Molecular
Biology and Genetics
|
Plant
Biotechnology
|
Electronic Journal of Biotechnology ISSN: 0717-3458
|
Vol. 6 No. 1, Issue of April 15, 2003 |
© 2003 by Universidad Católica de Valparaíso
-- Chile |
Received August 26, 2002 / Accepted April 7, 2003 |
Transient
gene expression in secondary somatic embryos from coffee tissues electroporated
with the genes gus and bar
Rafael
Fernandez-Da Silva
Laboratorio
de Clonación y Genética Vegetal
Instituto de Biología Experimental
Facultad de Ciencias
Universidad Central de Venezuela
Apartado 47114, Los Chaguaramos
Caracas 1041, Venezuela
Fax: 58 212 7535897
E-mail: rafaelfer@telcel.net.ve
Andrea
Menéndez-Yuffá*
Laboratorio
de Clonación y Genética Vegetal
Instituto de Biología Experimental
Facultad de Ciencias
Universidad Central de Venezuela
Apartado 47114, Los Chaguaramos
Caracas 1041, Venezuela
Tel: 58 212 7510111
Fax: 58 212 7535897
E-mail: amenendez@cantv.net
* Corresponding author
Financial support: FONACIT-Venezuela
Project S1-98003209.
Keywords: bar,
Coffea arabica, electroporation, genetic transformation, gus,
secondary somatic embryos, somatic embryos.
Different electroporation
conditions were evaluated, toward the goal of transformation of Coffea
arabica cv. Catimor. The tissues assayed were: embryogenic calli,
leaf sections from in vitro plants, and somatic embryos in globular
and torpedo stage obtained from cell suspensions. The effect of 1 or
24-hour pretreatment with an enzymatic solution (2% cellulase, 1% macerozyme)
and electric field strength (375, 625, 875 V/cm) was evaluated. In all
the experiments the tissues were incubated in ASP buffer (potassium
aspartate) during three hours, and then one hour with plasmid DNA (pCambia3201,
containing gus and bar genes) at room temperature. The
electroporation was performed at a capacitance of 900 μF. The effect
of the parameters evaluated was determined by the transient expression
of the gus gene. The optimal conditions for electroporation were
one hour of enzymatic pretreatment of torpedo shape embryos, electroporation
at 375 V and 900 μF. The culture of electroporated tissues in liquid
media with 8 mg/l benzyladenine conducted to maximal regeneration through
secondary somatic embryogenesis. The secondary somatic embryos were
formed directly in the hypocotyl surface of the electroporated torpedo
shape primary somatic embryos, the production of secondary somatic embryos
was significantly greater than the production of primary embryos, therefore,
this is an excellent method to propagate the products of genetic transformation.
The secondary somatic embryos regenerated from electroporated torpedo
shape somatic embryos were positive for gus expression, and also
in the PCR analysis for the genes gus and bar.
|