Please use this identifier to cite or link to this item:
標題: 利用農桿菌轉殖冰花培養細胞系統之建立
Establishment of Agrobacterium-mediated transformation system in Mesembryanthemum crystallinum L.
作者: 陳曉慧
Chen, Hsiao-Huei
關鍵字: 冰花;Agrobacterium-mediated transformation;農桿菌轉殖;Mesembryanthemum crystallinum L.
出版社: 生命科學系所
引用: 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. 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. 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. 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. Plantarum 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. Belarmino, M.M., and Mii, M. (2000). Agrobacterium-mediated genetic transformation of a phalaenopsis orchid. Plant Cell Rep. 19: 435-442. Bohnert, H.J., and Cushman, J.C. (2000). The ice plant cometh: Lessons in abiotic stress tolerance. J. Plant Growth Regul. 19: 334-346. 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. Chakrabarty, R., Viswakarma, N., Bhat, S., Kirti, P., Singh, B., and Chopra, V. (2002). Agrobacterium-mediated transformation of cauliflower: Optimization of protocol and development of Bt-transformed cauliflower. J. Biosciences 27: 495-502. Chaudhury, D., Madanpotra, S., Jaiwal, R., Saini, R., Kumar, P.A., and Jaiwal, P.K. (2007). Agrobacterium tumefaciens-mediated high frequency genetic transformation of an Indian cowpea (Vigna unguiculata L. Walp.) cultivar and transmission of transgenes into progeny. Plant Sci. 172: 692-700. Cheng, M., Lowe, B.A., Spencer, T.M., Ye, X.D., and Armstrong, C.L. (2004). Factors influencing Agrobacterium-mediated transformation of monocotyledonous species. In Vitro Cell Dev. Bio. plant 40: 31-45. Chia, T.F., Chan, Y.S., and Chua, N.H. (1994). The firefly luciferase gene as a noninvasive reporter for dendrobium transformation. Plant J. 6: 441-446 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.M., Akella, V., and Lurquin, P.F. (1995). Electroporation-mediated gene transfer into intact nodal meristems in planta. Generating transformed 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., 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). Transformed maize plants by tissue electroporation. Plant Cell 4: 1495-1505. Depicker, A., Herman, L., Jacobs, A., Schell, J., and Montagu, M. (1985). Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium/plant cell interaction. Mol. Gen. Genet. 201: 477-484. Ebinuma, H., Sugita, K., Matsunaga, S., Endo, S., Yamada, K., and Komamine, A. (2001). Systems for the removal of a selection marker and their combination with a positive marker. Plant Cell Rep. 20: 383-392. Ellul, P., Garcia-Sogo, B., Pineda, B., Rios, G., Roig, L., and Moreno, V. (2003). The ploidy level of transformed plants in Agrobacterium-mediated transformation of tomato cotyledons (Lycopersicon esculentum L.Mill.) is genotype and procedure dependent. Theor. Appl. Gent. 106: 231-238. 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. Fromm, M., Taylor, L.P., and Walbot, V. (1985). Expression of genes transferred into monocot and dicot plant-cells by electroporation. Proc. Natl. Acad. Sci. USA 82: 5824-5828. Garabagi, F., and Strommer, J. (2000). Green fluorescent protein as an all-purpose reporter in Petunia. Plant Mol. Biol. Rep. 18: 219-226. Gao, Z.S., Xie, X.J., Ling, Y., Muthukrishnan, S., and Liang, G.H. (2005). Agrobacterium tumefaciens-mediated sorghum transformation using a mannose selection system. Plant Biotechnol J. 3: 591-599. Gelvin, S.B. (2000). Agrobacterium and plant genes involved in T-DNA transfer and integration. Annu. Rev. Plant Phys. 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, table of contents. Hanson, M.R., and Kohler, R.H. (2001). GFP imaging: methodology and application to investigate cellular compartmentation in plants. J. Exp. Bot. 52: 529-539. Hellens, R., Mullineaux, P., and Klee, H. (2000). Technical Focus: 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. Hood, E.E., Gelvin, S.B., Melchers, L.S., and Hoekema, A. (1993). New Agrobacterium helper plasmids for gene transfer to plants. Transformed Res. 2: 208-218. 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. 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 Tiss. Organ Cult. 57: 61-63. Kieran, P.M., MacLoughlin, P.F., and Malone, D.M. (1997). Plant cell suspension cultures: some engineering considerations. J. Biotechnol. 59: 39-52. 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. 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. Kofer, W., Eibl, C., Steinmuller, 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. 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. Lubeck, M., Knudsen, I.M.B., Jensen, B., Thrane, U., Janvier, C., and Jensen, D.F. (2002). GUS and GFP transformation of the biocontrol strain Clonostachys rosea IK726 and the use of these marker genes in ecological studies. Mycol. Res. 106: 815-826. Lopez, M., Humara, J., Rodriguez, R., and Ordas, R. (2000). Factors involved in Agrobacterium tumefaciens-mediated gene transfer into Pinus nigra Arn. ssp. salzmannii (Dunal) Franco. Euphytica 114: 195-203. Lazzeri, P.A., Brettschneider, R., Luhrs, R., and Lorz, 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. Lichtenthaler, K.H., and Schweiger, J. (1998). Cell wall bound ferulic acid, the major substance of the blue-green fluorescence emission of plants. J. Plant Physiol. 152: 272-282 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 Tiss. Organ Cult. 97: 225-235. 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. 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 Mohamed, S., Boehm, R., and Schnabl, H. (2006). Stable genetic transformation of high oleic Helianthus annuus L. genotypes with high efficiency. Plant Sci. 171: 546-554. Newell, C.A. (2000). Plant transformation technology - Developments and applications. Mol. Biotechnol. 16: 53-65. Opabode, J.T. (2006). Agrobacterium-mediated transformation of plants: emerging factors that influence efficiency. Biotechnol. Mol. Biol. Rev. 1: 12-20. Qiu, D., Diretto, G., Tavarza, R., and Giuliano, G. (2007). Improved protocol for Agrobacterium-mediated transformation of tomato and production of transformed plants containing carotenoid biosynthetic gene CsZCD. Sci. Hortic-Amsterdam 112: 172-175. Ritala, A., Aspegren, K., Kurten, U., Salmenkallio-Marttila, M., Mannonen, L., Hannus, R., Kauppinen, V., Teeri, T.H., and Enari, T.-M. (1994). Fertile transformed barley by particle bombardment of immature embryos. Plant Mol. Biol. 24: 317-325. Salas, Park, Srivatanakul, and Smith. (2001). Temperature influence on stable T-DNA integration in plant cells. Plant Cell Rep. 20: 701-705. Stewart, C.N., Jr. (2001). The utility of green fluorescent protein in transformed plants. Plant Cell Rep. 20: 376-382. Stewart, C., Millwood, R., Halfhill, M., Ayalew, M., Cardoza, V., Kooshki, M., Capelle, G., Kyle, K., Piaseki, D., McCrum, G., and Benedetto, J. (2005). Laser-induced fluorescence imaging and spectroscopy of GFP transgenic plants. J. Fluoresc. 15: 697-705. Subramaniam, S., and Rahman, A.Z. (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. Tang, W. (2001). Agrobacterium-mediated transformation and assessment of factors influencing transgene expression in loblolly pine (Pinus taeda L.). Cell Res. 11: 237-243. Treichel, S. (1986). The influence of NaCl on Δ-pyrroline-5-carboxylate reductase in proline-accumulating cell suspension cultures of Mesembryanthemum nodiflorum and other halophytes. Plant Physiol. 67: 173-181. Turk, S.C.H.J., Melchers, L.S., Dendulkras, H., Regensburgtuink, A.J.G., and Tzfira, T., and Citovsky, V. (2006). Agrobacterium-mediated genetic transformation of plants: biology and biotechnology. Curr. Opin. Biotech. 17: 147-154. Tzfira, T., Li, J.X., 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 Tiss. Organ Cult. 75: 151-157. Vancanneyt, G., Schmidt, R., Oconnorsanchez, A., Willmitzer, L., and Rochasosa, M. (1990). Construction of an intron-containing marker gene - splicing of the intron in transformed plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol. Gen. Genet. 220: 245-250. Velcheva, M., Faltin, Z., Flaishman, M., Eshdatb, Y., and Perl, A. (2005). A liquid culture system for Agrobacterium-mediated transformation of tomato (Lycopersicon esculentum L. Mill.). Plant Sci. 168: 121-130. Veluthambi, K. (2003). The current status of plant transformation technologies. Curr. Sci. India 84: 368-378. 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., Inze, 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. Walter, M., Chaban, C., Schutze, K., Batistic, O., Weckermann, K., Nake, C., Blazevic, D., Grefen, C., Schumacher, K., Oecking, C., Harter, K., and Kudla, J. (2004). Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J. 40: 428-438. Yong, W.T.L., Abdullah, J.O., and Mahmood, M. (2006). Optimization of Agrobacterium-mediated transformation parameters for Melastomataceae spp. using green fluorescent protein (GFP) as a reporter. Sci. Hortic-Amsterdam 109: 78-85. Zambryski, P., Joos, H., Genetello, C., Leemans, J., Montagu, M.V., 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.
為了測得冰花培養細胞轉殖之最佳條件,使用農桿菌菌系EHA105內含GUS (β-glucuronidase)報導基因與nptII (neomycin phosphotransferase II)基因之pBISN1質體感染冰花培養細胞,透過PCR與GUS組織化學染色法分析轉殖細胞。分別選用農桿菌濃度為1.25 x109、2.5x 109或5 x 109 cells mL-1和共培養24或48小時進行測試,發現農桿菌濃度2.5x 109 cells mL-1和共培養48小時可得2.4%的轉殖率。進而測試冰花培養細胞生長階段的影響,以生長5天大的培養細胞可獲得轉殖效率3%最高。且以懸浮培養的方式增加篩選轉殖冰花的能力,使原本只有3%的轉殖細胞提升為70%,並以PCR的方式證實轉基因成功插入基因組,說明此農桿菌系統可使GUS報導基因在冰花細胞內穩定表現。再利用農桿菌GV3101體內含有GFP (green fluorescence protein)報導基因與hpt (hygromycin phosphotransferase)篩選基因之pCAMBIA1302質體感染冰花培養細胞,以PCR方式確認GFP報導基因已插入植物基因組,進一步利用免疫標定方式偵測GFP蛋白的累積,證實GFP可累積在冰花細胞內。當以共軛焦螢光顯微鏡觀察轉殖GFP細胞,發現其螢光散發量微弱,與未轉殖細胞無法區分,故使用共軛焦螢光顯微鏡之全波段掃描技術,發現冰花原生質體的細胞膜與細胞質自體螢光波段分佈在520-540 nm,說明冰花細胞自體螢光與GFP綠色螢光之emisson重疊。為了證實已轉殖之冰花細胞可重覆進行農桿菌轉殖,將已轉殖的冰花利用農桿菌再次感染,以PCR的方式得知二次轉殖細胞同時有GUS和GFP報導基因存在,且觀察GUS染色與GFP綠色螢光,可見細胞質區域有亮點的分布,計算二次轉殖效率為11%,表示冰花細胞可以進行二次轉殖。

Halophyte Mesembryanthemum crystallinum L. (ice plant) is used as a model to study the physiology of osmotic-related abiotic stresses. The molecular studies are hampered by lack of efficient transformation and regeneration procedures in ice plant. To develop an efficient protocol for Agrobacterium-mediated transformation in cultured ice plant cells, the effects of three factors were examined. Factors which significantly affect the transformation frequency were the concentrations of Agrobacteria, the durations of co-culture time, and the growth stages of cells.
To determine the suitable transformation conditions of cultured ice plant cells, Agrobacterium tumefaciens strain EHA105 was used. The EHA105 harbors the pBISN1 plasmid containing a GUS (β-glucuronidase) reporter gene and an nptII (neomycin phosphotransferase II) gene. Cultured ice plant cells were incubated with Agrobacterium concentration of 2.5×109 cells mL-1 and co-cultured for 48 hours, β-glucuronidase assays showed transformation efficiency of 2.4%. As for the growth stages of cultured cells, 5-day-old cultured cells had the highest transformation efficiency (3%). Transformed cells were transferred into liquid culture media to increase the antibiotic selection efficiency and the percentage of transformed cells increased dramatically from 3% to 70%. GUS gene integration was confirmed by polymerase chain reaction (PCR). This result showed that the GUS reporter gene was stably integrated into ice plant genome. Another Argobacterium tumefaciens strain GV3101 harboring pCAMBIA1302 plasmid was also used to confirm the transformation procedures. The pCAMBIA1302 contains a GFP (green fluorescence protein) reporter gene and an hpt (hygromycin phosphotransferase) selection marker gene. The integration of GFP gene into ice plant genome was confirmed by PCR, and accumulation of GFP protein was detected by immunolabeling. Although the accumulation of GFP was found in transformed ice plant cells, the emission of green fluorescence was weak, indistinguishable from the untransformed cells. It is due to the strong autofluorescence emitted from cultured cells. The autofluorescence of cultured ice plant cells was still strong even when cell wall has been removed. Wavelength scanning of fluorescence emitted from plasma membrane and cytoplasm revealed that the wavelengths of autofluorescence and GFP emission were overlapped. Cultured ice plant cells were further tested whether cells can be transformed repeatedly. The double-transformed cultured cells containing both GUS and GFP reporter genes were confirmed by genomic PCR. Blue GUS staining and punctuate yellow-green GFP fluorescence were overlapped in cytosol, and the double transformation efficiency was estimated as 11%. The result indicated that cultured ice plant cells can be transformed repeatedly.
Based on the abovementioned results, I successfully established an efficient Agrobacterium-mediated transformation procedure for cultured ice plant cells, and also confirmed the cultured cells can be repeatedly transformed by Agrobacterium. The developed Agrobacterium-mediated transformation protocol will be helpful for exploration of the molecular mechanism of salt tolerance in ice plant.
其他識別: U0005-1401201115073300
Appears in Collections:生命科學系所

Show full item record

Google ScholarTM


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