摘要
药用植物长春花(Catharanthus roseus)中含有130余种萜类吲哚生物碱(Terpenoid indole alkaloids, TIAs),包括长春碱(Vinblastine)、长春新碱(Vincristine)等重要的抗肿瘤TIAs。但这些TIAs在天然长春花植株中含量较低,化学结构复杂使其化学人工合成十分困难,因此,仅从天然长春花植物中提取TIAs不能满足市场需求,使其价格较为昂贵。尽管通过毛状根和细胞培养系统能产生单萜类吲哚生物碱,但因含量太低且不能产生最有药用价值的双萜类吲哚生物碱—长春碱和长春新碱而不具有商业化应用前景。因此,利用代谢工程提高植物生物碱含量和改变生物碱组成成份对商业化生产生物碱是非常必要的。
本研究建立了适合于遗传转化的长春花再生体系,为了提高TIAs的含量,并在长春花中过量表达TIAs生物合成途径中的关键基因g10h和dat。本研究得到了如下结果:
(1)建立了高效的长春花下胚轴再生体系。
(2)构建了含TIAs生物合成关键酶基因g10h或dat的单元或双元植物表达载体(pCAMBIA2300-g10h, pCAMBIA2300-g10h-dat)。
(3)采用农杆菌介导法转化长春花下胚轴,获得了19株转g10h基因的长春花、25株共转g10h和dat基因的长春花。
(4) Real-time PCR分析结果表明外源DNA整合到长春花基因组中。
Catharanthus roseus contains a group of about 130 terpenoid indole alkaloids (TIAs). Many TIAs are true lead compounds for drug development, including two commercially important cytotoxic dimeric alkaloids; vinblastine and vincristine are utilized in a number of different cancer chemotherapies. Most of these TIAs are produced at low levels in the natural plants and are difficult to be chemically synthesized due to their complex structures. Hairy root culture system and cell culture system offer promising alternatives for the production of these valuable alkaloids. Although transgenic hairy roots and cell cultures can produce a number of TIAs, they seldom produce the most valuable dimeric alkaloids, vinblastine and vincristine,only monomeric alkaloid can be produced in cell and tissue cultures, but at levels too low for commercialization. Metabolic engineering offers one of the most promising approaches for improving the product composition and increasing alkaloids yield in plants.
In this study, an efficient and rapid somatic embryogenesis based plant regeneration systerm was standardized for large-scale genetic transformation in Catharanthus roseus. To increase TIAs production, key genes involved in the TIAs biosynthetic pathway, g10h and dat were overexpressed in transgenic Catharanthus roseus plants by using genetic engineering method. The results are as following.
(1) An efficient and rapid somatic embryogenesis based plant regeneration systerm from Catharanthus roseus hypocotyls was establishment.
(2) Monovalent or bivalent expression vectors (pCAMBIA2300-g10h, pCAMBIA2300-g10h-dat) were constructed which contained the rate-limiting enzyme genes, g10h, and/or dat.
(3) A series of transgenic Catharanthus roseus were obtained using agrobacterium-mediated transformation. 19 independent transgenic plants containing g10h alone, 25 independent transgenic plants containing g10h and dat together were obtained.
(4) Some transgenic plants were analyzed by real-time PCR. Results showed that foreign DNA fragments were randomly inserted into Catharanthus roseus genome.
引文
[1]陈晓亚,刘培.植物次生代谢的分子生物学及基因工程.生命科学, 1996, 8(2): 8-11.
[2]唐中华,于景华,杨逢建,祖元刚.植物生物碱代谢生物学研究进展.植物学通报, 2003, 20(6): 696-702.
[3] Hashimoto T, Yamada Y. New genes in alkaloid metabolism and transport. Curr Opin iotechnol, 2003, 14: 163-168.
[4] Raskin I, Ribnicky DM, Komarnytsky S, et al. Plants and human health in the twenty-first century. Trends Biotechnol, 2002, 20: 522-531.
[5]祖元刚,罗猛,牟瑶松,付玉杰,王俐.长春花生物碱成分及其药理作用研究进展.天然产物研究与开发, 2006, 18: 325-329, 294.
[6] Johnson JS, Wright HF, Svoboda GH. Experimental basis of clinical evalution of antitumor principles derived from Vinca rosea L. Lab Clin Med, 1959, 54: 380.
[7] Van der Heijden R, Jacobs DI., Snoeijer W, Hallard D, Verpoorte R. The Catharanthus alkaloids: pharmacognosy and biotechnology. Curr. Med. Chem., 2004, 11: 607-628.
[8]赵剑,朱蔚华.长春花生物碱生物合成途径和相关酶及其基因调控的研究进展.植物生理学通讯, 1999, 35(1): 60-68.
[9]卢懿,候世详,陈彤.长春花抗癌成分长春新碱研究的进展.中国中药杂志, 2003, (11): 1006-1009.
[10] De Carolis E, De Luca V. Purification, characterization, and kinetic analysis of a 2-oxoglutarate-dependent deoxygenate involved in vindoline biosynthesis from Catharanthus roseus. J Biol Chem, 1993, 268: 5504-5551.
[11] De Carolis E, De Luca V. A novel 20-oxoglutarate-dependent dioxygenese involved in vindole biosynthesis: characterization, purification, and kinetic properties. Plant Cell, Tiss and Org Cult, 1994, 38: 281-287.
[12] De Luca V, Balsevich J, Tyler RT, et al. Biosynthesis of indole alkaloids: developmental regulation of the biosynthetic pathway from tabersonine to vindoline in Catharanthus roseus. J Plant Physiol, 1986, 125: 147-156.
[13] Blom TJM, Sierra MI, Van Vliet TB, et al. Uptake and accumulation of ajmalicine into isolated vacules of cultures cells of Catharabthus roseus (L.) G. don and its conversion into serpentine. Planta, 1991, 183: 170-177.
[14] Endo T, Goodbody A, Vikovic J, et al. Enzymes from Catharanthus roseus cell cultures that couple vindoline and catharanthine to from 3’,4’-anhydrovinblastine. Phytochem, 1988, 27: 2147-2149.
[15] Kutney JP, Botta B, Boulet GA, et al. Alkaloid production in Catharanthus roseus (L.) G. don cell cultures. XVI. Biotransformation of 3, 4-anhydrovinblastine with Catharanthus roseus cell cultures and enzyme systems. Heterocycles, 1988, 27: 629-637.
[16] Sprenger GA, Sch?rken U, Wiegert T, et al. Identification of a thiamin-dependent synthase in Escherichia coli required for the formation of the 1-deoxy-D-xylulose 5-phosphate precursor to isoprenoids, thiamin, and pyridoxol. Proc Natl Acad Sci USA, 1997, 94: 12857-12862.
[17] Estévez JM, Cantero A, Reindl A, et al. 1-deoxy-D-xylulose-5-phosphate synthase, a limiting enzyme for plastidic isoprenoid biosynthesis in plants. J Biol Chem, 2001, 276: 22901-22909.
[18] Lois LM, Campos N, Putra SR, et al. Cloning and characterization of a gene from Escherichia coli encoding a transketolase-like enzyme that catalyzes the synthesis of D-1-deoxyxylulose 5-phosphate, a common precursor for isoprenoid, thiamin, and pyridoxol biosynthesis. Proc Natl Acad Sci USA, 1998, 95: 2105-2110.
[19] Bouvier F, d’Harlingue A, Suire C, et al. Dedicated roles of plastid transketolases during the early onset of isoprenoid biosynthesis in pepper fruits. Plant Physiol 1998, 117: 1423-1431.
[20] Lois LM, Rodriguez-Concepcion M, Gallego F, et al. Carotenoid biosynthesis during tomato fruit development: regulatory role of 1-deoxy-D-xylulose 5-phosphate synthase. Plant J, 2000, 22(6): 503-513.
[21] Chahed K, Oudin A, Guivarc’h N, et al. 1-Deoxy-D-xylulose 5-phosphate synthase from periwinkle: cDNA identification and induced gene expression in terpenoid indole alkaloid-producing cells. Plant Physiol Biochem, 2000, 38: 559-566.
[22] Carretero-Paulet L, Ahumada I, Cunillera N, et al. Expression and molecular analysis of the Arabidopsis DXR gene encoding 1-Deoxy-D-xylulose 5-phosphate reductoisomerase, the first committed enzyme of the 2-C-methyl-D-erythritol 4-phosphate pathway. Plant Physiol, 2002, 129: 1581–1591.
[23] Zhu XF, Suzuki K, Saito T, et al. Geranylgeranyl pyrophosphate synthase encoded by the newly isolated gene GGPS6 from Arabidopsis thaliana is localized in mitochondria. Plant Mol Biol, 1997, 35: 331-341.
[24] Bantignies B, Liboz T, Ambid C. Nucleotide sequence of a Catharanthus roseus geranylgeranyl pyrophosphate synthase gene. Plant Physiol, 1995, 110: 336-336.
[25] Liao ZH, Chen M, Gong YF, et al. A new geranylgeranyl diphosphate synthase gene from Ginkgo biloba, which intermediates the biosynthesis of the key precursor for ginkgolides. DNA Sequence, 2004, 15(2): 153-158.
[26] Hefner J, Ketchum REB, Croteau R. Cloning and functional expression of a cDNA encoding geranylgeranyl diphosphate synthase from Taxus canadensis and assessment of the role of this prenyltransferase in cells induced for taxol production. Arch Biochem Biophys, 1998, 360: 62-74.
[27] Liao ZH, Gong YF, Kai GY, et al. An intron-free methyl jasmonate inducible geranylgeranyl diphosphate-synthase gene from Taxus media and its functional identification in yeast. Mol Biol, 2005, 39(1): 11-17.
[28] van der Fits L, Memelink J. ORCA3, a jasmonate-responsive transcriptionalregulator of plant primary and secondary metabolism. Science, 2000, 289: 295-297.
[29] Meijer AH, De Wall A, Verpoorte R, et al. Purification of the cytochrome P-450 enzyme geraniol-10-hydroxylase from cell cultures of Catharanthus roseus. J Chromatogr, 1993, 653: 237-249.
[30] Collu G, Unver N, Peltenburg-Looman AMG, et al. Geraniol 10-hydroxylase1, a cytochrome P450 enzyme involved in terpenoid indole alkaloid biosynthesis. FEBS Lett, 2001, 508: 215-220.
[31] Meijer AH, Lopes Cardoso MI, Voskuilen JTH, et al. Isolation and charac- terization of a cDNA clone from Catharanthus roseus encoding NADPH: cytochrome P-450 reductase, an essential for reactions catalysed by cytochrome P-450 monoxygenase. Plant Mol Biol, 1993, 22: 379-383.
[32] Whitmer S, Canel C, Hallard D, et al. Influence of precursor availability on alkaloid accumulation by transgenic cell line of Catharanthus roseus. Plant Physiol, 1998, 116: 853-857.
[33] Collu G, Garcia AA, van der Heijden R, et al. Activity of the cytochrome P450 enzyme geraniol 10-hydroxylase and alkaloid production in plant cell cultures. Plant Sci, 2002, 162: 165-172.
[34] Mc Farlane J, Madyastha KM, Coscia CJ. Regulation of secondary metabolism in higher plants. Effect of alkaloids on a cytochrome P450 dependent monooxygenase. Biochem Biophys Res Commun, 1975, 66: 1263-1269.
[35] Schiel O, Witte L, Berlin J. Geraniol 10-hydroxylase activity and its relation to monoterpene indole alkaloid accumulation in cell suspension cultures of Catharanthus roseus. Z Naturforsch, 1987, 42: 1075-1081.
[36] Gong YF, Liao ZH, Pi Y, et al. Engineering terpenoid indole alkaloids bio- synthetic pathway in Catharanthus roseus hairy root cultures by overexpressing the geraniol 10-hydroxylase gene. J Shanghai Jiao Tong Univ, 2005, accepted.
[37] Poulsen C, Bongaerts RJ, Verpoorte R. Purification and characterization of anthranilate synthase from Catharanthus roseus. Eur J Biochem, 1993, 212: 431-440.
[38] Meijer AH, Verpoorte R, Hoge JHC. Regulation of enzymes and genes involved in terpenoid indole alkaloid biosynthesis in Catharanthus roseus. J Plant Res, 1993, 3: 145-164.
[39] Hughes E, Hong SB, Gibson SI, et al. Metabolic engineering of the indole pathway in Catharanthus roseus hairy roots and increased accumulation of tryptamine and serpentine. Metab Eng, 2004, 6: 268-276.
[40] De Luca V, Marineau C, Brisson N. Molecular cloning and analysis of a cDNA encoding a plant tryptophan decarboxylase: comparison with animal dopa decarboxylases. Proc Natl Acad Sci USA, 1989, 86: 2582-2586.
[41] Lopez-Meyer M, Nessler CL. Tryptophan decarboxylase is encoded by two autonomously regulated genes in Camptotheca acuminata which are differentially expressed during development and stress. Plant J, 1997, 11(6): 1167-1175.
[42] Yamazaki Y, Sudo H, Yamazaki M, et al. Camptothecin biosynthetic genes in hairy roots of Ophiorrhiza pumila: cloning, characterization and differential expression in tissues and by stress compounds. Plant Cell Physiol, 2003, 44: 395-403.
[43] Nef C, Rio B, Chrestin H, et al. Induction of catharanthine synthesis and stimulation of major indole alkaloids production in Catharanthus roseus cells under non-growth altering treatment with Pythium vexans extracts. Plant Cell Rep, 1991, 10: 26-29.
[44] Pasquali G, Goddijn OJM, De vaal A, et al. Coordinated regulation of two indole alkaloid biosynthetic genes from Catharanthus roseus by auxin and elicitors. Plant Mol Biol, 1992, 18: 1121-1131.
[45] Canel C, Lopes-Cardoso MI, Whitmer S, et al. Effects of over-expression ofstrictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus. Planta, 1998, 205: 414-419.
[46] Kutchan TM. Strictosidine: from alkaloid to enzyme to gene. Phytochem, 1993, 32: 493-506.
[47] Yao K, De Luca V, Brisson N. Creation of a metabolic sink for tryptophan alters the phenylpropanoid pathway and the susceptibility of potato to phytophthora infestans. Plant Cell, 1995, 7: 1787-1799.
[48] McKnight TD, Roessner CA, Devagupta R, et al. Nucleotide sequence of a Cdna encoding the vacuolar protein strictosidine synthase from Catharanthus roseus. Nucleic Acids Res, 1990, 18(16): 4939.
[49] Stevens LH, Giroud C, Pennings EJM, et al. Purification and characterization of strictosidine synthase from a suspension culture of Cinchona robusta, Phytochem, 1993, 33: 99-106.
[50] Geerlings A, Hallard D, Caballero AM, et al. Alkaloid production by a Cinchona officinalis‘Ledgeriana’hairy root culture containing constitutive expression constructs of tryptophan decarboxylase and strictosidine synthase cDNAs from Catharanthus roseus. Plant Cell Rep, 1999, 19: 191-196.
[51] Geerlings A, Martínez-Lozano Iba?ez M, Memelink J, et al. Molecular cloning and analysis of strictosidineβ-Dglucosidase, an enzyme in terpenoid indole alkaloid biosynthesis in Catharanthus roseus. J Biol Chem, 1999, 275: 3051-3056.
[52] Gerasimenko I, Sheludko Y, Ma X, et al. Heterologous expression of a Rauvolfia cDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoterpenoid indole alkaloids. Eur J Biochem, 2002, 269: 2204-2213.
[53] Zárate R, Bonavia M, Geerlings A, et al. Expression of strictosidineβ-D-glucosidase cDNA from Catharanthus roseus, involved in the monoterpene indole alkaloid pathway, in a transgenic suspension culture of Nicotiana tabacum.Plant Physiol Biochem, 2001, 39: 763-769.
[54] Vazquez-Flota F, De Carolis E, Alarco AM, et al. Molecular cloning and characterization of desacetoxyvindoline-4-hydroxylase, a 2-oxoglutarate dependent-dioxygenase involved in the biosynthesis of vindoline in Catharanthus roseus (L.) G. Don. Plant Mol Biol, 1997, 34: 935-948.
[55] Schroder G, Unterbusch E, Kaltenbach M, et al. Light-induced cytochrome P450-dependent enzyme in indole alkaloid biosynthesis: tabersonine 16-hydroxylase. FEBS Lett, 1999, 458: 97-102.
[56] St-Pierre B, De Luca V. A cytochrome P-450 monooxygenase catalyzes the first step in the conversion of tabersonine to vindoline in Catharanthus roseus. Plant Physiol, 1995, 109: 131-139.
[57] De Carolis E, Chan F, Balsevich J, et al. Isolation and characterization of a 2-oxoglutarate dependent dioxygenase involved in the second-to-last step in vindoline biosynthesis. Plant Physiol, 1990, 94: 1323-1329.
[58] De Luca V, Balsevich J, Tyler RT, et al. Biosynthesis of indole alkaloids: Developmental regulation of the biosynthetic pathway from taberonine to vindoline in Catharanthus roseus. J Plant Physiol, 1986, 125: 147-156.
[59] Kurz WGW, Chatson KB, Constabel F, et al. Alkaloid production in Catharanthus roseus cell cultures: Initial studies on cell lines and their alkaloid content. Phytochem, 1980, 19: 2583-2587.
[60] Kutney JB, Choi LSL, Kolodziejczyk P, et al. Alkaloid production in Catharanthus roseus cell cultures: Isolation and characterization of alkloids from one cell line. Phytochem, 1980, 19: 2589-2595.
[61] De Luca V, Balsevich J, Tyler RT, et al. Characterization of a novel N-methyltransferase (NMT) from Catharanthus roseus. Plant Cell Rep, 1987, 6: 458-461.
[62] St-Pierre B, Vazquez-Flota FA, De Luca V. Multicellular compartmentation ofCatharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathway intermediate. Plant Cell, 1999, 11: 887-900.
[63] Léon J.P. van Tegelen, Paolo R.H. Moreno, Anton F. Croes, Robert Verpoorte, George J. Wullems. Purification and cDNA Cloning of Isochorismate Synthase from Elicited Cell Cultures of Catharanthus roseus. Plant Physiol. 1999, 119(2): 705–712.
[64] Daniel E. Stafford, Kurt S. Yanagimachi, Philip A. Lessard, Sushil K. Rijhwani, Anthony J. Sinskey, and Gregory Stephanopoulos. Optimizing bioconversion pathways through systems analysis and metabolic engineering. Proc Natl Acad Sci U S A. 2002, 19; 99(4): 1801–1806.
[65] Facchini PJ, Huber-Allanach KL, Tari LW. Plant aromatic L-amino acid decarboxylases: evolution, biochemistry, regulation, and metabolic engineering applications. Phytochemistry. 2000, 54(2):121-138.
[66] Stefano Di Fiore, Qiurong Li, Mark James Leech, Flora Schuster, Neil Emans, Rainer Fischer, and Stefan Schillberg. Targeting Tryptophan Decarboxylase to Selected Subcellular Compartments of Tobacco Plants Affects Enzyme Stability and in Vivo Function and Leads to a Lesion-Mimic Phenotype. Plant Physiol. 2002, 129(3): 1160–1169.
[67] Verpoorte R, van der Heijden R, ten Hoopen HJG, et al. Metabolic engineering of plant secondary metabolite pathways for the production of fine chemicals. Biotech Lett, 1999, 21: 467-479.
[68]赵剑,朱蔚华,胡秋.长春花叶片中愈伤组织诱导及培养过程中生物碱合成类型的变化调节及有关酶的分析.中草药, 1999, 30(7): 533-538.
[69] Goswami R, Tyayi BR, Rain A, et al. Colchicine induced autotertraploids in perwinkle Catharanthus roseus. J Medicinal Aromatic Plant Sci, 1996, 18(1): 38-45.
[70] Endo T, Goodbody A, Vikovic J, et al. Enzymes from Catharanthus roseus cellculutres that couple vindoline and catharanthine to from 3′,4′-anhydrovinblastine. Phytochem, 1988( 27): 2147-2149.
[71]张向飞,张秀省,王勇.植物生长调节物质对长春花细胞中吲哚生物碱积累的影响.植物生理学通讯, 2004, 40(3): 303-304.
[72] Schlatmann JE, Moreno PRH, Ten Hoopen HJG, et al . Effect of oxygen and nutrient limitation on ajamalicine production and related enzyme actives in high density cutures of Catharanthus roseus. Biotechnol Bioeng, 1994, 44 (4) :461-468.
[73] Drapeau D, Blanch HW, Wilke CR. Ajmalicine, serpentine and catharanthine accumulation in Catharanthus roseus bioreactor cultures. Planta Med, 1987(53): 373-376.
[74]郑珍贵,缪红,杨文杰.营养和环境因子对长春花激素自养型细胞生长和阿吗碱生成的影响.植物学报, 1999, 41(2): 184-189.
[75]王宁宁,王淑芳,田俊英.土壤农杆菌转化的长春花冠瘿细胞培养.生物工程学报, 1994, 10(3): 244-239.
[76] Hallard D, Van der Heijden R, Verpoorte R, et al. Suspension cultured transgenic cells of nicotiana tabacum expressing tryptophan decarboxylase and strictosidine synthase cDNAs from Catharanthus roseus produce strictosidine upon secologanin feeding. Plant Cell Reports, 1997, 17: 50-54.
[77]龚一富,孙小芬,唐克轩.生物碱生物合成的代谢调控(英文).中国生物工程杂志, 2005, (增刊): 289-396.
[78] Oksman-Caldentey KM, Inze D. Plant cell factories in the post-genomic era: new ways to produce designer secondary metabolism. Trends in Plant Sci, 2004, 9(9): 433-440.
[79] Lloyd AM, Walbot V, Davis RW. Arabidopsis and Nicotiana anthocyanin production activated by maize regulators R and C1. Science, 1992, 258: 1773-1775.
[80] Bradley JM, Davies KM, Deroles KM, et al. The maize Lc regulatory gene up-regulates the flavonoid biosynthetic pathway of Petunia. Plant J, 1998, 13: 381-392.
[81] Mooney M, Desnos T, Harrison K, et al. Altered regulation of tomato and tobacco pigmentation genes caused by the delila gene of Antirrhinum. Plant J 1995, 7: 333-339.
[82] Goddijn OJM, Pennings EJM, van der Helm P, et al. Overexpression of a tryptophan decarboxylase cDNA in Catharanthus roseus crown gall calluses results in increased tryptamine levels but not in increased terpenoid indole alkaloid production. Transgenic Res, 1995, 4: 315-323.
[83] Chintapakorn Y, Hamill JD. Antisense-mediated downregulation of putrescine N-methyltransferase activity in transgenic Nicotiana tabacum L. can lead to elevated levels of anatabine at the expense of nicotine. Plant Mol Biol, 2003, 53: 87-105.
[84] Davuluri GR, van Tuinen A, Fraser PD, et al. Fruit-specific RNAi-mediated suppression of DET1 enhances carotenoid and flavonoid content in tomatoes. Nat Biotech, 2005, 23: 890-895.
[85] Segal G, Song RT, Messing J. A new opaque variant of maize by a single dominant RNA-interference-inducing transgene. Genetics, 2003, 165: 387-397.
[86] Goossens A, Hakkinen ST, Laakso I, et al. Secretion of secondary metabolites by ATP-binding cassette transporters in plant cell suspension cultures. Plant Physiol, 2003, 131: 1161-1164.
[87] Shitan N, Bazin I, Dan K, et al. Involvement of CjMDR1, a plant multidrug resistance-type ATP-binding cassette protein, in alkaloid transport in Coptis japonica. Proc Natl Acad Sci USA, 2003, 100: 751-756.
[88] De Luca V, St-Pierre B. The cell and developmental biology of alkaloid biosynthesis. Trends Plant Sci, 2000, 5: 168-173.
[89] Sottomayor M, De Pinto MC, Salema R, et al. The vacuolar localization of a basic peroxidase isoenzyme responsible for the synthesis ofα-3’, 4’-anhydrovinblastine in Catharanthus roseus (L.) G. don leaves. Plant Cell Environ, 1996, 19: 761-767.
[90]黄玉仙.药用植物毛状根培养与次生代谢产物的研究.海峡药学, 2008, 20(8): 80-82.
[91]孙小芬,陈雨,唐克轩,潘俊松,王玉亮,孙克兴,蔡润.长春花主要农艺性状与文多灵含量的相关及通径分析.植物学报, 2009, 44 (1): 96-102.
[92] Kurz WG, Chatson KB, Constabel F, Kutney JP, Choi LS, Kolodziejczyk P, Sleigh SK, Stuart KL, Worth BR. Alkaloid production in Catharanthus roseus cell cultures VIII1. Planta Med., 1981, 42(5): 22-31.
[93] Bhadra R, Vani S, Shanks JV. Production of indole alkaloids by selected hairy root lines of Catharanthus roseus. Biotechnol. Bioeng., 1993, 41: 581-592.
[94] Hong SB, Peebles C, Shanks JV, San KY, Gibson SI. Terpenoid indole alkaloid production by C. roseus hairy roots induced by Agrobacterium tumefaciens harboring rol ABC genes. Biotechnol. Bioeng., 2006, 93(2): 386-390.
[95] Murashige T, Skoog F. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant, 1962, 15: 473-497.
[96] Kim SW, In DS, Choi PS, Liu JR. Plant regeneration from immature zygotic embryo-derived embryogenic calluses and cell suspension cultures of Catharanthus roseus. Plant Cell Tiss. Org. Cult., 2004, 76: 131-135.
[97] Junaid A, Mujib A, Sharma MP, Tang W. Growth regulators affect primary and secondary somatic embryogenesis in Madagaskar periwinkle (C. roseus (L.) G. Don) at morphological and biochemical levels. Plant Growth Regul., 2007, 51: 271-281.
[98] Dhandapani M, Kim DH, Hong SB. Efficient plant regeneration via somatic embryogenesis and organogenesis from the explants of Catharanthus roseus. In Vitro cell dev. Biol.-Plant, 2008, 44: 18-25.
[99]曲庆云,赵晓梅.统计分析方法——SAS实例精选.北京清华大学出版社, 2004, 97-102.
[100]郭素娟,任鹏,王桂云.燕山红栗下胚轴再生体系的建立.西南林学院学报, 2005, 25: 102-105.
[101] Junaid A, Mujib A, Bhat MA, Sharma MP, Samaj J. Somatic embryogenesis and plant regeneration in Catharanthus roseus. Biologia Plantarum, 2007*, 51 (4): 641-646.
[102] Halperin W, Wetherell WF. Ammonium requirement for embryogenesis in vitro. Nature, 1965, 205: 519-520.
[103]付凤玲,李晚忱,刘玉贞.玉米幼穗培养及植株再生.四川农业大学学报, 1999, 3: 278-281.
[104]李忠光,龚明.水解酪蛋白对烟草愈伤组织和悬浮培养细胞生长的促进作用.云南师范大学学报, 2006, 4: 60-61.
[105] Sanderst ME, Burkholder PR. Influence of amino acids on growth of Datura embryos in culture. Botany, 1948, 34: 516-526.
[106]向发云,宋志红,曾祥国,吴金平,刘凯,王新刚,吴润玲,冯小明,游艾青,顾玉成.外源脯氨酸及水分胁迫对籼稻花药培养效率的影响.湖北农业科学, 2007, 46: 861-863.
[107]张永华,辛建华,苑育文.番茄花药愈伤组织诱导中脯氨酸含量的变化.北方园艺, 2007, 6: 15-16.
[108] Bartlett JG, Alves SC, Smedley M, Snape JW, Harwood WA. High-throughput Agrobacterium-mediated barley transformation. Plant Methods, 2008, 4: 22-27.
[109] Green CE, Phillips RL, Kleese RA. Tissue culture of maize (Zea mays L): initiation, maintenance, and organic growth factors. Crop Sci., 1974, 14: 54-58.
[110] Jain RK, Peterson WL, Buchholz WG. Development and availability of germplasm with high type II culture formation response. Plant Cell Rep., 1996, 15: 449- 454.
[111] Firoozabady E, DeBoer DL. Plant regeneration via somatic embryogenesis in manycultivars of cotton (Gossypium hirsutum L.). In Vitro cell dev. Biol., 1993, 299: 166-173.
[112] Sakhanokho HF, Zip FA, Rajasekaran K, Saha S, Sharma GC. Induction of highly embryogenic calli and plant regeneration in Upland (Gossypium hirsutum L.) and Pima (Gossypium barbadense L.) Cottons. Crop Sci., 2001, 41: 1240-1246.
[113] Qayyum RA, Sarfraz HS, Saqib SM, Abbas BSY. Somatic embryogenesis in wild relatives of cotton (Gossypium Spp.). J. Zhejiang Univ. Sci. B, 2006, 7(4): 291-298.
[114] Choi PS, Lee SY, Chung HJ, In DS, Choi DW, Liu JR. Assessment of competence for adventitious shoot formation in hypocotyl explant cultures of 22 cultivars of Catharanthus roseus. J. Plant. Biol., 2003, 46 (2): 90-94.
[115] Lee SY, Choi PS, Chung HJ, In DS, Choi DW, Liu JR. Comparison of adventitious shoot formation in petiole explant cultures of 20 cultivars of Catharanthus rosesus. J. Plant Biotechnol., 2003, 5: 59-61.
[116] Joshua S Yuan, Ann Reed, Feng Chen,C Neal Stewart Jr. Statistical analysis of real-time PCR data. BMC Bioinformatics, 2006, 7: 1471-2105.
[117] Ririe KM, Rasmussen RP, Wittwer CT. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem, 1997, 245(2): 154-160.