Coffee is a very important crop in Latin America. As all crops, coffee is susceptible to different kinds of biotic stresses that significantly affect yield. In the cases where coffee does not have natural resistance for pests such as the leaf miner (Leucoptera coffeella) or the coffee berry borer (Hypothenemus hampei), it would be very useful to have a genetic transformation system allowing  the incorporation of resistance to those insects. The main objective of our work is to develop a method for coffee genetic transformation by electroporation. The electroporation is a physical process which produces a transient permeabilization of the cellular membrane by an electrical pulse, permitting incorporation of some biological molecules, among them the DNA to the cell. This method permits in certain cases the incorporation of a DNA fragment to the genome of certain cells, which may have a characteristic of interest, for example, insect resistance.  In this research the DNA fragment used contained the genes named gus and bar; serves as a marker of the transformed tissues (the tissues that incorporated the DNA fragment to their genome); bar confers resistance to the herbicide ammonium gluphosinate.  These genes were used with the aim of establishing the appropriate conditions for coffee transformation (foreign DNA incorporation) by electroporation.

Electroporation. Electroporation assays were carried out on different tissues of coffee (Coffea arabica cv. Catimor) cultivated in vitro following the procedure of embryogenic cell suspensions of Hermoso-Gallardo and Menéndez-Yuffá (2000). Leaf sections were sowed in an appropriate medium used to originate embryogenic cell suspension cultures, these are cell cultures in liquid medium that produce embryos from somatic cells. The tissues were incubated in a solution (buffer) containing the DNA to be introduced into coffee. This mix was then treated with a pulse generator equipment (Gene Pulser II system BIO-RAD) to apply electric field pulses. The DNA used was the plasmid pCambia3201 (Center for the Application of Molecular Biology to the International Agriculture of Canberra, Australia [CAMBIA]; E-mail: vectors@cambia.org).

The effect of different factors related to the electroporation procedure on the transient gus gene expresssion in coffee plants was evaluated: type of tissue, pre-treatment of the tissues with an enzymatic solution (cellulase and macerozyme) which digests the plant cell walls, electrical field strength and incubation temperature.

gus detection. The presence of the gus gene was detected using an histochemical reaction [method of Jefferson et al. 1987 with the modification of Kosugi et al. 1990 and Lacorte, 1998]. When the gene is expressed the tissue shows a blue color.

Regeneration. The regeneration of the electroporated explants was obtained in a medium for the induction of secondary somatic embryogenesis, composed by 1/2 strength Murashige and Skoog, 1962 salts, 100 mg/l mio-inositol, 35 mg/l cysteine, 10 mg/l thiamine, 30 g/l sucrose and 8 mg/l benzyladenine, in solid media (8 g/l agar powder extrapure HI-MEDIA) or liquid. The liquid cultures were maintained in a gyratory shaker at 150 rpm, cultures were incubated in the dark at room temperature (28 + 2ºC).

Polymerase Chain Reaction (PCR). To detect the presence of the genes introduced in the coffee tissues, a reaction called the polymerase chain reaction was used. This reaction permits to multiply a fragment of DNA which contains a certain DNA sequence (in this case part of the genes gus or bar). After the amplification the amplifyied fragment can be seen as a band in a gel.

Our results showed that electroporation is a promising technique for the transformation of intact tissues of coffee. Among the coffee plant material tested the somatic embryos in torpedo stage were the more appropriate material for the electroporation process, and showed the best gus expression and the best regeneration response through secondary somatic embryo formation. The optimal conditions previous to the electroporation of torpedo-shape somatic embryos, were: one hour of enzymatic pretreatment, incubation or the tissues in buffer ASP during three hours, with changes every hour and incubation of the tissues with DNA in buffer ASP at 28ºC (room temperature) during one hour. Electroporation conducted at 900 μF, 375 V/cm, and culture of the electroporated tissues in liquid medium (with 8 mg/l benzyladenine) to induce secondary somatic embryogenesis. The tests showed that the secondary somatic embryos regenerated had gus expression, and according to the PCR analysis, the genes gus and bar were present in two of the eight embryos assayed.

To Dr. Ana Herrera and Dr. Carlos F. Quiros for the revision of the manuscript and to Dr. Ariadne Vegas for the donation of the plasmid pCambia 3201.

HERMOSO-GALLARDO, L. and MÉNENDEZ-YUFFA, A. Multiplicación masiva del café (Coffea arabica L. cv. Catimor) mediante cultivo de suspensiones celulares embriogénicas. Acta Científica Venezolana, January 2000, vol. 51, p. 90-95.

JEFFERSON, R.A.; KAVANAGH, T.A. and BEVAN, M.W. GUS fusions: b-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO Journal, December 1987, vol. 6, no. 13, p. 3901-3907.

KOSUGI, S.; OHASHI, Y.; NAKAJIMA, K. and ARAI, Y. An improved assay for b-glucuronidase in transformed cells: methanol almost completely suppresses a putative endogenous for b-glucuronidase activity. Plant Science, 1990, vol. 70, p. 133-140.

LACORTE, G. b-glucuronidase (GUS). In: MIRANDA B., A.C. and DE CAMPOS, V.T. eds. Manual de transformacao genética de plantas. Embrapa servicio de producao de informacao, SPI, Brasilia, Brasil, 1998, chapter 8, p. 127-141.

MURASHIGE, T. and SKOOG, F. A revised medium for rapid growth on bioassays with tobacco tissue culture. Physiologia Plantarum, March 1962, vol. 15, p. 473-497.