Plant Cell Reports

, 28:387

High-efficiency Agrobacterium-mediated transformation of citrus via sonication and vacuum infiltration

  • Maria Luiza Peixoto de Oliveira
  • Vicente J. Febres
  • Marcio Gilberto Cardoso Costa
  • Gloria A. Moore
  • Wagner Campos Otoni
Genetic Transformation and Hybridization


An improved method for the Agrobacterium infiltration of epicotyl segments of ‘Pineapple’ sweet orange [Citrus sinensis (L.) Osbeck] and ‘Swingle’ citrumelo [Citrus paradisi Macf. X Poncirus trifoliata (L.) Raf.] was developed in order to increase transformation frequency. Sonication-assisted Agrobacterium-mediated transformation (SAAT), vacuum infiltration, and a combination of the two procedures were compared with conventional Agrobacterium-mediated inoculation method (‘dipping’ method). It was observed that the morphogenic potential of the epicotyl segments decreased as the duration of SAAT and vacuum treatments increased. Transient GUS expression was not affected by the different SAAT treatments, but it was significantly enhanced by the vacuum infiltration treatments. The highest transformation efficiencies were obtained when the explants were subjected to a combination of SAAT for 2 s followed by 10 min of vacuum infiltration. PCR and Southern blot analysis of the uidA gene were used to confirm the integration of the transgenes. The transformation frequencies achieved in this study (8.4% for ‘Pineapple’ sweet orange and 11.2% for ‘Swingle’ citrumelo) are the highest ones reported for both cultivars.


Agrobacterium tumefaciens Orange SAAT Agroinfiltration 





Naphtaleneacetic acid




Neomycin phosphotransferase II


Sonication-assisted Agrobacterium-mediated transformation


  1. Acereto-Escoffié POM, Chi-Manzanero BH, Echeverría-Echeverría S, Grijalva R, Kay AJ, González-Estrada T, Castaño E, Rodrígues-Zapata LC (2005) Agrobacterium-mediated transformation of Musa acuminata cv. ‘Grand Nain’ scalps by vacuum infiltration. Sci Hortic 105:359–371. doi:10.1016/j.scienta.2005.01.028 CrossRefGoogle Scholar
  2. Amoah BK, Wu H, Sparks C, Jones HD (2001) Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue. J Exp Bot 52:1135–1142PubMedCrossRefGoogle Scholar
  3. An G, Ebert PR, Miltra A, Ha SB (1988) Binary vectors. In: Gelvin SB, Schilperoort RA, Verma DPS (eds) Plant molecular biology manual, A3. Kluwer, Dordrecht, pp 1–19Google Scholar
  4. Ananthakrishnan G, Xia X, Amutha S, Singer S, Muruganantham M, Yablonsky S, Fischer E, Gaba V (2007) Ultrasonic treatment stimulates multiple shoot regeneration and explant enlargement in recalcitrant squash cotyledon explants in vitro. Plant Cell Rep 26:267–276. doi:10.1007/s00299-006-0235-1 PubMedCrossRefGoogle Scholar
  5. Beranová M, Rakouský S, Vávrová Z, Skalický T (2008) Sonication assisted Agrobacterium-mediated transformation enhances the transformation efficiency in flax (Linux usitatissimum L.). Plant Cell Tiss Org Cult 94:253–259. doi:10.1007/s11240-007-9335-z CrossRefGoogle Scholar
  6. Bidney D, Scelonge C, Martich J, Burrus M, Sims L, Huffman G (1992) Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens. Plant Mol Biol 18:301–313. doi:10.1007/BF00034957 PubMedCrossRefGoogle Scholar
  7. Bond JE, Roose ML (1998) Agrobacterium-mediated transformation of the commercially important citrus cultivar Washington navel orange. Plant Cell Rep 18:229–234CrossRefGoogle Scholar
  8. Canche-Moo RLR, Ku-Gonzalez A, Burgeff C, Loyola-Vargas VM, Rodríguez-Zapata LC, Castaño E (2006) Genetic transformation of Coffea canephora by vacuum infiltration. Plant Cell Tiss Org Cult 84:373–377. doi:10.1007/s11240-005-9036-4 CrossRefGoogle Scholar
  9. Cervera M, Juárez J, Navarro A, Pina JA, Duran-Vila N, Navarro L, Peña L (1998) Genetic transformation and regeneration of mature tissue of woody fruit bypassing the juvenile stage. Transgenic Res 7:51–59CrossRefGoogle Scholar
  10. Charity JA, Holland L, Donaldson SS, Grace L, Walter C (2002) Agrobacterium-mediated transformation of Pinus radiata organogenic tissue using vacuum-infiltration. Plant Cell Tiss Org Cult 70:51–60. doi:10.1023/A:1016009309176 CrossRefGoogle Scholar
  11. Chee PP, Fober KA, Slightom JL (1989) Transformation of soybean (Glycine max) by infecting germinating seeds with Agrobacterium tumefaciens. Plant Physiol 91:1212–1218PubMedCrossRefGoogle Scholar
  12. Cheng M, Fry JE, Zhou H, Hironaka CM, Duncan DR, Coner TW, Wan Y (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115:971–980PubMedGoogle Scholar
  13. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743PubMedCrossRefGoogle Scholar
  14. Costa MGC, Otoni WC, Moore GA (2002) An evaluation of factors affecting the efficiency of Agrobacterium-mediated transformation of Citrus paradisi (Macf.) and the production of transgenic plants containing carotenoid biosynthetic genes. Plant Cell Rep 21:365–373. doi:10.1007/s00299-002-0533-1 CrossRefGoogle Scholar
  15. Duan YX, Guo WW, Meng HJ, Tao DD, Deng XX (2007) High efficient transgenic plant regeneration from embryogenic calluses of Citrus sinensis. Biol Plant 51:212–216. doi:10.1007/s10535-007-0043-7 CrossRefGoogle Scholar
  16. Fleming GH, Olivares-Fuster O, Fatta Del-Bosco S, Grosser JW (2000) An alternative method of genetic transformation of sweet orange. In Vitro Cell Dev Biol Plant 36:450–455CrossRefGoogle Scholar
  17. Flores Solís JI, Mlejnek P, Studená K, Procházka S (2007) Application of sonication-assisted Agrobacterium-mediated transformation in Chenopodium rubrum L. Plant Soil Environ 49:255–260Google Scholar
  18. Gaba V, Kathiravan K, Amutha S, Singer S, Xiaodi X, Ananthakrishnan G (2006) The use of ultrasound in plant tissue culture. In: Gupta SD, Ybaraki Y (eds) Plant tissue culture engineering. Springer, Netherlands, pp 417–426Google Scholar
  19. 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. doi:10.1007/s002990050313 CrossRefGoogle Scholar
  20. Hidaka T, Omura M, Ugaki M, Tomiyama M, Kato A, Ohshima M, Motoyoshi F (1990) Agrobacterium-mediated transformation and regeneration of Citrus spp. from suspension cells. Jpn J Breed 40:199–207Google Scholar
  21. Horsch RB, Fry JE, Hoffman NL, Eicholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231CrossRefGoogle Scholar
  22. Humara JM, Lópes M, Ordás RJ (1999) Agrobacterium tumefaciens-mediated transformation of Pinus pinea L. cotyledons: an assessment of factors influencing the efficiency of uidA gene transfer. Plant Cell Rep 19:51–58. doi:10.1007/s002990050709 CrossRefGoogle Scholar
  23. Ikram-Ul-Haq (2004) Agrobacterium-mediated transformation of cotton (Gossypium hirsutum L.) via vacuum infiltration. Plant Mol Biol Rep 22:279–288CrossRefGoogle Scholar
  24. Jefferson RA, Kanavagh TA, Bevan MW (1987) GUS fusion: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3001–3907Google Scholar
  25. Kayim M, Koc NK (2005) Improved transformation efficiency in citrus by plasmolysis treatment. J Plant Biochem Biotech 14:15–20Google Scholar
  26. Leelavathi S, Sunnichan VG, Kumria R, Vijaykanth GP, Bhatnagar RK, Reddy VS (2004) A simple and rapid Agrobacterium-mediated transformation protocol for cotton (Gossypium hirsutum L.): embryogenic calli as a source to generate large numbers of transgenic plants. Plant Cell Rep 22:465–470. doi:10.1007/s00299-003-0710-x PubMedCrossRefGoogle Scholar
  27. Liu Z, Park B-J, Kanno A, Kameya T (2005) The novel use of a combination of sonication and vacuum infiltration in Agrobacterium-mediated transformation of kidney bean (Phaseolus vulgaris L.) with lea gene. Mol Breed 16:189–197. doi:10.1007/s11032-005-6616-2 CrossRefGoogle Scholar
  28. Liu Y, Yang H, Sakanishi A (2006) Ultrasound: mechanical gene transfer into plant cells by sonoporation. Biotech Adv 24:1–16. doi:10.1016/j.biotechadv.2005.04.002 CrossRefGoogle Scholar
  29. Luth D, Moore GA (1999) Transgenic grapefruit plants obtained by Agrobacterium tumefaciens-mediated transformation. Plant Cell Tiss Org Cult 57:219–222. doi:10.1023/A:1006387900496 CrossRefGoogle Scholar
  30. Meurer CA, Dinkins RD, Collins GB (1998) Factors affecting soybean cotyledonary node transformation. Plant Cell Rep 18:180–186. doi:10.1007/s002990050553 CrossRefGoogle Scholar
  31. Molinari HBC, Bespalhok JC, Kobayashi AK, Pereira LFP, Vieira LGE (2003) Agrobacterium tumefaciens-mediated transformation of ‘Swingle’ citrumelo (Citrus paradisi Macf. X Poncirus trifoliata L. Raf.) using thin epicotyl sections. Sci Hortic 99:375–379. doi:10.1016/S0304-4238(03)00111-0 Google Scholar
  32. Moore GA, Jacono 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. doi:10.1007/BF00235073 CrossRefGoogle Scholar
  33. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  34. Pathak MR, Hamzah RY (2008) An effective method of sonication-assisted Agrobacterium-mediated transformation of chickpeas. Plant Cell Tiss Org Cult 93:65–71. doi:10.1007/s11240-008-9344-6 CrossRefGoogle Scholar
  35. Peña L, Cervera M, Navarro A, 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. doi:10.1007/BF00232724 CrossRefGoogle Scholar
  36. 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 104:183–191CrossRefGoogle Scholar
  37. 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. doi:10.1007/s002990050311 CrossRefGoogle Scholar
  38. Pérez-Molphe-Balch E, Ochoa-Alejo N (1998) Regeneration of transgenic plants of Mexican lime from Agrobacterium rhizogenes transformed tissues. Plant Cell Rep 17:591–596. doi:10.1007/s002990050448 CrossRefGoogle Scholar
  39. 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 Tiss Org Cult 93:97–106. doi:10.1007/s11240-008-9347-3 CrossRefGoogle Scholar
  40. Santarém ER, Trick HN, Essing JS, Finer JJ (1998) Sonication-assisted Agrobacterium-mediated transformation of soybean immature cotyledons: optimization of transient expression. Plant Cell Rep 17:752–759. doi:10.1007/s002990050478 CrossRefGoogle Scholar
  41. Stachlel SE, Messens E, Van Montagu M, Zambryski P (1985) Identification of the signal molecules produced by wounded plant cells which activate the T-DNA transfer process in Agrobacterium tumefaciens. Nature 318:624–629CrossRefGoogle Scholar
  42. Tang W (2003) Additional virulence genes and sonication enhance Agrobacterium tumefaciens-mediated loblolly pine transformation. Plant Cell Rep 21:555–562. doi:10.1007/s00299-002-0550-0 PubMedGoogle Scholar
  43. Tang W, Sederoff R, Whetten R (2001) Regeneration of transgenic loblolly pine (Pinus taeda L.) from zygotic embryos transformed with Agrobacterium tumefaciens. Planta 213:981–989PubMedCrossRefGoogle Scholar
  44. Trick H, Finer JJ (1997) SAAT: sonication-assisted Agrobacterium-mediated transformation. Transgenic Res 6:329–336CrossRefGoogle Scholar
  45. Trick H, Finer JJ (2000) Use of Agrobacterium expressing green fluorescent protein to evaluate colonization of sonication-assisted Agrobacterium-mediated transformation-treated soybean cotyledons. Lett Appl Microbiol 30:406–410CrossRefGoogle Scholar
  46. Wordragen MF, Dons HJM (1992) Agrobacterium tumefaciens-mediated transformation of recalcitrant crops. Plant Mol Biol 10:12–36CrossRefGoogle Scholar
  47. Xue R-O, Xie H-F, Zhang B (2006) A multi-needle-assisted transformation of soybean cotyledonary node cells. Biotechnol Lett 28:1551–1557. doi:10.1007/s10529-006-9123-6 PubMedCrossRefGoogle Scholar
  48. Zaragozá C, Muñoz-Bertomeu J, Arrilaga I (2004) Regeneration of herbicide-tolerant black locust transgenic plants by SAAT. Plant Cell Rep 22:832–838. doi:10.1007/s00299-004-0766-2 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Maria Luiza Peixoto de Oliveira
    • 1
  • Vicente J. Febres
    • 2
  • Marcio Gilberto Cardoso Costa
    • 3
  • Gloria A. Moore
    • 2
  • Wagner Campos Otoni
    • 1
  1. 1.Plant Biology Department, Plant Tissue Culture Laboratory/BIOAGROFederal University of ViçosaViçosaBrazil
  2. 2.Horticultural Sciences Department, Institute of Food and Agricultural Sciences, Plant Molecular and Cellular Biology ProgramUniversity of FloridaGainesvilleUSA
  3. 3.Biological Sciences DepartmentState University of Santa Cruz (UESC)IlhéusBrazil

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