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標題: 蝴蝶蘭誘導擬原球體與轉殖ictB (inorganic carbon transporter B)基因之研究
Studies on protocorm-like body (PLB) induction and genetic tansformation ictB (inorganic carbon transporter B) of Phalaenopsis
作者: 陳俞廷
Chen, Yu-Tin
關鍵字: Phalaenopsis;蝴蝶蘭;PLB;genetic transformation;擬原球體;基因轉殖
出版社: 生命科學系所
引用: 王珍韶,1990. 蝴蝶蘭營養繁殖之研究。國立台灣大學園藝研究所碩士論文。 李哖,1990. 蘭之胚培養。中國園藝, 36:223-244. 李哖,1995. 蘭科植物。台灣農家要覽, 665-684. 李雪莉,2002. 農業新危機:台灣品種,海外盜版。天下雜誌,250:168-169。 李興進,1988. 蝴蝶蘭花梗苗葉片組織分生之研究。蘭花生產改進研討會專集, 49-53. 林金其,1988a. 萬代蘭花梗節間誘導形成癒合組織、體胚、擬原球體與植株過程形態學研究。洋蘭月刊, 23:84-90. 林金其,1988b. 蝴蝶蘭花梗節間培養之擬原球體誘導增生過程形態學研究。洋蘭月刊, 20:85-89. 林讚標,1988. 台灣蘭科植物。 林美華,1997. 農桿菌媒介法轉殖香蕉ACC合成酶反義基因之研究。國立台灣大學園藝學研究所碩士論文. 林雅亭,1999. 蝴蝶蘭苯基苯乙烯酮合成酶基因轉殖之研究。國立台灣大學園藝學研究所碩士論文. 郭慧蘭,2002. 文心蘭與蝴蝶蘭葉片培養之體胚發生。國立台灣大學園藝學研究所碩士論文. 徐善德,1995. 香蕉轉殖系統之建立及ACC合成酶反義基因之構築。國立台灣大學園藝學研究所碩士論文. 徐善德、黃鵬林,1995. 農桿菌媒介轉殖法。中國園藝, 41:237-250 黃聖佑,1997. 蝴蝶蘭基因轉殖系統之建立及ACC 合成反義基因之構築。 國立台灣大學園藝學研究所碩士論文. 黃萱,2001. 蝴蝶蘭體胚再生形式與基因轉殖之研究。國立成功大學生物科技研究所碩士論文. 陳建忠,1999. 文心蘭之體胚發生與植株再生。國立台灣大學園藝學研究所博士論文. 陳福旗、陳采晴,1998. 無機鹽類濃度及有機添加物對文心蘭擬原球體及組培苗生長之影響。中國園藝, 44:403-412. 陳健忠、張唯勤,2002. 蘭花之試管內形態發生。科學農業, 50:150-155 許家嘉、陳福旗,2003. 以蝴蝶蘭白化葉片誘導擬圓球體與植株再生。中國園藝, 49(4):335-342 詹明才、張新雄,1991. 農桿菌轉殖系統之影響因素。 科學農業, 39:249-255 鄭芳宜,2003. 蝴蝶蘭及文心蘭基因轉殖之研究。國立台灣大學園藝學研究所碩士論文. 潘秋燕,2000. 文心蘭懸浮細胞系之建立與體細胞變異之誘導。國立高雄師範大學生物科學研究所碩士論文. 謝永祥、黃鵬林,1995. 應用花粉管法於蝴蝶蘭基因轉殖之研究。中國園藝,41(4):309-324. 謝永祥、許聰耀、黃鵬林,1995. 應用基因槍法於蝴蝶蘭基因轉殖之研究。 中國園藝, 41(3):174-185. 蘇益正,2000. 植物生長調節劑對文心蘭葉培植體直接體胚發生之影響。中國文化大學生物科技研究所碩士論文. Arditti, J. 1992. Fundamentals of Orchid Biology. John Wiley and Sons. New York. Bagde, P. and Sharon, M. 1997. In vitro regeneration of Oncidium Gower Ramsey be high frequency protocorm like bodies proliferation. Indian J. Plant Physiol. 2:10-14. Belarmino, M. M. and Mii, M. 2000. Agrobacterium-mediated genetic transformation of a Phalaenopsis orchid. Plant Cell Rep. 19:435-442. David, J. B., Michal, R.T., Dieter, S., Judy, L. H., Daniella, S. and Aaron K.1998. A putative HCO3 transporter in the cyanobacterium Synechococcus sp.strain PCC 7942. FEBS Letters. 430:236-240 Bytebier, B. F., Deboeck, F., Greve, H. D., Van Montagu, M. and Hernalsteens, J.-P. 1987. T-DNA organization in tumor cultures and transgenic plants of the monocotyledon Asparagus officinalis. Proc. Natl. Acad. Sci. USA 88: 5345-5349 Chen, W. H. and Wang, Y. T. 1996. Phalaenopsis orchid culture. Taiwan Sugar Research Institute. 43: 11-16 Chen, J. T., Chang, C. and Chang, W. C. 1999. Direct somatic embryogenesis on leaf explants of Oncidium ‘Gower Ramsey’ and subsequent plant regeneration. Plant Cell Rep. 19:143-149. Chen, J. T., and Chang, W. C. 2000a. Efficient plant regeneration through somatic embryogenesis from callus cultures of Oncidium (Orchidaceae). Plant Sci 160:87-93 . Chen, J. T., and Chang, W. C. 2000b. Plant regeneration via embryo and shoot bub formation from flower-stalk explants of Oncidium Sweet Sugar. Plant Cell Tiss. Org. Cult. 62:95-100. Chen, J. T., and Chang, W. C. 2002. Effects of tissue culture conditions and explants characeteristics on direct somatic embryogenesis in Oncidium ‘Gower Ramsey’. Plant Cell Tiss. Org. Cult. 69:41-44. Colby, S. M., and Meredith, C. P. 1990. Kanamycin sensitivity of cultured tissues of Vitus. Plant Cell Rep. 9:237-240. Dellaporta, S. L., Wood, J. and Hicks, J. B. 1983. A plant DNA minipreparation: Version II. Plant Mol. Biol. Rep. 1:19-21. Delbreil, B., Guerche, P. and Jullien, M. 1993. Agrobacterium-mediated transformation of Asparagus officinalis L. long-term embryogenic callus and regeneration of transgenic plant. Plant Cell Rep. 12:129-132. Endo, M., and Ikuusima, I. 1989. Diurnal rhythm and characteristics of photosynthesis and respiration in the leaf and root of a Phalaenopsis plant. Plant Cell Physiol.30: 43-47 Grimsley, N., Hohn, T., Dacies, J. W. and Hohn, B. 1987. Agrobacterium-mediated delivery of infectious maize streak virus into maize plants. Nature 325: 177-179 Hodgson, M., Paine, R. and Anderson, N. 1991. Letts Guide to Orchid of the World. Charles Letts & Co. Homme, Y. and Asahira, T. 1985, New mean of Phalaennopsis propagation with internodal section of flower stalk. J. Jpn. Soc. Hort. Sci. 54: 379-397 Kors, F. T. M. 1991. Antibiotics, mode of action, spectrum, resistance, solubility, sterilization, stability. Haarlem:Duchefa Biochemicals B. V. Lieman-H. J., Rachmilevitch, S., Mittler, R., Marcus, Y. and Kaplan, A. 2003. Enhanced photosynthesis and growth of transgenic plants that express ictB, a gene involved in HCO3- accumulation in cyanobacteria. Plant Biotech J. 1:43-50. Lieman-H., Maya, H., Gali, S. M., Ai, I., Yukako, H., Ariel, G., Mario, L. and Aaron, K. 2008. A cyanobacterial AbrB-like protein affects the apparent photosynthetic affinity for CO by modulating low-CO-induced gene expression. Environ Microbiol. 11 Lin,Y. S., Hsieh, R. M., Chen, W. H., Tsai, W. T., Wu, C. C., Chyou, M. S. 1995. Kanamycin sensitivity of Phalaenopsis. Rept. Taiwan Sugar Res. Inst.147:11-19. Kerbauy, G. B. 1984a. Plant regeneration of Oncidium varicosum(Orchidaceae) by means of root tip culture. Plant Cell Rep. 3:27-29. Kerbauy, G. B. 1984b. In vitro flowering of Oncidium varicosum mericlones(Orchidaceae). Plant Sci. Lett. 35:73-75. Kumria, R., Waie, B. and Rajam, M. V. 2001. Plant regeneration from transformed embryogenic callus of an elite indica rice via Agrobacterium. Plant Cell Tissue Organ Cult. 67:63-71. Luo, Z. and Wu, R. 1989. A simple method for the transformation of rice via the pollen-tube pathway. Plant Mol. Biol. Rep. 7: 69-77. May, G.D., Afza, R., Mason, H. S., Wiecko, A., Novak, F. J. and Arntzen,C. J. 1995. Generation of transgenic banana (Musa acuminata) plants via formation of Agrobacterium-mediated transformation. Bio. Technol. 13: 486-492 Ohta, Y. 1986. High-efficiency genetic transformation of maize by a mixture of pollen and exogenous DNA. Proc.Natl. Acad. Cci. USA.83: 715-719. Rashid, H., Yokoi, S., Toriyama, K. and Hinata, K. 1996. Transgenic plant production mediated by Agrobacterium in Indica rice. Plant Cell Rep, 15: 727-730 Shimamoto, K., Terada, R. T., Izawa., and Fuijmoto, H. 1989. Fertile transgenic rice plants regenerated from transformed protoplasts. Nature 338:274-276. Somers, D. A., Rines, H. W., Gu, W., Kaeppler, H. F. and Bushnell, W. R. 1992. Fertile transgenic oat plant. Bio.Technology 10:1589-1594. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. Sweet, H. R. 1980. The genus Phalaenopsis. By Day Priting Corp. Pomon California, 91776, U.S.A. Tanaka, M. 1990. Micropropagation of Phalaenopsis throught leaf segment culture. Proc. Nagoya Inter. Orchid Show’90: 113-120 Tanaka, M. 1992. Micropropagation of Phalaenopsis. Biotechnology in Agriculture and Forestry, vol. 20, Springer-Verlag, Berlin:246-268. Teruo, O. 1991. Cloning and Inactivation of a Gene Essential to Inorganic Carbon Transport of Synechocystis PCC6803. Plant Physiol. 96, 280-284 Teob, E. S. 1980. Orchids of Asia. Times Books International, Singapore. Tyagi, A. K., and Mohanty, A. 2000. Rice transformation for crop improvement and functional genomics. Plant Sci. 158:1-18. Watson, W and Chapman, H. J. 1903. Orchids: Their culture and management. London and Country Printing Works, London. Yu, Z., Chen, M., Nie, L., Lu, H. X., Zheng, M. H., Qu, L. J. and Chen, Z. 1999. Recovery of transgenic orchid plants with hygromycin selection by particle bombardment to protocorms. Plant Cell Tissue Organ Cult. 58:87-92. Zhou, G. Y., Weng, J., Zeng, Y., Huang, S. Q. and Liu, G. 1983. Introduction of exogenous DNA into cotton embryos. Methods Enzymol. 101: 433-481
蘭花是一種高經濟價值的作物,可是栽培過程費時費力,成本高昂,栽培業者不斷嘗試栽培方式,希望能夠降低生產成本,縮短生長期,可是傳統的育種方法無法達成此目標,本研究擬利用基因轉殖方法,轉殖inorganic carbon transporter B (ictB)基因進入蝴蝶蘭與文心蘭中,期能提高蘭花生長效率。選用蝴蝶蘭品系Phal. amadinal "Taida"及文心蘭品系 Onc. Gower Ramsey 為實驗材料。首先是誘導蝴蝶蘭PLB (protocorm-like body)的形成,利用α-Naphthaleneacetic acid (NAA)與Benzylaminopurine (BAP)組合共16 種分別以蝴蝶蘭根尖、小葉及生長點進行誘導,結果發現小葉僅使用BAP 2 mg/L 的效果最好;接著改以Thidiazuron (TDZ)進行相同實驗,發現TDZ 效果更佳,誘導時間短且PLB 數量多,其中以TDZ 1 mg/L 的效果最好,PLB的誘導產率高達85%。其次是PLB 的再生培養條件的探討,利用1/2MS (Murashige & Skoog)培養基、NDM (New Dogashima)培養基、以及HP2B(hyponex-potato bacto)培養基等三種培養基。結果發現以HP2B 培養對蝴蝶蘭的再生效果最好,可以達到95%的再生率,HP2B 培養基同時對文心蘭的PLB 也有50%的再生效率。農桿菌基因轉殖,蝴蝶蘭PLB 及文心蘭PLB為材料,載體是帶有cyanobacterium ictB gene 的Poe1515 (11kb)。農桿菌感染七天後移入含有hygromycin 50 mg/L 的培養基進行一個月的篩選培養,
所得到的轉殖株分別進行GUS 組織染色、萃取genomic DNA 與PCR 分析。結果發現GUS 活性在植株根莖葉都有表達,表示ictB 的活性為全株表現,進一步萃取genomic DNA 進行PCR (polymeraise chain reaction)反應,電泳後可以得到預期大小的hptII 產物662 bp 及ictB 產物507 bp。再將PCR 產物純化並進行定序,定序結果於NCBI BLAST 上分析,結果得到99%的相似度,證明轉殖的外源基因確實進入轉植株中。分析轉殖株生長情形,經過三個月的培養後,轉殖株鮮重比未轉植株高,顯示轉殖ictB 確實可以提高蘭花的生長效率。至於是否能縮短蘭花幼年期則有待近一步觀察。

Orchid is a high-economic value crop. Breeders always choose the better character to reduce production cost. However, using traditional breeding processes to improve a character could take a lot of time. The objects of this study are to establish a technological protocol of gene transfer into Phalaenopsis and Oncidium to raise their growth rates. Cultivars of Phal. amadinal Taida and Onc. Gower Ramsey were used in this study. Application of α-Naphthaleneacetic acid (NAA) and Benzylaminopurine (BAP) to induce protocorm-like body(PLB), from cutting root tips, leaves and shoot apex, Sixteen concentration combinations of NAA and BAP were tested. The result indicated that only with BAP 2 mg/L in leaves have the bigest PLB production. Instead of Thidiazuron (TDZ) to BAP, we found that TDZ
have better efficiency than NAA with BAP. Moreover, it took less time and generated more PLBs. The best concentration of TDZ in PLBs induction was 1 mg/L, which has 80% induction efficiency. To test the Phalaenopsis PLBs regeneration efficiency, 1/2MS (Murashige & Skoog) medium,NDM (New Dogashima) medium,and HP2B (hyponex-potato bacto) medium were used. The result indicated that the best regeneration efficiency is using HP2B medium, which has 95% regenerate efficiency in Phalaenopsis and 50% regenerate efficiency in oncidium. Plasmid poe1515 which carried cyanobacterium ictB gene was transformed into Agrobacterium GV310. This strain was used to infecte PLBs for 7 days and selected with hygromycin 50 mg/L for one month. Transgenic plant has GUS activity which express in all parts of plant. Moreover, PCR analysis of genomic DNA showed two bands which is 662 bp for hptII and 507 bp for ictB, we checking the PCR products and BLAST search in the NCBI website, thas obtained 99% similarity with the origenal genes. The results demonstrated that have transferred ictB in Orchid
successfully. Finalily, comparison the growth rate of transgenic and untransgenic plants, that transgenic plant has higher fresh weight than untransgenic plants after 3
months.The possibility to short vegetative period needs further study.
其他識別: U0005-1901200923034800
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