1.Introduction

Alfalfa(Medicago sativa L.)is a perennial forage crop,and it does not only have high yield,good quality,and high protein content,but also can be planted on marginal land[1,2].Alfalfa is the largest artificial pastures planted in China,and the vast majority of alfalfa is grown in the drought-stricken northern China.Drought is one of the major environmental determinants of plant growth and productivity[3].In general,exposure to a drought environment causes many common reactions in plants.It can lead to cell dehydration with concomitant osmotic changes,where the removal of water from the cytoplasm into the extracellular space leads to a decrease in cytoplasmic and vacuolar volume[4];thus,the germination of alfalfa seeds is poor under drought conditions.Ding[5]investigated the effects of drought stress on germination and early seedling growth of weedy rice by using polyethyleneglycol-6000(PEG 6000)to simulate drought stress.Gao[6]put forward an evaluation method of maize drought resistance during germination,where PEG 6000 was applied to simulate drought stress.Drought stress was applied through incubation in four different concentrations of PEG 6000 in the research of Goraghani[7].Guo[8]studied the seed germination characteristics and drought resistance of legume forage species under different concentrations(5%,10%,and 15%)of PEG 6000.Therefore,the 0%,5%,10%,and 15%of PEG 6000 were chosen to simulate the different drought stresses for alfalfa seeds in this research.

Cold plasma technology(CPT)is an alternative to the traditional pretreatment methods in agricultural science,such as physical scratching,heat treatment,and chemical treatment.The vitality of seeds can be activated by CPT before sowing[9].Cold plasma,a type of non-thermal plasma,is an electrically energized substance in a gaseous state that is not in thermodynamic equilibrium,and can be generated through electric discharge in a gaseous environment[10].Non-thermal plasma,as opposed to thermal plasma,which is characterized by high temperatures,can work at low temperatures.Therefore,non-thermal plasma works effectively without causing thermal damage to surfaces[11].A large number of related studies on the effects of plasma treatment on seeds were carried out.Dobrin et al[12]reported that the plasma treatment had a positive effect on early wheat growth,and it influenced growth parameters.Matra et al[13]confirmed that plasma treatment could enhance the growth rate of radish seeds and the dry weight of radishes.Zahoranová et al[14]studied on the effect of plasma treatment on the surface wettability and germination index.Sarinont et al[15]studied the action of dielectric barrier discharge(DBD)plasma irradiation using various feeding gases on seeds of Raphanus sativus L.,and analyzed their growth.Sera et al[16]researched the influence of plasma treatment on wheat and oat germination,and early growth.Kitazaki et al[17]found that oxygen plasma irradiation could promote radish growth better than air and argon radio frequency plasma.Jiang et al[18]found that seed germination potential,germination rate,plant height,root length,fresh weight,plant height,stem diameter,leaf area,and leaf thickness were significantly improved by a CPT of 80 W,indicating that CPT could promote the growth of wheat.Shao[19]found that CPT could improve alfalfa seed germination,water absorption ability,and seedling growth.This may be because when the seeds passed through the cold plasma glow discharge zone,the cold plasma and seed surface would interact.CPT could improve the permeability,strengthen the imbibition to promote water and other necessary chemicals permeating into the seeds quickly,and provide a better condition for seed germination[14].At the same time,the biological macromolecules in seeds were affected,and internal transfer of energy occurred.For instance,a part of the energy absorbed can transfer electronic vibrations to heat.When the molecule absorbs the photon,its electron will transform from a low energy state(the ground state)to a higher energy state(excited state);this makes the molecules absorb energy,resulting in energy level transition.The effect of plasma treatment on many different kinds of seeds have been reported,and they concluded that germination potential,germination rate,roots,growth,yield,quality,and other indicators were improved after plasma treatment.However,these features of alfalfa seeds have not been studied under different drought conditions.The objectives of this research were to investigate the effect of CPT on alfalfa seeds under different drought conditions and to provide theoretical evidence for the application of plasma treatment on alfalfa seeds under these drought conditions.The long term goal of this research is to increase the quality and quantity of alfalfa on different drought lands through pretreating alfalfa seeds using suitable technology.

Figure 1.Schematic of plasma seed processing device.1 sample disk,2 vacuum chamber,3 vacuum gate,4 valves,5 pump,6 radio frequency(RF)power,7 bleed valve,8 gas flow meter,and 9 helium gas cylinders.

2.Materials and methods

2.1.Tested material

Alfalfa seeds were selected for this study since alfalfa represents one of the most important forage grasses in the world.The alfalfa seeds ‘Zhongmu 6’were obtained from Shandong Province Seeds Group Co.Ltd Seeds,harvested in 2015.Through visual selection,only healthy seeds without obvious defects were selected.

2.2.Seed processing device and principles

A plasma seed processing device(2SZ-10 RF,made by our team),as shown in figure 1,was used to activate crop seeds.The device was made of a vacuum system,gas distribution system,and plasma generation system.The vacuum system consisted of a vacuum chamber,a vacuum gate,valves,a pump,and a bleed valve.The gas distribution system consisted of gas cylinders,a gas flow meter,a gas valve,and so on.The plasma generation system consisted of RF power,electrode plates,RF connecting wire,and so on.The gas medium in the closed vacuum chamber of the device was a mixture of air and helium gas.By the glow discharge of gas ionization,gas molecules and atoms that obtained extra energy were decomposed and ionized.Then,they became negatively charged electrons and positively charged ions,and formed a beam of plasma.

Table 1.The treatment with different powers and PEG 6000 solutions.

Power(W)PEG 6000(%)　0　20　40　60　80　100　120　140　160　180　200　220　240　260　280 0 CK0-0　T0-20　T0-40　T0-60　T0-80　T0-100　T0-120　T0-140　T0-160　T0-180　T0-200　T0-220　T0-240　T0-260　T0-280 5 CK5-0　T5-20　T5-40　T5-60　T5-80　T5-100　T5-120　T5-140　T5-160　T5-180　T5-200　T5-220　T5-240　T5-260　T5-280 10　CK10-0　T10-20　T10-40　T10-60　T10-80　T10-100T10-120T10-140T10-160T10-180T10-200T10-220T10-240　T10-260　T10-280 15　CK15-0　T15-20　T15-40　T15-60　T15-80　T15-100T15-120T15-140T15-160T15-180T15-200T15-220T15-240　T15-260　T15-280

2.3.Processing method

Fifteen-second CPT was conducted for the seeds after winnowing,and the power levels were 0(untreated as control sample),20,40,60,80,100,120,140,160,180,200,220,240,260,and 280 W.The weight of seeds treated with each same power level was 20.00 ± 0.10 g,and the treated seeds were stored in a cool and dry place where temperature ranged from 10 °C to 23 °C with about 20%relative humidity.The seeds were treated on October 10,2016,and the germination test was carried out on October 15,2016.

2.4.Experimental design

Experimental variables included the mass fraction of PEG 6000 and the power level of the plasma seed processing device.The mass fractions of 0%,5%,10%,and 15%of PEG 6000 were applied to simulate the drought environment.The seeds to be treated with the plasma processing device at each power level were divided into four treatments,representing four PEG 6000 treatments.These four treatments were sown on filter paper with 0%,5%,10%,and 15%PEG 6000 solution,respectively.Each treatment was further separated into four repetitions.Meanwhile,each repetition included 100 disease-free and uniform seeds in a 12 cm Petri dish.All treatments in this test are shown in table 1.All Petri dishes with seeds were placed in an incubator with 20°C constant temperature,1250 Lx light intensity,and 45%–58%relative humidity,for 10 days[20].The germination of seeds was observed every day,and the number of germinations recorded.Filter papers with the corresponding solutions were renewed in time to maintain a stable environment.After the germination test,the seedling height and root length were measured,and the germination potential,germination rate,germination index,and vigor index were calculated.

2.5.Germination rate,germination potential,and germination index

GR values with 40W CPT under different PEG 6000 stresses were close to the highest values,and no Significant differences were detected between this value and higher GR values at higher power levels.Thus,considering the power consumption of seed processing,40W was selected as the best processing power.In PEG 6000-simulated drought stress,the appropriate power of CPT could improve the GR of alfalfa seeds.

2.6.Method for measuring seedling height and root length

在亦舒的小说《朝花夕拾》中，就讲述了这样一个故事。生活在2035年的女主人公生活乏善可陈，和母亲关系尤其不佳，她总是嫌母亲太过守旧啰嗦。偶然的一次车祸让她穿越到多年以前，那时母亲尚是一个不足5岁的幼儿。真奇怪，她嫌弃自己年老的母亲，对于幼年的母亲却无比体贴。

2.7.Statistical analysis

Cold plasma seed pretreatment technology is a modern agricultural technology,and it is known as a stimulation treatment to improve seed germination[23].In this study,the germination rate of seeds was improved,like in the study by Sera[24].Plasma treatment could produce ultraviolet radiation,free radicals,and chemical reactions,all of which have important influences on the treatment effect.The plasma provides light over a wide spectral range.In the wavelength range,due to the high photon energy,UV light has an important direct impact on the photochemical reaction[25].UV light also has an indirect effect,where the free radicals are generated by UV light in the gas or liquid phase,and react with the organism[15].

When the processing power was greater than or equal to 160W,the VI under 0–10%PEG 6000 stresses were almost all higher than those of CK,but the amplitude decreased with the increase of PEG 6000 stress.Under the 15%PEG 6000 stress,a negative effect appeared,and the VI of seeds under all CPT powers was lower than CK.The trends about the VI of the seeds with CPT under different drought conditions were almost same(as shown in figure 6),and thus CPT application could enhance the drought resistance of alfalfa seeds.

3.Test results and analysis

3.1.Effect of CPT on the germination of alfalfa seeds under different PEG 6000 stresses

GP is an important index to measure seed germination,and presents the germination speed and uniformity.As shown in table 2,with the increase of treatment power under the same PEG 6000 stress,the GP of alfalfa seeds first increased,then decreased,increased,and decreased again;these were characterized by bimodal curves.When increasing power from 0 to 20 and then to 40 W,GP values increased significantly for all drought levels;however,this increasing trend became in Significant for power levels 60 W and higher.Considering power consumption,40W was considered as the best treatment power for the alfalfa seeds studied.With the increase of the mass fraction of PEG 6000,GP of the seeds with the same processing power almost showed the tendency of decrease.The decrease in GP under the 15%PEG 6000 stress was greater than those under 0%,5%,and 10%PEG 6000 stresses.Under the 15%PEG 6000 stress,the GP of seeds processed with more than 180 W were significantly lower than those for CK15-0.This indicated that the germination ability of seeds treated with high power and high mass Friction of PEG 6000 dropped,that is to say,the seeds treated with higher power had relatively poorer adaptability in the drought condition.Under the 0%,5%and 10%PEG 6000 stresses,the GP of the seeds with different CPT power levels almost increased compared to those for CK0-0,CK5-0,and CK10-0,respectively.The GP of seeds with 40 W CPT had the best performance under the different PEG 6000 stresses.

Figure 2.ANOVA of the seedling height and root length under 0%PEG 6000 stress with different treatment powers.

Figure 3.ANOVA of the seedling height and root length under 5%PEG 6000 stress with different treatment powers.

GR is another important index to measure the quality of seeds.A higher GR represents better embryo development and seed vitality.As shown in table 3,the effect of CPT on alfalfa seeds was different under different PEG 6000 stresses.Under 0%PEG 6000 stress,the GR of alfalfa seeds with 20–260 W CPT were all higher than that of CK0-0.Under the 5%and 10%PEG 6000 stresses,the GR of alfalfa seeds treated with 20–280 W CPT were higher than those of CK5-0 and CK10-0,respectively.Under the 15%PEG 6000 stress,the GR of alfalfa seeds treated with 40–120 W and 160 W CPT were higher than that of CK15-0;a decrease was observed in the other treatment groups.This indicated that the promoting effect of CPT with low power on GR was more Significant under 0%or lower PEG 6000 stresses.The alfalfa seeds treated by lower-power CPT showed better adaptability under lower degrees of drought stresses.

Figure 4.ANOVA of the seedling height and root length under 10%PEG 6000 stress with different treatment powers.

Figure 5.ANOVA of the seedling height and root length under 15%PEG 6000 stress with different treatment powers.

The calculation formulas were as follows.Here,GP is the germination potential,GR is the germination rate,GI is the germination index,VI is the vigor index,n1is the germination number on the fourth day of treatment,n2is the germination number on the tenth day,N is the total number of seeds,Gtis the number of seed germination on the tth day,Dtis the number of days of germination,and S is the average length of seedlings[21].

As shown in table 4,the best processing power,40 W,corresponded to the maximum GI value under the different PEG 6000 stresses.The only exception is the GI value at 10%of PEG 6000;however,its maximum(39.48)is not significantly lower than the GI value(39.88)at 120W.Thus,40 W remains as the best processing power for the highest GI.The GI of alfalfa seeds treated by the same CPT almost decreased with increasing PEG 6000 concentration,and this trend was consistent with those seen for GP and GR.When the treatment power was 40 W,the GI was the highest(or close to the highest)under the different PEG 6000 stresses,and was significantly higher than that of CK0-0,CK5-0,CK10-0,and CK15-0;thus,40 W was considered as the best treatment power.The GI of alfalfa seeds under drought could be improved by using the appropriate power of CPT;the GI with CPT might even be higher than that under the no drought condition(CK0-0).

在我国现行的司法体制中，宪法、民法、刑法、相关司法解释和法规条例都对公民隐私权有一定的规定，但仍不完善[4]。

3.2.Effect of CPT on seedling growth of alfalfa seeds under different PEG 6000 stresses

Ten strains of uniform growing seedlings were selected from each repetition to measure the seedling height and root length at the last count.Variance analysis using a single factor randomized block test was carried out,and the results are shown in figures 2–5,where seedling height was positive,and root length was negative.The seedling height and root length were affected to different degrees by the CPT under the different PEG 6000 stresses.Under the 0%PEG 6000 stress,the seedling height with 40 and 140 W CPT increased significantly,and the root length with 40,60,80,120,and 160W CPT increased remarkably,compared with those of CK0-0.The seedling height and root length with 40 W CPT were better than those of CK0-0.Under the 5%PEG 6000 stress,the CPT had little effect on seedling height;it mainly affected root length,especially of seeds with 40,80,and 100W CPT.Under the 10%PEG 6000 stress,the seedling growth of alfalfa seeds reached the best growth condition after CPT,and the seedling height and root length showed great growth advantages compared with those for CK10-0.The seedling height with 20,40,80,100,140,160,240,and 280W CPT treatment was significantly better than those for CK10-0;heights under the other treatment powers(except 200W)were slightly higher than those for CK10-0.Meanwhile,the root length of alfalfa seeds with CPT was higher than that of CK10-0;the effects of 20,60,80,100,120,180,200,220,and 240 W power were particularly obvious.Under the 15%PEG 6000 stress,the seedling height with 80,180,and 200 W CPT,and the root length with 40 and 140 W,increased.

向南，深化以东盟为重点的“一带一路”沿线国家的旅游合作，对接海南自贸区，联手海南，深耕东盟。全面加强与泛北部湾地区各国的旅游务实合作，完善合作机制，大力发展滨海度假旅游，积极开发海上丝绸之路跨国旅游环线和泛北部湾区域内旅游线路，着力打造以海上跨国邮轮度假旅游为主体的国际精品旅游线路；加快北海区域性国际邮轮母港建设，利用涠洲岛西北部天然良港的优势，打造国际游轮中转港，大力发展“一程多站”式的邮轮度假产品，深度开发环岛度假慢行游和海岛生态休闲游。

3.3.Effect of CPT on the vigor index of alfalfa seeds under different PEG 6000 stresses

As shown in figure 6,the VI of alfalfa seeds with 20–140 W CPT were higher than CK under 0–15%PEG 6000 stresses,indicating that low CPT power could promote the germination of alfalfa seeds,and provided better adaptability under different drought environments.The VI of seeds with 40 W CPT under 0%,5%,10%,and 15%PEG 6000 stresses increased by 38.68%,43.91%,74.34%,and 39.20%,respectively,compared to CK0-0,CK5-0,CK10-0,and CK15-0;these increases uniformly achieved the best state of VI.

“保供工作做好了，每年春耕、秋收提供的油品可以达到50万吨，几乎占到黑龙江销售全年柴油销量的三分之一，从这个角度说，保供不仅是体现社会责任，更是体现经济责任。”黑龙江销售加油站管理处处长刘延奎说。

Ten uniform growing seedlings were selected from each repetition at the last count.Seedling height and root length were determined with a millimeter-scale ruler.

Figure 6.Vigor indexes of alfalfa seeds under CPT and different PEG 6000 stresses.

4.Discussion

Statistical analysis was carried out by SPSS program,and single factor random block analysis was used.Multiple comparisons were performed using Duncan post hoc tests.The results showed mean and standard deviation for each treatment.The correlation coefficients among each group were analyzed by a Pearson test,and the difference was Significant at the level of P ≤ 0.05[22].

During interaction between CPT and seeds,modification of the seed surface is important,which could improve the seed coat permeability,enhance imbibitions,and ensure that moisture and other necessary materials permeate into the seed quickly to provide active materials for seed germination[16,26].However,the best treatment effect of different crops might depend on seed size,seed coat hardness,and so on.

随着我国城市化发展步伐的逐步加快，人们开始源源不断地涌入城市，使得出行成为了一大难题，一系列交通运输问题随之而来。而城市地铁具有运输量大、速度快、节能、准时等诸多特点，所以近几年来已经成为了城市生活的一种主要出行工具，随着城市地铁线路的设计范围逐渐扩大，同时也拓宽了城市地铁网络系统的搭建范围，因此使得地铁换乘站的建筑设计也成为了当前地铁运输的重点内容，并且实际建筑效果也会对地铁服务质量产生重要影响，因此要致力于研究城市地铁换乘站的建筑设计。为此，本文主要对当前城市地铁换乘存在的问题进行了分析，并对城市地铁换乘站的建筑设计进行了初步探究。

There was no linear relationship between improvement effect and treatment power in different groups.In addition,previous studies found that high power or a long processing time resulted in inhibition of seed germination[27–30].Therefore,it is important to select a suitable power to process the seeds.The germination of alfalfa seeds with 40 W CPT was significantly improved,and yielded the best VI.The 40 W CPT was applied to the alfalfa seeds to show the better adaptability under the different PEG 6000 stresses;our results were consistent with previous results that the plasma could promote the drought resistance of alfalfa seeds.The GI of alfalfa seeds with CPT improved,which might be related to an increase in cell membrane permeability.Due to the effect of CPT,the seed coat structure became slightly loose,which increased the plasma membrane permeability,and thus the energy metabolism inside and outside the seed coat could be accelerated.This promoted germination and seedling growth.However,whether CPT can induce enzyme gene expression related to drought resistance or improve the drought resistance through other mechanisms are still unknown.The mechanism　through which CPT　enhances the drought resistance of plants will be further revealed with the continuous efforts of researchers.

5.Conclusion

The GP,GR,GI,seedling height,and root length of the alfalfa seeds were affected by different powers of CPT under different PEG 6000 stress.The VI was related to treatment power and water supply.The improvement of alfalfa seeds with lower power CPT was obvious,and these seeds had better adaptability to different drought conditions.On the other hand,the VI of alfalfa seeds with high-power CPT was decreased and the drought resistance was poor.The GP,GR,GI,and VI of the alfalfa seeds with 40 W CPT were significantly　improved,and　VI respectively　increased　by 38.68%,43.91%,74.34%,and 39.20%under the 0%,5%,10%,and 15%PEG 6000 stresses,compared with CK sample groups.The increase of VI at this power was high,and its best value was reached.In conclusion,the 40 W CPT was the best CPT power to adapt to different drought conditions.

①东北四省区节水增粮行动项目水资源论证工作坚定地贯彻执行最严格水资源管理制度，保障项目建设和布局与区域水资源承载能力相适应，合理利用水资源，提高了水资源利用效率和效益，确保节水增粮增效，成效显著。

This work was supported by China Agriculture Research System(CARS-34).

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