Skip to main content
SpringerLink
Log in

The use of the PMI/mannose selection system to recover transgenic sweet orange plants (Citrus sinensis L. Osbeck)

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

Abstract

A new method for obtaining transgenic sweet orange plants was developed in which positive selection (Positech) based on the Escherichia coli phosphomannose-isomerase (PMI) gene as the selectable marker gene and mannose as the selective agent was used. Epicotyl segments from in vitro-germinated plants of Valencia, Hamlin, Natal and Pera sweet oranges were inoculated with Agrobacterium tumefaciens EHA101-pNOV2116 and subsequently selected on medium supplemented with different concentrations of mannose or with a combination of mannose and sucrose as a carbon source. Genetic transformation was confirmed by PCR and Southern blot. The transgene expression was evaluated using a chlorophenol red assay and isoenzymes. The transformation efficiency rate ranged from 3% to 23.8%, depending on cultivar. This system provides an efficient manner for selecting transgenic sweet orange plants without using antibiotics or herbicides.

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. 1.
Fig. 2A–E.

Similar content being viewed by others

Abbreviations

BAP :

Benzylaminopurine

CPR :

Chlorophenol red

EGTA :

Ethylene glycol-0-0′- bis (2, aminoethyl) N′, N′, N′, N′ tetraacetic acid

MTT :

[3-(4,5-Dimethyl thiazol-2-YL)-2,5-diphenyl] tetrazolium bromide

PMI :

Phosphomannose isomerase (EC 5.3.1.8)

PMS :

Phenazine methosulphate

References

  • Alfenas AC, Peters I, Brune W, Passador, GC (1991) Eletroforese de proteínas e isoenzimas de fungos e essências florestais. Editora SIF, Viçosa

  • An YQ, McDowell JM, Huang S, McKinney EC, Chambliss S, Meagher RB (1996) Strong, constitutive expression of Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues. Plant J 10:107–121

    CAS  PubMed  Google Scholar 

  • Ashton GC, Braden AWH (1961) Serum β-globulin polymorphism in mice. Aust J Biol Sci 14:248–254

    CAS  Google Scholar 

  • Cervera M, Pina JA, Juárez J, Navarro L, Peña L (1998) Agrobacterium-mediated transformation of citrange: factors affecting transformation and regeneration. Plant Cell Rep 18:271–278

    CAS  Google Scholar 

  • Cervera M, Pina, JA, Juárez J, Navarro L, Peña L (2000a) A broad exploration of a transgenic population of citrus: stability of gene expression and phenotype. Theor Appl Genet 100:670–677

    CAS  Google Scholar 

  • Cervera M, Ortega C, Navarro A, Navarro L, Peña L (2000b) Generation of transgenic citrus plants with the tolerance-to-salinity gene HAL2 from yeast. J Hortic Sci Biotechnol 75:26–30

    CAS  Google Scholar 

  • Costa MGC, Otoni WC, Moore GA (2002) An evaluation of factors affecting the efficiency of Agrobacterium-mediated transformation of Citrus paradisi (Macf.) and production of transgenic plants containing carotenoid biosynthetic genes. Plant Cell Rep 21:365–373

    Article  CAS  Google Scholar 

  • Domínguez A, Guerri J, Cambra M, Navarro L, Moreno P, Peña L (2000) Efficient production of transgenic citrus plants expressing the coat protein gene of citrus tristeza virus. Plant Cell Rep 19:427–433

    CAS  Google Scholar 

  • Domínguez A, Mendoza AH, Guerri J, Cambra M, Navarro L, Moreno P, Peña L (2002a) Pathogen-derived resistance to Citrus tristeza virus (CTV) in transgenic Mexican lime [Citrus aurantifolia (Christ.) Swing.] plants expressing its p25 coat protein gene. Mol Breed 10:1–10

    Article  Google Scholar 

  • Domínguez A, Fagoaga C, Navarro L, Moreno P, Peña L (2002b) Regeneration of transgenic citrus plants under non-selective conditions results in high-frequency recovery of plants with silenced transgenes. Mol Genet Genomics 267:544–556

    Article  PubMed  Google Scholar 

  • Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15

    Google Scholar 

  • Fagoaga C, Rodrigo I, Conejero V, Hinarejos C, Tuset JJ, Arnau J, Pina JA, Navarro L, Peña L (2001) Increased tolerance to Phytophthora citrophthora in transgenic orange plantas constitutively expressing a tomato pathogenesis related protein PR-5. Mol Breed 7:175–185

    Article  CAS  Google Scholar 

  • Febres VJ, Niblett CL, Lee RF, Moore GA (2003) Characterization of grapefruit plants (Citrus paradisi Macf.) transformed with citrus tristeza closterovirus genes. Plant Cell Rep 21:421–428

    CAS  PubMed  Google Scholar 

  • Ghorbel R, Juárez J, Navarro L, Peña 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

    Article  Google Scholar 

  • Ghorbel R, López C, Fagoaga C, Moreno P, Navarro L, Flores R, Peña L (2001) Transgenic citrus plants expressing the citrus tristeza virus p23 protein exhibit viral-like symptoms. Mol Plant Pathol 2:27–36

    CAS  Google Scholar 

  • Gmitter FC, Grosser JW, Moore GA (1992) Citrus: In: Hammmerschlag FA, Litz RE (eds) Biotechnology of perennial fruit crops. CAB Int, Wallingford, pp 335–369

  • Grosser JW, Gmitter FG Jr (1990) Protoplast fusion and citrus improvement. Plant Breed Rev 8:339–374

    Google Scholar 

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

    CAS  Google Scholar 

  • Haldrup A, Petersen SG, Okkels FT (1998) The xylose isomerase gene from Thermoanaerobacterium thermosulfurogenes allows effective selection of transgenic plant cells using D-xylose as the selection agent. Plant Mol Biol 37:287–296

    Article  CAS  PubMed  Google Scholar 

  • Joersbo M, Okkels FT (1996) A novel principle for selection of transgenic plant cells: positive selection. Plant Cell Rep 16:219–221

    Article  CAS  Google Scholar 

  • Joersbo M, Donaldson I, Kreibeg J, Petersen SG, Brunstedt J, Okkels FT (1998) Analysis of mannose selection used for transformation of sugar beet. Mol Breed 4:111–117

    Article  CAS  Google Scholar 

  • Kaneyoshi J, Kobayashi S, Nakamura Y, Shigemoto N, Doi Y (1994) A simple and efficient gene transfer system of trifoliate orange (Poncirus trifoliate Raf.). Plant Cell Rep 13:541–545

    CAS  Google Scholar 

  • Lacorte C, Romano E (1998) Transferência de vetores para Agrobacterium In: Brasileiro ACM, Carneiro VTC (eds) Manual de Transformação Genética de Plantas. Embrapa, Brazil, pp 103–104

  • Lucca P, Ye X, Potrykus I (2001) Effective selection and regeneration of transgenic rice plants with mannose as selective agent. Mol Breed 7:43–49

    Article  CAS  Google Scholar 

  • Mendes BMJ, Mourão Filho FAA, Farias PCM, Benedito VA (2001) Citrus somatic hybridization with potential for improved blight and CTV resistance. In Vitro Cell Dev Biol Plant 37:490–495

    Google Scholar 

  • Mendes BMJ, Boscariol RL, Mourão Filho FAA, Almeida WAB (2002) Agrobacterium-mediated transformation of citrus Hamlin cultivar (Citrus sinensis L. Osbeck) epicotyl segments. Pesqui Agropecu Bras 37:955–961

    Google Scholar 

  • Miles JS, Guest JR (1984) Nucleotide sequence and transcriptional start point of the phosphomannose isomerase gene (manA) of Escherichia coli. Gene 32:41–48

    CAS  PubMed  Google Scholar 

  • Moore GA, Jacomo CC, Neidigh JL, Lawrence SD, Cline K (1992) Agrobacterium-mediated transformation of Citrus stem segments and regeneration of transgenic plants. Plant Cell Rep 11:238–242

    CAS  Google Scholar 

  • Moreira-Dias JM, Molina RV, Bordón Y, Guardiola JL, García-Luiz A (2000) Direct and indirect shoot organogenic pathways in epicotyl cuttings of Troyer citrange differ in hormone requirements and their response to light. Ann Bot 85:103–110

    Article  CAS  Google Scholar 

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

    CAS  Google Scholar 

  • Negrotto D, Jolley M, Beer S, Wenck AR, Hansen G (2000) The use of phosphomannose-isomerase as a selectable marker to recover transgenic maize plants (Zea mays L.) via Agrobacterium transformation. Plant Cell Rep 19:798–803

    Google Scholar 

  • Peña L, Navarro L (1999) Transgenic Citrus In: Bajaj YPS (ed) Biotechnology in agriculture and forestry—transgenic trees. Springer, Berlin Heidelberg New York, pp 39–54

    Google Scholar 

  • Peña L, Cervera M, Juárez J, Navarro L, Pina JA, Durán-Vila N, Navarro L (1995a) Agrobacterium-mediated transformation of sweet orange and regeneration of transgenic plants. Plant Cell Rep 14:616–619

    Google Scholar 

  • Peña L, Cervera M, Juárez J, Ortega C, Pina JA, Durán-Vila N, Navarro L (1995b) High efficiency Agrobacterium-mediated transformation and regeneration of citrus. Plant Sci 104:183–191

    CAS  Google Scholar 

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

    CAS  Google Scholar 

  • Peña L, Martín-Trillo M, Juárez J, Pina JA, Navarro L, Martínez-Zapater JM (2001) Constitutive expression of Arabidopsis LEAFY or APETALA1 genes in citrus reduces their generation time. Nat Biotechnol 19:263–267

    Article  PubMed  Google Scholar 

  • Pérez-Molphe-Balch E, Ochoa-Alejo N (1997) In vitro plant regeneration of Mexican lime and mandarin by direct organogenesis. HortScience 32:931–934

    Google Scholar 

  • Privalle LS, Wright M, Reed J, Hansen G, Dawson J, Dunder EM, Chang Y, Powell ML, Meghji M (1999) Phosphomannose isomerase, a novel selectable plant selection system: mode of action and safety assessment In: Fairbain C, Scoles G, Mcttughen A (eds) In: Proc 6th Int Symp Biosafety Genet Modified Organisms. University Extension Press, University of Saskatchewan, pp 171–178

  • Reed J, Privalle L, Powell ML, Meghji M, Dawson J, Dunder E, Suttie J, Wenck A, Launis K, Kramer C, Chang Y-F, Hansen G, Wright M (2001) Phosphomannose isomerase: an efficient selectable marker for plant transformation. In Vitro Cell Dev Biol Plant 37:127–132

    Google Scholar 

  • Wang AS, Evans RA, Altendorf PR, Hanten JA, Doyle MC, Rosichan JL (2000) A mannose selection system for production of fertile transgenic maize plants from protoplasts. Plant Cell Rep 19:654–660

    CAS  Google Scholar 

  • Wright M, Dawson J, Dunder E, Suttie J, Reed J, Kramer C, Chang Y, Novitzky R, Wang H, Artim-Moore L (2001) Efficient biolistic transformation of maize (Zea mays L.) using the phosphomannose isomerase gene, pmi, as the selectable marker. Plant Cell Rep 20:429–436

    Google Scholar 

  • Yang ZN, Ingelbrecht IL, Louzada ES, Skaria M, Mirkov TE (2000) Agrobacterium-mediated transformation of the commercially important grapefruit cultivar Rio Red (Citrus paradisi Macf.). Plant Cell Rep 19:1203–1211

    CAS  Google Scholar 

Download references

Acknowledgements

The authors acknowledge receiving financial support for this research from FAPESP (99/04073-3) and FUNDECITRUS. RLB and WABA both acknowledge FAPESP and CAPES/PICDT for receipt of a scholarship. BMJM and FAAMF acknowledge CNPq for research fellowships. The authors wish to thank Adriana P.M. Rodriguez for critical comments, Myriam R. Orsi for technical assistance and Syngenta for supplying pNOV2116.

Author information

Authors and Affiliations

  1. Laboratório de Biotecnologia Vegetal, Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Piracicaba 13 400-970, São Paulo, Brazil

    R. L. Boscariol & B. M. J. Mendes

  2. Laboratório de Melhoramento de Plantas, Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Piracicaba 13 400-970, São Paulo, Brazil

    M. T. V. C. Derbyshire

  3. Departamento de Produção Vegetal, Escola Superior de Agricultura "Luiz de Queiroz", Universidade de São Paulo, Piracicaba 13 418-900, São Paulo, Brazil

    W. A. B. Almeida & F. A. A. Mourão Filho

Authors
  1. R. L. Boscariol
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. W. A. B. Almeida
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. T. V. C. Derbyshire
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. F. A. A. Mourão Filho
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. B. M. J. Mendes
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to B. M. J. Mendes.

Additional information

Communicated by L. Peña

Rights and permissions

Reprints and permissions

About this article

Cite this article

Boscariol, R.L., Almeida, W.A.B., Derbyshire, M.T.V.C. et al. The use of the PMI/mannose selection system to recover transgenic sweet orange plants (Citrus sinensis L. Osbeck). Plant Cell Rep 22, 122–128 (2003). https://doi.org/10.1007/s00299-003-0654-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-003-0654-1

Keywords

Search

Navigation