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Biologia Plantarum

, Volume 49, Issue 4, pp 493–497 | Cite as

Genetic transformation of Coffea canephora by particle bombardment

  • A. F. Ribas
  • A. K. Kobayashi
  • L. F. P. Pereira
  • L. G. E. Vieira
Article

Abstract

Stable transformation of Coffea canephora P. was obtained by particle bombardment of embryogenic tissue. Leaf explants were cultured on medium supplemented with 5 µM isopentenyl-adenosine to induce direct embryogenesis. Explants with somatic embryos were transferred to half strength MS medium with 9 µM 2,4 dichlorophenoxyacetic acid. After 2 weeks, the explants with somatic embryos and embryogenic tissue were bombarded with tungsten particles (M-25) carrying the plasmid pCambia3301 (containing the bar and uidA genes) using a high pressure helium microprojectile device. The bombarded explants were submitted to selection on medium containing 5 µM ammonium glufosinate herbicide as selective agent. After 6 months, putative transgenic embryos were transferred to a growth regulator-free medium for germination. The regenerated plantlets were β-glucuronidase (GUS) positive whereas no GUS activity was observed in non-transgenic controls. Incorporation of the bar gene into the genome was confirmed by PCR and Southern blot analysis of the regenerated transformed plants. Greenhouse grown transgenic coffee plants were found to withstand the recommended level of the herbicide Finale™ for weed control.

Additional key words

biolistic transgenic coffee ammonium glufosinate 

Abbreviations

2iP

N6-(2-isopentenyl)-adenosine

2,4-D

2,4 dichlorophenoxyacetic acid

CTAB

cethyltrimethylamonium

GS

glutamine synthetase

GUS

β-glucuronidase

PAT

phosphinothricin acetyltransferase

PVP

polyvinylpyrrolidone

SDS

sodium dodecyl sulfate

SSC

sodium citrate

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Copyright information

© Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Praha 2005

Authors and Affiliations

  • A. F. Ribas
    • 1
  • A. K. Kobayashi
    • 2
  • L. F. P. Pereira
    • 3
  • L. G. E. Vieira
    • 1
  1. 1.IAPAR - Laboratorio de BiotecnologiaLondrina, PRBrazil
  2. 2.Embrapa - Mandioca e Fruticultura (CNPMF)Cruz das Almas, BABrazil
  3. 3.Embrapa - Cafe, Laboratorio de Biotecnologia, IAPARLondrina, PRBrazil

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