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Volume 65, Issue 4


Green fluorescent protein as a visual selection marker for coffee transformation

Manoj Mishra
  • The Norman Borlaug Institute for Plant Science Research, De Montfort University, Leicester, LE1 9BH, UK
  • Central Coffee Research Institute, Coffee Research Station, Chikmagalur District, Karnataka, 577117, India
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  • Other articles by this author:
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/ Santosini Devi / Alex McCormac / Nigel Scott
  • The Norman Borlaug Institute for Plant Science Research, De Montfort University, Leicester, LE1 9BH, UK
  • The Biomolecular Technology Group, Faculty of Health and Life Sciences, De Montfort University, Leicester, LE1 9BH, UK
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/ DongFang Chen / Malcolm Elliott / Adrian Slater
  • The Norman Borlaug Institute for Plant Science Research, De Montfort University, Leicester, LE1 9BH, UK
  • The Biomolecular Technology Group, Faculty of Health and Life Sciences, De Montfort University, Leicester, LE1 9BH, UK
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Published Online: 2010-06-10 | DOI: https://doi.org/10.2478/s11756-010-0078-7


The green fluorescent protein (GFP) was used as a visual selectable marker to produce transgenic coffee (Coffea canephora) plants following Agrobacterium-mediated transformation. The binary vector pBECKS 2000.7 containing synthetic gene for GFP (sgfp) S65T and the hygromycin phosphotransferase gene hph both controlled by 35S cauliflower mosaic virus CaMV35S promoters was used for transformation. Embryogenic cultures were initiated from hypocotyls and cotyledon leaves of in vitro grown seedlings and used as target material. Selection of transformed tissue was carried out using GFP visual selection as the sole screen or in combination with a low level of antibiotics (hygromycin 10 mg/L), and the efficiency was compared with antibiotics selection alone (hygromycin 30 mg/L). GFP selection reduced the time for transformed somatic embryos formation from 18 weeks on a hygromycin (30 mg/L) antibiotics containing medium to 8 weeks. Moreover, visual selection of GFP combined with low level of antibiotics selection improved the transformation efficiency and increased the number of transformed coffee plants compared to selection in the presence of antibiotics. Molecular analysis confirmed the presence of the sgfp-S65T coding region in the regenerated plants. Visual screening of transformed cells using GFP by Agrobacterium-mediated transformation techniques was found to be efficient and therefore has the potential for development of selectable marker-free transgenic coffee plants.

Keywords: coffee genetic transformation; Agrobacterium; antibiotic free; green fluorescent protein; visual selection

  • [1] Ahlandsberg S., Satish P., Sun C. & Jansson C. 1999. Green fluorescent protein as a reporter system in the transformation of barley cultivars. Physiol. Plant. 107: 194–200. http://dx.doi.org/10.1034/j.1399-3054.1999.100207.xCrossrefGoogle Scholar

  • [2] Barrueto C.L.P., Cruz A.R.R. & Castro L.H.R. 2004. Somatic embryogenesis from three coffee cultivars: ‘Rubi’, ‘Catuai Vermelho 81’, and ‘IAPAR 59’. HortScience 39: 130–131. Google Scholar

  • [3] Chiu W., Niwa Y., Zeng W. & Hirano T. 1996. Engineered GFP as a vital reporter in plants. Curr. Biol. 6: 325–330. http://dx.doi.org/10.1016/S0960-9822(02)00483-9CrossrefGoogle Scholar

  • [4] Davis A.P., Govaerts R., Bridson D.M. & Stoffelen P. 2006. An annotated taxonomic conspectus of the genus Coffea (Rubiaceae). Bot. J. Linnean Soc. 152: 465–512. http://dx.doi.org/10.1111/j.1095-8339.2006.00584.xCrossrefGoogle Scholar

  • [5] Dhekney S.A., Li Z.T., Dutt M. & Gray D.J. 2008. Agrobacterium-mediated transformation of embryogenic cultures and plant regeneration in Vitis rotundifolia Michx. (muscadine grape). Plant Cell Rep. 27: 865–872. http://dx.doi.org/10.1007/s00299-008-0512-2CrossrefGoogle Scholar

  • [6] Dominguez A., Guerri J., Cambra M., Navarro M. & Pena L. 2000. Efficient production of transgenic citrus plants expressing the coat protein gene of citrus tristeza virus. Plant Cell Rep. 19: 427–433. http://dx.doi.org/10.1007/s002990050751CrossrefGoogle Scholar

  • [7] Ebinuma H., Sugita K., Matsunaga E., Endo S., Yamada K. & Komanine A. 2001. Systems for the removal of a selection marker and their combination with a positive marker. Plant Cell Rep. 20: 383–392. http://dx.doi.org/10.1007/s002990100344CrossrefGoogle Scholar

  • [8] Eday C.C., Weld R.J. & Lister C.E. 2000. Agrobacterium tumefaciens-mediated transformation and transgenic plant regeneration of onion (Allium cepa L.). Plant Cell Rep. 19: 376–381. http://dx.doi.org/10.1007/s002990050743CrossrefGoogle Scholar

  • [9] Elliott A.R., Campbell J.A., Brettell R.I.S. & Grof C.P.L. 1998. Agrobacterium-mediated transformation of sugarcane using GFP as a screenable marker. Aust. J. Plant Physiol. 25: 739–743. http://dx.doi.org/10.1071/PP98066CrossrefGoogle Scholar

  • [10] Escobar A.M., Park J., Polito V.S., Leslie C.A., Uratsu S.L., McGranahan H.G. & Dandekar A. 2000. Using GFP as a scorable marker in walnut somatic embryo transformation. Annals Bot. 85: 831–835. http://dx.doi.org/10.1006/anbo.2000.1143CrossrefGoogle Scholar

  • [11] Ghorbel R., Juarez J., Navarro L. & Pena L. 1999. Green fluorescent protein as a screenable marker to increase the efficiency of generating transgenic woody fruit plants. Theor. Appl. Genet. 99: 350–358. http://dx.doi.org/10.1007/s001220051244CrossrefGoogle Scholar

  • [12] Haseloff J. & Siemering K.R. 1998. The uses of GFP in plants, pp. 191–220. In: Chalfie M. & Kain S.R. (eds) Green Fluorescent Protein: Properties, Applications and Protocols, Wiley, Chichester. Google Scholar

  • [13] Haseloff J., Siemering K.R., Prasher D.C. & Hodge S. 1997. Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc. Natl. Acad. Sci. USA 94: 2122–2127. http://dx.doi.org/10.1073/pnas.94.6.2122CrossrefGoogle Scholar

  • [14] Hatanaka T., Choi Y.E., Kusano T. & Sano H. 1999. Transgenic plants of coffee Coffea canephora from embryogenic callus via Agrobacterium tumefaciens mediated transformation. Plant Cell Rep. 19: 106–110. http://dx.doi.org/10.1007/s002990050719CrossrefGoogle Scholar

  • [15] Hood E.E., Gelvin S.B., Melchers L.S. & Hoekema A. 1993. New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res. 2: 208–218. http://dx.doi.org/10.1007/BF01977351CrossrefGoogle Scholar

  • [16] Hraska M., Rakousky S. & Curn V. 2006. Green fluorescence protein as a vital marker for non destructive detection of transformation events in transgenic plants. Plant Cell Tissue Organ Cult. 86: 303–318. http://dx.doi.org/10.1007/s11240-006-9131-1CrossrefGoogle Scholar

  • [17] Hraska M., Rakousky S. & Curn V. 2008. Tracking of the CaMV-35S promoter performance in GFP transgenic tobacco, with a special emphasis on flowers and reproductive organs, confirmed its predominant activity in vascular tissues. Plant Cell Tissue Organ Cult. 94: 239–251. http://dx.doi.org/10.1007/s11240-007-9312-6Web of ScienceCrossrefGoogle Scholar

  • [18] Kaeppler H.F., Menon G.K., Skadsen R.W., Nuutila A.M. & Carlson A.R. 2000. Transgenic oat plants via visual selections of cells expressing green fluorescent protein. Plant Cell Rep. 19: 661–666. http://dx.doi.org/10.1007/s002999900167CrossrefGoogle Scholar

  • [19] Leffel S.M., Mabon S.A. & Stewart C.N., Jr. 1997. Applications of green fluorescent protein in plants. Biotechniques 23: 912–918. Google Scholar

  • [20] Leroy T., Henry A.M., Royer M., Altosaar I., Frutos R., Duris D. & Philippe R. 2000. Genetically modified coffee plants expressing Bacillus thuringiensis cry1AC gene for resistance to leaf minor. Plant Cell Rep. 19: 382–389. http://dx.doi.org/10.1007/s002990050744Google Scholar

  • [21] McCormac A.C., Elliott M.C. & Chen D.F. 1998. A simple method for the production of highly competent cells for Agrobacterium for transformation via electroporation. Mol. Biotechnol. 9: 155–159. http://dx.doi.org/10.1007/BF02760816CrossrefGoogle Scholar

  • [22] McCormac A.C., Elliott M.C. & Chen D.F. 1999. pBECKS-2000: a novel plasmid for the facile creation of complex binary vectors which incorporates “clean gene” facilities. Mol. Gen. Genet. 261: 226–235. http://dx.doi.org/10.1007/s004380050961CrossrefGoogle Scholar

  • [23] Mishra M.K., Sandhyarani N., Santaram A., Sreenath H.L. & Jayarama. 2008. A simple method of DNA isolation from coffee seeds suitable for PCR analysis. Afr. J. Biotechnol. 7: 409–413. Google Scholar

  • [24] Mishra M.K. & Sreenath H.L. 2004. High-efficiency Agrobacterium-mediated transformation of coffee (Coffea canephora Pierre ex. Frohner) using hypocotyls explants, pp 792–796. In: Proceedings of the 21st International Association for Coffee Science (ASIC), Bangalore, India. Google Scholar

  • [25] Mishra M.K., Sreenath H.L., Jayarama., McCormac A.C., Devi S., Elliott M.C. & Slater A. 2009. Two critical factors: Agrobacterium strain and antibiotics selection regime improve the production of transgenic coffee plants, pp 843–850. In: Proceedings of the 22nd International Association for Coffee Science (ASIC), Campinas, Brazil. Google Scholar

  • [26] Molinier J., Himber C. & Hanhe G. 2000. Use of green fluorescent protein for detection of transformed shoots and homozygous offspring. Plant Cell Rep. 19: 219–223. http://dx.doi.org/10.1007/s002990050002CrossrefGoogle Scholar

  • [27] Murashige T. & Skoog F.A. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473–497. http://dx.doi.org/10.1111/j.1399-3054.1962.tb08052.xCrossrefGoogle Scholar

  • [28] Murray F., Brettell R., Mathews P., Bishop D. & Jacobson J. 2004. Comparison of Agrobacterium-mediated transformation of four barley cultivars using GFP and GUS reporter genes. Plant Cell Rep. 22: 397–402. http://dx.doi.org/10.1007/s00299-003-0704-8CrossrefGoogle Scholar

  • [29] Padilla I.M.G., Golis A., Gentile A., Damiano C. & Scorza R. 2006. Evaluation of transformation in peach Prunus persica explants using green fluorescent protein (GFP) and betaglucuronidase (GUS) reporter genes. Plant Cell Tissue Organ Cult. 84: 309–314. http://dx.doi.org/10.1007/s11240-005-9039-1CrossrefGoogle Scholar

  • [30] Pang S.Z., DeBoer D.L., Wan Y., Ye G., Layton J.G., Neher M.K., Armstrong C.L., Fry J.E., Hinshee M.A. & Fromm M.E. 1996. An improved green fluorescent protein gene as a vital marker in plants. Plant Physiol. 112: 893–900. http://dx.doi.org/10.1104/pp.112.3.893CrossrefGoogle Scholar

  • [31] Paz M.M., Shou H., Guo Z., Zhang Z., Banerjee A.K. & Wang K. 2004. Assessment of conditions affecting Agrobacterium-mediated soybean transformation using cotyledonary node explant. Euphytica 136: 167–179. http://dx.doi.org/10.1023/B:EUPH.0000030670.36730.a4CrossrefGoogle Scholar

  • [32] Sheen J., Hwang S., Niwa Y., Kobyashi H. & Galbraith D.W. 1995. Green fluorescent protein as a new vital marker in plant cells. Plant J. 8: 777–784. http://dx.doi.org/10.1046/j.1365-313X.1995.08050777.xCrossrefGoogle Scholar

  • [33] Siemering K.R., Golbik R., Sever R. & Haseloff J. 1996. Mutations that suppress the thermostability of green fluorescent protein. Curr. Biol. 6: 1653–1663. http://dx.doi.org/10.1016/S0960-9822(02)70789-6CrossrefGoogle Scholar

  • [34] Stewart C.N., Jr. 2001. The utility of green fluorescent protein in transgenic plants. Plant Cell Rep. 20: 376–382. http://dx.doi.org/10.1007/s002990100346CrossrefGoogle Scholar

  • [35] Tian L., Levee V., Mentag R., Charest P.J. & Seguin A. 1999. Green fluorescent protein as a tool for monitoring transgene expression in forest tree species. Tree Physiol. 19: 541–546. CrossrefGoogle Scholar

  • [36] Vain P., Worland B., Kohli A., Snape J. & Christou P. 2000. The green fluorescent protein (GFP) as a vital screenable marker in rice transformation. Theor. Appl. Genet. 96: 164–169. http://dx.doi.org/10.1007/s001220050723CrossrefGoogle Scholar

  • [37] Yancheva S.D., Shlizerman L.A., Golubowiez S., Yabloviz P.A., Hanania U. & Flaishman M.A. 2006. The use of green fluorescent protein (GFP) improves Agrobacterium-mediated transformation of ’spadona’ pear (Pyrus communis L.). Plant Cell Rep. 25: 183–189. http://dx.doi.org/10.1007/s00299-005-0025-1CrossrefGoogle Scholar

  • [38] Zhang C.L., Chen D.F., Mc Cormac A.C., Scott N.W., Elliott M.C. & Slater A. 2001. Use of the GFP reporter as a vital marker for Agrobacterium-mediated transformation of sugar beet (Beta vulgaris L). Mol. Biotechnol. 17: 109–117. http://dx.doi.org/10.1385/MB:17:2:109CrossrefGoogle Scholar

  • [39] Zhu Y.J., Agbayani R. & Moore P.H. 2004. Green fluorescent protein as a visual selection marker for papaya (Carcia papaya L) transformation. Plant Cell Rep. 22: 660–667. http://dx.doi.org/10.1007/s00299-004-0755-5CrossrefGoogle Scholar

About the article

Published Online: 2010-06-10

Published in Print: 2010-08-01

Citation Information: Biologia, Volume 65, Issue 4, Pages 639–646, ISSN (Online) 1336-9563, ISSN (Print) 0006-3088, DOI: https://doi.org/10.2478/s11756-010-0078-7.

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© 2010 Slovak Academy of Sciences. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. BY-NC-ND 3.0

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