Skip to main content
SpringerLink
Log in

Jellyfish green fluorescent protein as a useful reporter for transient expression and stable transformation in Medicago sativa L.

  • Genetic Transformation and Hybridization
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

The aim of the experiments reported herein was to transiently test different gene constructs using green fluorescent protein (GFP) as a reporter gene for a future localization of the maize β-zein in the chloroplast of alfalfa (Medicago sativa L.). The transient expression of two GFP genes was compared in alfalfa leaves to determine which of these two mutants is the easier to detect. Based on the intensity of fluorescence emitted, the GFP S65C gene was used to assemble a chloroplast-targeted GFP to verify the efficiency of the transit peptide for chloroplast targeting. A chloroplast-targeted fusion protein between β-zein and GFP was then assembled, and this protein was observed to accumulate in small aggregates into the chloroplasts of transiently transformed cells. To the best of our knowledge, this is the first report of the GFP S65C gene being used to obtain transformed alfalfa plants expressing GFP.

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

Fig. 1A–F.
Fig. 2A–H.
Fig. 3A–C.
Fig. 4A–D.
Fig. 5. A

Similar content being viewed by others

References

  • Bagga S, Adams HP, Rodriguez FD, Kemp JD, Sengupta-Gopalan C (1997) Coexpression of the maize δ−zein and β−zein genes results in stable accumulation of δ−zein in endoplasmic reticulum-derived protein bodies formed by β−zein. Plant Cell 9:1683–1696

    CAS  Google Scholar 

  • Bellucci M, Arcioni S (2002) Correct balance of the protein content in food and fodder crops through genetic engineering. In: Pandalai SG (ed) Recent research developments in plant biology. Research Signpost 2:137–146

    Google Scholar 

  • Bellucci M, Alpini A, Arcioni S (2002) Coexpression of γ-zein:KDEL and β-zein:KDEL polypeptides in tobacco leaf and zein accumulation in forage species (Lotus corniculatus and Medicago sativa). Plant Cell Rep 20:848–856

    CAS  Google Scholar 

  • Brown DCW, Atanassov A (1985) Role of genetic background in somatic embryogenesis in Medicago. Plant Cell Tissue Organ Cult 4:111–122

    Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Elliott AR, Campbell JA, Dugdale B, Brettel RIS, Grof CPL (1999) Green-fluorescent protein facilitates rapid in vivo detection of genetically transformed plant cells. Plant Cell Rep 18:707–714

    CAS  Google Scholar 

  • Ferro M, Salvi D, Riviere-Rolland H, Vermat T, Seigneurin-Berny D, Grunwald D, Garin J, Joyard J, Rolland R (2002) Integral membrane proteins of the chloroplast envelope: identification and subcellular localization of new transporters. Proc Natl Acad Sci USA 99:11487–11492

    CAS  PubMed  Google Scholar 

  • Geest AHM van der, Petolino JF (1998) Expression of a modified green fluorescent protein gene in transgenic maize plants and progeny. Plant Cell Rep 17:760–764

    Article  Google Scholar 

  • Guerineau F, Woolston S, Brooks L, Mullineaux P (1988) An expression cassette for targeting foreign proteins into chloroplasts. Nucleic Acids Res 16:11380

    CAS  PubMed  Google Scholar 

  • Harper BK, Mabon SA, Leffel SM, Halfhill MD, Richards HA, Moyer KA, Stewart CN Jr (1999) Green fluorescent protein as a marker for expression of a second gene in transgenic plants. Nat Biotechnol 17:1125–1129

    Article  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  • Haseloff J, Siemering KR (1998) The uses of GFP in plants. In: Chalfie M, Kain SR (eds) Green fluorescent protein: properties, applications, and protocols. Wiley, Chichester, pp 191–220

  • Haseloff J, Siemering KR, Prasher DC, 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

    CAS  PubMed  Google Scholar 

  • Heim R, Prasher DC, Tsien RY (1994) Wavelength mutation and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci USA 91:12501–12504

    CAS  PubMed  Google Scholar 

  • Heim R, Cubitt AB, Tsien RY (1995) Improved green fluorescence. Nature 373:663–664

    CAS  Google Scholar 

  • Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42:819–832

    Article  CAS  PubMed  Google Scholar 

  • Hibbered JM, Linley PJ, Khan MS, Gray JC (1998) Transient expression of green fluorescent protein in various plastid types following microprojectile bombardment. Plant J 16:627–632.

    Article  Google Scholar 

  • Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68

    CAS  PubMed  Google Scholar 

  • Jang IC, Nahm BH, Kim JK (1999) Subcellular targeting of green fluorescent protein to plastids in transgenic rice plants provides a high-level expression system. Mol Breed 5:453–461

    Article  CAS  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3908

    CAS  PubMed  Google Scholar 

  • Kim CS, Woo Y, Clore A, Burnett RJ, Carneiro NP, Larkins BA (2002) Zein protein interactions, rather than the asymmetric distribution of zein mRNAs on endoplasmic reticulum membranes, influence protein body formation in maize endosperm. Plant Cell 14:655–672

    CAS  PubMed  Google Scholar 

  • Köhler RH, Cao J, Zipfel WR, Webb WW, Hanson MR (1997) Exchange of protein molecules through connection between higher plant plastids. Science 276:2039–2042

    PubMed  Google Scholar 

  • Leffel SM, Mabon SA, Stewart CN Jr (1997) Green fluorescent protein as a marker for monitoring transgenic plants. In: McLean GD, Waterhouse PM, Evans G, Gibbs MJ (eds) Commercialisation of transgenic crops: risk, benefit and trade considerations. CRC for Plant Science and Bureau of Resources Sciences, Canberra, pp 193–199

  • Mercuri A, Sacchetti A, De Benedetti L, Schiva T, Alberti S (2001) Green fluorescent flowers. Plant Sci 161:961–968

    CAS  Google Scholar 

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

    CAS  Google Scholar 

  • Nitsch JP, Nitsch C (1969) Haploid plants from pollen grains. Science 163:85–87

    Google Scholar 

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

    CAS  PubMed  Google Scholar 

  • Pereira LF, Erickson L (1995) Stable transformation of alfalfa (Medicago sativa L.) by particle bombardment. Plant Cell Rep 14:290–293

    CAS  Google Scholar 

  • Ponappa T, Brzozowski AE, Finer JJ (1999) Transient expression and stable transformation of soybean using the jellyfish green fluorescent protein. Plant Cell Rep 19:6–12

    CAS  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  • Reichel C, Mathur J, Eckes P, Langenkemper K, Koncz C, Schell J, Reiss B, Maas C (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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  • Stewart CN Jr (2001) The utility of green fluorescent protein in transgenic plants. Plant Cell Rep 20:376–382

    CAS  Google Scholar 

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

    CAS  Google Scholar 

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

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Contribution no. 35 from the Institute of Plant Genetics, Research Division of Perugia, CNR. We wish to thank all members of our laboratory, particularly Dr. O. Calderini, for their stimulating comments during the experiments. We are also grateful to G. Carpinelli, F. Calderini and A. Bolletta for their technical assistance; to Ulrike Bechtold, Roger Hellens and Phil Mullineaux for providing us with the pGreen plasmid; to Dr. E.T. Bingham for providing us with seeds of the cultivar Regen SY. This work has been supported by MIUR, Project FIRB no. RBNE01TYZF.

Author information

Authors and Affiliations

  1. Institute of Plant Genetics, Research Division of Perugia, CNR, Via Madonna Alta, 130, 06128, Perugia, Italy

    M. Bellucci, F. De Marchis & S. Arcioni

  2. Department of Clinical Experimental Medicine, Section Internal Medicine and Oncology, Perugia University Medical School , Policlinico Monteluce, 06122, Perugia, Italy

    R. Mannucci

Authors
  1. M. Bellucci
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. F. De Marchis
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. R. Mannucci
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. S. Arcioni
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M. Bellucci.

Additional information

Communicated by D. Dudits

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bellucci, M., De Marchis, F., Mannucci, R. et al. Jellyfish green fluorescent protein as a useful reporter for transient expression and stable transformation in Medicago sativa L.. Plant Cell Rep 22, 328–337 (2003). https://doi.org/10.1007/s00299-003-0693-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-003-0693-7

Keywords

Search

Navigation