Development of a naturally miniaturised testing method based on the mitochondrial activity of fern spores: A new higher plant bioassay

One of the main concerns of current environmental toxicology is the low number of taxa used for standard bioassays. Ferns, with more than 10 000 living species, are the second largest group of vascular plants and are important components of numerous plant communities. Fern spores and gametophytes have long been recognized as useful models for plant research since they constitute a naturally miniaturised and economic higher plant model. Mitochondria are the main energy source in eukaryotic cells and any toxic damage will affect the whole organism. The reduction of tetrazolium salts to water-insoluble coloured formazan salts by the respiratory chain has been used for more than 50 years as a measure of cell mitochondrial activity and viability in eukaryotic organisms. Here, the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) by mitochondria is adapted and optimized to measure fern spore or gametophyte viability. Procedures selected as optimum in the model species Dryopteris guanchica are as follows: bleach sterilization, incubation without shaking at 20 °C in the dark for 1–4 h with 0.05–1.5% TTC in Dyer medium supplemented with 0.001–0.005% Tween 20 at pH 8. We conclude that this method constitutes a promising low cost bioassay for higher plant toxicity during development.