Plant Cell Reports

, Volume 22, Issue 2, pp 122–128 | Cite as

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

  • R. L. Boscariol
  • W. A. B. Almeida
  • M. T. V. C. Derbyshire
  • F. A. A. Mourão Filho
  • B. M. J. Mendes
Genetic Transformation and Hybridization

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.

Keywords

Agrobacterium tumefaciens Citrus transformation Sweet orange Mannose selection marker 

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

  1. Alfenas AC, Peters I, Brune W, Passador, GC (1991) Eletroforese de proteínas e isoenzimas de fungos e essências florestais. Editora SIF, ViçosaGoogle Scholar
  2. 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 10:107–121PubMedGoogle Scholar
  3. Ashton GC, Braden AWH (1961) Serum β-globulin polymorphism in mice. Aust J Biol Sci 14:248–254Google Scholar
  4. Cervera M, Pina JA, Juárez J, Navarro L, Peña L (1998) Agrobacterium-mediated transformation of citrange: factors affecting transformation and regeneration. Plant Cell Rep 18:271–278Google Scholar
  5. 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 100:670–677Google Scholar
  6. 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 75:26–30Google Scholar
  7. 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 21:365–373CrossRefGoogle Scholar
  8. 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 19:427–433Google Scholar
  9. 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 10:1–10CrossRefGoogle Scholar
  10. 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 267:544–556CrossRefPubMedGoogle Scholar
  11. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  12. 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 7:175–185CrossRefGoogle Scholar
  13. 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 21:421–428PubMedGoogle Scholar
  14. 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 99:350–358CrossRefGoogle Scholar
  15. 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 2:27–36Google Scholar
  16. Gmitter FC, Grosser JW, Moore GA (1992) Citrus: In: Hammmerschlag FA, Litz RE (eds) Biotechnology of perennial fruit crops. CAB Int, Wallingford, pp 335–369Google Scholar
  17. Grosser JW, Gmitter FG Jr (1990) Protoplast fusion and citrus improvement. Plant Breed Rev 8:339–374Google Scholar
  18. 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 16:745–753Google Scholar
  19. 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 37:287–296CrossRefPubMedGoogle Scholar
  20. Joersbo M, Okkels FT (1996) A novel principle for selection of transgenic plant cells: positive selection. Plant Cell Rep 16:219–221CrossRefGoogle Scholar
  21. 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 4:111–117CrossRefGoogle Scholar
  22. 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 13:541–545Google Scholar
  23. 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 103–104Google Scholar
  24. Lucca P, Ye X, Potrykus I (2001) Effective selection and regeneration of transgenic rice plants with mannose as selective agent. Mol Breed 7:43–49CrossRefGoogle Scholar
  25. 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 37:490–495Google Scholar
  26. 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 37:955–961Google Scholar
  27. Miles JS, Guest JR (1984) Nucleotide sequence and transcriptional start point of the phosphomannose isomerase gene (manA) of Escherichia coli. Gene 32:41–48PubMedGoogle Scholar
  28. 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 11:238–242Google Scholar
  29. 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 85:103–110CrossRefGoogle Scholar
  30. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol Plant 15:473–497Google Scholar
  31. 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 19:798–803Google Scholar
  32. Peña L, Navarro L (1999) Transgenic Citrus In: Bajaj YPS (ed) Biotechnology in agriculture and forestry—transgenic trees. Springer, Berlin Heidelberg New York, pp 39–54Google Scholar
  33. 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 14:616–619Google Scholar
  34. 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 104:183–191Google Scholar
  35. 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 16:731–737Google Scholar
  36. 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 19:263–267CrossRefPubMedGoogle Scholar
  37. Pérez-Molphe-Balch E, Ochoa-Alejo N (1997) In vitro plant regeneration of Mexican lime and mandarin by direct organogenesis. HortScience 32:931–934Google Scholar
  38. 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 171–178Google Scholar
  39. 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 37:127–132Google Scholar
  40. 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 19:654–660Google Scholar
  41. 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 20:429–436Google Scholar
  42. 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 19:1203–1211Google Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • R. L. Boscariol
    • 1
  • W. A. B. Almeida
    • 3
  • M. T. V. C. Derbyshire
    • 2
  • F. A. A. Mourão Filho
    • 3
  • B. M. J. Mendes
    • 1
  1. 1.Laboratório de Biotecnologia Vegetal, Centro de Energia Nuclear na AgriculturaUniversidade de São PauloSão PauloBrazil
  2. 2.Laboratório de Melhoramento de Plantas, Centro de Energia Nuclear na AgriculturaUniversidade de São PauloSão PauloBrazil
  3. 3.Departamento de Produção Vegetal, Escola Superior de Agricultura "Luiz de Queiroz"Universidade de São PauloSão PauloBrazil

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