摘要
柑橘是世界上最重要的水果之一,果实品质一直是柑橘育种追求的主要目标。长期以来,童期长、珠心胚、性器官败育以及遗传上的高度杂合等因素一直严重影响着柑橘育种工作的开展。基因工程的发展为柑橘的育种工作开辟了一条新的途径,从而使利用单个基因改造柑橘的品质成为可能。本研究利用童期较短的早实枳作为转化材料,建立并优化了其高效再生和遗传转化的体系,以期使其成为柑橘基因功能验证和研究的平台。利用该转化体系和RNA干涉技术,获得了一批与柑橘类胡萝卜素代谢相关基因的遗传转化材料,并对这些材料进行了分子鉴定和基因表达的初步分析,本研究主要取得了以下结果:
1.建立了早实枳(Poncirus trifoliata[L.]Raf.)和Cocktail葡萄柚(C.paradisiMacf.×C.reticulata Blanco.)的高效再生及遗传转化体系,对若干影响再生和转化效率的因素,如外植体的绿化、共培养条件,激素的组合和用量,筛选的方法等进行了优化。对黄化的早实枳茎段或Cocktail上胚轴进行4-5d的光照处理,可以显著提高外植体的再生能力和转化率;共培养过程中,0.4 mg L~(-1)的2,4-D可显著提高外植体的转化率,而过高的2,4-D浓度(0.6-0.8 mg L~(-1))则抑制不定芽的再生,降低转化率。培养基G(MT+1 mg L~(-1) BA+0.1 mg L~(-1) NAA)对正常芽的再生最有利,过高的BA浓度(大于1.5 mg L~(-1))虽然能提高芽的数量,但也造成大量畸形芽的出现。实验中还发现,潮霉素对早实枳茎段的再生有强烈的抑制作用。在早实枳再生试验中,通过嫁接再生的早实枳有36.4%(8/22)只需要9个月就能开花,而通过生根再生的植株在第12个月时有32%(8/25)的植株开花,第20个月时,这些再生植株有87.2%(41/47)开花。这表明早实枳早花早果的特性可以经过组织培养和再生过程稳定地遗传给后代,可以用于柑橘功能基因的研究。
2.构建了BCH基因片段的反义表达载体pCABCH,并用于转化伏令夏橙(C.sinensis Osb.cv.Valencia)的胚性愈伤组织,得到了6个独立的转化株系,并测定了抗性愈伤组织中类胡萝卜素含量的变化,结果表明,大部分转化愈伤中叶黄素和α-胡萝卜素的含量有所增加。50-60 mg L~(-1)的潮霉素对伏令夏橙愈伤组织的再生影响较大,强烈抑制再生植株的生长。
3.构建了CRTISO基因片段的反义表达载体pBICI,分别转化了早实枳和山金柑,得到了23株早实枳再生植株,其中12株PCR检测呈阳性;得到转化山金柑4株。
4.分别以LCYB的基因序列片段,和LCYE基因的5'和3'端片段,构建了pLCYB,pLCYE1和pLCYE2的RNAi表达载体,并用于转化早实枳和Cocktail葡萄柚。Real-time PCR结果表明,在LCYE基因被抑制的的早实枳植株中,PDS基因的表达量显著增加,LCYB和ZEP基因的表达量下降。HPLC分析表明,发生LCYE基因抑制的植株叶片中,α-胡萝卜素含量的下降,β-胡萝卜素的含量和紫黄质的含量增加,而叶黄素的含量没有显著的变化。但β-胡萝卜素和紫黄质的含量却与其基因的表达水平呈反比!据此我们推测,对LCYE基因的干涉可能通过某种机制激活了上游PDS基因的活性。而β-胡萝卜素和紫黄质的含量增加可能与代谢物质的反馈调节有关:随着PDS基因表达量增加,上游ζ-类胡萝卜素和番茄红素的含量增加,导致下游的代谢产物β-胡萝卜素和紫黄质的含量显著增加,而这些代谢产物对其本身的合成基因LCYB和ZEP又产生了反馈抑制作用。
在抑制LCYB表达的早实枳植株中,PSY,ZDS,CRTISO和ZEP基因的表达量呈下降趋势。同时,β-胡萝卜素和下游紫黄质的含量在转基因植株叶片中的含量下降,α-胡萝卜素和叶黄素的含量没有显著的变化。β-胡萝卜素的含量和LCYB基因的表达量呈正相关,即LCYB基因的表达量越高,叶片中β-胡萝卜素的含量也越高。而这一过程可能会导致番茄红素的积累。
Citrus is one of the most important fruits all over the world and the fruit quality is always the main goal for citrus breeding. Citrus breeding has been hindered seriously by several factors, such as long juvenility, micellar embryo, female/male abortion and high hetetozygosis. Genetic engineering opens a new avenue for citrus breeding and makes it possible for using a single gene to improve the fruit quality in citrus. In this study, the regeneration and transformation conditions of the precocious trifoliate orange have been built and optimized to establish a short juvenility genetic transformation system for gene functional verification related to citrus fruits. On the basis of the transformation system, a group of regenerated plantlets containing citrus carotenoids metabolic genes were obtained. Molecular identification and gene expression analysis were also conducted. The main results of this study are as follows:
1. High efficient Agrobacterium-mediated transformation and regeneration sytems for precocious trifoliate orange (Poncirus trifoliata [L.] Raf.) and Cocktail (C. paradisi Macf.×C. reticulata Blanco.) were established. Some factors which affect regeneration and transformation were optimized, such as, the illumination of etiolated explants, the coculture conditions, the selection media and the mode of selection etc. Higher regeneration frequencies and transformation frequencies were achieved in all 4-day-illuminated precocious trifoliate orange stem segments and 5-day-illuminated Cocktail epicotyl segment compared to etiolated controls. The highest transformation frequency obtained in coculture medium, which is MT basal medium supplemented with 0.4 mg L~(-1) 2, 4-D. However, increasing concentrations of 2, 4-D from 0.6 to 0.8 mg L~(-1) suppressed this effect. It was proper for precocious trifoliate orange to produce normal shoots on medium G (MT + 1 mg L~(-1) BA + 0.1 mg L~(-1) NAA). For precocious trifoliate orange, when over 1.5 mg L~(-1) BA was added to SRM, although more visible shoots were produced, most of these shoots were abnormal. Regeneration of precocious trifoliate orange was completely suppressed when hygromycin B was used as selection agent. In recovery shoots, 9 months after grafting, 36.4% of the plantlets flowered (8/22). For the rooting lines (8/25), the flower process was delayed for 12 months. 20 months after being grafted or transferred to greenhouse, 87.2% (41/47) of the transformants flowered. The results implies the in vitro regeneration plants could reserved the precocious traits of precocious trifoliate orange and the transformation system could be used for gene functional verification related to citrus fruits.
2. The anti-sense expression vector pCABCH containing BCH gene fragment was constructed and used to transform the embryonic callus of Valencia (C. sinensis Osb. cv. Valencia). 6 independent transgenic callus lines were obtained. The carotenoid contents were detected by HPLC. The result revealed that lutein andα-carotene content increased in most of transgenic callus lines. And the growth of Valencia transgenic calluses was greatly suppressed by 50-60 mg L~(-1) Hyg B.
3. The anti-sense expression vector pBICI containing CRTISO gene fragment was constructed, and used to transform precocious trifoliate orange and Hongkong kumquat (Fortunella japonica [champ.] Swing.). 4 independent transgenic Hongkong kumquat lines and 23 independent transgenic precocious trifoliate orange lines were obtained respectively.
4. The RNAi expression vector pLCYB containing LCYB gene fragment, pLCYE1 and pLCYE2 containing LCYE gene 5'-end and 3'-end fragment were constructed respectively, and the three vectors was used in precocious trifoliate orange and Cocktail tansformation experiments. Results of Real-time PCR revealed that the expression of PDS gene markedly increased in LCFE-suppressed transgenic lines. The expression of LCYB and ZEP decreased. HPLC analysis revealed that in LCYE-suppressed transgenic plants, the content ofα-carotene decreased andβ-carotene and Violaxanthin contents increased, there were no significant changes in respect of lutein content. Unexpectedly, both ofβ-carotene and Violaxanthin increases were inversely proportional to the expression of LCYB and ZEP. According to these findings, we hypothesized that PDS gene was activated by some mechanisms in LCYE-suppressed transgenic plants. And the feedback effects should be responsible for the improvement ofβ-carotene and Violaxanthin:ζ-carotene and lycopene contents upstream improved with the increased expression of PDS gene, and this led to the dramatical increase of metabolic products such asβ-carotene and Violaxanthin downstream. These synthesis genes LCYB and ZEP were suppressed by their metabolic products conversely.
In LCYB suppressed transgenic precocious trifoliate orange lines, the expression of genes PSY, ZDS , CRTISO and ZEP presented a downward tendency. At the same time, the content ofβ-carotene and Violaxanthin downstream decreased in the transgenic leaves and there were no significant changes in respect ofα-carotene and lutein contents. The results showed positive correlation between the conten of LCYB expression and β-carotene: the content ofβ-carotene elevated with the increasing of LCYB expression. This process may lead to the accumulation of lycopene.
引文
1.曹庆芹,孟海军,文晓鹏,伊华林,邓秀新.利用SSR标记分析柑橘雄性不育及低育资源的遗传多样性.农业生物技术学报,2006,14(6):937-941
2.陈锦清,郎春秀,胡张华,刘智宏,黄锐之.反义PEP基因调控油菜籽粒蛋白质/油脂含量比率的研究.农业生物技术学报,1999,7(4):316-320
3.程运江.柑橘体细胞胞质遗传及叶绿体SSR引物开发研究.[博士学位论文].武汉:华中农业大学图书馆,2004
4.邓秀新,郭文武,伊华林,刘继红,夏仁学,孙中海.一种生产瘪籽柚果实的方法.2004,专利号:ZL00109615.X
5.邓秀新,章文才,万蜀渊.柑橘原生质体分离及再生成株的研究.园艺学报,1988,15(2):99-102
6.邓秀新,章文才.柑橘原生质体融合及遗传转化的研究进展.果树科学,1991,8(1):51-58
7.邓秀新,章文才.柑橘原生质体培养与融合研究.自然科学进展:国家重点实验室通讯,1995,5(1):35-41
8.邓秀新.国内外柑橘产业发展趋势与柑橘优势区域规划.广西园艺,2004,15(4):6-10
9.邓秀新.世界柑橘品种改良的进展.园艺学报,2005,32(6):1140-1146
10.邓秀新,伊华林,李锋,郭文武,叶文明.以异源四倍体体细胞杂种为父本杂交培育三倍体柑橘植株.植物学报,1996,38(8):141-144
11.邓占鳌,章文才,万蜀渊.诱发柑橘珠心愈伤组织及原生质体高频率体细胞胚胎发生及植株再生.实验生物学报,1990,23(2):135
12.段艳欣,根癌农杆菌介导的LFY、AP1基因转化柑橘的研究及转基因植株的培育.[博士学位论文].武汉:华中农业大学图书馆,2006
13.段艳欣,郭文武.木本植物开花调节基因的分离克隆及其童期控制.中国生物工程杂志,2004,24(10):22-26
14.贺红,潘瑞炽,韩美丽,李耿光.枳壳外植体离体再生及农杆菌介导的遗传转化.云南植物研究,1998,20(4):459-463
15.洪柳,刘永忠,邓秀新.桠柑成熟种子胚培养获得四倍体植株.园艺学报,2005,32 (4):688-690
16.黄建昌,肖艳,赵春香,陈赞盛,王心燕.少核沙田柚的辐射选育研究.核农学报,2003,17(3):171-174
17.蒋迪,徐昌杰,陈大明,张上隆.柑橘转基因研究的现状及展望.果树学报,2002,19(1):48-52
18.李鼎立,柑橘遗传转化受体系统优化与抗溃疡病转基因植株培育.[博士学位论文].武汉:华中农业大学图书馆,2008
19.李东栋.根癌农杆菌介导愈伤组织遗传转化获得带有雄性不育基因的柑橘植株.[博士学位论文].武汉:华中农业大学图书馆,2002
20.梁遂权,王学炷,万同香.早实枳生物学特性及其砧木实验.浙江柑橘,1999,16(3):2-4
21.刘继红,胡春根,邓秀新.我国果树辐射诱变育种研究进展.中国果树,1999,(4):48-50
22.刘庆.暗柳'甜橙红色突变体性状形成的分子机理研究.[博士学位论文].武汉:华中农业大学图书馆,2008
23.鲁玉洋.利用SSR和RAPD技术构建柑橘分子遗传图谱.[硕士学位论文].重庆:西南大学图书馆,2006:
24.马瑞娟,俞明亮.油桃育种进展,果树学报,2000,17(3):214-219
25.沈德绪,王元裕,陈力耕.柑橘遗传育种学.科学出版社,1998,274-283
26.宋健坤.以异源四倍体体细胞杂种为父本与二倍体杂交创造柑橘三倍体的研究.[博士学位论文].武汉:华中农业大学图书馆,2006
27.陶俊,张上隆,徐建国,刘春荣.柑橘果实主要类胡萝卜素成分及含量分析.中国农业科学,2003,36(10):1202-1208
28.陶能国.甜橙(Citrus sinensis Osbeck)红肉突变体类胡萝卜素合成相关基因的克隆与特性分析.[博士学位论文].武汉:华中农业大学图书馆,2006
29.王峰,朱祯,李向辉,章文才.不同调控序列控制下的GUS基因在柑橘原生质体中的瞬间表达.福建农业学报,1998,13(2):1-5
30.王子成,邓秀新.柑橘转基因研究进展.华中农业大学学报,2002,21(5):494-499
31.徐娟.几个柑橘产区果实色泽评价及红肉脐橙(Citrus sinensis L.cv.Cara cara)果肉呈色机理初探.[博士论文].武汉:华中农业大学图书馆,2002
32.徐昌杰,张上隆.植物类胡萝卜素的生物合成及其调控.植物生理学通讯,2000,36(1):64-70
33.徐昌杰.柑橘果实类胡萝卜素研究进展.园艺学进展(第六辑).陕西科学技术出版社,2004,6:60-65
34.徐小勇,刘继红,邓秀新.柑橘生物技术研究进展.中国生物工程杂志,2003,23(8):35-38
35.叶荫民.柑橘品种改良的世纪回顾与展望.中国南方果树,2000,29(3):15-17
36.叶自行,曾泰,林顺权等.无子沙糖橘(十月橘)的选育.果树学报,2006,23(1):149-150
37.伊华林,覃伟,李长藻,邓秀新.桠柑新品种‘华柑2号'.园艺学报,2005,32(5):962-962
38.易干军,诏小浪,彭成绩等.柑橘原生质体培养的影响因子研究.广东农业科学,1997,4:20-22
39.张敏,邓秀新.柑橘芽变选种以及芽变性状形成机理研究进展.果树学报,2006,23(6):871-876
40.张敏.柑橘果实扇形嵌合体的分离及两组嫁接嵌合体的遗传研究.[博士学位论文].武汉:华中农业大学图书馆,2006
41.张俊娥.柑橘愈伤组织DNA含量变异、体细胞胚胎发生及同源四倍体的诱导研究.[博士学位论文],武汉:华中农业大学图书馆,2005
42.朱长甫,陈星,王英典.植物类胡萝卜素生物合成及其相关基因在基因工程中的应用.植物生理与分子生物学学报,2004,30(6):609-618
43.Agrawal N,Dasaradhi P V N,Mohmmed A,Malhotra P,Bhatnagar R K,Mukherjee S K.RNA Interference:Biology,Mechanism,and Applications.Microbiol Mol Biol Rev,2003:657-685
44.Alba R,Cordonnier-Pratt M M,Pratt L H.Fruit-localized phytochromes regulate lycopene accumulation independently of ethylene production in tomato.Plant Physiol,2000,123:363-370
45.Almeida W A B,Mourao Filho F A A,Mendes B M J,Pavan A,et al.Agrobacterium-mediated transformation of Citrus sinensis and Citrus limonia epicotyl segments.Scientia Agricola,2003,60(1):23-29
46. Alos E, Roca M, Iglesias D J, Minguez-Mosquera M I, Damasceno C M B, et al. An evaluation of the basis and consequences of a stay-green mutation in the navel negra (nan) citrus mutant using transcriptomic and proteomic profiling and metabolite analysis. Plant Physiol, 2008, 147: 1300-1315
47. Alquezar B, Rodrigo M J, Zacarias L. Regulation of carotenoid biosynthesis during fruit maturation in the red-fleshed orange mutant Cara Cara. Phytochemistry, 2008,69: 1997-2007
48. Alvarez R, Alonso P, Cortizo M, Celestino C, Hernandez I, Toribio M, Ordas R.Genetic transformation of selected mature cork oak (Quercus suber L.) trees. Plant Cell Rep, 2004, 23, 218-223
49. Atkinson R G and Gardner R C. Regeneration of transgenic tamarillo plants. Plant Cell Rep, 1993, 12:347-351
50. Bartley G E, Scolnik P A. cDNA cloning, expression during development and genome mapping of Psy-2, a second tomato gene encoding phytoene synthase. J Biol Chem, 1993, 268: 25718-25721
51. Bartley G E, Scolnik P A. Plant carotenoids: Pigments for photoprotection, visual attraction, and human health. Plant Cell, 1995, 7: 1027-1038
52. Bartley G E, Viitanen P V, Pecker I, Chamovitz D, Hirschberg J, Scolnik P A.Molecular cloning and expression in photosynthetic bacteria of a soybean cDNA coding for phytoene desaturase, an enzyme of the carotenoid biosynthesis pathway.Proc Natl Acid Sci USA, 1991, 88: 6532-6536
53. Bernhardt A E, Lechner P H V, Schade M D et al. CUL4 associates with DDB1 and DET1 and its downregulation affects diverse aspects of development in Arabidopsis thaliana. Plant J, 2006, 47: 591-603
54. Bird C R, Ray J A, Fletcher J D, Boniwell J M, Bird A, Teulieres C, Blain I, Bramley P M, Schuch W. Using antisense RNA to study gene function: inhibition of carotenoid biosynthesis in transgenic tomatoes. Bio Technology, 1991, 9: 635-639
55. Bolton G W, Nester E W and Gordon M P. Plant phenolic compounds induce expression of the Agrobacterium tumefaciens loci needed for virulence. Science,1986, 232: 983-985
56. Botella-Pavia P, Besumbes O, Phillips M A, Carretero-Paulet L, Boronat A,Rodr(?)guez-Concepcion M. Regulation of carotenoid biosynthesis in plants: evidence for a key role of hydroxymethylbutenyl diphosphate reductase in controlling the supply of plastidial isoprenoid precursors. plant J, 2004, 40(2): 188-199
57. Botella-Pavia P, Rodriguez-Concepcion M. Carotenoid biotechnology in plants for nutritionally improved foods. Physiol Plant, 2006, 126: 369-381
58. Bramley P M. Regulation of carotenoid formation during tomato fruit ripening and development. J Exp Bot, 2002, 377: 2107-2113
59. Bramley P, Teulieres C, Blain I, Bird C, Schuch W. Biochemical characterization of transgenic tomato in which carotenoid biosynthesis has been inhibited through the expression of antisense RNA to pTOM5, Plant J, 1992, 2: 343-349
60. Breedt H J, Snyman J C, Koekemoer P J J. Production seedless citrus cultivars with gamma irridation. Manicom B eds. Proc. Inter. Soc. Citriculture, 8th Inter Citrus Congress May. Sun City, South Africa, 1996, 1: 164-166
61. Bugos R C, Yamamoto H Y. Molecular cloning of violaxanthin de-epoxidase from romaine lettuce and expression in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 1996, 93: 6320-6325
62. Busch M, Seuter A, Hain R. Functional analysis of the early steps of carotenoid biosynthesis in tobacco. Plant Physiol, 2002, 128: 439-453
63. Byzova M, Verduyn C, De Brouwer D, De Block M. Transforming petals into sepaloid organs in Arabidopsis and oilseed rape: implementation of the hairpin RNA-mediated gene silencing technology in an organ-specific manner. Planta, 2004,218: 379-387
64. Cai Q, Guy C L, Moore G A. Extension of the genetic linkage map in Citrus using random amplified polymorphic DNA (RAPD) markers and RFLP mapping of cold-acclimation responsive loci. Theor Appl Genet, 1994, 89: 606-614
65. Cai X D, Liu X, Guo W W. GFP expression as an indicator of somatic hybrids between transgenic Satsuma mandarin and calamondin at embryoid stage. Plant Cell Tiss Org Cult, 2006, 87(3): 245-253
66. Cervera M, Juarez J, Navarro A, Pina J A, Duran-Vila, N, Navarro L, Pena, L.Genetic transformation and regeneration of mature tissues of woody fruit plants bypassing the juvenile stage. Transgenic Res, 1998a, 7: 51-59
67. Cervera M, Lopez M, Navarro L. Virulence and supervirulence of Agrobacterium tumefaciens in woody fruit plants. Physiol Mol Plant Pathol, 1998b, 52: 67-78
68. Cervera M, Navarro A, Navarro L, Pe(?)a L. Production of transgenic adult plants from Clementine mandarin by enhancing cell competence for transformation and regeneration. Tree Physiol, 2008, 28: 55-66
69. Cervera M, Pina J A, Juarez J, Navarro A, Navarro L, Pena L. Agrobacterium-mediated transformation of citrange: factors affecting transformation and regeneration. Plant Cell Rep, 1998c, 18: 271-278
70. Cervera M, Juarez J, Navarro L. Genetic transformation of mature citrus plants. Methods Mol Biol, 2005, 286: 177-188
71. Cevik B V, Lee R, Niblett C L. Genetic Transformation of Citrus paradise with Antisense and Untranslatable RNA-dependent RNA Polymerase Genes of Citrus tristeza closterovirus. Turk J Agric For, 2006, 30: 173-182
72. Chamovitz D, Pecker I, Hirschberg J. The molecular basis of resistance to the herbicide norflurazon. Plant Mol Biol, 1991, 16(6): 967-74
73. Chaparo J X. An early ripening grapefruit hybrid. In: Albrigo L G eds. Proc. Inter.Soc. of Citricuhure 9~(th) Congress, Orlando, 2003, 1: 28
74. Cheng Y J, Guo W W, Yi H L, Pang X M, and Deng X X. An efficient protocol for genomic DNA extraction from Citrus species. Plant Mol Biol Rep, 2003, 21:177a-177g
75. Cogoni C and Macino G. Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase. Nature, 1999, 399: 166-169
76. Cogoni G, Macino G. Isolation of quelling-defective (qde) mutants impaired in posttranscriptional transgene-induced gene silencing in Neurospora crassa. Proc Natl Acad Sci USA, 1994, 91: 10233-10238
77. Costa M G C, Otoni W C, Moore G A. An evaluation of factors affecting the efficiency of Agrobacterium-mediated transformation of Citrus paradisi (Macf.) and production of transgenic plants containing carotenoid biosynthetic genes. Plant Cell Rep, 2002, 21 (4): 365-373
78. Cunningham F X, Gantt E. Genes and enzymes of carotenoid biosynthesis in plants.Annu Rev Plant Phys, 1998,49: 557-583
79. Cunningham F X, Gantt E. One ring or two? Determination of ring number in carotenoids by lycopene e-lycopene cyclase. Proc Natl Acid Sci USA, 2001, 98(5):2905-2910
80. Cunningham F X, Gantt E. A portfolio of plasmids for identification and analysis of carotenoid pathway enzymes: Adonis aestivalis as a case study. Photosynth Res, 2007,92: 245-259
81. Cunningham F X, Pogson B, Sun Z, McDonald K A, Dallapenna D, Gantt E.Functional analysis of the β and s-lycopene cyclase enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid formation. Plant Cell, 1996, 8(9): 1613-1626
82. Curl A L. The Occurrence of beta-Citraurin and of beta-Apo-8'-Carotenal in the Peels of California Tangerines and Oranges. J Food Sci, 1965, 30(1): 13-18
83. Curtis M D, Grossniklaus U. A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol, 2003, 133: 462-469
84. D'Ambrosio C, Giorio G , Marino I, Merendino A, Petrozza A, Salfi L, Stigliani A L,Cellini F. Virtually complete conversion of lycopene into β-carotene in fruits of tomato plants transformed with the tomato lycopene β-cyclase (tlcy-b) cDNA. Plant Sci, 2004, 166(1): 207-214
85. Davison P A, Hunter C N, Horton P. Overexpression of beta-carotene hydroxylase enhances the stress tolerance in Arabidopsis. Nature, 2002, 418 (6894): 203-206
86. Davuluri G R, van Tuinen A, Fraser P D, Manfredonia A, Newman R, et al.Fruit-specific RNAi-mediated suppression of DET1 enhances carotenoid and flavonoid content in tomatoes. Nat Biotechnol, 2005, 23(7): 890-895
87. Davuluri G R, van Tuinen A, Mustilli A C et al. Manipulation of DET1 expression in tomato results in photomorphogenic phenotypes caused by post-transcriptional gene silencing. Plant J, 2004,40 (3): 344-354
88. De Bondt A, Eggermont K, Penninckx I, Goderis I, Broekaert W F.Agrobacterium-mediated transformation of apple (Malus domestica Borkh.): an assessment of factors affecting regeneration of transgenic. Plant Cell Rep, 1996, 15:549-554
89. Deng X X and Duan Y X. Modification of perennial fruit trees. In Fladung M and Ewald D (eds), Tree transgenesis: Recent Developments. Springer-Verlag Berlin Heidelberg, Germany, 2006, 4766
90. Deng X X, Grosser J W, Gmitter F G. Intergeneric somatic hybrid plants from protoplast fusion of Fortunella crassifolia 'Meiwa' with Citrus sinensis cv. 'Valencia'.Sci Hortic, 1992,49: 55-62
91. Deng X X, Guo W W, Yu G H. Citrus somatic hybrids regenerated from protoplat electrofusion. Acta Hortic, 2000, 1: 535
92. Deng Z, Huang S, Ling P, Yu C, Tao Q, Chen C, Wendell M K, Zhang H B, Gmitter F.Fine genetic mapping and BAC contig development for the citrus tristeza virus resistance gene locus in Poncirus trifoliata (Raf.). Mol Genet Genomics, 2001, 265:739-747
93. Deroles S C, Bradley J M, Schwinn KE, Markham K R, Bloor S, Manson D G, Davies K M. An antisense chalcone synthase cDNA leads to nover colour patterns in lisianthus (Eustoma grandiflorum) flower. Mol Breeding, 1998, 4 (1): 59-66
94. Dharmapuri S, Rosati C, Pallara P, Aquilani R, Bouvier F, Camara B, Giuliano G Metabolic engineering of xanthophyll content in tomato fruits. FEBS Lett, 2002, 519(1-3): 30-34
95. Diretto G, Tavazza R, Welsch R, Pizzichini D, Mourgues F, Papacchioli V, Beyer P,Giuliano G Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase. BMC Plant Biol, 2006, 6: 13
96. Diretto G, Welsch R, Tavazza R, Mourgues F, Pizzichini D, Beyer P, Giuliano G.Silencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato tubers. BMC Plant Biol, 2007, 7: 11
97. Duan Y X, Liu X, Fan J, Li D L, Wu R C, Guo W W. Multiple shoot induction from seedling epicotyls and transgenic citrus plant regeneration containing the green fluorescent protein gene. Botanical Studies, 2007, 48: 165-171
98. Ducreux L J, et al. Metabolic engineering of high carotenoid potato tubers containing enhanced levels of beta-carotene and lutein. J Exp Bot, 2005, 56: 81-89
99. Durham R E, Liou P C, Gmitter F G, Moore G A. Linkage of restriction fragment length polymorphisms and isozymes in Citrus. Theor Appl Genet, 1992, 84: 39-48
100.Durzan D J, Adult vs juvenile explants: directed totipotency. In: Rodriguez R,Sanchez Tames R and Durzan D J, eds., Plant Aging: Basic and Applied Approaches.NATO-ASI on Molecular Basis of Plant Aging, vol. 186, Plenum Press, New York,1990, pp. 19-25
101.Elbashir S M, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev, 2001, 15(2): 188-200
102.Endo T, Shimada T, Fujii H, Kobayashi Y, Araki T, Omura M. Ectopic expression of an FT homolog from Citrus confers an early flowering phenotype on trifoliate orange (Poncirus trifoliata L. Raf.). Trans Res, 2005, 14: 703-712
103.Fagoaga C, Lopez C, Mendoza A H, Moreno P, Navarro L, Flores R, Pena L.Post-transcriptional gene silencing of the p23 silencing suppressor of Citrus tristeza virus confers resistance to the virus in transgenic Mexican lime. Plant Mol Biol, 2006,60: 153-165
104.Fanciullino A L, Dhuique-Mayer C, Luro F, Morillon R, Ollitrault P. Carotenoid biosynthetic pathway in the Citrus genus: Number of copies and phylogenetic diversity of seven genes. J Agric Food Chem, 2007, 55: 7405-7417
105.Fankhauser C, Casal J J. Phenotypic characterization of a photomorphogenic mutant.Plant J, 2004, 39: 747-760
106.Fire A, Xu S, Montgomery M K, Kostas S A, Driver S E, Mello C C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature, 1998, 391:806-811
107.Fleming G H, Olivares-Fuster O, Fatta Del-Basco S, Grosser J W. An alternative method for the genetic transformation of sweet orange. In Vitro Cell Dev Biol Plant,2000, 36: 450-455
108.Fraser P D, Bramley P M. The biosynthesis and nutritional uses of carotenoids. Prog Lipid Res, 2004, 43: 228-265
109.Fraser P D, Enfissi E M, Halket J M, Truesdale M R, Yu D, Gerrish C, Bramley P M. Manipulation of phytoene levels in tomato fruit: effects on isoprenoids, plastids, and intermediary metabolism. Plant Cell, 2007, 19(10): 3194-3211
110.Fraser P D, Kiano J W, Truesdale M R, Schuch W, Bramley P M. Phytoene synthase-2 enzyme activity in tomato does not contribute to carotenoid synthesis in ripening fruit. Plant Mol Biol, 1999, 40: 687-698
111.Fraser P D, Romer S, Shipton C A, Mills P B, Kiano J W, Misawa N, Drake R G,Schuch W, Bramley P M. Evaluation of transgenic tomato plants expressing an additional phytoene synthase in a fruit specific manner. Proc Natl Acad Sci USA,2002, 99 (2): 1092-1097
112.Fraser P D, Truesdale M R, Bird C R, Schuch W, Bramley P M. Carotenoid biosynthesis during tomato fruit development (Evidence for tissue-specific gene expression). Plant physiol, 1994, 105(1): 405-413
113.Fray R G, Wallace A, Fraser P D, Valero D, Hedden P, Bramley P M, Grierson D.Constitutive expression of a fruit phytoene synthase gene in transgenic tomatoes causes dwarfism by redirecting metabolites from the gibberellinpathway. Plant J,1995, 8 (5): 693-701
114.Frey A, Audran C, Marin E, Sotta B, Marion-Poll A. Engineering seed dormancy by the modification of zeaxanthin epoxidase gene expression. Plant Mol Biol, 1999, 39(6): 1267-1274
115.Fujisawa M, Watanabe M, Choi S K, Teramoto M, Ohyama K, Misawa N.Enrichment of carotenoids in flaxseed (Linum usitatissimum) by metabolic engineering with introduction of bacterial phytoene synthase gene crtB. J Biosci Bioeng, 2008, 105(6):636-641
116.Galpaz N, Wang Q, Menda N, Zamir D, Hirschberg J. Abscisic acid deficiency in the tomato mutant high-pigment 3 leading to increased plastid number and higher fruit lycopene content. Plant J, 2008, 53(5): 717-730
117.Garfinkel D J and Nester E W. Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism. J Bacteriol, 1980, 144: 732-743
118.Ghorbel R, JuaHrez J, Navarro L, Pena L. Green fluorescent protein as a screenable marker to increase the efficiency of generating transgenic woody fruit plants. Theor Appl Genet, 1999, 99: 350-358
119.Giliberto L, et al. Manipulation of the blue light photoreceptor cryptochrome 2 in tomato affects vegetative development, flowering time, and fruit antioxidant content.Plant Physiol, 2005, 137: 199-208
120.Giuliano G, Bartley G E, Scolnik P A. Regulation of carotenoid biosynthesis during tomato development. Plant Cell, 1993, 5: 379-387
121.Giuliano G, Diretto G. Of chromoplasts and chaperones. Trends Plant Sci, 2007,12(12): 529-531
122.Giuliano G, Tavazza R, Diretto G, Beyer P, Taylor M A. Metabolic engineering of carotenoid biosynthesis in plants. Trends in Biotechnol, 2008, 26(3): 139-145
123.Gmitter F G, Wang Y, Wang L, Wendell M K, Zhou P, Grosser J W. Triploid fresh citrus fruit breeding at the UF-citrus research and education centre. In: E. Mohamed eds. Program and Abstract, 10~(th) Inter. Soc. Citriculture, Agadir, Morocco, 2004, 37-38
124.Gmitter F G Jr, Xiao S Y, Huang S, Hu X L, Garnsey S M, Deng Z. A localized linkage map of the citrus tristeza virus resistance gene region. Theor Appl Genet,1996, 92: 688-695
125.Gmitter F G, Ling X. Embryogenesis in vitro and nonchimeric tetraploid plant recovery from undeveloped Citrus ovules treated with colchicine. J Am Soc Hort Sci,1991,116:317-321
126.G(?)mez-Lim M A, Litz R E. Genetic transformation of perennial tropical fruits. In Vitro Cellular & Developmental Biology Plant, 2004, 40: 442-449
127.Goyal A, Chopra M, Lwaleed B A, Birch B and Cooper A J. The effects of dietary lycopene supplementation on human seminal plasma. Br J Urol 2007, 99 (6):1456-1460
128.Grosser J W, Chandler J L. Production of twelve new allotetraploid somatic hybrid citrus breeding parents with emphasis on late maturity and cold-hardiness. J Am Pomol Soc, 2004, 58:21-28
129.Grosser J W, Chandler J L. Somatic hybridization of high yield, cold-hardy and disease resistant parents for citrus rootstock improvement. J Hort Sci Biotech, 2000,75(6): 641-644
130. Guo S, Kemphues K J. par-1, a gene required for establishing polarity in C. elegans embryos, encodes putative Ser/Thr kinase that is asymmetrically distributed. Cell,1995, 81(4): 611-620
131 .Guo W W, Duan Y X, Olivares-Fuster O, Wu Z C, Arias C R, Burns J K, Grosser J W.Protoplast transformation and regeneration of transgenic Valencia sweet orange plants containing the juice quality related pectin methylesterase gene. Plant Cell Rep, 2005,24: 482-486
132.Guo W W, Prasad D, Cheng Y J, Serrano P, Deng X X, Grosser J W. Targeted cybridization in Citrus: transfer of Satsuma cytoplasm to seedy cultivars for potential seedlessness. Plant Cell Rep, 2004, 22: 752-758
133.Hannon G J. RNA interference. Nature, 2002, 418: 244-251
134.He X Y, Zhang Y L, He Z H, Wu Y P, Xiao Y G, Ma C X, Xia X C, Characterization of phytoene synthase 1 gene (Psyl) located on common wheat chromosome 7A and development of a functional marker. Theor Appl Genet, 2008, 116: 213-221
135.Heiser V, Rasmusson A G, Thicck O, et al. Antisense repression of the mitochondril NADH-binding subunit of complex I in transgenic potato affects male fertility. Plant Sci, 1997, 127: 61-69
136.Hensz R A. Mutation breeding and the development of the 'Star Ruby' grapefruit.Proc. Int. Soc. Citriculture, 1977, 2: 582-585
137.Hidaka T, Omura M, Ugaki M. Agrobacterium tumefaciens mediated transformation and regeneration of Citrus spp. from suspension cells. Japan J Breed, 1990, 40:199-207
138.Hidaka T, Omura M. Transformation of Citrus by electroporation. J Jpn Soc Hort Sci,1993, 62: 371-376
139.Hugueney P, Romer S, Kuntz M, Camara B. Characterization and molecular-cloning of a flavoprotein catalyzing the synthesis of phytofluene and zeta-carotene in capsicum chromoplasts. Eur J Biochem, 1992, 209: 399-407
140.Ikoma Y, Komatsu A, Kita M, Ogawa K, Omura M, Yano M, Moriguchi T.Expression of a phytoene synthase gene and characteristic carotenoid accumulation during citrus fruit development. Physiol Plant, 2001, 111 (2): 232-238
141.Isaacson T, Ohad I, Beyer P, Hirschberg J. Analysis in vitro of the enzyme CRTISO establishes a poly-cis carotenoid biosynthesis pathway in plants. Plant Physiol, 2004,136: 4246-4255
142.Isaacson T, Ronen G, Zamir D, Hirschberg J. Cloning of tangerine from tomato reveals a carotenoid isomerase essential for the production of β-carotene and xanthophylls in plants. Plant Cell, 2002, 14: 333-342
143.Izant J G, Weintraub H. Inhibition of thymidine kinase gene expression by anti-sense RNA: a molecular approach to genetic analysis. Cell, 1984, 36(4): 1007-1015
144.James D J, Passey A J, Baker S A, Wilson F M. Transgenes display stable patterns of expression in apple fruit and Mendelian segregation in the progeny. Bio Technology,1996, 14: 56-60
145.James D J, Uratsu S, Cheng J, Negri P, Viss P, Dandekar A M. Acetosyringone and osmoprotectants like betaine or praline synergistically enhance Agrobacterium-mediated transformation of apple. Plant Cell Rep, 1993, 12: 559-563
146.Jansson S, Douglas C J. Populus: a model system for plant biology. Annu Rev Plant Biol, 2007, 58: 435-458
147.Jarrell D C, Roose M L, Traugh S N, Kupper R S. A genetic map of citrus based on the segregation of isozymes and RFLPs in an intergeneric cross. Theor Appl Genet,1992, 84: 49-56
148.Jefferson, R A. Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol, 1987, 5: 387-405
149.Juarez J. Aleza P, Olivares-Fuster O. NavarroL. Recovery of tetraploid Clementine plants(Citrus Clementina Hort. Ex. ran)by in vitro colchicines treatment of shoot tips.In: Mohamed E eds. Program and Abstract, 10~(th) Inter. Soc. Citricuhure, Agadir,Morocco, 2004, 80
150.Kajiwara S, Fraser P D, Kondo K, Misawa N. Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid biosynthesis in Escherichia coli. Biochem J, 1997, 324: 421-426
151.Kaneyoshi J, Kobayashi S, Nakamura Y, Shigemoto N, Doi Y. A simple and efficient gene transfer system of trifoliate orange (Poncirus trifoliata Raf.). Plant Cell Rep,1994, 13:541-545
152.Kato M, Ikoma Y, Matsumoto H, Sugiura M, Hyodo H, Yano M. Accumulation of carotenoids and expression of carotenoid biosynthetic genes during maturation in Citrus fruit. Plant Physiol, 2004, 134: 824-837
153.Kato M, Ikoma Y, Matsumoto H, Sugiura M, Hyodo H, Yano M. Accumulation of Carotenoids and Expression of Carotenoid Biosynthetic Genes and Carotenoid Cleavage Dioxygenase Genes during Fruit Maturation in the Juice Sacs of 'Tamami,''Kiyomi' Tangor, and 'Wilking' Mandarin. J Japan Soc Hort Sci, 2007, 76 (2):103-111
154.Kato M, Matsumoto H, Ikoma Y, Okuda H, Yano M. The role of carotenoid cleavage dioxygenases in the regulation of carotenoid profiles during maturation in citrus fruit.J Exp Bot, 2006, 57: 2153-2164
155.Katsumoto Y, Fukuchi-Mizutani M, Fukui Y, Brugliera F, Holton T A, et al.Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-hued flowers accumulating delphinidin. Plant Cell Physiol, 2007, 48(11):1589-1600
156.Khawale R N, Singh S K, G Garg G, Baranwal V K, Alizadeh Ajirlo S.Agrobacterium-mediated genetic transformation of Nagpur mandarin (Citrus reticulata Blanco). Curr Sci, 2006, 91(12): 1700-1705
157.Kim I J, Ko K C, Kim C S, Chung W L. Isolation and expression patterns of a cDNA encoding phytoene synthase in Citrus. J Plant Physiol, 2001a, 158 (6): 795-800
158.Kim, I J, Ko K C, Kim C S, Chung W I. Isolation and characterization of cDNAs encoding P-carotene hydroxy lase in Citrus. Plant Sci, 2001b, 161(5): 1005-1010
159.Kishimoto S, Ohmiya A. Regulation of carotenoid biosynthesis in petals and leaves of chrysanthemum (Chrysanthemum morifolium). Physiol Plant, 2006, 128: 436-447
160.Kita M, Kato M, Ban Y, Honda C, Yaegaki H, Ikoma Y, Moriguchi T. Carotenoid accumulation in japanese apricot (Prunus mume Siebold & Zucc): Molecular analysis of carotenogenic gene expression and ethylene regulation. J Agric Food Chem,2007, 55: 3414-3420
161.Kita M, Komatsu A, Omura M, Yano M, Ikoma Y, Moriguchi T. Cloning and expression of CitPDSl, a gene encoding phytoene desaturase in citrus. Biosci Biotechnol Biochem, 2001, 65: 1424-1428
162.Kobayashi A K, Bespalhok J C, Pereira L F P, Vieira L G E. Plant regeneration of sweet orange (Citrus sinensis) from thin sections of mature stem segments. Plant Cell Tissue and Organ Cult, 2003, 74: 99-102
163.Kobayashi S, Goto-Yamamoto N, Hirochika H. Retrotransposon-induced mutations in grape skin color. Science, 2004, 304: 982
164.Kobayashi S, Ishimaru M, Ding C K, Yakushiji H, Goto-Yamamoto N. Comparison of UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) gene sequences between white grapes (Vitis vinifera) and their sports with red skin. Plant Sci, 2001, 160:543-550
165.Kobayashi S, Ohgawara T, Ohgawara E, Oiyama I, Ishii S. A somatic hybrid plant obtained by protoplast fusion between navel orange (Citrus sinensis) and satsuma mandarin (C. unshiu). Plant Cell Tiss Org Cult, 1988, 14: 63-69
166.Kobayashi S, Uchimiya H. Expression and integration of a foreign gene in orange (Citrus sinensis Osb.) protoplasts by direct DNA transfer. Jpn J Genet, 1989, 64:91-97
167.Kuntz M, Romer S, Suire C, Hugueney P, Weil J J, Schantz R, Camara B.Identification of a cDNA for the plastid-located geranylgeranyl pyrophosphate synthase from Capsicum annuum: correlative increase in enzyme activity and transcript level during fruit ripening. Plant J, 1992, 2: 25-34
168.Landrum J T, Bone R A. Lutein, zeaxanthin, and the macular pigment. Arch Biochem Biophys, 2001, 385 (1): 28-40
169.Lee H S. Characterization of carotenoids in juice of red navel orange (Cara Cara). J Agr Food Chem, 2001,49: 2563-2568
170.Lee H S. Objective measurement of red grapefruit juice color. J Agr Food Chem,2000,48: 1507-1511
171.Leitner-Dagan Y, Ovadis M, Zuker A, Shklarman E, Ohad I, Tzfira T, Vainstein A.CHRD, a plant member of the evolutionarily conserved YjgF family, influences photosynthesis and chromoplastogenesis. Planta, 2006, 225: 89-102
172.Li D D, Shi W, Deng X X. Agrobacterium-mediated transformation of embryogenic calluses of Ponkan mandarin and the regeneration of plants containing the chimeric ribonuclease gene. Plant Cell Rep, 2002, 21: 153-156
173.Li D D, Shi W, Deng X X. Factors influencing Agrobacterium-mediated embryogenic callus transformation of Valencia sweet orange (Citrus sinensis) containing the pTA29-barnase gene. Tree Physiol, 2003, 23: 1209-1215
174.Li F, Murillo C, Wurtzel E T. Maize Y9 encodes a product essential for 15-cis-zeta-carotene isomerization. Plant Physiol, 2007, 144: 1181-1189
175.Li L, Lu S, Cosman K M, Earle E D, Garvin D F, O'Neill J. beta-Carotene accumulation induced by the cauliflower OR gene is not due to an increased capacity of biosynthesis. Phytochemistry, 2006, 67 (12): 1177-1184
176.Li L, Van Eck J. Metabolic engineering of carotenoid accumulation by creating a metabolic sink. Transgenic Res, 2007,16(5): 581-585
177.Lilley C J, Bakhetia M, Charlton W L, Urwin P E. Recent progress in the development of RNA interference for plant parasitic nematodes. Mol Plant Pathol,2007, 8(5): 701-711
178.Lin S Q, Zhang Q Y. 'Hongyou'—A red color mutant of Pummelo. Proceedings of the Global Citrus Germplasm Network Meeting, 2002, 158
179.Liu E, Page J E. Optimized cDNA libraries for virus-induced gene silencing (VIGS) using tobacco rattle virus. Plant Methods, 2008,4: 5
180.Liu Q, Xu J, Liu Y Z, Zhao X L, Deng X X, Guo L L, Gu J Q. A novel bud mutation that confers abnormal patterns of lycopene accumulation in sweet orange fruit (Citrus sinensis L. Osbeck). J Exp Bot, 2007, 58: 4161-4171
181.Liu Y, Roof S, Ye Z, Barry C, van Tuinen A, Vrebalov J, Bowler C, Giovannoni J.Manipulation of light signal transduction as a means of modifying fruit nutritional quality in tomato. Proc Natl Acad Sci USA, 2004, 101: 9897-9902
182.Liu Y, Schiff M, Dinesh-Kumar S P. Virus-induced gene silencing in tomato. Plant J,2002, 31(6): 777-786
183.Lopez A B, Van Eck J, Conlin B J, Paolillo D J, O'Neill J, Li L. Effect of the cauliflower OR transgene on carotenoid accumulation and chromoplast formation in transgenic potato tubers. J Exp Bot, 2008, 59 (2): 213-223
184.Lu S and Li L. Carotenoid Metabolism: Biosynthesis, Regulation, and Beyond.Journal of Integrative Plant Biology, 2008, 50 (7): 778-785
185.Lu S, Van Eck J, Zhou X, Lopez A B. The cauliflower Or gene encodes a DnaJ cysteine-rich domain-containing protein that mediates high levels of beta-carotene accumulation. Plant Cell, 2006, 18(12): 3594-3605
186.Maheswaran, G, Welander, M, Hutchinson J F, Graham M W, Richards D.Transformation of apple rootstock M26 with Agrobacterium tumefaciens. J Plant Phys, 1992, 139: 560-568
187.Marty I, Bureau S, Sarkissian G, Gouble B, Audergon J M, Albagnac G. Ethylene regulation of carotenoid accumulation and carotenogenic gene expression in colour-contrasted apricot varieties (Prunus armeniaca). J Exp Bot, 2005, 56:1877-1886
188.Matzke M A, Birchler J A. RNAi-mediated pathways in the nucleus. Nat Rev Genet,2005, 6(1): 24-35
189.Moore G A, Jacono C C, Neidigh J, Lawrence S D, Cline K. Agrobacterium-mediated transformation of Citrus stem segments and regeneration of transgenic plants. Plant Cell Rep, 1992, 11:238-242
190.Morris W L, Ducreux L, Griffiths D W, Stewart D, Davies H V, Taylor M A.Carotenogenesis during tuber development and storage in potato. J Exp Bot, 2004, 55:975-982
191.Murashige T, Tucker D P H. Growth factor requirements for citrus tissue culture,Proceedings of the First International Citrus Symposium 3, 1969, 1115-1161
192.Mustilli A C, Fenzi F, Ciliento R, Alfano F, Bowler C. Phenotype of the tomato high pigment-2 mutant is caused by a mutation in the tomato homolog of DEETIOLATED1. Plant Cell, 1999, 11(2): 145-157
193.Napoli C, Lemieux C, Jorgensen R. Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans.Plant Cell, 1990, 2(3): 279-289
194.Nelson A, Roth D A, Johnson J D. Tobacco mosaic virus infection of transgenic Nicotiana plants is inhibited by antisense constructs directed at the 5' region of viral RNA. Gene, 1993, 127(2): 227-232
195.Oeller P W, Lu M W, Tanlor M L et al. Reversible inhibition of tomoto fruit senescence by antisense RNA. Science, 1991, 254: 437-439
196.Ogita S, Uefuji H, Yamaguchi Y, Koizumi N, Sano H. RNA interference: producing decaffeinated coffee plants. Nature, 2003,423 (6942): 823
197.Ohmiya A, Kishimoto S, Aida R, Yoshioka S, Sumitomo K. Carotenoid cleavage dioxygenase (CmCCD4a) contributes to white color formation in chrysanthemum petals. Plant Physiol, 2006, 142(3): 1193-1201
198.Okada K, Saito T, Nakagawa T, Kawamukai M, Kamiya Y. Five geranylgeranyl diphosphate synthases expressed in different organs are localized into three subcellular compartments in Arabidopsis. Plant Physiol, 2000, 122(4): 1045-1056
199.Olson J A, Krinsky N I, Faseb J. The colorful, fascinating world of the carotenoids: important physiologic modulators. FASEB J, 1995, 9(15): 1547-1550
200.Ossowski S, Schwab R, Weigel D. Gene silencing in plants using artificial microRNAs and other small RNAs. Plant J, 2008, 53(4): 674-690
201.Paine J A, Shipton C A, Chaggar S, et al. Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nat Biotechnol, 2005, 23(4): 482-487
202.Park H, Kreunen S S, Cuttriss A J, DellaPenna D, Pogson B J. Identification of the carotenoid isomerase provides insight into carotenoid biosynthesis, prolamellar body formation, and photomorphogenesis. Plant Cell, 2002, 14: 321-332
203.Pena L, Cervera M, Juarez J, Navarro A, Pina J A, Navarro L. Genetic transformation of lime (Citrus aurantifolia Swing.): factors affecting transformation and regeneration.Plant Cell Rep, 1997, 16: 731-737
204.Pena L, Cervera M, Ghorbel R, Dominguez A, fagoaga C, Juarez J, Pina A, Navarro L. Modification of Perennial Fruit Trees. In: Khan I A., (ed) Citrus Genetics,Breeding and Biotechnology. CAB International Publishing, 2007, pp, 329-344
205.Pena L, Martin-Trillo M, Juarez J, Pina J A, Navarro L, Martinez-Zapater J M.Constitutive expression of Arabidopsis LEAFY or APETALA1 genes in citrus reduces their generation time. Nature Biotech, 2001, 19: 263-267
206.Pena L, Perez R M, Cervera M, Juarez J A, Navarro L. Early events in Agrobacterium-mediated genetic transformation of citrus explants. Ann Bot, 2004, 94:67-74
207.Rapisarda P, Fallico B, Izzo R, MacCarone E. A simple and reliable method for determining anthocyanins in blood orange juices. Agrochimica, 1994, 38: 157-164
208.Ray J, Bird C R, Maunders M, Grierson D, Schuch W. Sequence of pTOM5, a ripening related cDNA from tomato. Nucl Acids Res, 1987, 15: 10587
209.Reforgiato R G, Russo G, Recupero S. New promising Citrus triploid hybrids selected from crosses between monoembryonic diploid female and tetraploid male parents.HortSci, 2005, 40: 516-520
210.Rodrigo M J, Marcos J F, Alferez F, Mallent M D, Zacarias L. Characterization of Pinalate, a novel Citrus sinensis mutant with a fruit-specific alteration that results in yellow pigmentation and decreased ABA content. J Exp Bot, 2003, 54: 727-738
211.Rodrigo M J, Marcos J F, Zacarias L. Biochemical and molecular analysis of carotenoid biosynthesis in flavedo of orange (Citrus sinensis L.) during fruit development and maturation. J Agr Food Chem, 2004, 52: 6724-6731
212.Romer S, Fraser P D, Kiano J W, Shipton C A, Misawa N, Schuch W, Bramley P M.Elevation of the provitamin A content of transgenic tomato plants. Nat Biotechnol,2000, 18: 666-669
213.R(?)mer S, Fraser P D. Recent advances in carotenoid biosynthesis, regulation and manipulation. Planta, 2005, 221: 305-308
214.R(?)mer S, Hugueney P, Bouvier F, Camara B, Kuntz M. Expression of the genes encoding the early carotenoid biosynthetic enzymes in Capsicum annuum. Biochem Biophys Res Communication, 1993, 196: 1414-1421
215.Romer S, Lubeck J, Kauder F, Steiger S, Adomat C, Sandmann G Genetic engineering of a zeaxanthin-rich potato by antisense inactivation and co-suppression of carotenoid epoxidation. Meta Eng, 2002,4 (3): 263-272
216.Ronen G, Carmel G L, Zamir D, Hirschberg J. An alternative pathway to beta-carotene formation in plant chromoplasts discovered by map-based cloning of Beta and old-gold color mutations in tomato. Proc Natl Acid Sci USA, 2000, 97(20):11102-11107
217.Roose M, Williams T E. Citrus scion breeding in California. In: Albrigo L G eds.Proc. Inter. Soc. of Citriculture 9~(th) Congress, Orlando, 2003, 1: 34-36
218.Rosati C, Aquilani R, Dharmapuri S, Pallara P, Marusic C, Tavazza R, Bouvier F,Camara B. Metabolic engineering of beta-carotene and lycopene content in tomato fruit. Plant J, 2000, 24 (3): 413-419
219.Rouseff R, Raley L, Hofsommer H J. Application of diode array detection with a C-30 reversed phase column for the separation and identification of saponified orange juice carotenoids. J Agr and Food Chem, 1996,44: 2176-2181
220.Russo G, Recupero S, Recupero G R. The development of citrus triploid hybrids. In:Albrigo L G eds. Proc. Liter. Soc. of Citriculture 9th Congress, Orlando, 2003, I:198-199
221.Sambrook J, David W R. Molecular Clone: A Laboratory Manual (3rd ed) New York:Cold Spring Harbor Laboratory Press, 2001
222.Sandmanna G, Romer S, Fraser P D. Understanding carotenoid metabolism as a necessity for genetic engineering of crop plants. Meta Eng, 2006, 8: 291-302
223.Scolnik P A, Bartley G E. Nucleotide swquence of lycopene cyclase (GenBank L40176) from Arabidopsis (PGR-019). Plant Physiol, 1995, 108: 1343
224.Senthil Kumar M, Hema R, Anand A, Kang L, Udayakumar M, Mysore K S. A systematic study to determine the extent of gene silencing in Nicotiana benthamiana and other Solanaceae species when heterologous gene sequences are used for virus-induced gene silencing. New Phytologist, 2007,176: 782-791
225.Senthil Kumar M, Rame Gowda H V, Hema R, Mysore K S, Udayakumar M.Virus-induced gene silencing and its application in characterizing genes involved in water-deficit-stress tolerance. J Plant Physiol, 2008, 165(13): 1404-1421
226.Sharp PA. RNA interference-2001. Genes Dev, 15: 485-490
227.Shewmaker C K, Sheehy J A, Daley M, Colburn S, Ke D Y. Seed specific overexpression of phytoene synthase: increase in carotenoids and other metabolic effects. Plant J, 1999, 20 (4): 401-412
228.Simkin A J, Gaffe J, Alcaraz J P, Carde J P, Bramley P M, Fraser P D, Kuntz M.Fibrillin influence on plastid ultrastructure and pigment content in tomato fruit.Phytochemistry, 2007, 68: 1545-1556
229.Simkin A J, Uhu C, Kuntz M, Sandmann G Light-dark regulation of carotenoid biosynthesis in pepper (Capsicum annuum) leaves. J. Plant Physiol, 2003, 160:439-443
230.Soost R K and Cameron J W. Citrus. In: Janick J and Moore J N. Advances in Fruit Breeding. West Lafayetete, Indiana, Purdue University Press, 1975, pp, 507-540
231.Srinivas A, Behera R K, Kagawa T, Wada M, Sharma R. High pigment1 mutation negatively regulates phototropic signal transduction in tomato seedlings. Plant Physiol, 2004, 134(2): 790-800
232.Stachel S E, Nester E W and Zambryski P C. A plant cell factor induces Agrobacterium tumefaciens vir gene expression. Proc Natl Acid Sci USA, 1986, 83:379-383
233.Starrantino A. Use of triploid for production of seedless cultivars in citrus improvement programs. In: Russo G Proc. Inter. Soc. Citriculture, 1992,1: 117-121
234.Stoutjesdijk P A, Singh S P, Liu Q, Hurlstone C J, Waterhouse P A, Green A G hpRNA-mediated targeting of the Arabidopsis FAD2 gene gives Highly efficient and stable silencing. Plant Physiol, 2002, 129(4): 1723-1731
235.Talon M, Gmitter F G Citrus Genomics. Int J Plant Genomics 2008, 2008: 528361
236.Tao N G, Xu J, Cheng Y J, Deng X X. Lycopene-e-cyclase pre-mRNA is alternatively spliced in Cara Cara navel orange (Citrus sinensis Osbeck). Biotechnol Lett, 2005, 27:779-782
237.Tao N G, Hu Z Y, Liu Q, Xu J, Cheng Y J, Guo L L, Guo W W, Deng X X.Expression of phytoene synthase gene (Psy) is enhanced during fruit ripening of Cara Cara navel orange (Citrus sinensis Osbeck). Plant Cell Reports, 2007, 26: 837-843
238.Taylor M and Ramsay G. Carotenoid biosynthesis in plant storage organs: recent advances and prospects for improving plant food quality. Physiol Plant, 2005, 124:143-151
239.Tchurikov N A, Chistyakova L G, Zavilgelsky G B, Manukhov I V, Chernovi B K ,Golovai Y B. Gene-specific silencing by expression of parallel complementary RNA in Escherichia coli. J Biol Chem, 2000, 275 (34): 26523-26529
240.Tracewell C A, Vrettos J S, Bautista J A, Frank H A, Brudvig G W. Carotenoid Photooxidation in Photosystem II Arch Biochem Biophys, 2001, 385: 61-69
241.Van Vliet T, van Schaik F, Schreurs W H, van den Berg H. In vitro measurement of (3-carotene cleavage activity: methodological considerations and the effect of other carotenoids on P-carotene cleavage. Int J Vitam Nutr Res, 1996, 66: 77-85
242.Vardi A, Bleichman S, Aviv D. Genetic transformation of citrus protoplasts and trgeneration of transgenic plants. Plant Sci, 1990, 69: 199-206
243.Vardi A. Isolation of protoplasts in Citrus. Proc. Int. Soc. Citriculture, 2, 1977,575-578
244.Vishnevetsky M, Ovadis M, Vainstein A. Carotenoid sequestration in plants: the role of carotenoid-associated proteins. Trends Plant Sci, 1999, 4: 232-235
245.Volkov R A, Panchuk I I, and Schoffl F. Heat-stress-dependency and developmental modulation of gene expression: the potential of house-keeping genes as internal standards in mRNA expression profiling using real-time RT-PCR. J Exp Bot, 2003,54 (391): 2343-2349
246.Von Lintig J, Welsch R, Bonk M, Giuliano G, Batschauer A, Kleinig H.Light-dependent regulation of carotenoid biosynthesis occurs at the level of phytoene synthase expression and is mediated by phytochrome in Sinapis alba and Arabidopsis thaliana seedlings. Plant J, 1997, 12: 625-634
247.Von Lintig J, Wyss A. Molecular analysis of vitamin A formation: cloning and characterization of P-carotene 15, 15'-dioxygenase. Arch Biochem Biophys, 2001 385 (1): 47-52
248.Wang F, Jiang J G, Chen Q. Progress on molecular breeding and metabolic engineering of biosynthesis pathways of C (30), C (35), C (40), C (45), C (50) carotenoids. Biotechnol Adv, 2007, 25(3): 211-222
249.Wang M B, Waterhouse P M. Application of gene silencing in plants. Curr Opin Plant Biol, 2002, 5(2): 146-150
250.Wang N, Fang W, Han H, Sui N, Li B, Meng Q W. Overexpression of zeaxanthin epoxidase gene enhances the sensitivity of tomato PSII photoinhibition to high light and chilling stress. Physiol Plant, 2008, 132: 384-396
251.Weigel D and Nilsson O. A developmental switch sufficient for flower initiation in diverse plants. Nature, 1995, 377: 495-500
252.Wesley S V, Helliwell C A, Smith N A, Wang M B, Rouse D T, Liu Q, et al.Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J,2001,27: 581-90
253.Wianny F, Zernicka-Goctz M. Specific interference with gene function by double-stranded RNA in early mouse development. Nat Cell Biol, 2000, 2: 70-75
254.Wielopolska A, Townley H, Moore I, Waterhouse P, Helliwell C. A high-throughput inducible RNAi vector for plants. Plant Biotechnol J, 2005, 3: 583-590
255.Wong W S, Li G G, Ning W, Xu Z F, Wendy Hsiao W L, Zhang L Y, Li N. Repression of chilling-induced ACC accumulation in transgenic citrus by over-production of antisense 1-aminocyclopropane-l-carboxylate synthase RNA. Plant Sci, 2001, 161:969-977
256.Wu J H, Mooney P. Autotetraploid tangor plant regeneration fromin vitro Citrus somatic embryogenic callus treated with colchicines. Plant Cell Tiss Org Cult, 2002,70: 99-104
257.Yao J L, Cohen D, Atkinson R, Richardson K, Morris B. Regeneration of transgenic plants from the commercial apple cultivar Royal Gala. Plant Cell Rep, 1995, 14:407-412
258.Yao J L, Wu J H, Gleave A P, Morris B A M. Transformation of citrus embryogenic cells using particle bombardment and production of transgenic embryos. Plant Sci,1996, 113: 175-183
259.Ye X D, Al-Babili S, Kloti A, Zhang J, Lucca P, Beyer P, Potrykus I. Engineering the provitamin A (P-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm.Science, 2000, 287: 303-305
260.Yi C E, Bekker J M, Miller G, Hill K L; Crosbie R H. Specific and potent RNA interference in terminally differentiated myotubes. J Biol Chem, 2003, 278: 934-939
261.Yu B, Lydiate D J, Young L W, Schafer U A, Hannoufa A. Enhancing the carotenoid content of Brassica napus seeds by downregulating lycopene epsilon cyclase.Transgenic Res. 2008, 17: 573-585
262.Zamore P D, Tuschl T, Sharp P A, Bartel D P. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell, 2000,101: 25-33
263.Zeng S H, Chen C W, Hong L Liu J H, Deng X X. In vitro induction, regeneration and analysis of autotetraploids derived from protoplasts and callus treated with colchicine in Citrus. Plant Cell Tiss Organ Cult, 2006, 87: 85-93
264.Zhang W, Dubcovsky J. Association between allelic variation at the Phytoene synthase 1 gene and yellow pigment content in the wheat grain. Theor Appl Genet, 2008,116:635-645
265 .Zhou X, Van Eck J, Li L. Use of the cauliflower Or gene for improving crop nutritional quality. Biotechnol Annu Rev, 2008, 14: 171-190
266.Zhu C, Yamamura S, Koiwa H, Nishihara M, Sandmann G cDNA cloning and expression of carotenogenic genes during flower development in Gentiana lutea.Plant Mol Biol, 2002,48: 277-285
267.Zhu X F, Suzuki K, Okada K, Tanaka K, Nakagawa T, Kawamukai M, Matsuda K.Cloning and functional expression of a novel geranylgeranyl pyrophosphate synthase gene from Arabidopsis thaliana in Escherichia coli. Plant Cell Physiol, 1997, 38:357-361