Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20244
標題: 利用農桿菌轉殖冰花培養細胞與植株系統之改良
Refinement of Agrobacterium-mediated stable transformation system in cultured cells and intact plants of Mesembryanthemum crystallinum L.
作者: 何佳芳
Ho, Jia-Fang
關鍵字: 農桿菌轉殖法;Agrobacterium-mediated transformation;冰花;Mesembryanthemum crystallinum L.
出版社: 生命科學系所
引用: 王詠中 (2006) 耐鹽植物冰花SNF-1基因之分離與分析。國立中興大學生命科學系學士論文。 林亞君 (2009) 以酵母菌雙雜交法分析冰花鹽誘導蛋白mcSKD1與mcSNF1和mcCPN1之間的交互作用。國立中興大學生命科學系碩士論文。 周映孜 (2007) 以蛋白質交互作用分析耐鹽植物冰花腎形細胞累積耐鹽相關mcSKD1蛋白及其參與之高等植物耐鹽機制。國立中興大學生命科學系博士論文。 陳曉慧 (2010) 利用農桿菌轉殖冰花培養細胞系統之建立。國立中興大學生命科學系碩士論文。 黃女娟 (2010) 耐鹽植物冰花sucrose non-fermenting related kinase mcSNF1自行及受質磷酸化之活性探討。國立中興大學生命科學系碩士論文。 楊邡郁 (2006) 冰花訊息傳導相關mcSNF1在鹽逆境下基因表現及蛋白累積之分析。國立中興大學生命科學系碩士論文。 Adams, P., Nelson, D.E., Yamada, S., Chmara, W., Jensen, R.G., Bohnert, H.J., and Griffiths, H. (1998). Growth and development of Mesembryanthemum crystallinum (Aizoaceae). New Phytol. 138: 171-190. Adams, P., Thomas, J.C., Vernon, D.M., Bohnert, H.J., and Jensen, R.G. (1992). Distinct cellular and organismic responses to salt stress. Plant Cell Physiol. 33: 1215-1223. Ahmed, M.B., Akhter, M.S., Hossain, M., Islam, R., Choudhuty, T.A., Hannan, M.M., Razvy, M.A., and Ahmad, I. (2007). An efficient Agrobacterium-mediated genetic transformation method of lettuce (Lactuca sativa L.) with an aphidicidal gene, pta (Pinellia ternata agglutinin). Middle-East J. Sci. Res. 2: 155-160. Altpeter, F., Baisakh, N., Beachy, R., Bock, R., Capell, T., Christou, P., Daniell, H., Datta, K., Datta, S., Dix, P.J., Fauquet, C., Huang, N., Kohli, A., Mooibroek, H., Nicholson, L., Nguyen, T.T., Nugent, G., Raemakers, K., Romano, A., Somers, D.A., Stoger, E., Taylor, N., and Visser, R. (2005). Particle bombardment and the genetic enhancement of crops: myths and realities. Mol. Breed. 15: 305-327. Andolfatto, P., Bornhouser, A., Bohnert, H.J., and Thomas, J.C. (1994). Transformed hairy roots of Mesembryantemum crystallinum: gene expression patterns upon salt stress. Physiol. Plant. 90: 708-714. Armstrong, C.L., Petersen, W.L., Buchholz, W.G., Bowen, B.A., and Sulc, S.L. (1990). Factors affecting PEG-mediated stable transformation of maize protoplasts. Plant Cell Rep. 9: 335-339. Bernaerts, M.J., and De Ley, J. (1963). A biochemical test for crown gall bacteria. Nature 197: 406-407. Bohnert, H.J., and Cushman, J.C. (2000). The ice plant cometh: lessons in abiotic stress tolerance. J. Plant Growth Regul. 19: 334-346. Caplan, A.B., Van Montagu, M., and Schell, J. (1985). Genetic analysis of integration mediated by single T-DNA borders. J. Bacteriol. 161: 655-664. Cardoza, V., and Stewart, C.N. (2003). Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants. Plant Cell Rep. 21: 599-604. Celenza, J., and Carlson, M. (1986). A yeast gene that is essential for release from glucose repression encodes a protein kinase. Science 233: 1175-1180. Chen, W.-S., Chiu, C.-C., Liu, H.-Y., Lee, T.-L., Cheng, J.-T., Lin, C.-C., Wu, Y.-J., and Chang, H.-Y. (1998). Gene transfer via pollen-tube pathway for anti-fusarium wilt in watermelon. IUBMB Life 46: 1201-1209. Cheng, M., Lowe, B.A., Spencer, T.M., Ye, X., and Armstrong, C.L. (2004). Factors influencing Agrobacterium-mediated transformation of monocotyledonous species. In Vitro Cell. Dev. Biol.-Plant 40: 31-45. Choi, I.-R., Stenger, D.C., Morris, T.J., and French, R. (2000). A plant virus vector for systemic expression of foreign genes in cereals. Plant J. 23: 547-555. Chowrira, G., Akella, V., and Lurquin, P.F. (1995). Electroporation-mediated gene transfer into intact nodal meristems in planta. Generating transgenic plants without in vitro tissue culture. Mol. Biotechnol. 3: 17-23. Citovsky, V., Kozlovsky, S.V., Lacroix, B., Zaltsman, A., Dafny-Yelin, M., Vyas, S., Tovkach, A., and Tzfira, T. (2007). Biological systems of the host cell involved in Agrobacterium infection. Cell Microbiol. 9: 9-20. Cushman, J.C., Meyer, G., Michalowski, C.B., Schmitt, J.M., and Bohnert, H.J. (1989). Salt stress leads to differential expression of two isogenes of phosphoenolpyruvate carboxylase during crassulacean acid metabolism induction in the common ice plant. Plant Cell 1: 715-725. Cushman, J.C., Wulan, T., Kuscuoglu, N., and Spatz, M.D. (2000). Efficient plant regeneration of Mesembryanthemum crystallinum via somatic embryogenesis Plant Cell Rep. 19: 459-463. D''Halluin, K., Bonne, E., Bossut, M., De Beuckeleer, M., and Leemans, J. (1992). Transgenic maize plants by tissue electroporation. Plant Cell 4: 1495-1505. Escobar, M.A., and Dandekar, A.M. (2003). Agrobacterium tumefaciens as an agent of disease. Trends Plant Sci. 8: 380-386. Finer, J.J., and McMullen, M.D. (1990). Transformation of cotton (Gossypium hirsutum L.) via particle bombardment. Plant Cell Rep. 8: 586-589. Gelvin, S.B. (2000). Agrobacterium and plant genes involved in T-DNA transfer and integration. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51: 223-256. Gelvin, S.B. (2003). Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol. Mol. Biol. Rev. 67: 16-37. Golds, T., Maliga, P., and Koop, H.-U. (1993). Stable plastid transformation in PEG-treated protoplasts of Nicotiana tabacum. Nat. Biotechnol. 11: 95-97. Hellens, R., Mullineaux, P., and Klee, H. (2000). A guide to Agrobacterium binary Ti vectors. Trends Plant Sci. 5: 446-451. Hiei, Y., Komari, T., and Kubo, T. (1997). Transformation of rice mediated by Agrobacterium tumefaciens. Plant Mol. Biol. 35: 205-218. Hofmann, K., and Bucher, P. (1996). The UBA domain: a sequence motif present in multiple enzyme classes of the ubiquitination pathway. Trends Biochem. Sci. 21: 172-173. Hood, E.E., Gelvin, S.B., Melchers, L.S., and Hoekema, A. (1993). New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res. 2: 208-218. Hrabak, E.M., Chan, C.W.M., Gribskov, M., Harper, J.F., Choi, J.H., Halford, N., Kudla, J., Luan, S., Nimmo, H.G., Sussman, M.R., Thomas, M., Walker-Simmons, K., Zhu, J.-K., and Harmon, A.C. (2003). The Arabidopsis CDPK-SnRK superfamily of protein kinases. Plant Physiol. 132: 666-680. Hu, C.-Y., and Wang, L. (1999). In planta soybean transformation technologies developed in China: procedure, confirmation and field performance. In Vitro Cell. Dev. Biol.-Plant 35: 417-420. Ishida, Y., Saito, H., Ohta, S., Hiei, Y., Komari, T., and Kumashiro, T. (1996). High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat. Biotechnol. 14: 745-750. Ishimaru, K. (1999). Transformation of a CAM plant, the facultative halophyte Mesembryanthemum crystallinum by Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult. 57: 61-63. Kado, C.I., and Heskett, M.G. (1970). Selective media for isolation of Agrobacterium, Corynebacterium, Erwinia, Pseudomonas, and Xanthomonas. Phytopathology 60: 969-976. Klein, T.M., Kornstein, L., Sanford, J.C., and Fromm, M.E. (1989). Genetic transformation of maize cells by particle bombardment. Plant Physiol. 91: 440-444. Klein, T.M., Wolf, E.D., Wu, R., and Sanford, J.C. (1987). High-velocity microprojectiles for delivering nucleic acids into living cells. Nature 327: 70-73. Kofer, W., Eibl, C., Steinmüller, K., and Koop, H.-U. (1998). PEG-mediated plastid transformation in higher plants. In Vitro Cell. Dev. Biol.-Plant 34: 303-309. Koncz, C., and Schell, J. (1986). The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet. 204: 383-396. Kondo, T., Hasegawa, H., and Suzuki, M. (2000). Transformation and regeneration of garlic (Allium sativum L.) by Agrobacterium-mediated gene transfer. Plant Cell Rep. 19: 989-993. Konieczny, R., Obert, B., Bleho, J., Novák, O., Heym, C., Tuleja, M., Müller, J., Strnad, M., Menzel, D., and Šamaj, J. (2011). Stable transformation of Mesembryanthemum crystallinum (L.) with Agrobacterium rhizogenes harboring the green fluorescent protein targeted to the endoplasmic reticulum. J. Plant Physiol. 168: 722-729. Kost, B., Spielhofer, P., and Chua, N.-H. (1998). A GFP-mouse talin fusion protein labels plant actin filaments in vivo and visualizes the actin cytoskeleton in growing pollen tubes. Plant J. 16: 393-401. Kumlehn, J., Serazetdinova, L., Hensel, G., Becker, D., and Loerz, H. (2006). Genetic transformation of barley (Hordeum vulgare L.) via infection of androgenetic pollen cultures with Agrobacterium tumefaciens. Plant Biotechnol. J. 4: 251-261. Lacroix, B., Kozlovsky, S.V., and Citovsky, V. (2008). Recent patents on Agrobacterium-mediated gene and protein transfer, for research and biotechnology. Recent Pat. DNA Gene Seq. 2: 69-81. Lai, E.-M., and Kado, C.I. (1998). Processed VirB2 is the major subunit of the promiscuous pilus of Agrobacterium tumefaciens. J. Bacteriol. 180: 2711-2717. Lazzeri, P.A., Brettschneider, R., Lührs, R., and Lörz, H. (1991). Stable transformation of barley via PEG-induced direct DNA uptake into protoplasts. Theor. Appl. Genet. 81: 437-444. Lee, L.-Y., and Gelvin, S.B. (2008). T-DNA binary vectors and systems. Plant Physiol. 146: 325-332. Li, F.-F., Wu, S.-J., Chen, T.-Z., Zhang, J., Wang, H.-H., Guo, W.-Z., and Zhang, T.-Z. (2009). Agrobacterium-mediated co-transformation of multiple genes in upland cotton. Plant Cell Tissue Organ Cult 97: 225-235. Lippincott-Schwartz, J., and Patterson, G.H. (2003). Development and use of fluorescent protein markers in living cells. Science 300: 87-91. López, M., Humara, J.M., Rodríguez, R., and Ordás, R.J. (2000). Factors involved in Agrobacterium tumefaciens-mediated gene transfer into Pinus nigra Arn. ssp. salzmannii (Dunal) Franco. Euphytica 114: 195-203. Luo, Z.-x., and Wu, R. (1988). A simple method for the transformation of rice via the pollen-tube pathway. Plant Mol. Biol. Rep. 6: 165-174. Lurquin, P.F. (1997). Gene transfer by electroporation. Mol. Biotechnol. 7: 5-35. Mannan, A., Syed, T.N., and Mirza, B. (2009). Factor affecting Agrobacterium tumefaciens mediated transformation of Aremisia absinthium L. Pak. J. Bot. 41: 3239-3246. McCullen, C.A., and Binns, A.N. (2006). Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer. Annu. Rev. Cell Dev. Biol. 22: 101-127. Michalczuk, B., and Wawrzyńczak, D. (2004). Effect of medium composition and date of explant drawing on effectiveness of Agrobacterium-mediated transformation in the petunia (Petunia hybrida pendula). J. Fruit and Ornamental Plant Res. 12: 5-16. Miki, B., and McHugh, S. (2004). Selectable marker genes in transgenic plants: applications, alternatives and biosafety. J. Biotechnol. 107: 193-232. Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15: 473-497. Nair , G.R., Lai, X., Wise, A.A., Rhee, B.W., Jacobs, M., and Binns, A.N. (2011). The Integrity of the periplasmic domain of the VirA sensor kinase is critical for optimal coordination of the virulence signal response in Agrobacterium tumefaciens. J. Bacteriol. 193: 1436-1448. Narasimhulu, S.B., Deng, X.-b., Sarria, R., and Gelvin, S.B. (1996). Early transcription of Agrobacterium T-DNA genes in tobacco and maize. Plant Cell 8: 873-886. Neuhaus, G., and Spangenberg, G. (1990). Plant transformation by microinjection techniques. Physiol. Plant. 79: 213-217. Neuhaus, G., Spangenberg, G., Mittelsten-Scheid, O., and Schweiger, H.-G. (1987). Transgenic rapeseed plants obtained by the microinjection of DNA into microspore-derived embryoids. Theor. Appl. Genet. 75: 30-36. Newell, C.A. (2000). Plant transformation technology, developments and applications. Mol. Biotechnol. 16: 53-65. Ni, M., Cui, D., Einstein, J., Narasimhulu, S., Vergara, C.E., and Gelvin, S.B. (1995). Strength and tissue specificity of chimeric promoters derived from the octopine and mannopine synthase genes. Plant J. 7: 661-676. Opabode, J.T. (2006). Agrobacterium-mediated transformation of plants: emerging factors that influence efficiency. Biotechnol. Mol. Biol. Rev. 1: 12-20. Rakoczy-Trojanowska, M. (2002). Alternative methods of plant transformation-a short review. Cell. Mol. Biol. Lett. 7: 849-858 Ritala, A., Aspegren, K., Kurtén, U., Salmenkallio-Marttila, M., Mannonen, L., Hannus, R., Kauppinen, V., Teeri, T.H., and Enari, T.-M. (1994). Fertile transgenic barley by particle bombardment of immature embryos. Plant Mol. Biol. 24: 317-325. Salas, M.G., Park, S.H., Srivatanakul, M., and Smith, R.H. (2001). Temperature influence on stable T-DNA integration in plant cells. Plant Cell Rep. 20: 701-705. Saunders, J.A., and Matthews, B.F. (1995). Pollen electrotransformation in tobacco. Meth. Mol. Biol. 55: 81-88. Scholthof, H.B., Scholthof, K.-B.G., and Jackson, A.O. (1996). Plant virus gene vectors for transient expression of foreign proteins in plants. Annu. Rev. Phytopathol. 34: 299-323. Sharma, K.K., Bhatnagar-Mathur, P., and Thorpe, T.A. (2005). Genetic transformation technology: status and problems. In Vitro Cell. Dev. Biol.-Plant 41: 102-112. Su, H., Balderas, E., Vera-Estrella, R., Golldack, D., Quigley, F., Zhao, C., Pantoja, O., and Bohnert, H.J. (2003). Expression of the cation transporter McHKT1 in a halophyte. Plant Mol.Biol. 52: 967-980. Subramaniam, S., and Rahman, Z.A. (2010). Early GFP gene assessments influencing Agrobacterium tumefaciens-mediated transformation system in Phalaenopsis violacea orchid. Emir. J. Food Agric. 22: 103-116. Sunagawa, H., Agarie, S., Umemoto, M., Makishi, Y., and Nose, A. (2007). Effect of urea-type cytokinins on the adventitious shoots regeneration from cotyledonary node explant in the common ice plant, Mesembryanthemum crystallinum. Plant. Prod. Sci. 10: 47-56. Tassan, J.-P., and Le Goff, X. (2004). An overview of the KIN1/PAR-1/MARK kinase family. Biol. Cell 96: 193-199. Tjokrokusumo, D., Heinrich, T., Wylie, S., Potter, R., and McComb, J. (2000). Vacuum infiltration of Petunia hybrida pollen with Agrobacterium tumefaciens to achieve plant transformation. Plant Cell Rep. 19: 792-797. Treichel, S. (1986). The influence of NaCl on Δ1-pyrroline-5-carboxylate reductase in proline-accumulating cell suspension cultures of Mesembryanthemum nodiflorum and other halophytes. Physiol. Plant. 67: 173-181. Turk, S.C.H.J., Melchers, L.S., Dulk-Ras, H., Regensburg-Tuïnk, A.J.G., and Hooykaas, P.J.J. (1991). Environmental conditions differentially affect vir gene induction in different Agrobacterium strains. Role of the VirA sensor protein. Plant Mol. Biol. 16: 1051-1059. Tzfira, T., and Citovsky, V. (2006). Agrobacterium-mediated genetic transformation of plants: biology and biotechnology. Curr. Opin. Biotechnol. 17: 147-154. Tzfira, T., Li, J., Lacroix, B., and Citovsky, V. (2004). Agrobacterium T-DNA integration: molecules and models. Trends Genet. 20: 375-383. Uchida, A., Nagamiya, K., and Takabe, T. (2003). Transformation of Atriplex gmelini plants from callus lines using Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult. 75: 151-157. Van Wert, S.L., and Saunders, J.A. (1992). Reduction of nuclease activity released from germinating pollen under conditions used for pollen electrotransformation. Plant Sci. 84: 11-16. Vancanneyt, G., Schmidt, R., O''Connor-Sanchez, A., Willmitzer, L., and Rocha-Sosa, M. (1990). Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol. Gen. Genet. 220: 245-250. Veluthambi, K., Gupta, A.K., and Sharma, A. (2003). The current status of plant transformation technologies. Curr. Sci. 84: 368-380. Vera-Estrella, R., Barkla, B.J., Bohnert, H.J., and Pantoja, O. (1999). Salt stress in Mesembryanthemum crystallinum L. cell suspensions activates adaptive mechanisms similar to those observed in the whole plant. Planta 207: 426-435. Vergauwe, A., Van Geldre, E., Inzé, D., Van Montagu, M., and Van den Eeckhout, E. (1998). Factors influencing Agrobacterium tumefaciens-mediated transformation of Artemisia annua L. Plant Cell Rep. 18: 105-110. Weigel, D., and Glazebrook, J. (2002). Arabidopsis: A Laboratory Manual. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press). Yen, H.E., Wu, S.-M., Hung, Y.-H., and Yen, S.-K. (2000). Isolation of 3 salt-induced low-abundance cDNAs from light-grown callus of Mesembryanthemum crystallinum by suppression subtractive hybridization. Physiol. Plant. 110: 402-409. Zambryski, P., Joos, H., Genetello, C., Leemans, J., Van Montagu, M., and Schell, J. (1983). Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity. EMBO J. 2: 2143-2150. Zhao, S.-Z., Ruan, Y., Sun, H.-Z., and Wang, B.-S. (2008). Highly efficient Agrobacterium-based transformation system for callus cells of the C3 halophyte Suaeda salsa. Acta Physiol. Plant. 30: 729-736. Zuo, J., Niu, Q.-W., and Chua, N.-H. (2000). An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J. 24: 265-273.
摘要: 
冰花(Mesembryanthemum crystallinum L.)為耐鹽模式植物,利用分生與生化的策略,已鑑定出許多耐鹽相關基因,但因冰花的轉殖與再生不易,使得目前尚無高效率的轉殖系統,故目前冰花耐鹽基因的功能性分析,不易在冰花體內進行須利用其它模式植物,因此本論文利用農桿菌轉殖法,嘗試建立冰花的穩定轉殖系統。依先前建立的冰花培養細胞轉殖條件,使用β-glucuronidase (GUS)報導基因,進行農桿菌轉殖冰花培養細胞系統的改良。欲加速轉殖冰花培養細胞的增殖,將農桿菌與冰花培養細胞於全光照下共培養;而為了降低實驗成本,在去除農桿菌過程中以無菌水進行清洗,僅在最後一次時加入timentin,結果轉殖的冰花培養細胞經過一年多的繼代培養,仍能表現GUS基因,顯示T-DNA可穩定的存在冰花的基因體中。進一步嘗試建立冰花植株的轉殖系統,以完整或不同部位的冰花小苗進行感染,結果以3天大剪去根尖的冰花小苗轉殖率最高為90%,GUS染色多分布於子葉和下胚軸。由於GUS染色無法觀測活細胞的動態變化,進一步選用黃色螢光蛋白(yellow fluorescence protein, YFP)報導基因,進行冰花轉殖並觀察螢光表現,結果顯示感染3天的培養細胞無法分辨YFP所釋放的螢光或是冰花細胞的自體螢光,持續將轉殖的培養細胞進行篩選培養,至長出新的癒傷組織,首先確認轉殖培養細胞的基因體DNA含有YFP基因,進一步觀察螢光的釋放,發現轉殖YFP培養細胞的細胞核、細胞質、細胞膜和細胞壁皆有黃色螢光分布,且在細胞質、細胞膜和細胞壁上有明顯的黃色亮點,顯示轉殖冰花培養細胞能穩定表現YFP基因。為了以冰花進行基因功能分析,嘗試將冰花培養細胞轉入逆境相關蛋白激酶sucrose non-fermenting 1 (SNF1)基因,並以誘導型啟動子驅動,由於轉殖的培養細胞生長緩慢,目前細胞量不足進行大規模耐鹽性測試。預備實驗則是進行未轉殖冰花培養細胞生長的耐鹽性測試,結果發現在適當的鹽分(100 mM NaCl)環境中,冰花培養細胞的生長狀況良好,而200 mM以上的鹽濃度會延緩冰花培養細胞的生長速度。綜合以上結果得知,利用農桿菌轉殖法,可成功地穩定轉殖冰花培養細胞和小苗,未來可利用冰花轉殖株,進行耐鹽基因功能的分析,以進一步了解高等植物的耐鹽機制。

Ice plant (Mesembryanthemum crystallinum L.) is a model plant for halophytes. Many salt tolerant-related genes have been identified using molecular and biochemical approaches. Because ice plant is difficult to transform and regenerate, it lacks efficient transformation system and needs to use other model plants to analyze salt tolerant-related genes. In this thesis, I used Agrobacterium-mediated transformation to establish a stable transformation system of ice plant. In order to refine Agrobacterium-mediated stable transformation system in cultured ice plant cells, I used β-glucuronidase (GUS) reporter system previously established in the lab. For increasing the growth of transformed cultured cells, Agrobacterium and cultured ice plant cells were co-cultured under continuous light. For reducing the cost of antibiotics, Agrobacterium was washed with sterile water, and timentin was added in the final round of washing. As a result, transformed cultured cells continued to express GUS gene after being subcultured over one year indicating that T-DNA was stably integrated into ice plant genome. Furthermore, I established an Agrobacterium-mediated stable transformation system in seedlings of ice plant using intact or parts of seedlings for infection. Three-day-old cut root tip seedlings of ice plant yielded the best transformation rate of 90% with GUS staining distributed in cotyledon and hypocotyl. Yellow fluorescence protein (YFP) reporter gene was transformed into ice plant to observe fluorescence in lived cells. The result revealed that YFP fluorescence and autofluorescence of ice plant were hard to distinguish in cells infected with Agrobacterium for 3 days. Newly emerged callus transformed with YFP was obtained and the integration of YFP gene into ice plant genome was confirmed. When these cells were used to observe the fluorescence, yellow fluorescence was detected in the nucleus, cytosol, cell membrane and cell wall of YFP-transformed cultured cells with punctate spots found in cytosol, cell membrane and cell wall. The result showed that cultured ice plant cells were able to express YFP gene stably. Furthermore, in order to establish a system to study gene function in ice plant, a stress-related protein kinase sucrose non-fermenting 1 (SNF1) gene were transformed into cultured ice plant cells under the control of an inducing promoter. Because transformed cultured cells grew slowly, it was unable to perform a large-scaled salt tolerance test due to insufficient amount of cells on hand. The preliminary experiment for salt tolerance assay was done in non-transformed cultured cells. Cultured cells grew well in 100 mM NaCl condition, and the growth of cultured cells started to decrease in 200 mM NaCl or higher. In conclusion, I have successfully established an Agrobacterium-mediated transformation procedure for cultured cells and intact plants in ice plant. This is one step towards making transgenic ice plant for analyses the functions of salt-tolerance genes.
URI: http://hdl.handle.net/11455/20244
其他識別: U0005-2108201215293400
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