Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/28556
標題: 小白菜組織培養再生與基因轉殖之研究
Studies on the In Vitro Tissue Regeneration and Gene Transformation of Pak-Choi (Brassica campestris L. ssp. chinensis L. Makino)
作者: 許家言
Hsu, Jia-Yan
關鍵字: Pak-Choi
小白菜
In Vitro Tissue Regeneration
Gene Transformation
組織培養再生
基因轉殖
出版社: 園藝學系所
引用: 尤進欽、曾夢蛟、陳良築。1996。蘇力菌殺蟲晶體蛋白基因轉移到青花菜、花椰菜及小白菜。中國園藝42(4):312-330。 尤進欽。1994。鎘結合蛋白基因、抗凍蛋白基因及蘇力菌殺蟲晶體蛋白轉移至三種蕓薹屬蔬菜之研究。國立中興大學園藝學系碩士論文。 尤進欽。2000。超氧化歧化酵素與過氧化氫酵素基因之轉殖與選殖。國立中興大學園藝學系博士論文。260頁。 方紅筠。1997。轉豇豆胰蛋白酶抑制劑基因抗蟲甘藍植株的獲得。植物學報39(10):940-945。 王淩健。1999。青菜組織培養和轉化系統的初步建立。實驗生物學報32(1):93-95. 丘應模。1994。清潔蔬菜。渡假出版社有限公司。p.57。 朱祝軍。2003。芸薹屬植物高效再生體系的建立。中國農業科學 2(11): 1239-1245。 何其仁。1993。四種蕓薹屬蔬菜作物原生質體再生及基因轉移的研究。國立中興大學園藝學系碩士論文。 何婉芬。2002。植物組織培養。152頁。復文書局,台南市。 于建明。1996。結球甘籃的離體再生及基因轉化條件研究。江蘇農業學報12(3):6-9. 吳佳春。2002。結球白菜的超氧化歧化酵素與過氧化氫酵素基因轉移至甘藍及結球白菜之研究。國立中興大學園藝學系碩士論文。125頁。 呂鋒洲。1998。體內自由基掃除劑---穀胱甘鈦。 健康世界叢書 p.1-24。健康世界雜誌社,臺北市。 李國梁。1999。青花菜子葉和下胚軸原生貭體的遺傳轉化系統。上海農業學報15(1):28-32。 林志輝、鄭榮宗、陳良築。2004。葉綠體基因轉殖。植物基因轉殖之原理與應用-第九章:111-120。植物生物技術教學資源中心主編。 林傳琦、高景輝。2000。 植物之氧化性傷害與鹽分逆境。 科學農業 48(9,10):244-248。 林照能、陳甘澍、劉政道。2004。小白菜新品種’臺農三號’簡介。農業世界250:34-35。 林榮芳 。1995。 縐葉菸草異源染色體添加系的建立與鑑定 國立臺灣大學植物學研究所博士論文。94頁。 高景輝。1987a。植物荷爾蒙。華香園出版社。586頁。 高景輝。1987b。植物生長與分化。國立編譯館。731頁。 張有明。1997。苦瓜組織培養再生、農桿菌媒介轉殖法及花粉電穿孔法之基因轉殖研究。國立臺灣大學園藝學研究所博士論文。233頁。 張清俊。2002。高等植物葉綠體的基因轉殖與植物生物技術。科學農業50(5,6):243-248。 許圳塗、鍾仁彬。1997。甘藍組織培養再生繁殖及組培苗建化技術。「園藝作物組織培養研究與應用」技術手冊p.21-31。農林廳種苗改良繁殖場編印。 郭昭麟、羅淑芳、李鎮宇、蔡新聲。2004。植物組織培養再生技術與基因轉殖。植物基因轉殖之原理與應用-第三章:25-46。植物生物技術教學資源中心主編。 陳志宏(-b)。2004。熱休克蛋白基因與過氧化氫酵素基因轉移到甘藍及結球白菜葉綠體之研究。國立中興大學園藝學系碩士論文。75頁。 陳淑惠、曾夢蛟。1998。不同啟動子對鎘結合蛋白基因在轉殖青花菜中表現的影響。中國園藝44(2):190-204。 陳榮芳。2004。青花菜之基因轉殖技術。植物基因轉殖與分子檢測技術-第二章:十字花科作物-第二節:p.57-74。植物生物技術教學資源中心主編。 曾夢蛟、劉程煒。2004。甘藍之基因轉殖技術。植物基因轉殖與分子檢測技術-第二章:十字花科作物-第三節:p.75-89。植物生物技術教學資源中心主編。 葉元純。2003。共同轉殖蘇力菌殺蟲晶體蛋白、熱休克蛋白、超氧化歧化酵素及過氧化氫酵素等基因至結球白菜及甘藍之研究。國立中興大學園藝學系碩士論文。123頁。 詹明才。2004。農桿菌轉殖技術。植物基因轉殖之原理與應用-第五章:59-72。植物生物技術教學資源中心主編。 詹富智、王曉俐。2004。菸草-農桿菌轉殖。植物基因轉殖與分子檢測技術-第一章:茄科作物-第二節: p.17-26。植物生物技術教學資源中心主編。 廖芳心。1998。應用花粉管基因導入法輔助甘藍抗黑腐病育種之研究。中國園藝44(1):55-63。 趙權才、墨峰主編。1991。家庭食品全書。天津: 天津科技翻譯出版公司。P.12,602。 劉程煒。2003。水稻農桿菌基因轉殖系統與甘藍及水稻葉綠體基因轉殖系統之建立及利用。國立中興大學園藝學系博士論文。 蔡小寧。1997。建立青菜農桿菌介導法基因轉化體系。江蘇農業學報13(2):110-114. 蔡小寧。1999。影響大白菜離體再生和基因轉化的因素。江蘇農業學報20(2):1-4. 蔡小寧。2003。芸苔屬蔬菜轉基因研究新進展。江蘇農業學報專文。共6頁。 張智奇。1998。小白菜子葉誘導不定芽再生植株。上海農業學報14(2):25-28。 Agnihotri, A., V. Upta and V. Jafannathan. 1990. Plant regeneration from callus and protoplasts of Brassica nigra (IC257) through somatic embryogenesis. Plant Cell Rep. 9:427-430. Allen, R. D. 1995. Dissection of oxidative stress tolerance using transgenic plants. Plant Physiol. 107:1049-1054. Alscher, R. G., J. L. Donahue and C. L. Cramer. 1997. Reactive oxygen species and antioxidants: relationships in green cells. Physiol. Plant. 100:224-233. Ammirato, P. V. 1973. Some effects of abscisic acid on the development of embryos from caraway cells in suspension culture. Am. J. Bot. 60:22-23. Andersson, B., M. Ponticos, J. Barber, A. Koivuniemi, E-M. Aro, A. Hagman, A. H. Salter,Y. Dan-Hui and M. Lindahl. 1994. Photoinhibition of photosynthesis. In: Molecular Mechanisms to the Field. N. R. Baker and J. R. Bowyer,(eds.) p. 143-159. Bios, Oxford. Aro, E-M., I. Virgin and B. Andersson. 1993. Photoinhibition of photosystem II-- Inactivation, protein damage and turnover. Biochemi. Biophys. Acta 1143:113-134. Asada, K. 1980. Formation and scavenging of superoxide in chloroplasts with relation to injury by sulfur dioxide. Res. Rep. Natl. Inst. Environ. Stud. 11:165-179. Asada, K. 1992. Ascorbate peroxidase- a hydrogen peroxide scavenging enzyme in plants. Physiol. Plant. 85:235-241. Asada, K. 1994. Mechanisms for scavenging reactive molecules generated in chloroplasts under light stress. In: Photoinhibition of photosynthesis. N. R. Baker and J. R. Bowyer, (eds.) p. 129-142. Bios, Oxford. Asada, K. and M. Takahashi. 1987. Production and scavenging of active oxygen in photosynthesis. In: Photoinhibition. Kyle D. J., C. B. Osmond and C. J. Arntzen. (eds.) pp.227-287. Elsevier Science Publishers, Amsterdam. Asada, K. and M. Takahashi. 1987. Production and scavenging of active oxygen in photosynthesis. In: Photoinhibition. Kyle D.J., C.B. Osmond and C.J. Arntzen.(eds.),p.227-287. Elsevier Science Publishers, Amsterdam. Attree, S. M. and L. C. Fowke. 1993. Somatic embryogenesis and synthetic seeds of conifers. Plant Cell Tiss. Org. Cult. 35:1-35. Banzet, N. 1998. Accumulation of small heat shock proteins, including mitochondria HSP 22, induced by oxidative stress and adaptive response in tomato cells. Plant J. 13: 519-527. Barwale U. B., H. R. Kerns and J. M. Widholm. 1986. Plant regeneration from callus cultures of several soybean genotypes via embryogenesis and organogenesis. Planta 167:473-481. Beers, R. F. Jr. and I. W. Sizer. 1952. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. 195: 133-140. Beyer, E. Jr. 1976a. Silver Ion: A potent anti-ethylene agent in cucumber and tomato. HortScience 11:195-196. Beyer, E. Jr. 1976b. A potent inhibitor of ethylene action in plants. Plant Physiol. 58:268-271. Bhojwani, S. S. and M. K. Razdan. 1983. Plant Tissue Culture: Theory and Practice. pp.502. Elsevier Science Publishers B. V., Amsterdam, The Netherlands. Bogorad, L. 2000. Engineering chloroplasts: An alternative site for foreign genes, proteins, reactions and products. Trends Biotechnol. 18(6):257-263. Bolwell, G. P. 1999. Role of active oxygen species and NO in plant defence responses. Curr. Opin. Plant Biol. 2; 287-294. Bonner J. and P. S. Devirian. 1939. Growth factor requirements of four species of isolated roots. Am. J. Bot. 26:661-665. Bowler, C. and R. Fluhr. 2000. The role of calcium and activated oxygens as signals for controlling cross-tolerance. Trends in Plant Sci. 5(6):241-246. Bowler, C., M., Von Montagu and D. Inz’e. 1992. Superoxide dismutase and stress tolerance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43:83-116. Bowyer, J. R. and R. C. Leegood. 1997. Photosynthesis. In: Plant Biochemistry . P.M. Dey and J. B. Harborne, (eds.), p.49-110.Academic Press. Inc., USA. Boynton, J. E., N. W. Gillham, E. H. Harris, J. P. Hosier, A. M. Johnson, A. R. Jones, and J. C. Sanford. 1988. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240(4858):1534-1538. Bradford, K. J. and S. F. Yang. 1981. Physiological responses of plants to waterlogging. HortScience 16:25-30. Brown, C. L. and R. H. Lawrence. 1968. Culture of pine callus on a defined medium. Forest Sci. 14:62-64. Brownleader, M.D., J.B. Harborne and P.M. Dey.1997. Carbohydrate metabolism: primary metabolism of monosaccharides. In: Plant Biochemistry . P.M. Dey and J. B. Harborne, (eds.), p.111-141. Academic Press. Inc., USA. Bruce, B. D. 2000. Chloroplast transit peptides: structure, function and evolution. Trends in Cell Biol. 10:440-447. Budavari, S., M. J. O’Neil, A. Smith P. E. Heckelman, J. F. Kinneary. 1996. The Merck Index. (twelfth edition). Merck Research Laboratories. Merck & Co., Inc. Whitehouse Station, NJ. Burnet, G. and R. K. Ibrahim. 1973. Tissue culture of Citrus peel and its potential for flavonoid synthesis. Z. Pflanzenphysiol.69:152-162. Cai, X. N., J. M. She, Z. Zhu, W. M. Zhu, X. H. Yuan and X. J. Su. 1997. Establishment of Agrobacterium-mediated genetic transformation system of common Chinese cabbage (Brassica chinensis). Jiangsu Journal of Agricultural Science 13(2):110-114. Casano, L. M., M. Martin and B. Sabater. 1994. Sensitivity of superoxide dismutase transcript level and activities to oxidative stress is lower in mature-senescent than in young barley leaves. Plant Physiol. 106:1033-1039. Chamnongpol, S. 1998. Defense activation and enhanced pathogen tolerance induced by H2O2 in transgenic tobacco. Proc. Natl. Acad. Sci. U.S.A. 95: 5818-5823. Chen, L. F. O., J.Y. Hwang, Y. H. Wang, Y. T. Chen and J. F. Shaw. 2004. Ethylene insensitivity and post-harvest yellowing retardation in mutant ethylene response sensor (boers) gene transformed broccoli (Brassica oleracea var. Italica). Mol. Breed. 14:199-213. Chen, L. F. O., J.Y. Hwang, Y. Y. Charng, C. W. Sun and S F. Yang. 2001. Transformation of broccoli (Brassica oleracea var. Italica) with isopentenyltransferase gene via Agrobacterium tumefaciens for postharvest yellowing retardation. Mol. Breed. 7:243-257. Chen, X. and D. J. Schnell. 1999. Protein import into chloroplasts. Trend in Cell Biol. 9:222-227. Corneille, S., K. Lutz, Z. Svab and P. Maliga. 2001. Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system. Plant J. 27(2):171-178. Cross, C. E., A. van der Vliet, S. Louie, J. J. Thiele and B. Halliwell. 1998. Oxidative stress and antioxidants at biosurfaces: Plants, skin and respiratory tract surfaces. Environ. Health Perspect. 106(5):1241-1251. Daniell, H. 1999. Environmentally friendly approaches to genetic engineering. In Vitro Cellular & Developmental Biology Plant. 35: 361-368. Daniell, H., B. Muthukumar and S. B. Lee. 2001. Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection. Curr. Genet. 39(2):109-116. Daniell, H., R. Datta, S. Varma, S. Gray and S. B. Lee. 1998. Containment of herbicide resistance through genetic engineering of the chloroplast genome. Nat. Biotechnol. 16:345-348. Del Rio, L. A., L.M. Sandalio, J. M. Palma, P. Bueno and F. J. Corpas. 1992. Metabolism of oxygen radicals in peroxysomes and cellular implications. Free Radical Biol. Med. 13:557-580. Dellaporta, S.L., J. Wood and J.B. Hicks. 1983. A plant DNA Minipreparation : VersionⅡ. Plant Mol. Biol. Rep. 1:19-21. Deng, X. W., P. A. Wing and W. Gruissem. 1989. The chloroplast genome exists in multimeric forms. Proc. Natl. Acad. Sci. USA. 86: 4156-4160. Ding. 1998. Development of insect-resistant transgenic cauliflower plants expressing the trypsin inhibitor gene isolated from local sweet potato. Plant Cell Report 17:854-860. Doba, T., G.W. Burton and K.U. Ingold. 1985. Antioxidant and coantioxidant effect of vitamin C. The effect of vitamin C, either alone or in the presence of vitamin E or a water soluble vitamin E analogue, upon the peroxidation of aqueous multilamellar phospholipid liposome. Biochem. Biophys. Acta 835:298-303. Dodds, J. H. and L. W. Roberts. 1995. Experiments in Plant Tissue Culture. 3rd Ed. pp.256. Press Syndicate of University of Cambridge. Eeuwens, C. J. 1976. Mineral requirements for growth and callus initiation of tissue explants excised from mature coconut palms(Cocos nucifera) and cultured in vitro. Physiol. Plant. 36:23-28. Elstner, E.F. 1987. of activated oxygen species. In: The Biochemistry of Plants,Vol.11: Biochemistry of Metabolism. D. D. David, (eds), p.253-315. Academic Press, San Diego, CA. Feierabend, J. and S. Engel. 1986. Photoinactivation of catalase in vivo in leaves. Arch. Biochem. Biophys. 251:567-576. Feierabend, J., C. Schaan and B. Hertwig. 1992. Photoinactivation of catalase occurs under both high- and low-temperature stress conditions and accompanies photoinhibition of photosystemⅡ. Plant Physiol. 100:1554-1561. Finer, J. J. and A. Nagasawa. 1988. Development of an embryogenic suspension culture of soybean(Glycine max Merrill.). Plant Cell Tiss. Organ Cult. 15:125-136. Finkel, T. and N. J. Holbrook. 2000. Oxidants, oxidative stress and the biology of aging. Nature 408:239-247. Fisher, N., O. Stampacchia, K. Redding and J. D. Rochaix. 1996. Selectable marker recycling in the chloroplast. Mol. Gen. Genet. 251(3):373-380. Foyer, C. H. 1997. Hydrogen peroxide- and glutathione-associated mechanisms of acclimatory stress tolerance and signalling. Physiol. Plant 100: 241-254. Frank, H. A. and R. J. Cogdell. 1993. Carotenoids. In: Carotenoids in Photosynthesis. A. J. Young and G. Britton, (eds.) p. 252-326. Chapman & Hall, London. Fridovich I. 1986. Superoxide dismutases. Adv. Enzymol. 58:62-97. Fridovich I. 1995. Superoxide radical and superoxide dismutase. Annu. Rev. Biochem. 64:97-112. Fryer, M. J. 1992. The antioxidant effects of thylakoid vitamin E (a-tocopherol). Plant, Cell Environ. 15:381-392. Gamborg O. L. 1966. Aromatic metabolism in plants. Ⅱ. Enzymes of the shikimate pathway in suspension cultures of plant cells. Can. J. Biochem. 44:791-799. George, E. F. 1993. Plant propagation by tissue culture. Part Ⅰ&Ⅱ (2nd Ed.) pp.1361. Exegetics Ltd., Wilts, England. George, E. F. and P. D. Sherrington. 1984. Plant propagation by tissue culture. In: Handbook and Dictionary of Commercial Alboratories. pp.125-330. Eastern Press, Reading, Berks. England. Giraudat, J., F. Parcy, F. Bertauche, F. Gosti, J. Leung, P. C. Morris, M. Bouvier-Durand and N. Vartanian. 1994. Recent advances in abscisic acid action and signaling. Plant Mol. Biol. 26:1557-1577. Green, C. E. 1982. Somatic embryogenesis and plant regeneration from the friable callus of Zea mays. pp.107-108 in Fujiwara A. (ed.) 1982 (q.v.). Guerineau, F. 1995. Tools for expressing foreign genes in plants. In: Jones H. (Ed) Methods in Molecular Biology, vol. 49: Plant Gene Transfer and Expression Protocols. (pp. 1-32). Humana Press Inc., Totowa, N. J. Halliwell, B. and J.M.C. Gutteridge. 1989. Free Radicals in Biology and Medicine. Clarendon Press, Oxford. Halliwell, B. and J.M.C. Gutteridge. 1999. Free Radicals in Biology and Medicine. Clarendon Press, Oxford. Harman, D. 1957. Aging: a theory based on free radical and radiation chemistry. J. Gerontol. 2:298-300. Harwood, J. L. 1997. Plant lipid metabolism. In: Plant Biochemistry . P.M. Dey and J. B. Harborne, (eds.), p.237-272. Academic Press. Inc., USA. Heifetz, P. 2000. Genetic engineering of chloroplast. Biochemie. 82(6-7):655-666. Herbert, S. K., G. Samson, D. C. Fork and D. E. Landenbach. 1992. Characterization of damage to photosystems ⅠandⅡ in a cyanobacterium lacking detectable iron O2- dismutase activity. Proc. Natl. Acad. Sci. USA. 89:8716-8720. Herouart, D., M. Van Montagu and D. Inze. 1993. Redox-activated expression of the cytosolic copper/zinc superoxide dismutase gene in Nicotiana. Proc. Natl. Acad. Sci. USA. 90:3108-3112. Hertwig, B., P. Streb and J Feierabend. 1992. Light dependence of catalase synthesis and degradation in leaves and in influence of interfering stress condition. Plant Physiol. 100:1547-1553. Hiraoka, N., T. Kodama and Y. Tomita. 1986. Selection of Bupleurum falcatum callus line producing anthocyanins in darkness. J. Nat. Prod. 49:470-474. Hodgson, J. 1999. Monarch Bt-corn paper questioned. Nat. Biotechnol. 17:627. Horsch, R. B., J. E. Fry, N. L. Hoffmann, D. Eichholtz, S. G. Rogers and R. T. Fraley. 1985. A simple and general method for transferring genes into plants. Science 277: 1229-1231. Hossain, M. A., Y. Nakano and K. Asada. 1984. Monodehydroascorbate reductase in spinach chloroplasts and its participation in regeneration of ascorbate for scavenging hydrogen peroxide. Plant Cell Physiol. 25:385-395. Hou, B. K., Y. H. Zhou, L. H. Wan, Z. L. Zhang, G. F. Shen, Z. H. Chen and Z. M. Hu. 2003. Chloroplast transformation in oilseed rape. Transgenic Research 12:111-114. Ingelbrecht, I., H. Van Houdt, M. Van Montagu and A. Depicker. 1994. Posttranscriptional silencing of reporter transgenes in tobacco correlates with DNA methylation. Proc. Natl. Acad. Sci. USA 91: 10502-10506. Inoue M. and E. Maeda. 1982. Control of organ formation in rice callus using two-step culture method. P.183-184. in Fujiwara A.(Ed.)1982(q.v.) Ishikawa, T., K. Sakai, K. Yoshimura, T. Takeda and S. Shigeoka. 1996. cDNAs encoding spinach stromal and thylakoid-bound ascorbate peroxidase, differing in the presence or absence of their 3’-coding regions. FEES Lett. 384:289-293. Jabs, T. 1997. Elicitor-stimulated ion fluxes and O2_ from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley. Proc. Natl. Acad. Sci. U.S.A. 94: 4800-4805. James, C. 2005. Global status of commercialized biotech/gm crops: 2005. ISAAA Briefs No. 34-2005. James, C. 2005. Global status of commercialized biotech/gm crops: 2005. ISAAA Briefs No. 34-2005. Jenny, A. C., A. P. Singh, K. J. Horgan and T. A. Thorpe. 1985. Explant development state and shoot formation in Pinus radiata cotyledons. Bot. Gaz. 146:196-203. Jorgensen, R. 1995. Cosuppression, flower color patterns and metastable gene expression states. Science 268:686-691. Kavathekar A. K., Ganapathy P. S. and Johri B. M. 1978.In vitro responses of embryoids of Eschscholzia californica. Biol. Plant. 20:98-106. Keegstra, K. and K. Cline. 1999. Protein import and routine system of chloroplast. Plant Cell 11:557-570. Keeler, K., C. Turner and M. Bolick. 1996. Movement of crop transgenes into wild plants. In: S. O. Duke. CRC Press. Herbicide Resistant Crops. pp.303-330. Khan, M. S. and P. Maliga. 1999. Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants. Nat Biotechnol. 17(9):910-915. Köhler, R. H. and M. R. Hanson. 2000. Plastid tubules of higher plants are tissue-specific and developmentally regulated. J. Cell Sci. 113:81-89. Köhler, R. H., J. Cao, W. R. Zipfel, W. W. Webb and M. R. Hanson. 1997. Exchange of protein molecules through connections between higher plant plastids. Science 276:2039-2042. Ku, H. H., U. T. Brunk and R. S. Sohal. 1993. Relationship between mitochondrial superoxide and hydrogen peroxide production and longevity of mammalian species. Free Radical Biol. Med. 15:621-627. Kunert, K. J. and M. Ederer. 1985. Leaf aging and lipid peroxidation--The role of the antioxidants vitamin C and E. Physiol. Plant. 65:85-88. Kuo, C. L., A. P. Sagare, S. F. Lo, C. Y. Lee, C. C. Chen and H. S. Tsay. 2002. Abscisic acid promotes development of somatic embryos on converted somatic embryos of Corydalis yanhusuo (Fumariaceae). J. Plant Physiol. 159:423-427. Lamb, C. and R. A. Dixon. 1997. The oxidative burst in plant disease resistance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:251-275. Lim, H-T.,H-J. Lee, E-J. Park and Y. N. Song. 2000. Factors influencing on the high efficient plant regeneration and genetic transformation in Brassica vegetable crops. 3rd ISHS International Symposium on Brassicas – 12th Crucifer Genetics Workshop (help at Horticulture Research International ),Wellesbourne, cv 35 9EF, UK. 5th-9th,Sep., 2000. Lin, J-N. and C. H. Kao. 1998. Effect of oxidative stress caused by hydrogen peroxidase on senescence of rice leaves. Bot. Bull. Acad. Sin. 39: 161-165. Liu, F., M. Q. Cao, L. Yao, Y. Li, C. Robaglia, C. Tourneur. 1998. In planta transformation of Pakchoi (Brassica campestris L. ssp. chinensis) by infiltration of adult plants with Agrobacterium. ISHS Acta Horticulturae 467: 187-192. III International Symposium Diversification of Vegetable Crops. Livne, A. and Y. Vaddia. 1972. Water deficits and hormone relations. In: T. T. Koglowski, ed. : Water Deficits and Plant Growth. Academic Press.pp.255-275. Lowe, K. C., M. R. Davey and J. B. Power. 1996. Plant tissue culture: past, present and future. Plant Tissue Culture and Biotechnology 2(4):175-186. Lutz, K. A., J. E. Knapp and P. Maliga. 2001. Expression of bar in the plastid genome confers herbicide resistance. Plant Physiol. 125(4):1585-1590. Madamanchi, N. R., J. L. Donahue, C. L. Cramer, R. G. Alscher and K. Pedersen. 1994. Differential response of Cu, Zn superoxide dismutases in two pea cultivars during a short-term exposure to sulfur dioxide. Plant Mol. Biol. 26:95-103. Malan, C. 1990. Correlation between Cu/Zn superoxide dismutase and glutathione reductase, and environmental and Xenobiotic stress tolerance in maize inbreds. Plant Sci. 69: 157-166. Mansfield, T. A., A. R. Wellburn and T. J. S. Moreiora. 1978. The role of abscisic acid and farnesol in the alleviation of water stress. Phil. Trans. R. Soc. Lond. B. 284:471-482. Margarita, C. and M. Margarita. 1991. Morphogenesis in leaf, hypocotyls and explants of Digitalis thapsi L. cultured in vitro. Plant Cell Tiss. Org. Cult. 25:117-123. Martinez, C. 1998. Apoplastic peroxidase generates superoxide anions in cells of cotton cotyledons undergoing the hypersensitive reaction to Xanthomonas campestris pv. Malracearum race 18. Mol. Plant-Microbe Interact. 11: 1038-1047. Mascio, P., T., P. A. Devasagayam, S. Kaiser and H. Sies. 1990. Carotenoids, tocopherols and thiols as biological singlet molecular oxygen quenchers. Biochem. Soc. Trans. 18:1050-1054. Matters, G. L. and J. G. Scandalios. 1986. Effect of the free radical-generating herbicide paraquat on the expression of the superoxide dismutase (SOD) genes in maize. Biochem. Biophys. Acta 882:29-38. Matters, G. L. and J. G. Scandalios. 1987. Synthesis of isozymes of superoxide dismutase in maize leaves in response to O3, SO2 and elevated O2. J. Exp. Bot. 38:842-852. Matzke, M. A. and A. J. M. Matzke. 1995. How and why do plants inactivate homologous (trans)genes ? Plant Physiol. 107:679-685. May, M. J. and C. J. Leaver. 1993. Oxidative stimulation of glutathione synthesis in Arabidopsis thaliana suspension cultures. Plant Phyiol. 103: 621-627. McCord, J. M. and I. Fridovich. 1969. Superoxide dismutase. An enzymatic function for erythrocuperin (hemocuperin). J. Biol. Chem. 244:6049-6055. Mehlorn, H., B. J. Tabner and A. R. Wellburn.1990. Electron spin resonance evidence for the formation of free radicals in plants exposed to ozone. Physiol. Plant 79:377-383. Metz, T. D., R. Dixit and E. D. Earle. 1995. Agrobacterium tumefaciens-mediated transformation of broccoli (Brassica oleracea var. italica) and cabbage (Brassica oleracea var. capitata). Plant Cell Rep. 15:287-292. Meyer, P. 1995. Understanding and controlling transgene expression. Trends Biotechnol. 13:332-337. Miborrow, B. V. 1968. Identification and measurement of (+)-abscisic acid in plants. In: Biochemistry and Physiology of Plant Growth Substances(ed. by F. Wightman and G. Setterfield), pp.1531-1545. Runge Press, Ottawa. Millerd, A., D. Spencer, W. F. Dudman and M. Stiller. 1975. Growth of immature pea cotyledons in culture. Aust. J. Plant Physiol. 2:51-59. Mittler, R. 1999. Transgenic tobacco plants with reduced capability to detoxity reactive oxygen intermediates are hyperresponsive to pathogen infection. Proc. Natl. Acad. Sci. U.S.A. 96:14165-14170. Mittler, R. and B. A. Zilinskas. 1991. Purification and characterization of pea cytosolic ascorbate peroxidase. Plant Physiol. 97:962. Mittler, R. and B. A. Zilinskas. 1994. Regulation of pea cytosolic ascorbate peroxidase and other antioxidant enzymes during the progression of drought stress and following recovery from drought. Plant J. 5:397-405. Miyake, C. and K. Asada. 1992. Thylakoid-bound ascorbate peroxidase in spinach chloroplasts and photoreduction of its primary oxidation product monodehydroascorbate radical in thylakoid. Plant Cell Physiol. 33:541-553. Mueller, F., J. Gilbert, G. Davenport, G. Birgnetic and D. C. Baulcombe. 1995. Homology-dependent resistance transgenic virus resistance in plants related to homology-dependent gene silencing. Plant J. 7:1001-1013. Murashige T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497. Nawrath, C., Y. Poirier and C. Somerville. 1994. Targeting of the polyhydroxybutyrate biosynthetic pathway to the plastids of Arabidopsis thaliana results in high levels of polymer accumulation. Proc. Natl. Acad. Sci. U.S.A. 91:12760-12764. Nickell, L. G. and A. Maretzkia. 1969. Growth of suspension cultures of sugarcane cells in chemically defined media. Physiol. Plant. 22:117-125. Panaud, O., G. Magpantay, and S. McCouch. 1993. A Protocol for nonradioactive DNA labelling and detection in the RFLP analysis of rice and tomato using single-copy probes. Plant Mol. Biol. Rep. 11:54-59. Perl-Treves, R. and E. Galun. 1991. The tomato Cu, Zn superoxide dismutase genes are developmentally regulated and respond to light and stress. Plant Mol. Biol. 17:745-760. Phillips, G. C. and G. B. Collins. 1981. Induction and development of somatic embryos from cell suspension cultures of soybean. Plant Cell Tiss. Organ Cult. 1:123-129. Pierik, R. L. M. 1997. In Vitro Culture of Higher Plants. pp.348. Kluwer Academic Publishers. Dordrecht, The Netherlands. Pitcher, L. H., E. Brennan and B.A. Zilinskas.1992. The antiozonant ethylenediurea does not act via superoxide dismutase induction in bean. Plant Physiol. 99:1388-1392. Poirier Y., C. Nawrath, and C. Somerville. 1995. Production of polyhydroxyalkanoates, a family of biodegradable plastics and elastomers, in bacteria and plants. Biotechnology (NY). 13: 142-150. Polle, A., K. Chakrabartii, W. Schurmann and H. Rennenberg.1990. Composition and properties of hydrogen peroxide decomposing systems in extracellular and total extracts from needles of Norway spruce (Picea abies L., Karst). Plant Physiol. 94:312-319. Potrykus, I. 1990. Gene transfer to plants: Assessment and perspectives. Physiol. Plant 79:125-134. Puddephat, I. J., T. J. Riggs and T. M. Fenning. 1996. Transformation of Brassica oleracea L.: a critical review. Mol. Breed. 2:185-210. Radin, J. W. and R. C. Ackerson. 1982. Does abscisic acid control stomatal closure during stress? What’s New in Plant Physiology 12:9-12. Rajasekaran, K., M. B. Hein and I. K. Vasil. 1987. Endogenous abscisic acid and indole-3-acetic acid and somatic embryogenesis in cultured leaf explants of Pennisetum purpureum Schum. Effects in vivo and in vitro of glyphosate, fluridone and paclobutrazol. Plant Physiol. 84:47-51. Rangan, T. S. 1974. Morphogenic investigations on tissue culture of Panicum miliaceum. Z. Pflanzenphysiol. 72:456-459. Reiert, J. and M. M. Yeoman. 1982. Plant Cell and Tissue Culture: A Laboratory Manual. pp.83. Springer-Verlag, Berlin, Heidelberg, Germany. Reiert, J. and Y. P. S. Bajaj. 1977. Applied and Fundamental Aspects of Plant Cell, Tissue, and Organ Culture. p.180-190. Springer-Verlag, Berlin, Heidelberg, Germany. Ritchie, G. A., K. C. Short and M. R. Davey. 1991. In vitro acclimatization of Chrysanthemum and sugar beet plantlets by treatment with paclobutrazol and exposure to reduced humidity. J. Exp. 42:1557-1563. Robbins W. J. and M. B. Schmidt. 1939a. Vitamin B6 , a growth substance for isolated tomato roots. Proc. Nat. Acad. Sci. Wash. 25:1-3. Robbins W. J. and M. B. Schmidt. 1939b. Further experiments on excised tomato roots. Am. J. Bot. 26:149-159. Roest S. and G. S. Bokelmann. 1975. Vegetative propagation of Chrysanthemum morifolium Ram. In vitro. Scientia Hort. 3:317-330. Rogers, S. G., H. Klee, M. Byren, R. B. Horsch and R. T. Fraey. 1988. Improved vector for plant transformation: expression cassette vectors and new selectable marker. Method in Enzymology. Academic Press. Romeis, T. 1999. Rapid Avr9- and Cf-9- dependent activation of MAP kinases in tobacco cell cultures and leaves: convergence of resistance gene, elicitor, wound, and salicylate responses. Plant Cell 11: 273-287. Ruf, S., M. Hermann, I. Berger, H. Carrer and R. Bock. 2001. Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nat. Biotechnol. 19(9):870-875. Rusterucci, C. 1999. Involvement of lipoxygenase-dependent production of fatty acid hydroperoxides in the development of the hypersensitive cell death induced by cryptogein on tobacco leaves. J. Biol. Chem. 17: 36446-36455. Saiki, R.K. 1989. The design and optimization of the PCR. In: PCR Technolgy. Erlich, H.A.M.(ed), Stockton Press. pp.7-16. Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A laboratory manual, 2nd ed, Cold Spring Harbor, New York. Sanford, J. C. 1990. Biolistic plant transformation. Physiol. Plant. 79:206-209. Scandalios, J. G. 1990. Response of plant antioxidant defense genes to environmental stress. Adv. Genet. 28: 1-41. Scandalios, J. G. 1993. Oxygen stress and superoxide dismutases. Plant Physiol. 101; 7-12. Sharma, Y. K. 1996. Ozone-induced responses in Arabidopsis thalima – the role of salicylic acid in the accumulation of defense –related transcripts and induced resistance. Proc. Natl. Acad. Sci. U.S.A. 93: 5099-5104. Shepard, J. F. and H. E. Street. 1974. The decline of embryogenic potential as callus and suspension cultures of carrot (Daucus carota L.) are serially subcultured. Ann. Bot. 38:223-241. Shepard, J. F.. 1980. Abscisic acid-enhanced shoot initiation in protoplast-derived calli of potato. Plant Sci. Lett. 18:327-333. Shiina, T., K. Hayashi, N. Ishii, K. Morikawa and Y. Toyoshima. 2000. Chloroplast tubules visualized in transplastomic plants expression green fluorescent protein. Plant Cell Physiol. 41:367-371. Shinozaki, K., M. Ohme, M. Tanaka. T. Wakasugi, N. Hayashida, T. Matsubayashi, N. Zaita, J. Chunwongse, J. Obokada, K. Yamaguchi-Shinozaki, C. Ohto, K. Torazawa, B.-Y. Meng, M. Sugita, H. Deno, T. Kamogashira, K. Yamada, J. Kusuda, F. Takaiwa, A. Kato, H. Tohdoh, H. Shimada and M. Suguira. 1986. The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. EMBO J. 5: 2043-2050. Sidorov, V. A., D. Kasten, S. Z. Pang, P. T. J. Hajdukiewicz, J. M. Staub and N. S. Nehra. 1999. Stable chloroplast transformation in potato: use of green fluorescent protein as a plastid marker. Plant J. 19(2):209-216. Skirvin, R. M., M. C. Chu, M. L. Mann, H. Young, J. Sullivan and T. Fenmamian. 1986. Stability of tissue culture medium pH as a function of autoclaving, time and cultured plant material. Plant Cell Rep. 5:292-294. Smith, D. L. and A. D. Krikorian. 1990. Somatic proembryo production from excised wounded zygotic carrot embryos on hormone-free medium: evaluation of the effect of pH, ethylene and activated charcoal. Plant Cell Rep. 9:34-37. Smith, R. H. 1992. Plant Tissue Culture Techniques & Experiments. pp.171. Academic Press, Inc. San Diego, California. U.S.A. Soll, J. and R. Tien. 1998. Protein translocation into and across the chloroplast envelope membranes. Plant Mol. Biol. 38:191-207. Streb, P., A. Michael-Knauf and J. Feierabend. 1993. Preferential photoinactivation of catalase and photoinactivation of photosystemⅡ are common early symptoms under various osmotic and chemical stress conditions. Physiol. Plant 88:590-598. Strother, S. 1988. The role of free radicals in leaf senescence. Gerontology 34: 151-156. Su, N., Y. M. Wu, B. Y. Sun and G. F. Shen. 2001. A new way of plant genetic engineering chloroplast transformation. Biotechnology Information 4:9-13. Svab, Z. and P.. Maliga. 1993. High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc. Natl. Acad. Sci. USA 90:913-917. Svab, Z., P. Hajdukiewicz, and P.. Maliga. 1990. Stable transformation of plastids in higher plants. Proc. Natl. Acad. Sci. USA 87(21):8526-8530. Tang, K., P. Tinjuangjun, Y. Xu, X. Sun, J. A. Gatehouse, P. C. Ronald, H. Qi, X. Lu, P. Christou and A. Kohli. 1999. Particle-bombardment-mediated co-transformation elite Chinese rice cultivars with genes conferring resistance to bacterial blight and sap-sucking insect pests. Planta 208:552-563. Thompson, J. E., R. L. Legge and R. F. Barber. 1987. The role of free radicals in senescence and wounding. New Phytol. 105;317-344. Tsai, L. Y., S. L. Tu and H. M. Li. 1999. Insertion of at Toc34 into the chloroplastic outer membrane is assisted by at least two proteinaceous components in the import system. J. Biol. Chem. 274:18735-18740. Vaddia, Y. 1976. Plant hormones and water stress. Phil. Trans. Soc. Lond. B. 273:513-522. Van Blokland, R. N. Van der Geest, J. N. M. Mol and J. M. Kooter.1994. Transgene-mediated suppression of chalcone synthase expression in Petunia hybrida results from an increase in RNA turnover. Plant J. 6:861-877. Vasil, V. and I. K. Vasil. 1981. Somatic embryogenesis and plant regeneration from suspension cultures of pearl millet (Pennisetum americanum). Ann. Bot. 47:669-678. Volk, S. and J. Feierabend. 1989. Photoinactivation of catalase at low temperature and its relevance to photosynthetic and peroxide metabolism in leaves. Plant, Cell Environ. 12:701-712. Walden, R. and R. Wingender. 1995. Gene-transfer and plant-regeneration techniques. Trends Biotechnol. 13:324-331. Walton, D. C. 1980. Biochemistry and physiology of abscisic acid. Ann. Rev. Plant Physiol. 31:453-489. Wang, J., H. Zhang and R. D. Allen. 1999. Overexpression of and Arabidopsis peroxisomal ascorbate peroxidase gene in tobacco increase protection against oxidative stress. Plant Cell Physiol. 40(7):725-732. Wegener, D., P. Steinecke, T. Herget, I. Petereit, C. Philipp and P. H. Schreier. 1994. Expression of a reporter gene is reduced by a ribozyme in transgenic plants. Mol. Gene. Gen. 245: 465-470. Weide, R., M. Koornneef and P. Zabel. 1989. A simple, nondestructive spraying assay for the detection of an active kanamycin resistance gene in transgenic tomato plants. Theor. Appl. Genet. 78: 169-172. Westneat, D.F., W.A. Noon, H.K. Reeve, and C.F. Aquadro. 1988. Improved hybridization conditions for DNA ‘fingerprints’ probed with M13. Nucleic Acids Res. 16:4161. White, P. R. 1963. The
摘要: 本論文之目的為:1. 建立小白菜組織培養再生系統;2. 建立不同株齡小白菜的組織培養再生系統;3. 建立小白菜之農桿菌基因轉殖系統;4. 建立小白菜之葉綠體基因轉殖系統;5. 利用基因轉殖技術培育出具有耐環境逆境及高品質小白菜。 本研究針對影響小白菜組織培養再生的培殖體來源及培養基成分等因子,作一系列的研究探討。試驗結果顯示,含有1 mg/L BA、0.5 mg/L NAA、0.25mg/L GA3、3mg/L AgNO3與5mg/L putrescine之2% sucrose的MS基本鹽類與有機成分配方對''台農一號''與''台農二號''小白菜誘導組織培養再生最佳,再生率達66.7%~70%;且子葉組織的再生率均優於下胚軸組織。本研究同時發現通常小白菜的下胚軸或子葉培殖體是經由誘導出癒傷組織,再依循分化出芽原體,抽出芽梢,形成再生植株等過程,亦即經由「器官形成再生路徑」(organogenesis pathway)。但若在再生培養基中添加多胺類,例如腐胺或亞精胺,會使得誘導癒傷組織的比率降低,誘導出胚狀體,再生長出芽梢及根,形成再生植株,此即經由「體細胞胚胎發生再生途徑」(somatic embryogenesis pathway),而再生成植株。 探討不同種類、年齡與部位之培殖體對''台農一號''與''台農二號''小白菜組織培養再生之影響及培養基中添加植物生長調節劑、胺基酸類、多胺類等物質對成熟或老化之小白菜葉片、莖段組織培養再生之影響。試驗結果顯示3天齡培殖體之不定芽再生率與發根率最高,隨著培殖體年齡的增加,其分化再生力逐漸下降,至21天齡培殖體降至最低,其不定芽再生率與發根率趨近於零,但27天齡培殖體之分化再生能力卻反而增加。在最適當之小白菜再生培養基 (1 mg/L BA、0.5 mg/L NAA、0.25mg/L GA3、3mg/L AgNO3之2% sucrose的MS基本鹽類與有機成分) 中添加50 ml/l coconut juice可顯著提高15天齡小白菜培殖體之不定芽再生與不定根形成率。在最適當之小白菜再生培養基中添加0.1 mg/L ABA亦可提高21天齡小白菜培殖體之分化再生能力。而27天齡培殖體,則僅需使用與3天齡、9天齡相同之小白菜再生培養基配方,即可達到高分化再生率。 利用農桿菌共同轉移法,同時轉移帶有rbcS為啟動子或/且攜帶有大豆葉綠體訊息transit peptide之結球白菜之超氧化歧化酵素 (sod62)及結球白菜之過氧化氫酵素 (cat78) 等二種基因至''台農一號''及''台農二號''小白菜。轉殖rbcS-sod62 (pKrScn) 與 rbcS-cat78 (pKrCcn) 基因到小白菜的子葉與下胚軸,植株再生率約為16%,但經20 mg/L kanamycin篩選後,篩選成活率約為1.6%。以PCR、南方墨點雜交分析之結果顯示rbcS-sod62及rbcS-cat78基因同時存在於2株轉殖''台農二號''小白菜植株的染色體內,共同轉殖效率為0.075%。轉殖 rbcS-TP-sod62與rbcS-TP-cat78基因到小白菜的子葉與下胚軸,於共同培養液中加入金鋼砂或添加200µM乙酰丁香酮,可提高基因轉殖效率。植株再生率約為18%,但經20 mg/L kanamycin篩選後,篩選成活率約為2.2%。以PCR、南方墨點雜交分析之結果顯示rbcS-TP-sod62及rbcS-TP-cat78基因同時存在於2株轉殖''台農一號''及4株轉殖''台農二號''小白菜植株的染色體內,共同轉殖效率為0.202%。採收轉殖 rbcS-TP-sod62與rbcS-TP-cat78基因之T0代自交種子。並將其播種於含有20 mg/L kanamycin 之MS基本培養基,調查抗kanamycin 的T1代苗株數。以PCR及RT-PCR分析共同轉殖rbcS-TP-sod62及rbcS-TP-cat78基因之小白菜的T1代後裔植株顯示,sod及cat基因已遺傳到T1代小白菜並表現出其mRNA。轉殖T1代小白菜葉片之SOD及CAT酵素活性均較未轉殖之對照組顯著增加,SOD活性最高者約為對照組之4.2倍,CAT活性最高者約為對照組之2.2倍。 將以prrn為啟動子的sod62基因 (pASCCSOD) 及cat78基因 (pASCCCAT) 的葉綠體基因轉殖載體,藉由基因槍法轉殖到''台農一號''及''台農二號''小白菜。本研究建議以Biolistic PDS-1000/He (Bio-Rad) 基因槍進行小白菜基因槍轉殖時採用直徑小於1.0 μm的金粒子、4mm高度墊圈、直徑4mm間隔條,射擊距離4.5cm、微粒子發射裝置到材料間的距離3 cm等組裝設定。槍擊時使用650psi~900psi壓力膜及以6天齡之葉片培殖體為材料可獲得較多之小白菜轉殖植株。小白菜槍擊後之受體,培養於含有10 mg/L spectinomycin之篩選培養基進行初步篩選,小白菜試管苗株移出健化後,每天澆20mg/L spectinomycin溶液以達到繼續篩選之效果。 以基因槍法轉殖sod62 (pASCCSOD) 及 cat78 (pASCCCAT) 基因到小白菜的子葉與下胚軸,經10 mg/L spectinomycin篩選後,篩選成活率約為3.86%。以PCR、南方墨點雜交分析之結果顯示sod62及cat78基因同時存在於3株轉殖''台農一號''及29株轉殖''台農二號''小白菜植株的染色體內,共同轉殖效率為0.43%。採收轉殖 sod62及 cat78基因 之T0代自交種子。並將其播種於含有10 mg/L spectinomycin 之MS基本培養基,調查抗spectinomycin 的T1代苗株數。以PCR及RT-PCR分析共同轉殖sod62及 cat78基因之小白菜的T1代後裔植株顯示,sod及cat基因已遺傳到T1代小白菜並表現出其mRNA。轉殖T1代小白菜葉片之SOD及CAT酵素活性均較未轉殖之對照組顯著增加,SOD活性最高者約為對照組之4.9倍,CAT活性最高者約為對照組之2.6倍。
The objectives of this study were to: 1. develop an in vitro tissue regeneration system for Pak-choi (Brassica campestris L. ssp. chinensis L. Makino), 2. explore the possibility for improving the in vitro tissue regeneration derived from mature or aging explants of Pak-choi, 3. establish the Agrobacterium transformation system of Pak-choi, 4. establish the gene-gun transformation system of Pak-choi, and 5. explore the possibility for improvement of Pak-choi with stresses resistance via Agrobacterium-mediated and gene-gun transformation. The sources of explants of Pak-choi and the components of culture medium were studied to develop a tissue culture system for Pak-choi. The results indicated that the MS basal medium (Murashige and Skoog, 1962) supplemented with 2% sucrose, 1 mg/L BA, 0.5 mg/L NAA, 0.25mg/L GA3, 3mg/L AgNO3 and 5mg/L putrescine was the best components of culture medium for the explants regeneration of ''Tainung No. 1'' and ''Tainung No. 2'' Pak-choi, and 66.7% to 70% of regeneration rate could be achieved. Higher regeneration rates were obtained from the explants derived from the cotyledons than those of from hypocotyls. In general, calli were first regenerated from the explants of Pak-choi, and then adventitious shoots and roots were regenerated on the regeneration medium subsequently, ie. via “organogenesis pathway”. However, embryoids rather than calli were regenerated on the regeneration medium supplemented with polyamines (e.g. putrescine or spermidine), ie. via “somatic embryogenesis pathway”. The effects of types, ages, and positions of explants of ''Tainung No. 1'' and ''Tainung No. 2'' Pak-choi on in vitro tissue regeneration were performed. Effects of plant growth regulators, amino acids, polyamines, casein hydrolysate, and silver nitrate on shoot regeneration and root formation derived from the 15-days-old and 21-days-old explants of Pak-choi was also conducted. The highest rates of shoot regeneration and root formation were found in the 3-days-old explants. As the ages increased, the rates of shoot regeneration and root formation decreased. However, the high differentiation capacity was also found in the 27-days-old explants. Significantly increased the rates of shoot regeneration and root formation was found in the 15-days-old explants cultivated in the regeneration medium of Pak-choi (MS basal medium containing 2% sucrose, 1 mg/L BA, 0.5 mg/L NAA, 0.25mg/L GA3, and 3mg/L AgNO3) supplemented with 50 ml/l coconut juice. Improving the in vitro tissue regeneration of 21-days-old explants could be achieved by addition of 0.1 mg/L ABA into the regeneration medium of Pak-choi. Nevertheless, the efficient regeneration medium for 27-days-old explants was the same culture medium as the 3-days-old and 9-days-old explants.Attempts had been made to co-transfer the superoxide dismutase (sod62), and catalase (cat78) genes of Chinese cabbage into the ''Tainung No.1'' and ''Tainung No.2'' Pak-choi. Cotyledon and hypocotyl explants of Pak-choi were co-infected with two Agrobacterium carrying a distinct disarmed T-DNA containing rbcS-sod62 (pKrScn) and rbcS-cat78 (pKrCcn) genes, respectively. Results indicate that regenerated rates of Pak-choi were 16%. After 20 mg/L of kanamycin selection, the survival rate was 1.6%, and co-transformation rate of two genes was 0.075%. The results of PCR and Southern bolt hybridization indicated that the rbcS-sod62 and rbcS-cat78 genes had been transferred into Pak-choi, and inserted into their genomes. Two co-transformed plants of ''Tainung No.2'' Pak-choi were obtained. Increases in the transformation efficiency were obtained in the co-culture medium supplemented with the emery or 200µM acetosyringone (AS). Cotyledon and hypocotyl explants of Pak-choi were co-infected with two Agrobacterium carrying a distinct disarmed T-DNA containing rbcS-TP-sod62 (pKrTScn) and rbcS-TP-cat78 (pKrTCcn) genes, respectively. Results indicate that regenerated rates of Pak-choi were 18%. After20 mg/L of kanamycin selection, the survival rate was 2.2%, and co-transformation rate of two genes was 0.202%. The results of PCR and Southern bolt hybridization indicated that the rbcS-TPsod62 and rbcS-TPcat78 genes had been transferred into Pak-choi, and inserted into their genomes. Two and four co-transformed plants of ''Tainung No.1'' and ''Tainung No.2'' Pak-choi were obtained, respectively. T0 plants of rbcS-TP-sod62 and rbcS-TP-cat78 genes transformed Pak-choi were allowed to self-pollinated, and seeds were collected at maturity. T0 seeds were germinated on MS medium containing 20 mg/L of kanamycin for two weeks. Survival T1 seedlings were subjected to PCR and Southern bolt hybridization analysis. The results of genetic analysis indicated that the transformed rbcS-TP-sod62 and rbcS-TP-cat78 genes had been stably inherited in T1 progeny plants. Significant higher SOD and CAT activities were detected in the T1 transgenic progeny of Pak-choi than those of control plants. Following settings were suggested when Pak-choi explants was bombarded with Biolistic PDS-1000/He (Bio-Rad) particle gun apparatus: gold particle size less than 1.0 μm in diameter, 4mm spacer ring in thickness, 4mm spacer bar in diameter, 4.5cm for distance of bombardment, 3cm for distance from microcarrier launch assembly to the recipient explants. High transformation efficiency was achieved from the 6-days old Pak-choi explants bombarded with 650psi~900psi rupture disks. Cotyledon and hypocotyl explants of Pak-choi were bombarded with two plasmids containing sod62 (pASCCSOD) and cat78 (pASCCCAT) genes, respectively. After 10 mg/L of spectinomycin selection, the survival rate was 3.86%, and co-transformation rate of two genes was 0.43%. The results of PCR and Southern bolt hybridization indicated that the sod62 and cat78 genes had been transferred into Pak-choi, and inserted into their genomes. Two and twenty-nine co-transformed plants of ''Tainung No.1'' and ''Tainung No.2''Pak-choi were obtained, respectively. T0 plants of sod62 and cat78 genes transformed Pak-choi were allowed to self-pollinated, and seeds were collected at maturity. T0 seeds were germinated on MS medium containing 10 mg/L of spectinomycin for two weeks. Survival T1 seedlings were subjected to PCR and Southern bolt hybridization analysis. The results of genetic analysis indicated that the transformed sod62 and cat78 genes had been stably inherited in T1 progeny plants. Significant higher SOD and CAT activities were detected in the T1 transgenic progeny of Pak-choi than those of control plants.
URI: http://hdl.handle.net/11455/28556
其他識別: U0005-0408200612585500
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0408200612593200
Appears in Collections:園藝學系

文件中的檔案:

取得全文請前往華藝線上圖書館



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.