Milan University


University of Pavia1

Glyphosate tolerance in cultured cells --Milvia L. Racchi, Roberta Pelanda, G. Forlani1, G. Musitelli1 and E. Nielsen1 Glyphosate exerts its action blocking the shikimate pathway and EPSP synthase, the herbicide target enzyme, has been characterized and purified.

In monocots the amount of information on glyphosate effects and EPSP synthase properties is very limited. We have investigated the effects of glyphosate on growth and EPSP synthase activity in maize tissue culture. Cell cultures and calli of Black Mexican Sweet (BMS) do not show a significant reduction of growth in presence of increasing doses of glyphosate up to 10 mM (Fig.1).

On the contrary, seedling growth is severely inhibited by only 0.5 mM glyphosate. The tolerance observed in BMS cells is also showed by cells of other strains. In embryogenic cultures tolerance is confined to the cell proliferation stage while it is lost during the regeneration phase (Fig.2).

Figure 1. Glyphosate effect on BMS cell growth after 30 days of culture. The growth is expressed as % of the control.

Figure 2. Glyphosate (0.2mM) effect on growth of callus and embryogenic cultures (A188xBMS) expressed as % of the control.

The glyphosate tolerance of maize cells is unusual since cells of other species are, in the same conditions, sensitive to the compound. The glyphosate inhibition kinetics of the target enzyme was investigated in plastid or cytoplasm-enriched extracts prepared from BMS cells. The results obtained reveal that the plastidial EPSP synthase activity is severely affected by micromolar concentrations of

Figure 3. EPSP synthase activity in presence of different concentrations of glyphosate in plastid or cytoplasm enriched extracts prepared from BMS cells.

glyphosate, while the cytosolic enzyme maintains a remarkable activity at 10 mM glyphosate (Fig. 3).

These results, confirmed in other maize cell lines, were not obtained in extracts prepared from cultured cells of carrot, where no tolerant EPSPs activity could be detected. The presence of two isoforms of EPSPs in monocots has been recently reported by Ream (1988) but no substantial differences in glyphosate sensitivity were described.

The existence in maize of a cytosolic glyphosate-resistant EPSPs form may allow cells to maintain a level of aromatic amino acids sufficient to sustain in vitro growth, thus explaining the tolerance to the herbicide.

Further evidence supporting the existence of two different EPSPs forms was recently obtained by means of the chromatographic separation of two EPSPs peaks from BMS cultured cell extracts. One of them is glyphosate-resistant and has a decreased affinity for the PEP substrate.

The presence and the expression level of the glyphosate insensitive EPSPs form need to be investigated at the plant level. In any case it could have significant implications relating to the study of the mechanism of glyphosate tolerance at cell level and during the differentiation processes and poses interesting questions concerning the physiological role of each EPSPs isoform.

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