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研究生:何慧敏
研究生(外文):Hui-min Ho
論文名稱:蝴蝶蘭花粉與農桿菌共培養基因轉殖系統之建立
論文名稱(外文):Development of the Genetic Transformation System in Phalaenopsis by Pollen Incubated with Agrobacterium
指導教授:蔡新聲蔡新聲引用關係
指導教授(外文):Hsin-Sheng Tsay
學位類別:碩士
校院名稱:朝陽科技大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:99
中文關鍵詞:蝴蝶蘭花粉農桿菌
外文關鍵詞:PhalaenopsisPollenAgrobacterium
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蝴蝶蘭為商業市場上最重要的蘭花,可供生產盆花及切花。目前蘭花品種的改良仍依賴傳統的雜交育種,但因雜交育種的有性世代過長而限制了改良的空間,近來發展的基因轉殖方式,可有效改善此缺點。本研究利用花粉與農桿菌共培養的方式,發展出一種創新的基因轉殖技術,試驗中使用農桿菌EHA105 品系,構築質體pCAMBIA1302、pCAMBIA1301 與pTAG-8。
利用pCAMBIA1302 質體內帶GFP 基因,分別構築花色基因DFR 、CHI;pCAMBIA1301 質體內帶GUS 基因,及花色基因F3′H 基因;以pTAG-8 質體內帶有GUS 報導基因與4 套的加強子(enchancers)能使基因大量表現。
轉殖後的蝴蝶蘭植株利用hygromycin 篩選系統,發現使用含有pTAG-8質體構築的農桿菌與花粉共培養,以 5×10PP cfu /ml農桿菌濃度搭配 0.1 mM AS共培養12hr,每個果莢平均可獲得6棵疑轉植株,為蝴蝶蘭最適轉殖條件。另外以 1×10P
P cfu /ml農桿菌搭配 0.1 mM AS濃度共培養12hr,每個果莢平均可獲得68棵疑轉植株,為朵麗蝶蘭最適轉殖條件。最適條件下所獲得之轉植株,經PCR檢測確實帶有GUS報導基因,在蝴蝶蘭外觀型態上觀察到轉入F3′H基因的植株葉片,其顏色產生由綠色轉變為粉紅色或紅色的現象。另外於朵麗蝶蘭的種子中則觀察到,有4.89%的GFP螢光蛋白表現,以PCR-Southern檢測確實帶有DFR, CHI, F3′H等花色基因。本研究為第一個成功的使用農桿菌配合花粉進行蝴蝶蘭基因轉殖。
Phalaenopsis is one of the most important orchids grown for commercial production of cut flowers and potted plants. Genetic improvement of
Phalaenopsis through sexual hybridization is however, restricted by long growth
and reproductive cycles. Genetic transformation has been used to create mutants
with attractive or new flower patterns. In the present study, a novel plant
transformation method has been developed by co-cultivation of pollen with
Agrobacterium tumefaciens strains EHA105 (pCAMBIA1301 or
pCAMBIA1302) and EHA105 with an activation vector (pTAG-8). The
construct pCAMBIA1302 had GFP and pigment biosynthetic gene DFR
(dihydroflavonol 4-reductase) or CHI (chalcone isomerase), while
pCAMBIA1301 had GUS and F3′H (flavanone 3′-hydroxylase) genes.
Using the hygromycin-selection system, it was found that the optimum
conditions for affecting transformation in Phalaenopsis were: A. tumefaciens
(pTAG-8) concentration of 5×10P
P cfu/ml, a supplement of 0.1mM
acetosyringone (AS) in the medium and a co-culture period with pollen for 12 h.
Under these conditions, 6 transformants were obtained that from one capsule
under the selection pressure of 5 mg/l hygromycin. In another Phalaenopsis
variety, Doritaenopsis, 68 transformants could be obtained that from one capsule
under 5 mg/l hygromycin selection medium using A. tumefaciens ( pTAG-8 )
concentration of 1×10PP cfu /ml and a co-culture with pollen for 12 h without AS.
Presence of GUS gene in transformants of Phalaenopsis and Doritaenopsis was
confirmed by PCR and Southern blotting analysis. The transformed Phalaenopsis showed change in leaf pigments from green to pink or red. In
Doritaenopsis, 4.89% of the transformants were found to have the GFP activity.
The presence of DFR, CHI and F3′H genes were also confirmed by Southern
blot analysis. Thus, in this thesis, for the first time, a new transformation
technology for orchid Phalaenopsis has been reported.
五、參考文獻
尤崇魁,蝴蝶蘭栽培技術,園藝世界出版社 (1989)。
李哖,蝴蝶蘭-繁殖、生育特性、產期調節及產後品質,財團法人台灣區花
卉發展協會(2002)。
李淑華,文心蘭體胚分化及基因轉殖研究系統之研究,國立成功大
學生物科技研究所碩士論文,(2000)。
余淑美,植物功能性基因體研究,後基因體時代之生物技術,第41-50頁
(2003)
周光宇、翁堅、龔蓁蓁、曾以申、楊曉霞、沈慰芳、王自芬、陶金洲,農
業分子育種-授粉後外源DNA導入植物的技術,中國科學 第21期,第
1-6頁(1988)。
林讚標,台灣蘭科植物,南天書局有限公司 (1988)。
林淑芳,文心蘭原生殖體與農桿菌轉殖系統之研究,國立高雄師
範大學生物科學研究所(2001)。
涂美智、李哖,蝴蝶蘭授粉適期與果莢成熟度對種子發芽之影響,中國園
藝,第33卷,第3期,第190-200頁(1987)。
陳福旗、G. Mangai Kasthuri、蔡宜容、黃建誌、李文立、羅元
宏、姜素芬、陳雅惠、鄭汀琦、Manju M. George, 台灣花卉
園藝月刊,第219 期,第16 頁 (2005)。
詹明才、張新雄,農桿菌轉殖系統之影響因素,科學農業,第39 卷,第11-12
期:,第249-255 頁(1991)。
蔡瑜卿,蘭花產業經營效率改善座談會紀實,台灣花卉園藝月刊,
第222 期,第54 頁 (2006)。
潘子明, 2003 年全球基因改造作物之發展,生物產業,第15卷,第1期,
第37-50頁(2003)。
83
關淑卿,有機添加物對蝴蝶蘭幼苗生長的影響及原球體增殖之探討,國立
台灣大學園藝研究所碩士論文,(1989)。
蝴蝶蘭栽培手冊,台灣糖業公司農務處和糖業研究所 合編 (1989)。
蕭翌柱、夏奇鈮、蔡新聲,彩葉蘭授粉期與果莢冷藏對種子發芽之影響,
中華農業研究,第53期,第193-200頁( 2004 )。
Aida, R., S. Kishimoto, Y. Tanaka, and M. Shibata. Modification of
flower color in torenia (Torenia fournieri Lind.) by genetic transformation.
Plant Sci.153:33-42 (2000a)
Aida, R., K. Yoshida, T. Kondo, S. Kishimoto, and M. Shibata.
Copigmentation gives bluer flowers on transgenic torenia plants with the
antisense dihydroflavonol-4-reductase gene. Plant Sci. 160:49-56 (2000b)
Anzai, H., Ishii Y., Shichinohe M., Katsumata K., Nojiri C., Morikawa
H.,Tanaka M. Transformation of Phalaenopsis by particle
bombardment. Plant Tissue Cult. Lett. 13:265-271 (1996).
Armstrong, C. L., W. L. Petersen, W.G. Bucholz, and B. A. Bowen. Factors
affecting PEG-mediated stable transformation of maize protoplasts.
Plant Cell Rep. 9:335-339.(1990).
Asen, S., R. N. Stewart, and K. H. Norris.. Co-pigmentation of anthocyanins
in plant tissue and its effect on color. Phytochemistry
11:1139-1144(1972).
Belarmino, M. M. and Mii, M. Agrobacterium-mediated genetic transformation
of a phalaenopsis orchid. Plant Cell Rep. 19: 435-442 (2000).
Booy G, Krens FA, Huizing HJ. Attempted pollenmediated transformation of
maize. J Plant Physiol 135:319–324(1989).
Chen, W.S., Chiu, C.C., Liu, H.Y., Lee, T.L., Cheng, J.T., Lin, C.C., Wu, Y.Y.
and Chang, H.Y. Gene transfer via pollen-tybe pathway for anti fusarium
wilt in watermelon. Bioch. Mol. Biol. Intern. 46:1201-1209 (1998).
Chen, J. T. and Chang, W. C. Effects of tissue culture conditions and
84
explant characteristics on direct somatic embryogenesis in Oncidium
‘Gower Ramsey’. Plant Cell Tiss. Org. Cult. 69: 41-44 (2002).
Cheng, M., J. E. Fry, S. Pang, H. Zhou, C. M. Hironaka, D. R. Duncan, T. W.
Conner, and Y. Wan. Genetic transformation of wheat mediated by
Agrobacterium tumefaciens. Plant Physiol. 115:971-980 (1997).
Christou, P. Genetic transformation of crop plants using microprojectile
bombardment. Plant Jou. Cell Mol. Biol. 2: 275-281 (1992).
Citovsky, V., B. Guralnick, M. Simor and J. S. Wall. The molecular
structure of Agrobacterium VirE2-Single stranded DNA complexes
involved in nuclear import. J. Mol. Biol. 271:718-727 (1997).
Chia T.F., Y.S. Chan and N. H. Chua. Genetic engineering of tolerance to
cymbidium mosaic virus. In: Bonham D. G., Kemohan J (eds.)
Proceeding of the 13th World Orchid Conference 1990. 13 WOC
Proceeding Trust, Auckland (1990).
De la Pena, A., Lörz, H. and Schell, J. Transgenic rye plants obtained by
injecting DNA into young floral tillers. Nature 235:274-276 (1987).
D’Halluin, K., E. Noone, M. Bossut, M. DE Beuckeleer, J. Leemans (1992).
Dressler, R. L. The orchids natural history and classfication. Harvard
University Press, Cambridge, USA (1981).
Elomaa, P.,Y. Helarirtta, R. J. Griesbach, M. Kotilainen, P. Seppanen, T. H.
Teeri. Transgenic inactivation in Petunia hybrida is influenced by the
properties of the foreign gene. Mol. Gen. Genet. 248:649-656.(1995).
Forkmann, G., and B. Dangelmayr. Genetic control of chalcone isomerase
activity in flowers of Dianthus caryophyllus. Biochem. Genet. 5:519-527
(1980).
Frame, B. R, H. Shou, R. K. Chikqamba, Z. Zhang, C. Xiang, T. M. Fonger, S.
E.K. Pegg, B. Li, D. S. Nettleton, D. Pei, and K. Wang. Agrobacterium
tumeraciens-mediated transformation of maize embryos using a standard
85
binary vector system. Plant Physiol. 129:13-22.(2002)
Goh, C. J., and Arditti, J. Orchidaceae. In: A. H. Halevy(ed) Handbook of
Flowering. Vol.1. CRC Press Inc., Boca Raton Florida. p309-336 (1985)
H. Yu · S.H. Yang · C.J. Goh. Agrobacterium-mediated transformation of
a Dendrobium orchid with the class 1 knox gene DOH1 .Plant Cell
Reports (2001)
Hess D, Dressler K, Nimmrichter R. Transformation experiments by
pipetting Agrobacterium into the spikelets of wheat (Triticum aestivum L.).
Plant Sci 72:233–244(1990)
Holton, T. A., and E. C. Conish. Genetics and biochemistry of
anthocyanin biosynthesis. The Plant Cell. 7:1071-1083.(1995)
Hu, C.Y. and Wang, L. In planta soybean transformation technologies
developed in China: procedure, confirmation and field performance. In
Vitro Cell. Dev. Biol.-Plant 35: 417-420. (1999)
Hiei, Y., T. Komari and T. Kubo. Transformation of rice mediated by
Agrobacterium tumefaciens. Plant Mol. Bio. 35:205-218.(1997)
Humara, J. M., M. Lopez, and R. J. Ordas. Agrobacterium
tumefaciens-mediated transformation of Pinus pinea L. cotyledons: an
assessment of factors influencing the efficiency of uidA gene transfer. Plant
Cell Rep. 19:51-58 (1999).
Hsieh, R. M., Chen, W. H., Hsu, H. M., Lin, Y. S., Tsai, W. T., Fu, Y. M.,
Chan, M. T. and Yu, S. M. Agrobacterium tumefaciens-mediated
transfomation of Phalaenopsis orchid. Rep. Taiwan Sugar Res. Inst. 155:
41-45.(1997)
James, C. Global status of commercialized biotech/GM crops: 2004.
ISAAA Briefs, No. 32.(2004)
Klein, T. M., E. D. Wolf, R. Wu, and J. C. Sanford. High-velocity micro
86
projectiles for delivering nucleic acids into living cells. Nature.
346:776-777.(1987)
Knapp, J. E., Kausch, A. P. and Chandlee, J. M. Transformation of
three genera of orchid using the bar gene as a selectable marker.
Plant Cell Rep. 19: 893-898.(2000)
Lazzeri, P. A., R. Brettschneider, R. Lûhrs, and H. Lörz. Stable
transformation of barley via PEG-induced direct DNA uptake into
protoplasts. Theor. Appl. Genet. 81:437-444.(1991)
Lohrke, S. M., H. Yang and S. Jin. Reconstitution of acetosyringone-mediated
Agrobacterium tumefaciens virulence gene expression in the heterologous
host Esxherichia coli. J. Bacteriol. 183:704-3711.(2001)
Langridge P, Brettschneider R, Lazzeri P, Lorz H. Transformation of cereals via
Agrobacterium and the pollen pathway: a critical assessment. Plant J
2 :631–638 (1992)
Liau, C.-H., Lu, J.-C., Prasad, V., Hsiao, H.-H., You, S.-J., Lee, J.-T., Yang,
N.-S., Huang, H.-E., Feng, T.-Y., Chen, W.-H. and Chan, M.-T.. The sweet
pepper ferredoxin-like protein (pflp) conferred resistance against soft rot
disease in Oncidium orchid. Transgenic Res. 12: 329-336.(2003)
Liau, C.-H., You, S.-J., Prasad, Hsiao, H.-H., Lu, J.-C., Yang, N.-S. and Chan,
M.-T. Agrobacterium tumefaciens-mediated trnasfomation of an Oncidium
orchid. Plant Cell Rep. 21: 993-998. (2003)
Michielse, C. B., P. J. J. Hooykaas, and C. A. M. J. J. van den Hondel.
Role of bacterial virulence protein in Agrobacterium-mediated
transformation of Aspergillus awamori. Fungal Gent. And Biol.
41:571-578.(2004)
Mu, H.M., Liu, S.J., Zhou, W.J., Wen, Y.X., Zhang, W.J. and Wei, R.X.
Transformation of wheat with insecticide gene of arrowhead proteinase
inhibitors by pollen tube pathway and analysis of transgenic plants. I
Chuan Hsueh Pao (Chinese) 26:634-642. (1999)
87
Michael Fromm, Loverine P. Taylor, and Virginia Walbot. Expression of Genes
Transferred into Monocot and Dicot Plant Cells by Electroporation PNAS
vol. 82:5824-5828 (1985).
Marrs, K. A., M. R. Alfenito, A. M. Lloyd, and V. Walbot. A glutathione
S-transferase involved in vacuolar transfer encoded by the maize gene
Bronze-2. Nature 375:397-400 (1995).
Nishihara, M., T. Nakatsukal, S. Yamamura. Flavonoid components and
flower color change in transgenic tobacco plants by suppression of
chalcone isomerase gene. FEBS Letters 579:6074–6078 (2005).
Neuhaus, G., G. Spangenberg, M. O. Scheid, and H. G. Schweiger. Transgenic
rapeseed plants obtained by the microinjection of DNA into
microspore-derived embryoids.(1987).
Powell-Abel et al.TMV virus-resistant tobacco and tomato transgenic plants
developed using Cdna of coat protein gene of TMV (1986).
Pelta, J. L., W. A. Doenesa, R. V. Schoborgb, and C. A. Mcintosha. Flavanone
3-hydroxylase expression in Citrus paradisi and Petunia hybrida seedlings.
Phytochemistry 64:435–444 ( 2003).
Rashid, H., S. Yokoi, K. Toriyama and K. Hinata. Transgenic plant production
mediated by Agrobacteium in Indica rice. Plant Cell Rep. 15:727-730
( 1996).
Schaaf, J. M. Schoneberg, M. Staebell, P. Flynn, J. Anderson, and J.Howard.
Transgenic tobacco plants and their progeny derived by micro-projectile
bombardment of tobacco leaves. Plant Mol.Biol. 14: 261-268 (1990).
Sato, S., C. Newell, K. Kolacz, L. Tredo, J. Finer, and M. Hinchee. Stable
transformation via particle bombardment in two different soybean
regeneration systems. Plant Cell Rep. 12: 408-413(1993).
Sweet, H. R.. The Genus Phalaenopsis. Orchid Digest Corp., Pomona,
California, USA(1980).
88
Stachel, S. E., E. Messens, , M. van Montagu,and P. Zambryski. Identification
of the signal molecules produced by wounded plant cells that activate
T-DNA transfer in Agrobacterium tumefaciens.Nature.318:624-629(1985).
Simo, P. W. Plant pigments for color and nutrition. Hot. Sci. 32:12-13(1997).
Tjokrokusumo, D., Heinrich, T., Wylie, S., Potter, R. and McComb, J. Vacuum
infiltration of Petunia hybrida pollen with Agrobacterium tumefaciens to
achieve plant transformation. Plant Cell Reports.19:792–797 (2000).
Tsuda, S., Y. Fukuil, N. Nakamural, Y. Katsumoto, K. Yonekura-Sakakibarala,
M. Fukuchi-Mizutanil, K. Ohiral, Y. Ueyama, H. Ohkaea, T. A. Holton, T.
Kusumi, Y. Tanakal. Flower color modification of Petunia hybrida
commercial varieties by metabolic engineering. Plant Biotechnol.
21(5):377–386.(2004).
Tinland, B. The integration of T-DNA inot plant genomes. Trend In Plant Sci.
1:178-184(1996).
Tzfira, T. and V. Citovsky. From host recognition to T-DNA integration:the
function of bacterial and plant genes in the Agrobacterium-plant cell
interaction. Mol. Plant Path. 1(4): 1-212(2000).
Todd, J. J., and Vodkin, L. O. Pigmented soybesn (Glycine max) seed coat
accumulate proanthocyanidins that bind RNA. Plant Physiol.102:663:670
(1933)
Tomes ,D. T., A. K. Weissinger, M. Ross, R. Higgins, B. J. Drummond, S.
Vain, P., M. D. McMullen, and J. F. Finer. Osmotic treatment enhances
particle bombardment-mediated transient and stable transformation of
maize. Plant Cell Rep. 12: 84-88(1993).
Valentine, L. Agrobacterium tumefaciens and the plant: David and Goliath of
Modern Genetics. Plant Physiol. 133:948-955(2003).
Van Wert,S.L.and J.A.Saunders. Reduction of nuclease activity release from
germinating pollen under condition used for pollen electrotransformation.
89
Plant Sci.84:11-16(1992).
Ward, D. V. and P. C. Zambryski. The six functions of Agrobacterium VirE2.
Proc. Natl. Acad. Sci. USA. 98:385-386.(2001)
Winkel-Shirley, B. Flavonoid biosynthesis. A colorful model for genetics,
biochemistry, cell biology and biotechnoloty. Plant Physiol.
126:485-493(2001)
You, S. J., Liau, C. H., Huang, H. E., Feng, T. Y., Prasad, V., Hsiao, H. H.,Lu, J.
C. and Chan, M. T. Sweet pepper ferredoxin-like protein (pflp) gene as a
novel selection marker for orchid transformation.Planta 217: 60-65(2003).
Yu, Z., M. Chen, N. Lin, L. Huafei, X. Ming, H. Zheng, L. J. Qu, and Z.Chen.
Recovery of transgenic orchid plants with hygromycin selection by particle
bombardment to protocorm. Plant Cell, Tissue Organ Cult.
58:87-92(1999).
Yu, H., Yang, S.H. and Goh, C. J. Agrobacterium-mediated transformation of a
Dendrobium orchid with the class 1 Knox gene DOH1. Plant Cell Rep. 20:
301-305(2001).
Yang J., H.-J. Lee, D.H. Shin, S.K. Oh, J.H. Seon, K.Y. Paek, K.-H. Han.
Genetic transformation of Cymbidium orchid by particle bombardment.
Plant Cell Rep. 18: 978-984(1999).
Zhao, Zuo-yu., W. Gu, T. Cai, L. Tagliani, D. Hondred, D. Bond, S. Schroeder,
M. Rudert, and D. Pierce. High throughput genetic transformation
mediated by Agrobacterium tumefaciens in maize. Mol. Breed.
8:323-333(2001).
Zeng J-Z, Wang D-J, Wu Y-Q, Zhang J, Zhou W-J, Zhu X-P, Xu N-Z
Transgenic wheat plants obtained with pollen-tube pathway method. Sci
China 37 :319–325(1994)
Zhao, Zuo-yu., W. Gu, T. Cai, L. Tagliani, D. Hondred, D. Bond, S. Schroeder,
M. Rudert, and D. Pierce. High throughput genetic transformation
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