Plant Biotechnology

Electronic Journal of Biotechnology ISSN: 0717-3458 Vol. 9 No. 5, Issue of October 15, 2006
© 2006 by Pontificia Universidad Católica de Valparaíso -- Chile Received June 6, 2005 / Accepted April 5, 2006
DOI: 10.2225/vol9-issue5-fulltext-4  
RESEARCH ARTICLE

Viability in protoplasts and cell suspensions of Coffea arabica cv. Catimor

Rafael Fernandez-Da Silva
Laboratorio de Clonación y Genética Vegetal
Instituto de Biología Experimental
Facultad de Ciencias
Universidad Central de Venezuela
Calle Suapure, Colinas de ello Monte
Caracas, Venezuela
Tel: 58 212 7510111
Fax: 58 212 7535897
E-mail: rfernandez2@uc.edu.ve

Andrea Menéndez-Yuffá*
Laboratorio de Clonación y Genética Vegetal
Instituto de Biología Experimental
Facultad de Ciencias
Universidad Central de Venezuela
Calle Suapure, Colinas de ello Monte
Caracas, Venezuela
Tel: 58 212 7510111
Fax: 58 212 7535897
E-mail: amenendez@cantv.net

*Corresponding author

Financial support: Project S1-98003209 FONACIT (Ministerio de Ciencia y Tecnología de Venezuela).

Keywords: cell suspensions, cell viability,coffee, Evans blue, MTT, protoplasts.

Abbreviations:

2,4-D: 2,4-dichlorophexyacetic acid
BA: Benzyladenine
FDA: fluorescein diacetate
MES: (2-[morpholino]ethanesulfonic acid)
MTT: (3-[4,5-Dimethyltiazol-2-yl]-2,5-diphenyltetrazolium bromide)
TTC: triphenyl tetrazolium chloride

Abstract
Full Text

Two methods were evaluated in order to assess the viability of protoplasts and cell suspensions of Coffea arabica cv. Catimor used in a protocol of transformation by electroporation. One method consisted of staining with 1 % Evans blue and the other staining with 1% 3-[4,5-dimethyltiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). When Evans blue was applied to viable cells and protoplasts they either did not stain or acquired a faint blue colour. However, it was difficult to distinguish the non-viable cells in the wide spectrum of clear blue tonalities. In contrast, with the MTT assay only the viable cells and protoplasts reduced this salt to the red coloured formazan; viable and non-viable cells were distinguished more clearly with MTT than with Evans blue. The optimal temperature for the reaction with MTT was 37ºC. The time of incubation was shown to be important, since longer times improved the reaction; the highest viability value was obtained after incubation for 120 min.

Supported by UNESCO / MIRCEN network
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