Skip to main content
SpringerLink
Log in

An alternative method for the genetic transformation of sweet organce

  • Published:
In Vitro Cellular & Developmental Biology - Plant Aims and scope Submit manuscript

Summary

An alternative method for transforming sweet organe [Citrus sinensis (L.) Osbeck] has been developed. Plasmid DNA encoding the non-destructive selectable marker enhanced green fluorescent protein gene was introduced using polyethylene glycol into protoplasts of ‘Itaborai’ sweet organe isolated from an embryogenic nucellar-derived suspension culture. Following protoplast culture in liquid medium and transfer to solid medium, transformed calluses were identified via expression of the green fluorescent protein, physically separated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. Transgenic plantlets were recovered from germinating somatic embryos and by in vitro rooting of shoots. To expedite transgenic plant recovery, regenerated shoots were also micrografted onto sour orange seedling rootstocks. Presence of the transgene in calluses and regenerated sweet organe plants was verified by gene amplification and Southern analyses. Potential advantages of this transformation system over the commonly used Agrobacterium methods for citrus are discussed.

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

  • Bond, J. E.; Roose, M. L. Agrobacterium-mediated transformation of the commercially important citrus cultivar Washington navel orange. Plant Cell Rep. 18:229–234; 1998.

    Article  CAS  Google Scholar 

  • Castle, W. S. Promising new selections of sweet orange cultivars. Citrus Ind. 80:24–28; 1999.

    Google Scholar 

  • Cervera, M.; Juarez, J.; Navarro, A.; Pina, J. A.; Duran-Vila, N.; Navarro, L.; Pena, L. Genetic transformation and regeneration of mature tissues of woody fruit plants bypassing the juvenile stage. Transgen. Res. 7:51–59; 1998.

    Article  CAS  Google Scholar 

  • Datla, R. S. S.; Bekkaoui, F.; Hammerlindl, J. K.; Pilate, G.; Dunstan, D. I.; Crosby, W. L. Improved high-level constitutive foreign gene expression in plants using an AMV RNA4 untranslated leader sequence. Plant Sci. 94:139–149; 1993.

    Article  CAS  Google Scholar 

  • Dellaporta, S. I.; Wood, J.; Hicks, J. B. A plant DNA minipreparation: version II. Plant Mol. Biol. Rep. 1:19–21; 1983.

    CAS  Google Scholar 

  • Deng, X. X.; Grosser, J. W.; Gmitter, F. G., Jr. Intergeneric somatic hybrid plants from protoplast fusion of Fortunella crassifolia cultivar Meiwa with Citrus sinensis cultivar Valencia. Scientia Hort. 49:55–62; 1992.

    Article  Google Scholar 

  • Ghorbel, R.; Juarez, J.; Navarro, L.; Pena, L. Green fluorescent protein as a valuable marker for efficient transformation and improved regeneration of recalcitrant woody fruit plants. Theor. Appl. Genet. 99:350–358; 1999.

    Article  Google Scholar 

  • Grosser, J. W. In vitro culture of tropical fruits. In: Vasil, I. K.; Thorpe, T. A., eds. Plant cell and tissue culture, 20. Dordrecht: Kluwer Academic; 1994; 475–496.

    Google Scholar 

  • Grosser, J. W.; Gmitter, F. G., Jr. Protoplast fusion and citrus improvement. Plant Breed. Rev. 8:339–374; 1990.

    Google Scholar 

  • Grosser, J. W.; Gmitter, F. G., Jr.; Chandler, J. L. Somaclonal variation in sweet orange: a tool for cultivar improvement. Proc. Int. Soc. Citriculture 1:203–206; 1996a.

    Google Scholar 

  • Grosser, J. W.; Gmitter, F. G., Jr.; Tusa, N.; Recupero, G. R.; Cucinotta, P. Further evidence of a cybridization requirement for plant regeneration from citrus leaf protoplasts. Plant Cell Rep. 15:672–676; 1996c.

    Article  CAS  Google Scholar 

  • Grosser, J. W.; Jiang, J.; Louzada, E. S.; Chandler, J. L.; Gmitter, F. G., Jr. Somatic hybridization, an integral component of citrus cultivar improvement: II. Rootstock improvement. HortSci. 33:1060–1061; 1998b.

    Google Scholar 

  • Grosser, J. W.; Jiang, J.; Mourao-Fo, F. A. A.; Louzada, E. S.; Baergen, K.; Chandler, J. L.; Gmitter, F. G., Jr. Somatic hybridization, an integral component of citrus cultivar improvement: I. Scion improvement. HortSci. 33:1057–1059; 1998a.

    Google Scholar 

  • Grosser, J. W.; Mourao-Fo, F. A. A.; Gmitter, F. G., Jr.; Louzada, E. S.; Jiang, J.; Baergen, K.; Quiros, A.; Cabasson, C.; Schell, J. L.; Chandler, J. L. Allotetraploid hybrids between Citrus and seven related genera produced by somatic hybridization. Theor. Appl. Genet. 92:557–582; 1996b.

    Google Scholar 

  • Gutierrez, E. M. A.; Luth, D.; Moore, G. A. Factors affecting Agrobacterium-mediated transformation in citrus and production of sour orange (Citrus aurantium L.) plants expressing the coat protein gene of the citrus tristeza virus. Plant Cell Rep. 16:745–753; 1997.

    Article  Google Scholar 

  • Haas, J.; Park, E. C.; Seed, B. Codon usage limitation in the expression of HIV-1 envelope glyocoprotein. Curr. Biol. 6:315–324; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Hidaka, T.; Omura, M. Transformation of citrus protoplasts by electoporation. J. Jpn Soc. Hort. Sci. 62:371–376; 1993.

    Article  CAS  Google Scholar 

  • Hidaka, T.; Omura, M.; Ugaki, M.; Tomiyama, M.; Kato, A.; Oshima, M.; Motoyoshi, F. Agrobacterium-mediated transformation and regeneration of Citrus spp. from suspension cells. Jpn. J. Breed. 40:199–207; 1990.

    Google Scholar 

  • Hodgson, R. W. Horticultural varieties of citrus. In: Reuther, W.; Webber, H. J.; Batchelor, L. D., eds. The citrus industry, 1. Berkeley: University of California Press; 1968;431–591.

    Google Scholar 

  • Kao, K. N.; Michayluk, M. R. Nutritional requirements for the growth of Vicia hajastana cells and protoplasts at a very low population density in liquid media. Planta 126:105–110; 1975.

    Article  CAS  Google Scholar 

  • Kobayashi, S.; Uchimaya, H. Expression and integration of a foreign gene in orange (Citrus sinensis Osb.) protoplasts by direct DNA transfer. Jpn J. Genet. 64:91–97; 1989.

    Google Scholar 

  • Moore, G. A.; Jacono, C. C.; Neidigh, J. L.; Lawrence, S. D.; Cline, K. Agrobacterium-mediated transformation of citrus stem segments and regeneration of transgenic plants. Plant Cell Rep. 11:238–242; 1992.

    Article  CAS  Google Scholar 

  • Murashige, T.; Tucker, D. P. H. Growth factor requirements of citrus tissue culture. Proc. First Int. Citrus Symp. 3:1155–1161; 1969.

    CAS  Google Scholar 

  • Navarro, L. Citrus shoot tip grafting in vitro. In: Bajaj, Y. P. S., ed. Biotechnology in agriculture and forestry, No. 18. High-tech and micropropagation. Berlin: Springer-Verlag; 1992;327–338.

    Google Scholar 

  • Pena, L.; Cervera, M.; Juarez, J.; Navarro, A.; Pina, J. A.; Duran-Vila, N.; Navarro, L. Agrobacterium-mediated transformation of sweet orange and regeneration of transgenic plants. Plant Cell Rep. 14:616–619; 1995b.

    Article  CAS  Google Scholar 

  • Pena, L.; Cervera, M.; Juarez, J.; Navarro, A.; Pina, J. A.; Navarro, L. Genetic transformation of lime (Citrus aurantifolia Swing.): factors affecting transformation and regeneration. Plant Cell Rep. 16:731–737; 1997.

    Article  CAS  Google Scholar 

  • Pena, L.; Cervera, M.; Juarez, J.; Ortega, C.; Pina, J. A.; Duran-Vila, N.; Navarro, L. High efficiency Agrobacterium-mediated transformation and regeneration of citrus. Plant Sci. 104:183–191; 1995a.

    Article  CAS  Google Scholar 

  • Perez, R. M.; Galiana, A. M.; Navarro, L.; Duran-Vila, N. Embryogenesis in vitro of several Citrus species and cultivars. J. Hort. Sci. Biotech. 73:796–802; 1998.

    Google Scholar 

  • Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular cloning, a laboratory manual, 2nd ed. NY: Cold Spring Harbor Laboratory Press; 1989.

    Google Scholar 

  • Vardi, A.; Bleichman, S.; Aviv, D. Genetic transformation of citrus protoplasts and regeneration of transgenic plants. Plant Sci. 69:199–206; 1990.

    Article  CAS  Google Scholar 

  • Vardi, A.; Spiegel-Roy, P.; Galun, E. Plant regeneration from Citrus protoplasts: variability in methodological requirements among cultivars and species. Theor. Appl. Genet. 62:171–176; 1982.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Citrus Research and Education Center, University of Florida, 700 Experiment Station Road, 33850, Lake Alfred, FL, USA

    G. H. Fleming, O. Olivares-Fuster, S. Fatta Del-Bosco & J. W. Grosser

Authors
  1. G. H. Fleming
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. O. Olivares-Fuster
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S. Fatta Del-Bosco
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. W. Grosser
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J. W. Grosser.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fleming, G.H., Olivares-Fuster, O., Del-Bosco, S.F. et al. An alternative method for the genetic transformation of sweet organce. In Vitro Cell.Dev.Biol.-Plant 36, 450–455 (2000). https://doi.org/10.1007/s11627-000-0081-8

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11627-000-0081-8

Key words

Search

Navigation