For many years coffee has been the subject of improvement by conventional methods; the application of biotechnology in the improvement of this crop has recently progressed. The application of biotechnological methods, such as cell culture, protoplasts (cells without the cell wall) and transformation (insertion of new genes) requires a reliable and efficient method to establish if the cells are alive (cell viability).

The methods used to evaluate viability can be classified in two groups: those that stain only the dead cells, and those in which only the living cells are coloured, because the colour is a product of cell metabolic activity (Widholm, 1972). The most used stains for dead cells are Evans blue, bromophenol blue, methylene blue and phenosafranin, whereas fluorescein diacetate (FDA) is used for living cells. Evans blue and methylene blue are reduced by the living cells turning colourless while dead cells remain blue. When FDA is used, the coloration (fluorescence) is the product of activity in the cells; the FDA can enter the cell, where it releases fluorescein (Huang et al. 1986) but requires a costly fluorescence microscope to be detected.

Viability can also be quantified with activity assays, using for example the tetrazolium salts 3-[4,5-dimethyltiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and triphenyl tetrazolium chloride (TTC). These salts are reduced in viable cells to a red coloured compound (Tisserat and Manthey, 1996; Verleysen et al. 2004). Black coloured or non-coloured cells are considered dead (Khatun and Flowers, 1995).

The main objective of this research was to establish a clear, efficient and cheap method to estimate the viability of protoplasts and cell suspensions of Catimor coffee, in order to  apply this method to cell suspensions, protoplasts isolation, and genetically transformed material.

Tissue culture

Coffee (Coffea arabica cv. Catimor) cell suspensions were obtained following Hermoso-Gallardo and Menéndez-Yuffá (2000).In the first stage callus formation was induced from leaf sections, in culture media M1, composed by ½ Murashige and Skoog (1962) salts, thiamine-HCl, myo-inositol, cysteine-HCl, pyridoxine, nicotinic acid, kinetin, 2,4-dichlorophexyacetic acid, sucrose and phytagel (SIGMA, St. Louis, USA) before sterilization. The leaf sections were incubated for 12 weeks in the dark at room temperature. The calli were then transferred to a medium similar to M1, benzyladenine (BA) as the only growth regulator. In the third phase the embryogenic calli were transferred to a liquid medium with BA under continuous shaking at 160 rpm for 15 days, and then was filtered through a steel sieve. The isolation and culture of protoplasts were based on the procedures described by Tahara et al. (1994) with modifications, a solution with macerozyme and cellulase was used to eliminate the cell walls.

Viability tests

Viability was determined by two methods, Evans blue and the MTT assay. The procedures were based on Widholm (1972) for Evans blue and on Tisserat and Manthey (1996) for MTT. Viability was determined by counting the coloured cells.

Control tests of viability were done with suspension cultures fixed in 70% ethanol or in FAA (5% of 37% formaldehyde, 5% glacial acetic acid, 90% of 70% ethanol).

NOTE: The experiments described in this article only can be performed by trained personnel, following safety procedures, because several of the above mentioned compounds are dangerous to health.

According to previous reports Evans blue is an indicator of non-viable cells; in the present study the viable cells and protoplasts were non-stained or clear blue (Figure 1a). In contrast, the non-viable cells and protoplasts, were distinguished by an intense blue colour (Figure 1b).

Viable cells treated with MTT were distinguished by a pink, red or purple colour in the cell cytoplasm or protoplasts (Figure 1c), and the non-viable cells either showed a clear yellow colour or did not change their colour (Figure 1d).

Evans blue stains only the dead cells because in the living cells the dye is reduced to a colourless form, but it is difficult to distinguish the dead cells in a blue background due to the low extinction coefficient of this dye (Huang et al. 1986). In agreement with these authors, we also observed that it was difficult to discriminate between living and dead cells. In contrast, the MTT assay based on the cell metabolic activity changes the colour of living cells by the reduction of the salt (MTT), forming an insoluble compound of red or purple colour (formazan) through the action of the mitochondrial dehydrogenase (Tisserat and Manthey, 1996), whereas the black colour or no change in colour is a sign of cell death (Khatun and Flowers, 1995).

We observed that incubation time was important when detecting viability, the MTT assay having an optimum incubation time of 120 min. These results are in accordance with previous work: the time for the MTT reaction can vary from 15 min for pollen (Khatun and Flowers, 1995) to 24-48 hrs for spores of phytopathogenic fungi (Sutherland and Cohen, 1983). These authors also consider that MTT concentration and incubation temperature and time are important factors in the assay. Gracia-Medrano and Miranda-Ham (2003) report that they used an incubation time of 8 hrs with MTT or TTC to evaluate changes in viability of Lycopersicon esculentum suspension cultures.

An incubation temperature of 37ºC was better than room temperature to detect viability with MTT, probably because this temperature was better for the activity of the mitochondrial dehydrogenase.

In this research we demonstrate the usefulness of the MTT assay to evaluate viability in protoplasts and cell suspensions of coffee; additionally, that of the two methods assayed, MTT is the best system to determine without ambiguity the viability of cells and protoplasts of coffee ‘Catimor’, since in appropriate conditions of temperature and time of incubation it allows the clear discrimination between viable and non-viable cells and avoids the need for expensive equipments of fluorescence required by the method of FDA.

To FONACIT for the financial support of this research (Project S1-98003209). To Dr. Ana Herrera for the revision of the manuscript and to Lic. Luis Hermoso-Gallardo for his assistance in tissue culture.

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