species [J]. Acta Physiologiae Plantarum,31(3):649–653
Somerville C R, Bonetta D.2001. Plants as factories for technical materials [J]. Plant Physiol,125:168-171
Southern E M.1975. Detection of specific sequences among DNA fragments separated by gelelectrophoresis [J]. Mol Biol,98:503-517
Srinnivas L, Ganapathi T R, Suprasanna P, Bapat V A.2006. Desiccation and ABA treatmentimproves conversion of somatic embryos to plantlets in banana (Musa SPP.) cv. Rasthal(iAAB)[J]. IndianJournal of biotechnology,5:521-526
Stoger, E, Vaquero C, Torres E, Sack M, Nicholson L, Drossard J, Williams S, Keen D, Perrin Y,Christou P.2000. Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies[J]. Plant Mol. Biol,42:583-590
Stoger E. Sack M, Fischer R, Christou P.2002a. Plantibodies: applications, advantages and bottlenecks[J]. Curr Opin Biotechnol,13:161-166
Stoger E, Sack M, Perrin Y, Vaquero C, Torres E, Twyman R M, Christou P and Fischer R.2002b.Practical considerations for pharmaceutical antibody production in different crop systems [J]. MolecularBreeding,9:149–158
Stone J M.1995. Plant protein kinase families and signal transduction [J]. Plant Physiol,108:451-458
Streatfield S J.2007. Approaches to achieve high level heterologous protein production in plants [J].Plant Biotechnology Journal,5:2-15
Sun J, Li W, Zhang H, Zhao J, Yin X, and Wang L.2009. Somatic embryogenesis and plantregeneration in glandless upland cotton (Gossypium hirsutum L.)[J]. Frontiers of Agriculture in China,3(3):279-283
Sun M, Qian K X, Su N.2003. Foot-and-mouth disease virus VP1protein fused with choler a toxin Bsubunit expressed in Chlamydomonas reinhardtii chloroplast [J]. Biot echnology Letters,25(13):1087-1092.
Sundararaj S G, Agrawal A, Tyagi R K.2010. Encapsulation for in vitro short-term storage andexchange of ginger (Zingiber officinale Rosc.) germplasm [J]. Sci Hortic,125(4):761–766
Tacket C O. Mason H S, Losonsky G, Clements J D, Levine M M, Arntzen C J.1998. Immunogenicityin humans of a recombinant bacterial-antigen delivered in transgenic potato [J]. Nat.Med,4,607-609
Tacket C O, Mason H S, Losonsky G.2000. Human immune responses to a novel Norwalk virusvaccine delivered in transgenic potatoes [J]. Infect Dis,82:302-305
Takahatta Y, Brown D C W, Keller W A, Kaizuma N.1993. Dry artificial seeds and desiccationtolerance induction in microspore-derived embryos of broccoli [J]. Plant Cell Tissue and Organ Cult,35:121-129
Taniguchi T, Ohmiya Y, Kurita M, Tsubomura M, Kondo T.2008. Regeneration of transgenicCryptomeria japonica D. Don after Agrobacterium tumefaciens-mediated transformation of embryogenictissue [J]. Plant Cell Rep,27:1461-1466
Tannourg M, Vintejoux C, Dereuddre J.1995. Cryopreservation of carnation shoot tips (Daintiescaryophyllus L.) by encapsulation dehydration [J]. Acta Botanica Gallica,142:415-424
Tetteroo FAA, Peters A H L J, Hoekstra F A.1995. ABA redues resiration and sugar metabolism indevelioing carrot embryoids [J]. Plant Physiol,145:477-482
Tian L, Wang H, Wu K, Latoszek-Green M, Hu M, Miki B,Brown D.2002. Efficient recovery oftransgenic plants through organogenesis and embryogenesis using a cryptic promoter to drive marker geneexpression [J]. Plant Cell Rep,20(12):1181-1187
Timbert R, Barbotin J N, Thomas D.1996. Effect of sole and combined pretreatments on reserveaccumulation, survival and germination of encapsulated and dehydrated carrot somatic embryos [J]. PlantScience Limerick,120(2):223-231
Tissa S.1995. Significance of the zygotic seed coat on quiescence and desiccation tolerance ofMedicago Sativa L. somatic embryos [J]. Plant Cell Reports,14:375-379
Tuholy T, Yu W, Bailey A.2000. Immunogenicity of porcine transmissible gastroenter it is virusspike protein expressed in plants [J]. Vaccine,18(19):2023-2028
Usha R, Rohll J B, Arntzen C J.1993. Expression of an animal virus antigenic site on the surface of aplant virus particle [J]. Virology,197:366-374
Vaeck M, Reynaerts A, Hofte H.1987. Transgenic plants protected from insect attack [J]. Nature,328:33-37
Vancanneyt G, Schmidt R, O’Conor-Sanchez A, Willmitzer L, Rocha-Sosa M.1990. Construction ofan intron-containing marker gene splicing of the intron in transgenic plants and its use in monitoring earlyevents in Agrobacterium-mediated plant transformation [J]. Mol Gen Genet,220:245-250
Varvara P G.2000.Increased ability of transgenic plants expressing the bacterial enzyme ACCdeaminase to accumulate Cd, Co,Cu, Ni, Pb and Zn [J]. Journal of Biotechnology,81:45-53
Vasil V, Castillo A M, Fromm M E.1992. Herbicide resistant fertile transgenic wheat plants obtainedby micro-project bombardment of regenerableem bryogenic callus [J]. Bio/Tech,10:667-674
Vengadesan G, Pijut P M.2009. Somatic embryogenesis and plante regeneration of northern red oak(Quercus rubra L.)[J]. Plant cell tissue organ culture,97:141-149
Vierling R A, Wilcox J R.1996. Microplate assay for soybean seed coat peroxidase activity [J]. SeedScience Technol,24:485-494
Von S, Margareta Z, Guido Z.1996. Ethanol from lignocellulosics: a review of the economy [J].Bioresource Technology,56:131-140
Wigdorovitz A, Carrillo C, Dus Santos M J.1999. Induction of a protective antibody response to footand mouth disease virus in mice following oral or parenteral immunization with alfalfa transgenic plantsexressing the viral structural protein VP1[J]. Virology,255(2):347-353
Witcher D R, Hood E E, Peterson D, Bailey M, Bond D, Kusnadi A, Evangelista R, Nikolov Z, WoogeC, Mehigh R.1998. Commercial production of β-glucuronidase (GUS): a model system for the productionof proteins in plants [J]. Mol Breed,4:301-312
Yang L, Ding J, Zhang C, Jia J, Weng H, Liu W, Zhang D.2005. Estimating the copy number oftransgenes in transformed rice by real-time quantitative PCR [J]. Plant Cell Rep,23:759-763
Yu J, Langridge W.2003. Expression of rotavirus capsid protein VP6in transgenic potato and its oralimmunogenicity in mice [J]. Transgenic Res,12:163-169.
Yusibov D C, Hooper S V, Spitsin N.2002. Expression in plants and immunogenicity of plantvirus-based experimental rabies vaccine [J]. Vaccine,20:3155-3164.
Zambryski P, Joos H, Genetello C.1983. Ti plasmid vector for the int roduction of DNA in to plantcells without alteration of their normal regeneration capacity [J].The MBOJ,2:2143-2150
Zare N, Valizadeh M, Tohidfar M, Mohammadi SA, Malboobi M A, and Habashi A A.2009. Selectionof regenerative genotypes from Iranian alfalfa cultivars [J]. Int J Food Agri Environ,7(2):567-572
Zeitlin L. Olmsted S S., Moench T R, Co M S, Martinell B J, Paradkar V M, Russell D R, Queen C,Cone R A, Whaley K J.1998. A humanized monoclonal antibody produced in transgenic plants forimmunoprotection of the vagina against genitalherpes [J]. Nat. Biotechnol,16:1361–1364
Zhang H, Huang Q, and Su J.2010. Development of alfalfa (Medicago sativa L.) regeneration systemand Agrobacterium-mediated genetic transformation [J]. Agri Sci China,9(2):170-178
Ziauddin, A, Lee R W H, Lo R, Shewen P, and Strommer J.2004. Transformation of alfalfa with abacterial fusion gene, Mannheimia haemolytica A1leukotoxin50-gfp: Response with Agrobacteriumtumefaciens strains LBA4404and C58[J]. Plant Cell Tissue Organ Cult,79(3):271-278
Ziegelhoffer T, Will J.1999. Expression of bacterial cellulase genes in transgenic alfalfa (Medicagosativa L.), potato (Solanum tuberosum L.), and tobacco (Nicotiana tabacum L.)[J]. Mol Breed,5:309-318