It has been shown by numerous studies [1-3] that exposure of plants to environmental stress can increase seed germination and promote morphometrical changes in plants. This study aimed to evaluate the effects of seed coat color on the seed germination of red clover (Trifolium pratense L.), seedling development, and the amount of flavonoids and root nodulation induced by electromagnetic field (EMF) and cold plasma (CP). The experiments were performed on seeds of red clover cultivar 'Sadūnai'. Before sowing, red clover seeds of different seed colors (yellow and dark purple) were treated with two types of physical stressors: CP for 5 and 7 minutes (CP5 and CP7) and EMF for 10 minutes (EMF10). The effect of treatment on seed germination was estimated by in vitro germination test. The kinetic parameters of germination were determined: Vi - the final germination percentage, Me - the median germination time, Qu – quartile deviation, uniformity germination in seed population. The morphometric changes, changes in the amount of flavonoids, and the number of root nodules for the cultivar that was growing in the substrate for 5 weeks were measured.
The results have shown that the effect of seed treatment with CP and EMF on the germination kinetics, seedling development of red clover seeds significantly depends on the color of the seeds. Control seeds of both colors showed Vi$\approx$100%, therefore, treatment of seeds with stressors could not have a positive effect on this indicator. However, CP5 reduced Vi of yellow seeds by 17%. Nonetheless, the stress-induced changes in the Me index were different for yellow and dark purple seeds. Stress treatment did not have a significant effect on Me index for yellow seeds, but the treatment of purple seeds with CP5, CP7 reduced Me by 24% and 56%, respectively. Treatment with EMF10 did not affect the germination rate of either yellow or purple seeds.
EMF10 seed treatment had positive effect (16%) on the height of the aboveground part of seedling grown from the yellow seeds and the length of their roots. None of the other stressors had a significant effect on these parameters. EMF10 promoted aboveground part weight gain by 30%, although it did not have a reliable effect on root weight. CP7 had a negative effect on root weight, which decreased by 50%. The EMF10 and CP5 exposure did not cause such a significant difference. Stressor treatment did not change the number of seedling leaves, but CP5 and EMF10 increased the number of nodules by 6.6 and 11.6 times, respectively. The height of the aboveground part of the control group of seedlings grown from the purple seeds was 20%, the weight was 29% higher, these parameters for the seedlings of the CP7 group were as much as 28% and 40% higher compared to the seedlings growing from the respective groups. Treatment of purple seeds with CP5 reduced the height of the aboveground part of seedlings by 20%. Stress treatment of purple seeds had no reliable effect on root length, the number of leaves, and the number of nodules.
Seed treatment with CP and EMF induced changes in the amount of flavonoids (except luteolin) in root exudates. Particularly strong was effect of CP7 - the amount of hesperetin, quercetin, 7-hydroxyphlavone, daidzein increased 2-3 times. The obtained results confirm the hypothesis that the changes in red clover nodulation activity caused by seed treatment stressors may be due to an active interaction with rhizobacteria due to the changed chemical composition of root exudates.
The study showed that pre-sowing treatment of red clover seeds with cold plasma and an electromagnetic field can induce seed coat color-dependent changes in germination, morphometrical parameters, nodulation, and the amount of flavonoids.