Effect of seed treatment with cold plasma and electromagnetic field on red clover germination, growth and content of major isoflavones

Pre-sowing treatment of red clover seeds with cold plasma (CP) and electromagnetic field (EMF) was applied aiming to evaluate changes induced in germination and early seedling growth under controlled laboratory conditions, and to estimate treatment effects on plant growth, biomass production, nutritional value and the amounts of major leaf secondary metabolites under long-term field experimental conditions. The effects of seed treatment using radio-frequency EMF (5, 10, 15 min) and CP (2, 5, and 7 min) on seed germination kinetics, morphometric parameters of seedlings were assessed for two cultivars of red clover—'Vyčiai' and 'Sadūnai'. Long-term observations revealed that the effects of seed treatments persist for the entire vegetation season, while the observed effects on germination (CP increased germination rate by 4%–20%) do not provide sufficient information to predict the impact on plants on a longer time scale. The effects on morphometric parameters were dependent on plant cultivar, and were much stronger (up to 24% for 'Sadūnai' and 49% for 'Vyčiai') after growing the plants in the field for 5 months in comparison to the effects (absent or below 10%) observed in the early growth stages. A minor improvement of nutritional value per biomass unit was observed for 'Vyčiai'. Protein yield per plant increased substantially (up to 70%). Pre-sowing seed treatment with CP and EMF had an obvious impact on the amounts of major isoflavones in leaves, and the nature of these changes strongly depended on the vegetation stage of plants (before flowering or at flowering stage). CP treatment for 5 min was found to be the most effective in this respect in not flowering plants, inducing a strong increase in biochanin A/formononetin ratio in leaves of both cultivars. However, an opposite effect was observed in the flowering plants, where the CP and EMF treatments decreased biochanin A/formononetin ratio. (paper)