Skip to main content
SpringerLink
Log in

Green fluorescent protein (GFP) as a marker during pollen development

  • Published:
Transgenic Research Aims and scope Submit manuscript

Abstract

The transient expression of three mutant forms of green fluorescent protein (GFP) genes, GFP4, GFP5ER, and GFP4S65C, under several constitutive and pollen‐specific promoters throughout pollen development in Nicotianatabacum, thaliana and Antirrhinummajus is described. Immature pollen of tobacco, Arabidopsis and snapdragon, isolated at different developmental stages, were bombarded with plasmids containing the GFP and cultured in vitro for several days until maturity. The expression of GFP was monitored every day during in vitro maturation, germination and pollination, as well as after in situ pollination. The expression pattern of each construct was compared in parallel experiments to that of ß‐glucuronidase (GUS) constructs expressed by the same promoters. The results show that the expression level of all three GFP mutant forms was dependent on the strength of the promoter used. The strongest promoter was the DC3 promoter, and no notable differences in the intensity and brightness of all three versions of GFP were observed. GFP‐expressing pollen from tobacco and snapdragon developed in vitro for several days until maturity and germinated in vitro as well as on the surface of stigmata, strongly suggesting that all three GFPs are not toxic for the development of functional pollen. Furthermore, stably transformed tobacco plants expressing GFP under the control of the strong pollen‐expressed DC3 and LAT52 promoters were not impaired in reproductive function, confirming that GFP can be used as a non‐destructive marker for plant reproductive biology and development.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Baulcombe DC, Chapman S and Santa Cruz S (1995) Jellyfish green fluorescent protein as a reporter for virus infections. Plant J 7: 1045-1053.

    Article  PubMed  Google Scholar 

  • Brewbaker JL and Kwack BH (1963) The essential role of calcium ion in pollen germination and pollen tube growth. Am J Bot 50: 859-865.

    Google Scholar 

  • Chalfie M, Tu Y, Euskirchen G, Ward WW and Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263: 802-805.

    PubMed  Google Scholar 

  • Cheng L, Fu J, Tsukamoto A, Hawley RG (1996) Use of green fluorescent protein variants to monitor gene transfer and expression in mammalian cells. Nat Biotechnol 14: 606-610.

    PubMed  Google Scholar 

  • Chiu W-L, Niwa Y, Zeng W, Hirano T, Kobayashi H and Sheen J (1996) Engineered GFP as a vital reporter in plants. Curr Biol 6: 325-330.

    PubMed  Google Scholar 

  • Christensen AH and Quail PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Trans Res 5: 213-218.

    Google Scholar 

  • Crameri A, Whitehorn E, Tate E and Stemmer W (1995) Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat Biotechnol 14: 315-318.

    Google Scholar 

  • Cubitt A, Heim R, Adams S, Boyd A, Gross L and Tsien R (1995) Understanding, improving and using green fluorescent proteins. Trends Biochem Sci 20: 448-455.

    PubMed  Google Scholar 

  • Davis, SJ and Viestra, RD (1998) Soluble, highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants. Plant Mol Biol 36: 521-528.

    Article  PubMed  Google Scholar 

  • Dumas C and Knox RB (1983) Callose and determination of pistil viability and incompatibility. Theor Appl Genet 67: 1-10.

    Google Scholar 

  • Grebenok RJ, Pierson E, Lambert GM, Gong F, Afonso CL, Haldeman-Cahill R, Carrington JC and Galbraith DW (1997) Green-fluorescent protein fusions for efficient characterization of nuclear targeting. Plant J 11: 573-586.

    Article  PubMed  Google Scholar 

  • Haseloff J and Amos B (1995) GFP in plants. Trends Genet 11: 328-329.

    PubMed  Google Scholar 

  • Haseloff J, Siemering KR, Prasher DC and 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.

    Google Scholar 

  • Heim R and Tsien Y (1995) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol 6: 178-182.

    Google Scholar 

  • Heim R, Prasher DC, Tsien RY (1994) Wavelength mutations and posttranslational autooxidation of green fluorescent protein. Biochemistry 91: 12501-12504.

    Google Scholar 

  • Horsh RB, Fry JE, Hoffman NL, Eichholtz D, Rogers SG and Fraley RT (1985) A simple and general method for transferring genes to plants. Science 227: 1229-1231.

    Google Scholar 

  • Hu W and Cheng CL (1995) Expression of Aequorea green fluorescent protein in plant cells. FEBS Lett 369: 331-334.

    PubMed  Google Scholar 

  • Jefferson R, Kavanagh T and Bevan M (1987) GUS fusions: ßglucuronidase activity as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901-3907.

    PubMed  Google Scholar 

  • Keller NL and Hamilton DA (1998) Transient expression of the green fluorescent protein in pollen. Sex Plant Reprod 11: 163-165.

    Google Scholar 

  • Kohler R, Cao J, Zipfel W, Webb W and Hanson MR (1997a) Exchange of protein molecules through connections between higher plant plastids. Science 276: 2039-2042.

    Article  PubMed  Google Scholar 

  • Kohler RH, Zipfel WR, Webb WW and Hanson MR (1997b) The green fluorescent protein as a marker to visualize plant mitochondria in vivo. Plant J 11: 613-621.

    Article  PubMed  Google Scholar 

  • Leffel SM, Marbon SA and Neal CS Jr (1997) Applications of green fluorescent protein in plants. BioTechniques 23: 912-918.

    PubMed  Google Scholar 

  • Mascarenhas JP (1990) Gene activity during pollen development. Annu Rev Plant Physiol Plant Mol Biol 41: 317-338.

    Article  Google Scholar 

  • Murashige T and Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473-497.

    Google Scholar 

  • Oparka KJ, Roberts AG, SantaCruz S, Boevink P, Prior D and Smallcombe A (1997) Using GFP to study virus invasion and spread in plant tissues. Nature 388: 401-402.

    Google Scholar 

  • Pang SZ, DeBoer DL, Wan Y, Guangning Y, Layton JG, Neher MK, Armstrong CL, Fry JE, Hinchee MAW and Fromm ME (1996) An improved green fluorescent protein gene as a vital marker in plants. Plant Physiol 112: 893-900.

    Article  PubMed  Google Scholar 

  • Park S-H and Raines RT (1997) Green fluorescent protein as a signal for protein-protein interactions. Protein Science 6: 2344-2349.

    PubMed  Google Scholar 

  • Pines JN (1995) Using GFP in mammalian cells. Trends Genet 11: 326-327.

    PubMed  Google Scholar 

  • Prasher DC (1995) Using GFP to see the light. Trends Genet 11: 320-323.

    Article  PubMed  Google Scholar 

  • Prasher DC, Eckenrode VK, Ward WW, Prendergast FG and Cormier MJ (1992) Primary structure of the Aequorea victoria green fluorescent protein. Gene 111: 229-233.

    Article  PubMed  Google Scholar 

  • Presley JF, Cole NB, Schroer TA, Hirschberg K, Zaal KJM and Lippincott-Schwartz J (1997) ER-to-Golgi transport visualized in living cells. Nature 389: 81-85.

    PubMed  Google Scholar 

  • Reichel C, Mathur J, Eckes P, Langenkemper K, Koncz C, Schell J, Reiss B and Maas Ch (1996) Enhanced green fluorescence by the expression of an Aequorea victoria green fluorescent protein mutant in mono-and dicotyledonous plant cells. Proc Natl Acad Sci USA 93: 5888-5893.

    Google Scholar 

  • Roberts MR, Foster GD, Blundell RP, Robinson S, Kumar A, Draper J and Scott R (1993) Gametophytic and sporophytic expression of an anther-specific Arabidopsis thaliana gene. Plant J 3: 111-120.

    Article  PubMed  Google Scholar 

  • Rouwendahl GJA, Mendes O, Wolbert EJH and de Boer DA (1997) Enhanced expression in tobacco of the gene encoding green fluorescent protein by modification of its codon usage. Plant Mol Biol 33: 989-999.

    PubMed  Google Scholar 

  • Sambrook J, Fritch EF and Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Lab. Press, 2nd edition, Cold Spring Harbor, NY, USA.

  • Sanford JC, Smith FD and Russell JA (1993) Optimizing the biolistic process for different biological applications. Methods Enzym 27: 483-509.

    Google Scholar 

  • Seffens WS, Almoguera C, Wilde DH, Vonder Haar RA and Thomas TL (1990) Molecular analysis of a phylogenetically conserved carrot gene: developmental and environmental regulation. Dev Genet 11: 65-76.

    PubMed  Google Scholar 

  • Sheen J, Hwang S, Niwa Y, Kobayashi H and Galbraith DW (1995) Green fluorescent protein as a new vital marker in plant cells. Plant J 8: 777-784

    Article  PubMed  Google Scholar 

  • Shivanna KR and Johri BM (1985) The Angiosperm Pollen: Structure and Function. Wiley Eastern Ltd, New Delhi.

    Google Scholar 

  • Siemering K, Golbik R, Sever R and Haseloff J (1996) Mutations that suppress the thermosensitivity of green fluorescent protein. Curr Biol 6: 1653-1663.

    PubMed  Google Scholar 

  • Stauber RH, Horie K, Carney P, Hudson EA, Tarasova NI, Gaitanaris GA and Pavlakis GN (1998) Development and applications of enhanced green fluorescent protein mutants. Biotechniques 24: 462-471.

    PubMed  Google Scholar 

  • Stöger E, Benito Moreno R-M, Ylstra B, Vicente O and Heberle-Bors E (1992) Comparison of different techniques for gene transfer into mature and immature tobacco pollen. Trans Res 1: 71-78.

    Google Scholar 

  • Takada T, Iida K, Awaji T, Itoh K, Takahashi R, Shibui A, Yoshida K, Sugano S and Tsujimoto G (1997) Selective production of transgenic mice using green fluorescent protein as a marker. Nat Biotechnol 15: 458-461.

    PubMed  Google Scholar 

  • Tian L, Sequin A and Charest PC (1997) Expression of the green fluorescent protein gene in conifer tissues. Plant Cell Rep 16: 267-266.

    Google Scholar 

  • Touraev A, Fink CS, Stöger E and Heberle-Bors E (1995) Pollen selection: a transgenic reconstruction approach. Proc Natl Acad Sci USA 92: 12165-12169.

    PubMed  Google Scholar 

  • Touraev A, Stöger E, Voronin V and Heberle-Bors E (1997) Plant male germ line transformation. Plant J 12: 949-956.

    Google Scholar 

  • Twell D (1994) The diversity and regulation of gene expression in the pathway of male gametophyte development. In: Scott RJ and Stead AD (eds) Molecular and Cellular Aspects of Plant Reproduction. Seminar Series 55, pp. 83-135. Cambridge University Press, Cambridge.

    Google Scholar 

  • Twell D, Yamaguchi J and Mc Cormick S (1990) Pollen specific gene expression in transgenic plants: coordinate regulation of two different tomato gene promoters during microsporogenesis. Development 109: 705-713.

    PubMed  Google Scholar 

  • Vain P, Worland B, Kohli A, Snape JW and Christou P (1998) The green fluorescent protein (GFP) as a vital screenable marker in rice transformation. Theor Appl Genet 96: 164-169.

    Google Scholar 

  • Wang J, Shi HZ, Zhou C, Yang HY, Zhang XL and Zhang RD (1998) beta-glucuronidase gene and green fluorescent protein gene expression in de-exined pollen of Nicotiana tabacum by microprojectile bombardment. Sex Plant Reprod 11: 159-162.

    Google Scholar 

  • Wang S and Hazelrigg T (1994) Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis. Science 369: 400-403.

    Google Scholar 

  • Yang TT, Cheng LZ and Kain SR (1996) Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. Nucl Acids Res 24: 4592-4593.

    PubMed  Google Scholar 

  • Yeh E, Gustafson K and Boulianne G (1995) Green fluorescent protein as a vital marker and reporter of gene expression in Drosophila. Genetics 92: 7036-7040.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Vienna Biocenter, Institute of Microbiology & Genetics, Vienna University, Dr. Bohrgasse 9, A‐1030, Vienna, Austria

    Iris Ottenschläger

  2. Institute for Genetics, University of Cologne, Weyertal 121, 50931, Cologne, Germany

    Ioulia Barinova, Viktor Voronin, Marlis Dahl, Erwin Heberle‐Bors & Alisher Touraev

Authors
  1. Iris Ottenschläger
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ioulia Barinova
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Viktor Voronin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marlis Dahl
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Erwin Heberle‐Bors
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Alisher Touraev
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ottenschläger, I., Barinova, I., Voronin, V. et al. Green fluorescent protein (GFP) as a marker during pollen development. Transgenic Res 8, 279–294 (1999). https://doi.org/10.1023/A:1008938728051

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1008938728051

Search

Navigation