Simple method of improving harvest by nonthermal air plasma irradiation of seeds of Arabidopsis thaliana (L.)

The application of atmospheric-pressure nonthermal discharge plasma to a living body allows us to control its activities from proliferation to apoptosis. The plasma can produce a wide density range of reactive species without any thermal damage. This nonequilibrium feature opens a wide variety of nonthermal plasma applications in medical^1–5) and agricultural areas.^6–10) The growth enhancement of plants, which is an important topic in agriculture, has been studied using the nonthermal atmospheric-pressure plasma^8–10) and other methods such as those involving low-pressure plasmas,^10–13) plasma-activated water,^14–16) and pulsed electric field.^17–20) Most of these studies have reported an improvement in seed germination rate and/or an increase in plant length. Thus far, we have shown that atmospheric-pressure air plasma irradiation of seeds of Raphanus sativus (L.) (radish sprout) can induce continuous growth enhancement for weeks after their germination.^9,10) In these studies, we have reported the time evolution of the plant length until the plant matures. To the best of our knowledge, there are no reports on the effects of plasma irradiation of seeds on crop yield, which is one of the most important topics in agricultural applications. Here, we report on the effects of plasma irradiation of seeds of Arabidopsis thaliana (L.) on their growth until their harvest and the improvement in crop yield due to the plasma irradiation. A. thaliana (L.) is an attractive eukaryotic model for biological research^21,22) because its genome has been sequenced and analyzed.²³⁾

Experiments were carried out using a scalable dielectric barrier discharge device, as shown in Fig. 1(a).⁹⁾ The device consisted of 20 electrodes of a stainless rod of 1 mm outer diameter and 60 mm length covered with a ceramic tube of 2 mm outer diameter. The electrodes were arranged parallel to each other at a distance of 0.2 mm. The discharge voltage and frequency were 7.96 kV and 9.2 kHz, respectively. The discharge power, which was obtained from Q–V Lissajous characteristics, was 2.17 W. As pretreatment of A. thaliana (L.) seeds, they were immersed in DI water for 1 min and kept at 4 °C for 4 days in a refrigerator to break dormancy. They were dried during that period in the refrigerator. Twenty seeds of the plant were set 3 mm under the electrodes, as shown in Fig. 1(b). Plasma was irradiated for 3 min under indoor conditions. The temperature and relative humidity were room temperature and 40–60%, respectively. After plasma irradiation, the seeds were sown in soil tab with a commercial mineral solution (Hyponex) diluted (1/1000) with deionized water. They were grown under a 12 h light/12 h dark cycle at 22 °C and 80% relative humidity from the beginning of cultivation to harvest. To study the growth enhancement of the plants, we measured the total stem length with an image analysis system every three days after their germination and the total weight of the harvested seeds with a microbalance. The cultivation experiments were carried out three times to confirm reproducibility. The measured data were analyzed by two-way analysis of variance.

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Figure 2 shows the time evolution of stem length for the plants (a) without plasma irradiation (control) and (b) with plasma irradiation (plasma). There are three growth stages, namely, initial growth stage, adult stage, and harvest stage. The plants grow rapidly from their germination in the initial growth stage and then slowly in the adult stage. In the harvest stage, the growth rate is almost the same as that in the adult stage. The growth rates of the three stages were obtained by linear fitting, as shown in Fig. 1. Germination day is defined as the x-intercept of the fitted line in the initial growth stage. The germination of 2.9 days for plasma is slightly faster than that of 3.8 days for the control. Maturity day is defined as the intersection of the two fitted lines. In the initial growth stage, the growth rates for the control and plasma are 1.42 and 1.78 cm/day, respectively. In the adult stage, those for the control and plasma are 0.10 and 0.09 cm/day, respectively. The maturity periods for the control and plasma are 22.0 and 17.5 days, respectively. These results indicate that plasma irradiation accelerates stem growth in the initial growth stage, while the acceleration effects are minimal in the adult stage. Harvest day is defined as the time from the beginning of cultivation to the first seed production. Table I shows the harvesting period and the total weight of harvested seeds for the control and plasma. The harvesting periods for the plasma and control are 62.4 ± 4.7 and 55.8 ± 3.7 days, respectively. The time difference between the periods for the plasma and control is 6.6 days, while the mature period difference is 4.5 days. These results indicate that plasma irradiation can accelerate growth in all the growth stages. Plasma irradiation not only enhances stem growth but also improves crop yield. It led to increases of 56% in the total weight of harvested seeds, 12% in the average weight of one seed, and 39% in seed number. These experiments were repeated three times and similar results were obtained.

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There are two major candidate growth enhancement mechanisms: modification of the husk of seeds^24,25) and activation of plant cells by plasma irradiation.²⁶⁾ Figure 3 shows scanning electron microscopy images of seeds for the control and plasma. There are no visible changes in the husk of seeds between the control and the plasma. This suggests that the effects of plasma-induced surface modification are minimal, and that the plasma-induced activation of plant cells is a predominant mechanism for growth enhancement. The nonthermal atmospheric-pressure plasma generates reactive oxygen species, reactive nitrogen species, ions, electric fields, and photons.^27,28) In previous studies on sterilization and plasma medicine, cell inactivation takes place after the application of oxidative stress.^29,30) From the results of our previous study,⁹⁾ the ozone concentration is 100 ppm at the position of seed irradiation, while the discharge is localized near the electrode because air with no gas flow was employed. Thus, the fluxes of short-lifetime and long-lifetime reactive species and ions might be lower than those of other atmospheric-pressure plasmas such as plasma jet. Therefore, plasma-induced cell activation might occur owing to relatively gentle oxidative stress.

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In summary, we have examined the growth enhancement of Arabidopsis thaliana (L.) from germination to harvest by nonthermal air plasma irradiation of their seeds. Three minute irradiation of the seeds led to an 11% shorter harvest period, a 56% increase in total seed weight, a 12% increase in each seed weight, and a 39% increase in seed number compared with the control. The species generated by the nonthermal atmospheric plasma has short-lifetime and local effects, indicating that the plasma is environmentally friendly. Thus, nonthermal plasma irradiation of seeds is a promising method of reducing the harvest period and improving the crop yield.

Acknowledgments