Acta Physiologiae Plantarum

, Volume 34, Issue 2, pp 471–477 | Cite as

Genetic transformation of three sweet orange cultivars from explants of adult plants

  • Pâmela Fávero
  • Francisco de Assis Alves Mourão Filho
  • Liliane Cristina Libório Stipp
  • Beatriz Madalena Januzzi Mendes
Original Paper

Abstract

The difficulty in adult tissue genetic transformation in woody species is still an obstacle to be overcome, including in most sweet orange cultivars of the Brazilian citrus industry. This work reports that, after in vitro culture adjustments, transgenic adventitious buds of ‘Hamlin’, ‘Pêra’, and ‘Valencia’ sweet oranges (Citrus sinensis L. Osbeck) were recovered using adult material as explant source, in genetic transformation experiments via Agrobacterium tumefaciens. The transgenic buds were identified by the GUS histochemical analysis and confirmed by PCR analysis, which indicated the presence of an amplified fragment of 817 bp corresponding to the uidA gene sequence. The efficiencies of genetic transformation for ‘Hamlin’, ‘Pêra’, and ‘Valencia’ sweet orange cultivars were 2.5, 1.4, and 3.7%, respectively. Media supplemented with auxins and cytokinins during co-culture, and media with high concentrations of cytokinins (3 mg L−1) during transgenic selection led to the transformation and, consequently, the regeneration of adequate number of adventitious buds for the three cultivars. The use of sonication during the explant disinfection was not effective to reduce endophytic contamination and reduced transformation efficiency.

Keywords

Citrus sinensis Tissue culture Biotechnology Genetic improvement Agrobacterium tumefaciens 

References

  1. Almeida WAB, Mourão Filho FAA, Pino LE, Boscariol RL, Rodriguez APM, Mendes BMJ (2003) Genetic transformation and plant recovery from mature tissues of Citrus sinensis L. Osbeck. Plant Sci 164:203–211CrossRefGoogle Scholar
  2. Bond JE, Roose ML (1998) Agrobacterium-mediated transformation of the commercially important citrus cultivar Washington navel orange. Plant Cell Rep 18:229–234CrossRefGoogle Scholar
  3. Boscariol RL, Almeida WAB, Derbyshire MTVC, Mourão Filho FAA, Mendes BMJ (2003) The use of the PMI/mannose selection system to recover transgenic sweet orange plants (Citrus sinensis L. Osbeck). Plant Cell Rep 22:122–128PubMedCrossRefGoogle Scholar
  4. Cervera M, Juárez J, Navarro A, Pina JA, Durán-Vila N, Navarro L, Peña L (1998a) Genetic transformation and regeneration of mature tissues of woody fruit plants bypassing the juvenile stage. Transgenic Res 7:51–59CrossRefGoogle Scholar
  5. Cervera M, Pina JA, Juárez J, Navarro L, Peña L (1998b) Agrobacterium-mediated transformation of citrange: factors affecting transformation and regeneration. Plant Cell Rep 18:271–278CrossRefGoogle Scholar
  6. Cervera M, Navarro A, Navarro L, Peña L (2008) Production of transgenic adult plants from clementine mandarin by enhancing cell competence for transformation and regeneration. Tree Physiol 28:55–66PubMedCrossRefGoogle Scholar
  7. Cervera M, Navarro L, Peña L (2009) Gene stacking in 1-year-cycling APETALA1 citrus plants for a rapid evaluation of transgenic traits in reproductive tissues. J Biotechnol 140:278–282PubMedCrossRefGoogle Scholar
  8. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  9. Duan Y-X, Fan J, Guo W-W (2010) Regeneration and characterization of transgenic kumquat plants containing the Arabidopsis APETALA1 gene. Plant Cell Tissue Organ Cult 100:273–281CrossRefGoogle Scholar
  10. Dutt M, Grosser JW (2009) Evaluation of parameters affecting Agrobacterium-mediated transformation of citrus. Plant Cell Tissue Organ Cult 98:331–340CrossRefGoogle Scholar
  11. Gentile A, Deng Z, La Malfa S, Distefano G, Domina F, Vitale A, Polizzi G, Lorito M, Tribulato E (2007) Enhanced resistance to Phoma tracheiphila and Botrytis cinerea in transgenic lemon plants expressing a Trichoderma harzianum chitinase gene. Plant Breed 126:146–151CrossRefGoogle Scholar
  12. Liu X, Pijut PM (2010) Agrobacterium-mediated transformation of mature Prunus serotina (Black cherry) and regeneration of transgenic shoots. Plant Cell Tissue Organ Cult 101:49–57CrossRefGoogle Scholar
  13. Lloyd G, McCown B (1980) Commercially feasible micropropagation of mountain laurel (Kalmia latifolia) by use of shoot tip culture. Combined Proc of Int Plant Propagator’s Soc 30:421–427Google Scholar
  14. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol Plan 15:473–497CrossRefGoogle Scholar
  15. Oliveira MLP, Febres VJ, Costa MGC, Moore GA, Otoni WC (2009) High-efficiency Agrobacterium-mediated transformation of citrus via sonication and vacuum infiltration. Plant Cell Tissue Organ Cult 28:387–395Google Scholar
  16. Oliveira MLP, Costa MGC, Da Silva CV, Otoni WC (2010) Growth regulators, culture media and antibiotics in the in vitro shoot regeneration from mature tissue of citrus cultivars. Pesq Agropec Bras 45:654–660CrossRefGoogle Scholar
  17. 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 19:263–267PubMedCrossRefGoogle Scholar
  18. Peña L, Pérez RM, Cervera M, Juárez JA, Navarro L (2004) Early events in Agrobacterium-mediated genetic transformation of citrus explants. Ann Bot 94:67–74PubMedCrossRefGoogle Scholar
  19. Poethig RS (1990) Phase change and the regulation of shoot morphogenesis in plants. Science 250:923–930PubMedCrossRefGoogle Scholar
  20. Puite K, Schaart J (1999) Agrobacterium-mediated transformation of the apple cultivars ‘Gala’, ‘Golden’ and ‘Elstar’, and the strawberry cultivars ‘Gariguette’, ‘Polka’ and ‘Elsanta’. Acta Hort 484:547–553Google Scholar
  21. Rodríguez A, Cervera M, Peris JE, Peña L (2008) The same treatment for transgenic shoot regeneration elicits the opposite effect in mature explants from two closely related sweet orange (Citrus sinensis (L.) Osb.) genotypes. Plant Cell Tissue Organ Cult 93:97–106CrossRefGoogle Scholar
  22. Tan B, Li D-L, Xu S-X, Fan G-E, Fan J, Guo W-W (2009) Highly efficient transformation of the GFP and MAC12.2 genes into precocious trifoliate orange (Poncirus trifoliata [L.] Raf), a potential model genotype for functional genomics studies in Citrus. Tree Genet Genome 5:529–537CrossRefGoogle Scholar
  23. Tong Z, Tan B, Zhang J, Hu Z, Guo W, Deng X (2009) Using precocious trifoliate orange (Poncirus trifoliata [L.] Raf.) to establish a short juvenile transformation platform for citrus. Sci Hortic 119:335–338CrossRefGoogle Scholar
  24. Yancheva SD, Druart P, Watillon B (2002) Agrobacterium-mediated transformation of plum (Prunus domestica L.). Acta Hortic 577:215–217Google Scholar
  25. Yang L, Xu C-J, Hu G-B, Chen K-S (2007) Establishment of an Agrobacterium-mediated transformation system for Fortunella crassifolia. Biol Plan 51:541–545CrossRefGoogle Scholar

Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2011

Authors and Affiliations

  • Pâmela Fávero
    • 1
  • Francisco de Assis Alves Mourão Filho
    • 1
  • Liliane Cristina Libório Stipp
    • 1
  • Beatriz Madalena Januzzi Mendes
    • 2
  1. 1.Escola Superior de Agricultura “Luiz de Queiroz”Universidade de São PauloPiracicabaBrazil
  2. 2.Centro de Energia Nuclear na AgriculturaUniversidade de São PauloPiracicabaBrazil

Personalised recommendations