摘要
棉花不仅是主要的纤维作物,还是重要的油料和蛋白质资源,是世界上主要的经济作物之一。棉花生产稳定与否,直接关系到整个农业生产、农民收入和纺织工业的发展。因此,对棉花品种进行不断的改良和创新,培育出更多高产、优质、抗逆的新品种,成为棉花育种家们不断追求的目标。棉花组织培养是育种研究中的有效手段,可以通过各种生物技术手段加速育种进程,提高育种效率,创造新的育种材料,并可为现代分子育种和基因工程研究提供有效的实验技术手段。棉花组织培养工作起步较晚,而且棉花是被公认的体细胞胚胎发生困难和难于体外遗传操作的作物之一。因此,研究亚洲棉体细胞胚胎发生、棉花体细胞胚胎发生的起源以及遗传规律,以及从分子水平揭示棉花细胞脱分化现象等方面的工作,是进一步完善棉花转基因体系的重要途径。本研究的主要结果如下:
1对四十个亚洲棉品种进行了愈伤诱导,以及后期的分化调控处理,为建立亚洲棉再生体系积累了资料。所有的亚洲棉品种在2,4-D+KT,NAA+KT,IBA+KT三种常用植物激素组合条件下都容易诱导出愈伤,出愈率均为100%。但在不同激素组合上的愈伤状态显著不同,继代处理表明NK处理更适合于后期的分化调控处理,能够获得状态较好的愈伤。此后,通过改变培养方式、培养基组分,以及其他处理等方式或组合上述处理等方式进行分化调控,使用了近300种培养基,仅在卡宝品红染色时发现了部分类似于胚性愈伤的细胞,但未获得体细胞胚。
2对YZ_1进行了不同激素组合条件下的体细胞胚胎发生研究,建立了高效快速的再生体系;并利用组织切片的方法观察了体细胞胚胎发生的过程,表明不同植物激素处理条件下的体细胞胚胎发生方式是不同的。YZ_1在8种植物激素(包括不含植物激素)处理上均能通过体细胞胚胎发生方式获得再生植株。但不同处理间的体细胞胚胎发生能力存在显著差异,IBA+KT处理表现出最高的分化率,可以达到97.6%,是常用植物激素组合2,4-D+KT处理中分化率28.6%的3倍。同时观察发现,整个体细胞胚胎发生过程(从外植体接种到体细胞胚的出现)仅为33天,是目前棉花体细胞胚胎发生耗时最短的报道。YZ_1也能够在不含植物激素的MSB培养基上也能够获得分化。此外,组织学观察表明,2,4-D+KT和IBA+KT处理中的体细胞胚胎发生模式不同,胚性细胞团分别起源于初生形成层和皮层细胞,从一定程度上解释了IBA+KT处理能够快速促进体细胞胚胎发生的原因。
3通过构建陆海杂种F_1及F_2群体,以及8个不同再生能力品种的完全双列杂交,研究棉花体细胞胚胎发生遗传规律。选择容易再生的陆地棉品种Coker201与再生困难的海岛棉品种Pima90杂交获得F_1,利用不同植物激素组合诱导两个亲本和F_1进行体细胞胚胎发生,Pima90和F_1均不能体细胞胚胎发生;次年自交获得F_2种子,F_2个体经过半年多的诱导调控处理未见一个个体分化。随后,对容易再生的陆地棉品种Coker201,Coker312,Y668,YZ_1,再生困难的陆地棉品种TM-1,EK4及海岛棉品种Pima90和Pima3-79,进行完全双列杂交,获得了47个杂种F_1代种子,同样进行愈伤诱导和分化调控处理,仅有陆地棉之间的两个杂种获得了再生,且其对应的反交没有获得再生。随后,选择正反交均不能分化的F_1(YZ_1和EK4杂交获得)进行自交,获得的F_2群体进行体细胞胚胎发生遗传规律分析;仅有YZ_1作父本的F_2群体中有14个个体获得了胚性愈伤,且胚性愈伤状态各异,胚胎发生能力间也存在显著差异。表明体细胞胚胎发生能力是一种复杂的遗传性状,可能同时受细胞质和细胞核内多个基因的互作控制。
4构建细胞脱分化相关基因的SSH文库,对部分相关基因进行了表达分析,并提出了可能的SAM-dependent转甲基调控细胞脱分化的分子机制,部分相关基因进行了初步的功能验证。利用来自三种不同激素处理诱导6,12,24,72,168小时的下胚轴的cDNA作为tester,以下胚轴的切段作为driver,进行差异表达基因的差减和两轮PCR扩增后,获得棉花细胞脱分化相关基因的SSH差减文库。对反向Northern杂交中的差异表达明显的克隆进行测序,共有286个差异表达的cDNA克隆被测序,112个独立的EST被分离鉴定,且其中有40.2%的EST是第一次被分离鉴定,可能为新发现的棉花细胞初始脱分化相关基因。对相关基因的表达分析表明,GST在植物激素诱导初期的6-24小时内高度表达,PRPs在不同处理中优势表达并表现出不同的表达模式,表明它们可能与棉花细胞脱分化关系密切。此外,棉花SAM代谢途径中假定的GhSAMS,GhSAMDC,GhSAHH和GhAC03被鉴定,realtime-PCR分析表明,它们的表达水平在激素诱导初期出现两次明显的上升/下降过程,且GhSAMS和GhSAHH的转录表达水平表现出高度正相关,进一步推测高表达水平的GhSAMS可能与脱分化细胞重新进入细胞周期密切相关。组织形态学观察进一步表明,2,4-D处理条件下的部分细胞在72小时内完成脱分化和分裂过程,SAM-dependent转甲基途径可能通过两次转甲基活性的改变调控棉花细胞脱分化过程。此外,差异表达基因在不同处理中的表达模式表明了2,4-D和激动素之间存在着复杂的互作关系。
此外,我们借助Gateway技术构建了Extensin和PRPL的RNAi干涉表达载体以及SAMS、EF1A4、PRPL的超表达载体,并利用农杆菌转化方法转化YZ_1下胚轴。其中PRPL的RNAi转化的下胚轴分化率较对照明显降低,初步推测可能是PRPL的干涉影响正常的细胞脱分化进程,进而导致正常的体细胞胚胎发生进程受限。此外,三个超表达基因都已经获得了胚性愈伤和再生植株,PCR检测阳性转化率为72.73%。上述基因的准确功能还需要进一步的验证。
Cotton is not only one of the most important fiber crops, but also is the most important sources for oils and proteins industry, and is one of the most important economic crops in the world. The steady production of cotton directly affects the whole agricultural production, farmer income and textile industry. Therefore, durative improvement and innovation of cotton variety, including high yield, quanlity and strong anti-adversity competence, were the persistent objective of cotton breeding. Cotton tissue culture is common and effective method for cotton breeding, which accelerates the breeding procedure with various biotechnologies, and improves breeding efficiency, and provides an effective tool for modern molecular breeding and gene engineering. As we know, cotton tissue culture began lately than other crops, and it is accredited that cotton is one of the most diffcult crop for plant regeneration via somatic embryogenesis. Based on above, broadening materials for cotton gene engineering, investigating the genetic controlling and origin of embryogenic cells, and disclosing the moleculr mechanism of cellular dedifferentiation are important to further improve cotton gene engineering. Besides, those studies also help us realize somatic embryogenesis in various aspects. And the results are as follows:
1 Callus induction and embryogenic callus differentiation of 40 asian cotton (Gossypium arboreum L.) were investigated, which laid a solid foundation for establishment of regeneration system in asian cotton. Calli were easily induced under 2, 4-D + KT, NAA + KT, IBA + KT treatments of all varieties, and the rates of callus induction were 100%. The configuration of callus in different treatments were remarkably different, the subculture of calli in different treatments indicated NK treatment was more suitable for durative subculture, and calli could maintain a advantaged state. Subsequently, alteration of culture mode, medium component and other treatments, or combinations of above treatments were used for embryogenic callus differentiation, almost 300 different mediums were used and the somatic embryo still did not emerge, and only morphology of some cells like embryogenic cell were observed.
2 Somatic embryogenesis of YZ_1 in different phytohormone combinations were investigated, a more effective and rapid regeneration system was established; procedure of somatic embryogenesis with histological observation was observed, which suggested the patterns of somatic embryogenesis were distinct in different treatments. Plant regenerations via somatic embryogenesis were represented in the eight treatments. Somatic embryogenesis competence were distinct in different treatments, and the highest ratio of embryogenic calli presented in IBA+KT treatment was 97.6%, which was three times the ratio of embryogenic calli in 2, 4-D+KT treatment. Furthermore, the whole procedure of somatice embryogenesis (from explant inoculated on medium to emergence of somatic embryo) only took 33 days, it was the most short time used for somatic embryogenesis in cotton. Somatic embryogenesis was also represented in MSB without phytohormone. Besides, histological observation suggested the patterns of somatic embryogenesis in DK and IK treatment were different, embryogenic cells were originated from primary meristem and cortex, which partly explained somatic embryogenesis could be rapidly induced in IK treatment.
3 To study the genetic controll of embryogenic competence, cross between upland cotton and island cotton, and corresponding F_2 individuals, complete diallel cross with 8 different regenerative competence varities were constructed. Embryogenic variety Coker201(upland cotton) and non-embryogenic variety Pima90(island cotton) were selected for crossing, the somatic embryogenesis of the parents and F_1 were investigated in different phytohormone treatments, Pima90 and F_1 cloud not take somatic embryogenesis in those treatments. Next year, corresponding F_2 seeds was got, none of them took somatic embryogenesis after callus induction and differentiation treatment for more than six months. Subsequently, we designed complete diallel cross with embryogenic varieties, including Coker201, Coker312, Y668, YZ_1 and non-embryogenic varieties, including upland cotton TM-1, EK4 and island cotton Pima90, Pima3-79, and 47 F_1 seeds were acquired, callus induction and differentiation treatment were performed for somatic embryogenesis as above, only two hybrids between upland cotton regenetated plantlet, but the correponding anti-cross hybrids did not. Subsequently, cross and anti-cross hybrids of nonembryogenic F_1 (YZ_1 and EK4) was selected for selfcrossing, F_2 family was used to genetic analysis for somatic embryogenesis competence. Only 14 individuals from F_2 seeds with YZ_1 as male parent represented somatic embryogenesis, and the characterization of embryogenic calli from differnt individuals were distinct, the embryogenic competence were also remarkable. Therefore, somatic embryogenesis competence in cotton was a complicated characterization, which might be controlled by multi-genes from cytoplasm and nucleous.
4 SSH library related cellular dedifferentiation was constructed, expression analysis were preformed with partly important genes, and provided a putative molecular mechanism for cellular dedifferentiation with SAM-dependent transmethylation. Functional validations of some related genes were also performed. cDNA of explants of three different treatments sampled at 6 h, 12 h, 24 h, 48 h, 72 h and 168 h as tester and 0 h(hypocotyl) as driver, subtract especially expressed genes and twice PCR, SSH library were constructed. Especially expressed clones were picked out and sequenced, a total of 286 differential cDNA clones were sequenced.. Among these clones, 112 unique ESTs were isolated and identified, and 40.2% of the ESTs were first identified, which might mean new genes related to cellular dedifferentiation. Expression analysis of related genes was performed, GST was highly expressed from 6 to 24 h after induction with phytohormone treatment. PRPs were predominantly expressed and exhibited distinct expression patterns in different treatments, suggested they are closely related to cellular dedifferentiation in cotton. Besides, Putative GhSAMS, GhSAMDC, GhSAHH and GhACO3 involvement in SAM metabolism was identified in this library. The analysis of realtime-PCR showed that two remarkable increased expressions of the four SAM-related genes happened during the early phase of phytohormone induction, and that a highly positive correlation existed between GhSAMS and GhSAHH, which further indicated the highest expression level of GhSAMS might be associated with reentry into the cell cycle. The histological observations further showed that some cells accomplished cellular dedifferentiation and division within 72 h in 2, 4-D treatment, and cellular dedifferentiation might be regulated through two alterations in SAM-dependent transmethylation activity in cotton. In addition, the expression patterns of differential genes in different treatments disclosed the complicated interaction between 2, 4-D and kinetin.
Furthermore, we constructed RNAi vector of Extensin and PRPL, and overexpression vector of SAMS, EF1A4 and PRPL with Gateway technology, and then infected hypocotyls of YZ_1. The differentiation rate of PRPL RNAi infected hypocotyls was remarkablely lower than CK, which might be associated with interference of cellular dedifferentiation, and the normal procedure of somatic embryogenesis was affected subsequently. Besides, embryogenic calli and regenerated plantlets were acquired with infected hypocotyls with three overexpression vectors, positive rate of transformation was 72.73% with PCR detection, exact functions needed further certification.
引文
1.迟吉娜,李喜焕,王省芬,张桂寅,马峙英.棉花体细胞胚胎发生和植株再生的影响因素.棉花学报,2004,16(1):55-61
2.迟吉娜,马峙英,张桂寅.中国棉花体细胞植株再生的基因型分析.分子植物育种,2005,3(1):75-82
3.崔凯荣,戴若兰.植物体细胞胚发生的分子生物学[M].北京:科学出版社,2000
4.董合忠.不同基因型棉花下胚轴离体培养胚状体发生的研究.莱阳农学院学报,1991,8(2):97-101
5.董合忠,焦改丽,陈志贤.2,4-D,KT对棉花愈伤组织的诱导和体细胞胚胎发生的影响.华北农学报,1993,8(3):87-91
6.丰嵘,王清连,张宝红,余学科.棉花组织培养植株再生的基因型分析.西北农业学报,1997,6(2):27-30
7.高鹏,王国英,赵虎基,樊立,陶亚忠.抑制差减杂交法分离玉米幼苗淹水诱导表达基因.植物学报,2003,45(4):479-483
8.宫相忠,黄健.苜蓿组织培养体细胞胚发生的细胞学观察.青岛海洋大学学报,1997,27(4):504-507
9.贺雅婷,曲延英,孔庆平,严勇亮,尚爽,郑良玉,陈全家.海岛棉愈伤组织诱导及分化的影响因素初探.分子植物育种,2008,6(3):597-602
10.黄鑫,戴思兰,孟丽,郑国生.抑制性差减杂交(SSH)技术在分离植物差异表达基因中的应用.分子植物育种,2006,4(5):735-746
11.金双侠.棉花遗传转化体系的优化及突变体的创制.[博士学位论文].武汉:华中农业大学图书馆,2006
12.冷春旭,李付广,刘传亮.亚洲棉再生体系研究进展.棉花学报,2005,17(3):178-181
13.李中奎,刘成运,刘文平.水稻愈伤组织内部胚性细胞的形成及发育.植物研究,2001,21(1):62-64
14.李子银,陈受宜.环境因子胁迫下水稻翻译延伸因子1A基因的诱导表达.植物学报,1999,41(8):800-806
15.刘桂云,吴敬音,吴鹤鸣,鲍维钊,奚元龄.棉花组织培养中胚状体发生及细胞学观察.江苏农业学报,1986,2(1):25-32
16.刘华英,萧浪涛,鲁旭东,吴顺.柑橘体细胞胚发生的组织细胞学研究.果树学报,2004,21(4):311-314
17.陆文梁,诸颖杰.风信子再生雄蕊的小孢子发生和花粉发育的细胞学观察.植物学报,1991,33:343-349
18.吕复兵,张献龙.陆地棉原生质体培养与植株再生.华北农学报,1999,14(1): 73-78
19.孙世孟,徐丽娟,何忠诚,姜伟.玉米胚性愈伤组织发生频率及其遗传规律的研究.莱阳农学院学报,1995,12(3):173-176
20.田振东,柳俊,谢从华.利用抑制差减杂交技术分离马铃薯晚疫病抗性相关基因.遗传学报,2003,30(7):597-605
21.王琦,张丕方.斑叶海棠薄层培养中细胞无丝分裂及2,4-D的特殊效应的研究.西北植物学报,1989,9(1):1-5
22.王喆之,李克勤,张大力,郝联芳,郭德志,刘全宏,肖娅萍,张苏锋.陆地棉胚性愈伤组织变异及高频胚胎发生.植物学报,1994,36(5):331-338
23.王武,张献龙.油菜素内酯对陆地棉体细胞胚胎发生的影响.植物生理学通讯,1994,28(1):15-18
24.亓建飞,李付广,张朝军,刘传亮.植物组织培养中畸形苗发生机理的研究进展.棉花学报,2004,16(4):243-248
25.吴燕,周嫦.萱草幼嫩花粉原生质体培养启动细胞分裂的超微结构研究.植物学报,1992,34(1):20-25
26.夏铭,王首锋,王小兵,李海波,吴平.利用抑制性扣除杂交技术克隆水稻磷饥饿诱导基因.植物学报,2003,45(6):736-741
27.谢德意,金双侠,郭小平,张献龙.长江和黄河流域棉区棉花品种体细胞胚胎发生和植株再生比较研究.作物学报,2007,33(3):394-400
28.许文亮,黄耿青,王秀兰,邰付菊,汪虹,李学宝.两个棉花HYPRP基因的分子鉴定和初步表达分析.作物学报,2007a,33(7),1146-1153
29.许文亮,黄耿青,王秀兰,汪虹,李学宝.一类新的编码PRPs基因的分离及其在棉花纤维等组织细胞中的表达.生物化学与生物物理进展,2007b,34(5):509-517
30.余迪求,洪维廉,陈睦传,汪德耀.甜菊叶肉细胞脱分化过程中超微结构的研究.植物学报,1993,35(7):499-505.
31.张宝红,李秀兰,李凤莲,李付广,王武.基因型在棉花花药培养中的效应.棉花学报,1994,6(增刊):36-38
32.张宝红,李秀兰.TDZ在棉花组织培养中的效应研究.棉花学报,1994,6(2):78-82
33.张宝红,刘方,刘志红,王红梅,姚长兵.外源激素对棉花体细胞胚胎发生及发育的调控作用 棉花学报,2000,12(1):17-21
34.张寒霜,李俊兰,高鹏,王海波.低酚陆地棉的体细胞胚胎发生和植株再生.河北农业大学学报,1999,22(1):9-12
35.张家明,孙雪飘,郑学勤,张献龙,赵燕,刘金兰,孙济中.陆地棉愈伤诱导及胚胎发生能力的遗传分析.中国农业科学,1997,30(3):36-43
36.张林,服部一三.水稻愈伤组织增殖力的遗传研究.华南农业大学学报(自然科 学版),2003,24(4):44-47
37.张丕方,王琦.烟草薄层培养中细胞早期动态的研究.植物学报,1989,31(6):422-426
38.张鹏,傅爱根,王爱国.AgNO3在植物离体培养中的作用及可能的机制.植物生理学通讯,1997,33(5):376-379
39.张文胜,张宝红,李秀兰,陈光荣,李凤莲,金玻,李学宝.糖源在棉花愈伤组织诱导、胚胎发生与发育中的作用.江西农业大学学报,1997,19(3):34-39
40.张献龙,孙济中,刘金兰.陆地棉体细胞胚胎发生与植株再生.遗传学报,1991,18(5):461-467
41.张新英,黄亚斌,尹光初.离体培养下大豆体细胞胚胎发生的组织学研究.植物学报,1992,34(3):214-218
42.中国农业科学院棉花研究所.中国的亚洲棉[M].北京:农业出版社,1989.124.
43.周大云,张宝红,刘方,刘志红.一种新型的棉花体细胞胚胎发生的快速诱导法.生命科学研究,2000,4(3):237-242
44.周小凤,张碧瑶,刘冠泽,高巍,余渝,邓福军,李保成,孔宪辉,张献龙,金双侠.新疆棉花高体细胞胚胎发生能力基因型的筛选.分子植物育种,2007,5(6):819-826
45.祝水金,季道藩.色素腺体和棉酚在陆地棉体细胞培养中的作用.科学通报,2001,46(16):1380-1384
46.Abe T,Futsuhara Y.Diallel analysis of callus growth and plant regeneration in rice seed-callus.Jpn J Genet,1991,66:129-140
47.Albrecht V,Ritz O,Linder S,Harter K,Kudla J.The NAF domain defines a novel protein-protein interaction module conserved in Ca2+-regulated kinases.EMBO J,2001,20:1051-1063
48.Aristarkhov A,Eytane E,Moghe A,Admon A,Hershko A,Ruderman J V.E2-C,a cyclin-selective ubiquitin carrier protein required for the destruction of mitotic cyclins.Proc Natl Acad Sci USA,1996,93:4294-4299
49.Armstron G C L,Green C E,Phillips R L,Stucker R E.Genetic control of plant regeneration from maize tissue culture.Maize Genet Cooper Newsl,1985,59:92-93
50.Asiegbu F O,Nahalkova J,Li G.Pathogen - inducible cDNA from the interacton of the root rot fungus Heterobasidion annosum with Scotspine.Plant Sci,2004,168:365-372
51.Axelos M,Bardet C,Liboz T,VanTha A L,Curie C,Lescure B.The gene family encoding the Arabidopsis thaliana translation elongation factor EF-1 alpha:molecular cloning,characterization and expression.Mol Gen Genet,1989,219:106-112
52.Bajaj Y P S,Gill M S.Micropropagation and germplasm preservation of cotton (Gossypium spp.) through shoot tip and meristem culture.Indian J Exp Biol,1986, 24: 581-583
53. Beasley C A. In vitro culture of fertilized cotton ovules. Bioscience, 1971, 21:906-907
54. Beasley J O. The origin of American tetraploid Gossypium species. Am Nat, 1940, 74:285-286
55. Borchert R. Time course and spatial distribution of phenylalanine Ammonia-Lyase and peroxidase activity in wounded potato tuber tissue. Plant Physiol, 1978, 62:789-793
56. Buyser J, Marcotte J L, Henry Y. Genetic analysis of in vitro wheat somatic embryogenesis. Euphytica, 1992, 63: 265-270
57. Carneiro N P, Hughes P A, Larkins B A. The eEFlAQ gene family is differentially expressed in maize endosperm. Plant Mol Biol, 1999, 41 (6): 801-813
58. Causevic A, Delaunay A, Ounnar S, Righezza M, Delmotte F, Brignolas F, Hagege D,Maury S. DNA methylating and demethylating treatments modify phenotype and cell wall differentiation state in sugarbeet cell lines. Plant Physiol Bioch, 2005, 43:681-691
59. Chen J , Varner J E. Isolation and characterizationof cDNA clones for carrot extensin and a proline -rich 33KD protein. Proc Natl Acad Sci USA, 1985, 82: 4399-4403
60. Chitteti B R, Peng Z H. Proteome and phosphoproteome dynamic change during cell dedifferentiation in Arabidopsis. Proteomics, 2007, 7: 1473-1500
61. Cionini P G, Bennici A, D'Amato F. Nuclear cytology of callus induction and development in vitro. I. Callus from Vicia faba cotyledons. Protoplasma, 1978, 96:101-112
62. Coenen C, Lomax T L. Auxin cytokinin interactions in higher plants: old problems,new tools. Trends Plant Sci, 1997, 2: 351-356
63. Crea F, Bellucci M, Damiani F, Arcioni S. Genetic control of somatic embryogenesis in alfalfa (Medicago sativa L. cv.Adriana). Euphytica, 1995, 81: 151-155
64. Davidonis G H, Hamilton R H. Plant regenerating from callus tissues of Gossypium hirsutum L. Plant Sci Lett, 1983, 32: 89-93
65. Denchev P D, Velcheva M R, Atanassov A I.A new approach to direct somatic embryogenesis in Medicago. Plant Cell Rep, 1991, 10: 338-341
66. Diatchenko L, Lau Y F, Campbell A P, Chenchik A, Moqadam F, Huang B,Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov E D, Siebert P D. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA, 1996, 93:6025-6030
67. Dovzhenko A, Bosco D C, Meurer J, Koop H U. Efficient regeneration from cotyledon protoplasts in Arabidopsis thaliana. Protoplasma, 2003, 222: 107-111
68. Faure O, Aarrouf J, Nougarede A. Ontogenesis, differentiation and precocious germination in anther-derived somatic embryos of grapevine {Vitis vinifera L): proembryogenesis. Ann Bot, 1996, 78: 23-28
69. Finer J J. Plant regeneration from somatic embryogenic suspension cultures of cotton (Gossypium hirsutum L). Plant Cell Rep, 1988, 7: 399-402
70. Finer J J, McMullen M. Transformation of cotton (Gossypium hirsutum L.) via particle bombardment. Plant Cell Rep, 1990, 8: 586-589
71. Finer J J, Smith R H. Initiation of callus and somatic embryos from explants of mature cotton (Gossypium klotzschianum Anderss). Plant Cell Rep, 1984, 3: 41-43
72. Firoozabady E, DeBoer D L. Plant regeneration via somatic embryogenesis in many cultivars of cotton (Gossypium hirsutum L.). In Vitro Cell Dev Biol-Plant, 1993, 29:166-173
73. Gamborg O L, Miller R A, Ojima K. Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res, 1968, 50: 151-158
74. Ganesan M, Jayabalan N. Evaluation of haemoglobin (erythrogen): for improved somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L. cv.SVPR 2). Plant Cell Rep, 2004, 23:181-187
75. Gawel N J, Robacker C D. Genetic control of somatic embryogenesis in cotton petiole callus cultures. Euphytica, 1990, 49: 249-253
76. Grafl G, Ben-Meir H, Avivi Y, Moshe M, Dahan Y, Zemach A. Histone methylation controls telomerase-independent telomere lengthening in cells undergoing dedifferentiation. Dev Biol, 2007, 306: 838-846
77. Grosset J, Marty I, Chattier Y, Meyer Y. mRNAs newly synthesized by tobacco mesophyll protoplasts are wound-inducible. Plant Mol Biol, 1990, 15: 485-496
78. Guo L, Wang Z Y, Lin H, Cui W E, Chen J, Liu M H, Chen Z L, Qu L J, Gu H Y.Expression and functional analysis of the rice plasma-membrane intrinsic protein gene family. Cell Res, 2006, 16: 277-286
79. Gyorgyey J, Nemeth K, Magyar Z, Kelemen Z, Alliotte T, Inze D, Dudits D.Expression of a novel-type small proline-rich protein gene of alfalfa is induced by 2,4-dichlorophenoxiacetic acid in dedifferentiated callus cells. Plant Mol Biol, 1997, 34:593-602
80. He C Y, Zhang J S, Chen S Y. A soybean gene encoding a proline-rich protein is regulated by salicylic acid, an endogenous circadian rhythm and by various stresses.Theor Appl Genet, 2002, 104: 1125-1131
81. Hein I, Campbell E I, Woodhead M, Hedley P E, Young V, Morris W L, Ramsay L, Stockhaus J, Lyon G D, Newton A C, Birch P R J. Characterisation of early transcrip tional changes involving multip le signaling pathways in the Mla13 barley interaction with powdery mildew. Planta, 2004, 218(5): 803-813
82. Jamet E, Durr A, Parmentier Y, Criqui M C, Fleck J. Is ubiquitin involved in the dedifferentiation of higher plant cells? Cell Differ Dev, 1990, 29: 37-46
83. Jin S X, Mushke R, Zhu H G, Tu L L, Lin Z X, Zhang Y X, Zhang X L. Detection of somaclonal variation of cotton (Gossypium hirsutum) through RAPD, SSR markers, cytogenetic and flow cytometer analysis. Plant Cell Rep, 2008, 27:1303-1316
84. Jin S X, Zhang X L, Nie Y C, Guo X P, Liang S G and Zhu H G Identification of a novel elite genotype for in vitro culture and genetic transformation of cotton. Biol Plant, 2006, 50(4): 519-524
85. Jones P L, Wolffe A P. Relationships between chromatin organization and DNA methylation in determining gene expression. Semin Cancer Biol, 1999, 9: 339-347
86. Jose-Estanyol M, Gomis-Ruth F X, Puigdomenech P. The eight-cysteins motif, a versatile structure in plant proteins. Plant physiol Biochem, 2004,42: 355-365
87. Karthikeyan M, Jayakumar V, Radhika K, Bhaskaran R, Velazhahan R, Alice D.Induction of resistance in host against the infection of leaf blight pathogen (Alternaria palandui) in onion (Allium cepa var aggregatum). Indian J Biochem Biophys, 2005,42 (6): 371-377
88. Kidou S, Ejiri S. Isolation, characterization and mRNA expression of four cDNAs encoding translation elongation factor 1A from rice (Oryza sativa L.). Plant Mol Biol,1998,36:137-148
89. Kloos D U, Oltmanns H, Dock C, Stahl D, Hehl R. Isolation and molecular analysis of six tap root expressed genes from sugar beet. J Exp Bot, 2002, 373 (53):1533-1534
90. Kochba J, Lavee S, Spiegel-Roy P. Differences in peroxidase activity and isoenzymes in embryogenic ane non-embryogenic 'Shamouti' orange ovular callus lines. Plant and Cell Physiol, 1977,18(2): 463-467
91. Koukalova B, Fojtova M, Lim K Y, Fulnecek J, Leitch A R, Kovarik A.Dedifferentiation of tobacco cells is associated with ribosomal RNA gene hypomethylation, increased transcription, and chromatin alterations. Plant Physiol,2005, 139: 275-286
92. Kumar S, Sharma P, Pental D. A genetic approach to in vitro regeneration of non-regenerating cotton {Gossypium hirsutum L.) cultivars. Plant Cell Rep, 1998, 18:59-63
93. Kumria R, Sunnichan V G, Das D K, Gupta S K, Reddy V S, Bhatnagar R K,Leelavathi S. High-frequency somatic embryo production and maturation into normal plants in cotton (Gossypium hirsutum) through metabolic stress. Plant Cell Rep, 2003,21:635-639
94. Kurczynska E U, Gaj M D, Ujczak A, Mazur E. Histological analysis of direct somatic embryogenesis in Arabidopsis thaliana (L.) Heynh. Planta, 2007, 226:619-628
95. Kwak K J, Kim J Y, Kim Y O, Kang H. Characterization of transgenic arabidopsis plants overexpressing High Mobility Group B Proteins under high salinity, drought or cold stress. Plant Cell Physiol, 2007, 48: 221-231
96. Lashari M I, Arshad M, Zafar Y, Asad S. Optimization of zeatin and explant types for efficient embryogenesis and plant regeneration of diploid cotton (Gossypium arboretum L.). J Agric Res, 2008, 46(1): 1-13
97. Li J M, Nam K H. Regulation of Brassinosteroid signaling by a GSK3/SHAGGY-Like kinase. Science, 2002, 295: 1299-1301
98. Liu J, Liu J D, Yuan Z Q, Qian X Y, Qian M, Yang J S. Isolation and identification of genes expressed differentially in rice inflorescence meristem with suppression subtractive hybridization. Chin Sci Bull, 2001, 46 (2): 98-101
99. Liu X J, Huang B B, Lin J, Fei J, Chen Z, Pang Y, Sun X.,Tang K. A novel pathogenesis-related protein (SsPR10) from Solanum surattense with ribonucleolytic and antimicrobial activity is stress and pathogen-inducible. Plant Physiol, 2006,163(5): 546-556
100.Liu Z, Haas A L, Diaz L A, Conrad C A, Giudice G J. Characterization of a novel keratinocyte ubiquitin carrier protein. J Biol Chem, 1996, 271: 2817-2822
101.Lurie S, Fallik E, Handros A, Shapira R. The possible involvement of peroxidase in resistance to Botrytis cinerea in heat treated tomato fruit. Physiol Mol Plant P, 1997,50: 141-149
102.Marrs K A. The functions and regulation of glutathione S-transferases in plants. Annu Rev Plant Physiol, 1996, 47: 127-158
103.McFadden H G, Chappie R, De Feyter R, Dennis E. Expression of pathogenesis-related genes in cotton stems in response to infection by Verticillium dahliae. Physiol Mol Plant P, 2001, 58: 119-131
104.Mei X H, Hao Y L, Zhu H L, Gao H Y, Luo Y B. Cloning of pectin methylesterase inhibitor from kiwi fruit and its high expression in Pichia pastoris. Enzyme Microb Tech, 2007, 40: 1001-1005
105.Merkle S A. Strategies for dealing with limitations of somatic embryogenesis in hardwood trees. Plant Cell Tiss Organ Cult, 1995, 1:112-121
106.Mishra R, Wang H Y, Yadav N R, Wilkins T A. Development of a highly regenerable elite Acala cotton (Gossypium hirsutum cv. Maxxa) - a step towards genotype-independent regeneration. Plant Cell Tiss Organ Cult, 2003, 73: 21-35
107.Morelli J K, Shewmaker C K, Vayda M E. Biphasic stimulation of translational activity correlates with induction of translation elongation factor 1 subunit A upon wounding in potato. Plant Physiol, 1994, 106: 697-903
108.Moriarty T, West R, Small G, Rao D, Ristic Z. Heterologous expression of maize chloroplast protein synthesis elongation factor (EF-Tu) enhances Escherichia coli viability under heat stress. Plant Sci, 2002, 163: 1075-1082
109.Muir W H, Hildebrandt A C, Riker A J. The preparation, isolation and growth in culture of single cells from higher plants. Am J Bot, 1958, 45: 589-597
110.Munsh R, Kandl K A, Carr-Schmid A, Whitacre J L, Adams A E M, Kinzy T G.Overexpression of translation elongation factor 1A affects the organization and function of the actin cytoskeleton in yeast. Genetics, 2001, 157: 1425-1436
111.Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissues cultures. Physiol Planta, 1962,15: 473-479
112.Nadolska-Orczyk A, Orczyc W. New aspects of soybean somatic embryogenesis.Euphytica, 1994, 80: 137-143
113.Nagata T, Ishida S, Hasezawa S, Takahashi Y. Genes involved in the dedifferentiation of plant cells. Int J Dev Biol, 1994, 38: 321-327
114.Nakano T, Nishiuchi T, Suzuki K, Fujimura T, Shinshi H. Studies on transcriptional regulation of endogenous genes by ERF2 transcription factor in tobacco cells. Plant Cell Physiol, 2006, 47: 554-558
115.Nobre J, Keith D J, Dunwell J M. Morphogenesis and regeneration from stomatal guard cell complexes of cotton (Gossypium). Plant Cell Rep, 2001, 20: 8-15
116.Okamoto S, Alexander L, Hosaka T, Okamoto-Hosoya Y, Ochi K. Enhanced expression of S-adenosylmethionine synthetase causes overproduction of actinorhodin in Streptomyces coelicolor A3(2). J Bacteriol, 2003, 185: 601-609
117.Park C J, Kim K J, Shin R, Park J M, Shin Y C, Paek K H. Pathogenesis-related protein 10 isolated from hot pepper functions as a ribonuclease in an antiviral pathway. Plant J, 2004, 37: 186-198
118.Parmentier Y, Durr A, Marbach J, Hirsinger C, Criqui M C, Fleck J, Jamet E. A novel wound-inducible extensin gene is expressed early in newly isolated protoplasts of Nicotiana sylvestris. Plant Mol Biol, 1995, 29: 279-292
119.Pereira L A, Todorova M, Cai X, Makaroff C A, Emery R J N, Moffatt B O. Methyl recycling activities are co-ordinately regulated during plant development. J Exp Bot,2007,58: 1083-1098
120.Pinto G, Park Y S, Neves L, Araujo C, Santos C. Genetic control of somatic embryogenesis induction in Eucalyptus globulus Labill. Plant Cell Rep, 2008, 27:1093-1101
121.Pokalsky A R, Hiatt W R, Ridge N, Rasmussen R, Houck C M, Shewmaker C K.Structure and expression of elongation factor 1 alpha in tomato. Nucleic Acids Res,1989, 17 (12): 4661-4673
122.Price H J, Smith R H. Somatic embryogenesis in suspension cultures of Gossypium klotzschianum Anderss. Planta, 1979, 145: 305-307
123.Quiroz F R, Cerda F J, Herrera R, Loyola V M. Histological studies on the developmental stages and differentiation of two different somatic embryogenesis systems of Coffea Arabica. Plant Cell Rep, 2002, 20: 1141-1149
124.Rajasekaran K, Sakhanokho H F, Zipf A, Saha S, Sharma G C, Chee P W. Somatic embryo initiation and germination in diploid cotton (Gossypium arboreum L.). In Vitro Cell Dev Biol-Plant, 2004, 40: 177-181
125.Ransom-Hodgkins W D, Brglez I, Wang X M, Boss W F. Calciumregulated proteolysis of eEF1A. Plant Physiol, 2000, 122: 957-965
126.Ransom-Hodgkins W D, Lao P C, Gage D A, Boss W F.Phosphoglycerylethanolamine posttranslational modification of plant eukaryotic elongation factor 1a. Plant Physiol, 1998, 117: 949-960
127.Rao T R., Slobin L I. The stability of mRNA for eukaryotic elongation factor Tu in friend erythroleukemia cells varies with growth rate. Mol Cell Biol, 1988, 8:1085-1092
128.Reinert J. Untersuchungen uber die morphogenese an gewebekulturen. Ber Dtsch Bot,1958,71:15
129.Rosenberry T L, Krall J A, Dever T E, Haa R S, Louvard D, Merrick W C.Biosynthetic incorporation of [~3H]ethanolamine into protein synthesis elongation factor la reveals a new posttranslational protein modification. J Biol Chem, 1989,264: 7096-7099
130.Roth W W, Bragg P W, Corrias M V, Reddy N S, Dholakia J N, Wahba A J.Expression of a gene for mouse eukaryotic elongation factor Tu during murine erythroleukemic cell differentiation. Mol Cell Biol, 1987, 7: 3929-3936
131.Sahyoun N, McDonald O B, Farrell F, Lapetina E G Phosphorylation of a ras-related GTP-binding protein, Rap-1b, by a neuronal Ca2+/calmodulin-dependent protein kinase, CaM kinase Gr. Proc Natl Acad Sci USA, 1991, 88: 2643-2647
132.Sakhanokho H F, Zipt A, Rajasekaran K, Saha S, Sharma G C. Induction of highly embryogenic calli and plant regeneration in upland (Gossypium hirsutum L.) and Pima (Gossypium barbadense L.) cottons. Crop Sci, 2001, 41: 1235-1240
133.Sakhanokho H F, Ozias-Akins P, May O L, Chee P W. Putrescine enhances somatic embryogenesis and plant regeneration in upland cotton. Plant Cell Tiss Organ Cult,2005,81:91-95
134.Sancho M A, Forchetti S M, Pliego F, Valpuesta V, Quesada M A. Peroxidase activity and isoenzymes in the culture medium of NaCl adapted tomato suspension cells.Plant Cell Tiss Organ Cult, 1996, 44(2):161-167
135.Sharma S K, Bryan G J, Millam S. Auxin pulse treatment holds the potential to enhance efficiency and practicability of somatic embryogenesis in potato. Plant Cell Rep, 2007, 26(7): 945-950
136.Sharp W R, Sondahl M R, Caldas L S, Maraffa S B. The physiology of in vitro asexual embryogenesis. Hort Rev, 1980, 2: 268-310
137.Shi Y H, Zhu S W, Mao X Z, Feng J X, Qin Y M, Zhang L, Cheng J, Wei L P, Wang Z Y, Zhu Y X. Transcriptome profiling, molecular biological, and physiological studies reveal a major role for ethylene in cotton fiber cell elongation. Plant Cell,2006,18:651-664
138.Shoemaker R C, Couche L J, Galbraith D W. Characterization of somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L.). Plant Cell Rep, 1986,3: 178-181
139.Showalter A M, Bell J N, Cramer C L, Bailey J A, Varner J E, Lamb C J.Accumulation of hydroxyproline-rich glycoprotein mRNAs in response to fungal elicitor and infection. Proc Natl Acad Sci USA, 1985, 82 (19): 6551-6555
140.Shumway L K, Yang V V, Ryan C A. Evidence for the presence of proteinase inhibitor I in vacuolar protein bodies of plant cells. Planta, 1976, 129, 161-165
141.Skoblinska B A, Kepczynski J, Szopa J. The identification of the ACC synthase and ACC oxidase genes in Amaranthus caudatus. Acta Biochim Pol, 2007, 54: 128
142.Skoog F, Miller C O. Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp Soc Exp Biol, 1957,11: 118-131
143.Smith R H, Price H J, Thaxton J B. Defined conditions for the initiation and growth of cotton callus in vitro (Gossypium arboreum). In vitro, 1977, 13(5): 329-334
144.Suhandono S, Hughes J, Brown K, Hughes M A. Expression and structure of an elongation factor-la gene (MeEF1) from cassava (Manihot esculenta Crantz).Euphytica, 2001,120 (1): 49-58
145.Sun Y, Carneiro N, Clore A M, Moro C, Habben J E, Larkins B A. Characterization of maize elongation factor 1A and its relationship to protein quality in the endosperm.Plant Physiol, 1997, 115: 1101-1107
146.Sun Y Q, Zhang X L, Guo X P, Huang C, Nie Y C. Somatic embryogenesis and plant regeneration from different wild diploid cotton {Gossypium) species. Plant Cell Rep,2006, 25(4): 289-296
147.Sun Y Q, Zhang X L, Jin S X, Liang S G, Nie Y C. Somatic embryogenesis and plant regeneration in wild cotton (Gossypium klotzschianum Anderss). Plant Cell Tiss Organ Cult, 2003, 75: 247-253
148.Steward F C, Mapes M O, Joan Smith. Growth and Organized Development of Cultured Cells. I Growth and Division of Freely Suspended Cells. Am J Bot, 1958,45(9) : 693-703
149. Steward F C, Mapes M O, Mears K. Growth and organized development of cultured cells. II: organization in cultures grown fro freely suspended cells. Am J Bot, 1958,45(10): 705-708
150.Steward F C, Mapes M O, Joan Smith. Growth and organized development of cultured cells1 III1 interpretations of the growth from free cell to carrot plant. Am J Bot, 1958, 45 (10): 709-713
151.Tang Z X, Wang X F, Hu Z Q, Yang Z F, Xu C W. Genetic dissection of cytonuclear epistasis in line crosses. Genetics, 2007, 177: 669-672
152.Trolinder N L, Goodin J R. Somatic embryogenesis in cotton (Gossypium). I. Effects of explants and hormone regime. Plant Cell Tiss Org Cult, 1988a, 12: 31-42
153.Trolinder N L, Goodin J R. Somatic embryogenesis in cotton.II.Requirements for embryo development and plant regeneration. Plant Cell Tiss Org Cult, 1988b, 12:43-53
154.Trolinder N L, Goodin J R. Somatic embryogenesis and plant regeneration in Gossypium hirsutum L. Plant Cell Rep, 1987, 6: 231-234
155.Trigiano R N, Gray D J, Conger B V, Mc Daniel J K. Origin of direct somatic embryos from cultured leaf segments of Dactylis glomerata. Bot Gaz, 1989, 150: 72-77
156.Taguchi-Shiobara F, Komatsuda T, Oka S. Comparison of two indices for evaluating regeneration ability in rice (Oryza sativa L.) through a diallel analysis. Theor Appl Genet, 1997,94:378-382
157.Takeuchi Y, Abe T, Sasahara T. Genetic analysis of plant regeneration from seed-derived calli in rice (Oryza sativa L.). Crop Sci, 1997, 37: 963-965
158.Tsukahahara M, Hirosawa T, Nagai E, Kato H, Ikeda R, Maruyama K. Genetic analysis of plant regeneration ability and cell suspension culture of rice (Oryza sativa L). Breeding Sci, 1995, 45: 425-428
159.Tan H, Creech R G, Jenkins J N. Cloning and expression analysis of two cotton (Gossypium hirsutum L.) genes encoding cell wall proline-rich proteins. DNA Sequence, 2001,12: 367-380
160.Trolinder N L, Chen Z X. Genotype specificity of the somatic embryogenesis response in cotton. Plant Cell Rep, 1989, 8: 133-136
161.Tu L L, Zhang X L, Liang S G, Liu D Q, Zhu L F, Zeng F C, Nie Y C, Guo X P, Deng F L, Tan J F, Xu L. Genes expression analyses of sea-island cotton (Gossypium barbadense L.) during fiber development. Plant Cell Rep, 2007, 26: 1309-1320
162.Vasil V, Hildebrandt A C. Differentiation of tobacco plants from single isolated cells in microcultures. Science, 1965,150: 889-892
163.Voo K S, Rugh C L, Kamalay J C. Indirect somatic embryogenesis and plant recovery from cotton. In vitro Cell Dev Bio-Plant, 1991, 27: 117-124
164.Watillon B, Kettmann R, Boxus P, Burny A. Elongation factor la (EF1α) transcript levels are developmentally and environmentally regulated in apple plants. Plant Physiol, 1998,104(1): 1-9
165.Wilk D J, Kurek I, Hogan P, Delmer D P. The cotton fiber zinc-binding domain of cellulose synthase A1 from Gossypium hirsutum displays rapid turnover in vitro and in vivo. Proc Natl Acad Sci USA, 2006, 103: 12191-12196.
166.Wu J H, Zhang X L, Nie Y C, Jin S X, Liang S G Factors affecting somatic embryogenesis and plant regeneration from a range of recalcitrant genetypes of Chinese cotton. In vitro Cell Dev Biol-Plant, 2004, 40: 371-375
167.Wu Y R, Wang Q Y, Ma Y M, Chu C C. Isolation and expressin analysis of salt up-regulated ESTs in upland rice using PCR-based subtractive suppression hybridization method. Plant sci, 2004, 168: 847-853.
168.Xiang Y, Huang Y M, Xiong L Z. Characterization of stress-responsive CBL-interacting protein kinase genes in rice for stress tolerance improvement. Plant Physiol, 2007, 144: 1416-1428
169.Xu W L, Wang X L, Wang H, Li X B. Molecular characterization and expression analysis of nine cotton GhEF1A genes encoding translation elongation factor 1A.Gene, 2007, 389: 27-35
170.Yang F, Demma M, Warren V, Dharmawardhane S, Condeelis J. Identification of an actin-binding protein from Dictyostelium as elongation factor 1a. Nature, 1990, 347:494-496
171 .Yang X Y, Tu L L, Zhu L F, Fu L L, Min L, Zhang X L. Expression profile analysis of genes involved in cell wall regeneration during protoplast culture in cotton by suppression subtractive hybridization and macroarray. J Exp Bot, 2008, 59(13):3661-3674
172.Zeng F C, Zhang X L, Zhu L F, Tu L L, Guo X P, Nie Y C. Isolation and characterization of genes associated to cotton somatic embryogenesis by suppression subtractive hybridization and macroarray. Plant Mol Biol, 2006, 60 (2): 167-183
173.Zhu S W, Sun J S. Rapid plant regeneration from cotton (Gossypiwn hirsutum L.).Chin Sci Bull, 2000, 45(19): 1771-1774
174.Zhao J, Morozova N, Williams L, Libs L, Avivi Y, Grafi G. Two phases of chromatin decondensation during dedifferentiation of plant cells: distinction between competence for cell fate switch and a commitment for S phase. J Biol Chem, 2001,276: 22772-22778
175.Zhang L, Hattorik K. Inheritance of high shoot regeneration ability from seed callus in a rice cultivar Joshu. Breeding Sci, 1998, 48(1): 41-44
176.Zhang B H, Feng R, Li X L. Anther culture and plant regeneration of cotton (Gossypiwn klotzschianum Anderss). Chin Sci Bull, 1996, 41: 1340-1343
177.Zhang X L, Ren Y, Zhao J. Roles of extensins in cotyledon primordium formation and shoot apical meristem activity in Nicotiana tabacum. J Exp Bot, 2008, 59(14):4045-4058