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

Abstract

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.

Seed treatment with cold plasma and electromagnetic field induces changes in red clover root growth dynamics, flavonoid exudation, and activates nodulation

“…Experiments performed while growing seedlings in rhizoboxes revealed that seed treatments induce greater changes in root development compared to the above‐ground part, while the most important finding was an enhanced amount of nine flavonoids in the root exudates and an increased number of nodules. Our study confirmed recently reported [ 28 ] results that CP treatment can stimulate legume root nodulation and that positive changes in seedling agronomic performance (like biomass production) [ 20,28 ] can be explained by CP‐ and EMF‐stimulated nodulation, leading to improved nitrogen fixation activity. Such a finding illustrates that the mechanisms of the complex response of plants to seed treatment consists not only of numerous internal changes in plant biochemical and physiological processes but due to changes in the secondary metabolism and root exudation also involve changes in plant communication with microorganisms, possibly resulting in both increased protection from pathogens and enhanced symbiotic interactions with beneficial species.…”

“…[ 28 ] We found that the number of root nodules increased after red clover seed treatment with CP and EMF, although we did not inoculate seeds and the seedlings were grown in multipurpose potting substrate; thus, nodular rhizobacteria originated either from red clover seeds or from the substrate, as they would in natural conditions. We did not observe any substantial effects on the growth of the above‐ground part of young (5‐week‐old) plants in this study, but we suppose that a large increase in red clover biomass production obtained after 5 months [ 20 ] can be explained by CP‐ and EMF‐stimulated nodulation and nitrogen fixation activity.…”

“…[ 20 ] In addition, substantial changes in the amounts of major isoflavones biochanin A and formononetin, in red clover leaves have been observed. [ 20 ] The latter finding implies that changes in the secondary root metabolism may be also induced in plants growing from CP and EMF treated seeds. Therefore, in this study, we aimed to estimate the effects of seed treatments on red clover root development to test the hypothesis that CP and EMF‐induced stimulation of growth and biomass production of red clover are related to changes in root development, the amounts of flavonoids in root exudates, and nodulation.…”

“…So far, the investigations have mostly focused on the effects of treatments on seed germination and early seedling growth; although some alterations of physiological and biochemical parameters have also been reported, such as changes in seed enzyme activities, [8][9][10] sugar content, [8,11] photosynthetic indices, [12,13] the amount of photosynthetic pigments, [14][15][16] and secondary metabolites [12,17] in seedling leaves. Long-term observations have revealed that the effects of presowing seed treatment with CP and EMF on plant growth may persist for the entire vegetation season [18][19][20][21] or even several seasons in perennials, [22,23] and the changes induced on a longer time scale can be much larger in comparison to those induced in germination. [18][19][20][21][22][23] Although the molecular mechanisms underlying a positive response of plants to seed exposure to CP and EMF still remain elusive, recent findings have shown that presowing seed treatment induces changes in the amount of seed phytohormones, [13,24,25] reactive oxygen species generation in germinating seeds, [23] and epigenetic modifications leading to changes in gene expression, [26] followed by numerous changes in the tissues of growing seedlings: phytohormone amount, [27,28] proteome, [25] photosynthetic efficiency, [13,25] and secondary metabolite amount [17,20] in the leaves of the growing seedling.…”

“…Long-term observations have revealed that the effects of presowing seed treatment with CP and EMF on plant growth may persist for the entire vegetation season [18][19][20][21] or even several seasons in perennials, [22,23] and the changes induced on a longer time scale can be much larger in comparison to those induced in germination. [18][19][20][21][22][23] Although the molecular mechanisms underlying a positive response of plants to seed exposure to CP and EMF still remain elusive, recent findings have shown that presowing seed treatment induces changes in the amount of seed phytohormones, [13,24,25] reactive oxygen species generation in germinating seeds, [23] and epigenetic modifications leading to changes in gene expression, [26] followed by numerous changes in the tissues of growing seedlings: phytohormone amount, [27,28] proteome, [25] photosynthetic efficiency, [13,25] and secondary metabolite amount [17,20] in the leaves of the growing seedling. Moreover, changes in the plant-associated-microbiome composition have been reported [29] indicating that the effects of stressors on plant growth may not be restricted to internal processes, but involve changes in plant communication with microorganisms that are highly important for shaping agronomic plant properties.…”