空气等离子体预处理提高穿心莲种子活力的研究
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摘要
种子活力(seed vigor)即种子健壮度,是种子内在的发芽、生长及生产的潜力。高活力的种子萌发迅速而整齐,幼苗健壮并能抵抗逆境的影响,植株充分生长发育,获得高产、稳产。本文针对由于种质退化等原因引起的穿心莲种子活力降低、质量参差不齐的问题开展研究,包括制订种子质量检验规程及种子质量分级标准,为种子质量评价提供依据；并选择一批质量合格且净度高的种子进行介质阻挡放电大气压空气等离子体预处理,考察其对种子活力的影响,以期找到促进穿心莲种子萌发、出苗、幼苗及植株生长,并提高产量的处理剂量。
1.穿心莲种子质量检验规程的制订：
按照《农作物种子检验规程》及《<规程>实施指南》的指导性原则制订了穿心莲种子的真实性鉴定、净度分析、重量测定、水分测定、生活力测定及发芽试验的具体检验规程。扦样种子批重量<50kg,送检样品最少重量>50g,净度分析样品≥5.0g；以百粒法进行重量测定；以130℃高恒温烘150min测定种子水分；发芽试验规程为：种子以室温水浸种18-20h,以纸上法(TP)、27.5℃进行光照培养；初次及末次计数时间分别为置床后第2d,第7d。
2.穿心莲种子质量分级标准的制订：
按照《农作物种子检验规程》及《<规程>实施指南》的指导性原则及本文制订的具体规程对我国穿心莲主产区的种子进行了质量检测。对国内常用的4大质量指标(发芽率、水分、千粒重及净度)检测结果进行K-means聚类,围绕聚类中心值,结合生产实际和可操作性制订穿心莲种子质量分级标准,把穿心莲种子分为3级。其中1级种子要求发芽率≥85%,水分≤14.0%,千粒重≥1.2g,净度≥85%,为优质种子；2级种子发芽率65.0-85.0%,水分≤14.0%,千粒重1.1-1.2g,净度80.0-85.0%,为合格种子；任何一项低于2级标准为不合格种子。
不同等级穿心莲种子重复育苗试验表明：质量不合格种子出苗率差,基本无种用价值。进行GAP生产应选择1级的优质种子进行育苗种植。
3.空气等离子体对穿心莲种皮透性、种皮结构及荫发率的改变：
发芽试验表明：5950V激发的空气等离子体处理穿心莲种子60s虽然对萌发率的影响与对照组比较未见显著性差异；然而,由于初生根长度显著小于对照组,从而引起萌发活力指数(vigor index)显著低于对照组；可见,较长时间等离子体处理有一定抑制萌发的作用。推测是由于种子表面受到等离子体再沉积(re-deposition)作用(扫描电镜观察)的影响,阻碍了吸胀的进程,使得发芽受到抑制。而较低的激发电压或较短的处理时间(30s)穿心莲种子萌发参数改变不明显。
后续的试验缩短了处理时间,处理剂量分别为3400V、4250V、5100V、5950V处理l0s以及20s。
4250V激发的空气等离子体处理10s以及3400V和5950V处理20s后,种子的发芽势及活力指数显著高于对照组；可见,较低剂量的空气等离子体处理能够促进穿心莲种子萌发。
种子浸出液电导率测定表明：空气等离子体确实能改变穿心莲种皮的透性,其中5950V处理10s种皮透性明显高于对照组,推测是等离子体刻蚀作用使得种子表面颗粒状的突起上产生多数细小的孔洞(扫描电镜观察)引起透性增强并促进萌发。3400V处理20s种皮被刻蚀的孔洞较少,透性明显低于对照组；推测是再沉积作用占优势。
4.空气等离子体预处理对穿心莲种子出苗率、幼苗生长量、蛋白质积累、抗氧化酶类活性及其同工酶表达的影响：
育苗试验表明：空气等离子体处理改变了穿心莲种子的出苗速率；不同剂量的效应不一致。5950V电压激发的空气等离子体处理种子10s能够提高出苗速率,使得穿心莲提前出苗,并提高壮苗率。5100V处理20s有相似的效应,但与对照组比较的差异不显著。而5100V处理l0s及3400处理20s对后期出苗有抑制作用。
空气等离子体预处理穿心莲种子对苗期株高及真叶面积具有后续效应。5950V处b理10s及3400V处理20s显著促进了株高的增长；3400V处理10s及4250V处理20s抑制了株高的增长。5950V处理20s对真叶面积增加有明显促进作用,然而对幼苗干重却无正面的影响；而3400V处理10s,4250V处理10s及4250V处理20s显著抑制了真叶面积的增长。处理时间为10s随着电压升高,对株高及叶面积的增长都有促进的作用；处理时间为20s,随着电压的升高,对幼苗干重的增加有抑制作用。综合看来,5100V处理10s、5950V处理10s及3400V处理20s促进幼苗生长效果较好。
通过测定穿心莲幼苗叶片SOD、POD、CAT活性,MDA含量以及可溶性蛋白含量表明：空气等离子体预处理种子改变了穿心莲50日龄幼苗抗氧化酶活性和细胞脂质过氧化程度；同时,对于苗期蛋白质的积累有促进作用。5950V电压处理种子10s能激发穿心莲幼苗的CAT酶活性,降低MDA含量,表明环境胁迫下细胞过氧化损伤程度减轻；换而言之,就是抵御外界环境的胁迫能力增强。
采用聚丙烯酰胺凝胶电泳分析表明：空气等离子体预处理穿心莲种子对幼苗的CAT、SOD、POD及APX同工酶的表达都有影响。其中5950s处理10s及3400V处理20s明显促进了幼苗期CAT-1表达。3400V,4250V,5100V处理10s和20s促进了SOD-2的表达。5950V处理10s和20s则分别(比对照组)各少了1条窄的酶条带POD-4和POD-1。等离子对各组APX的表达都有一定影响。
综合以上结果,大气压空气等离子体预处理穿心莲种子对幼苗活力具有后续影响。其中,5950V处理l0s能够促进穿心莲种子提前出苗,增加壮苗；对于苗期的株高及幼苗鲜重增长效应明显；促进苗期初生代谢产物(蛋白质)的积累；促进过氧化氢酶(CAT)同工酶的表达,提高过氧化氢酶的活性,并且细胞脂质过氧化程度轻。因此,5950V处理10s是较为理想的处理剂量。
3400V处理20s有促进幼苗株高增长,鲜重增加,蛋白质积累增加,以及促进CAT同工酶的表达的效应；然而,其出苗速度受到抑制,壮苗少,并不是理想的处理剂量。
5.空气等离子体预处理种子对穿心莲植株生长及药材产量的影响：
第1次栽培试验,种植40天后,各处理组的植株生长量与对照组比较总体差异不大。第2次栽培试验,种植30天后,处理组的株高、茎节数、叶片数均低于对照组；然而,第3、4、5组的第3对真叶面积大于对照组。两次栽培试验生长量结果的不尽一致可能是由于植株处在不同生长时期(不同植株年龄)引起的。
从最终药材产量看来,空气等离子体预处理种子10s,随着处理电压的升高,各组药材的鲜重、干重及折干率有逐渐升高的趋势,5950V处理10s与对照组比较达到显著性差异。
综上所述,空气等离子体预处理种子确实可以通过刻蚀种皮改善通透性等作用促进种子萌发、出苗及幼苗生长。其中5950V处理10s对于穿心莲种子活力的强化有显著的作用,是较好的处理剂量。空气等离子体所含的活性分子、带电粒子等是影响种子活力,继发使得萌发后幼苗及植株的生长发生改变的重要因素；然而其作用机理有待深入研究。
Seed vigor is the total sum of those properties of the seed which determine the potential level of activity and performance of the seed or seed lot during germination and seedling emergence. It reflects the potential activities of germination, seedling emergence, plant growth and production. That means seeds with high vigor level germinate rapidly and orderly, develope into strong seedlings that can resist environment stress, result in healthy plant growth and stable and high production. However, the seed vigor of Andrographis paniculata decline because of germ-plasm degeneration and other reasons, lead to variation of seed quality. This dissertation focuses on the following issues:establish the seed testing rules and seed quality grade standard for A. paniculata which mean testing method and quality evaluation. One qualified seed lot with high seed purity level was selected for pretreatment using atmospheric pressure dielectric barrier discharge air plasma with different dose. Seed invigoration effects and an optimal plasma treating dose which can increase seed vigor of A. paniculata, then accelerate the germination and seedling emergence, promote plant growth, finally increase yield, were investigated. Methods and results were as following:
1. Establishment of seed testing rules for A. paniculata:
Based on the Rules for agricultural seed testing and its implementation guide, testing rules of seed trueness, purity analysis, weight determination, moisture content determination, viability test by TTC method, and germination test were established. Weight of sampling seed lot, seed testing sample, and seed sample for purity analysis were prescribed as less than50kg, more than50g, and no less than5.0g, respectively;100seeds method was proposed for seed weight determination; high-temperature (130℃) baking method for moisture content determination. Germination test procedure was established as: seeds soaked in water at R.T. for18-20h, then cultured at27.5℃by TP method (top of paper) under light; germinated seed should be counted on the2nd and7th day after sowing for the first and last time, respectively.
2. Establishment of seed quality grade standard for A. paniculata:
Seed lots from main producing area in China were tested according to the testing rules for A. paniculata and based on the guideline of Rules for agricultural seed testing and its implementation guide. Data of germination percentage, moisture content,1000seeds weight, and seed purity were classified into3grades by K-means cluster analysis. Then, the seed quality grading standard was preliminarily established by cluster center in combination with actual institution, agricultural production needs and maneuverability. The germination percentage, moisture content,1000seeds weight, and seed purity of1st grade high quality seed were prescribed as no less than85%, no more than14.0%, no less than1.2g, and no less than85%, respectively; and2nd grade seeds prescribed as65.0-85.0%, no more than14.0%,1.1-1.2g, and80.0-85.0%, respectively; otherwise, the seed lot would be disqualified.
Different grade seeds lots were used to conduct seedling emergence test, results showed that seedling emergence percentage of unqualified seeds (3rd grade) was poor, and had no cultivated value. In good agricultural practice, the1st grade seed lot is suggested to be chosen for cultivation.
3. Effects of air plasma on germination, penetrability, ultra-microscopy surface characteristic of A. paniculata seeds:
Germination test showed that germination percentage of seeds pretreated by air plasma excitated in5950V for60s showed no significant difference compared with the Ctrl (untreated). However, the primary root length of the treated seeds was significantly shorter than that of the Ctrl, resulted in lower germination vigor index. That means long time air plasma treatment can lead to germination suppression. The seed coat re-deposition effect of plasma observed by SEM was presumed to be the reason of suppression. Germination of the other treated seeds by lower voltage or shorter time had no significant change.
The plasma exposure time in follow-up investigation was decreased as3400V.4250V.5100V,5950V treated for10s (group1-group4), and3400V,4250V,5100V,5950V for20s (group5-group8).
Results showed that the germination energy of group1,2,3,4,5,6and8were higher than Ctrl; and the germination vigor index of group2,5,8were both significantly higher than Ctrl. It indicates that lower air plasma treatment could promote the germination.
Electric conductivity (EC) determination of soaked seeds leakage showed that air plasma could change the permeability of A. paniculata seeds. The EC of seeds treated by5950V for10s was significant higher than that of Ctrl, which was explained as the etching effect of air plasma produced tiny holes on the seed coat observable by SEM, resulted in imbibition and germination acceleration. On the other side, EC of seeds treated by3400V for20s was significant lower than ctrl; and the presumable reason was predominant re-position effect by air plasma.
4. Effects of air plasma seed pretreatment on emergence, growth, and protein accumulation, antioxidant enzyme activities, antioxidant isoenzyme expression of A. paniculata seedling:
Results showed that seedling emergences were changed after air plasma seed pretreatment, effects varied in different dose. Seeds pretreated by air plasma excitated in5950V for10s achieved emergence acceleration, resulted in more amounts of strong seedlings. Seeds pretreated by5100V for20s had the similar effect, but had no significance compared with Ctrl. On the other side, pretreatment by5100V for10s and3400for20s presented inhibition effects.
There were follow-up effects on seedling height and leaf area after A. paniculata seeds pretreated by air plasma. The seedling heights were significantly increased after air plasma pretreating excitated by5950V for10s and3400V for20s, but decreased by3400V for10s and4250V for20s. Leaf areas of seedlings pretreated by5950V for20s were significantly increased, but no significant change was shown in dry weight of the seedlings. Leaf areas of seedlings pretreated by3400V for10s,4250V for10s, and4250V for20s were significantly decreased. There were also rise trend of the seedling height and leaf area when the voltage rising with10s of treating time; on the other hand, falling trend of seedling dry weight presented when voltage rising by20s of treating time. In summary, the seedling growth promotion effects of air plasma seed pretreatment by5100V for10s,5950V for10s, and3400V for20s were shown beneficial for growth of the herb.
After extraction of protein from50day seedlings, activities of SOD, POD, CAT, and content of MDA and protein were determined. Results showed that activities of antioxidant enzymes and cellular lipid peroxidation degree were change variously; protein accumulation was promoted by air plasma seed pretreatment. After seed pretreatment with5950V for10s, the CAT activity was increased and MDA content decreased, which meant alleviation of lipid peroxidation and enhancement of environment stress-resistant ability of the seedlings.
SDS-PAGE analysis of seedlings'protein appeared no significant change in protein expression. However, native-PAGE analyses showed that isoenzymes expression of CAT, SOD, POD, and APX were changed variously after plasma seed pretreatment. There was an additional clear band of CAT-1after air plasma pretreatment by5950V for10s and3400V for20s, compared with ctrl. There was one more band of SOD-2after air plasma pretreatment by3400V,4250V,5100V for10s and20s. However, there was one narrow band less of POD-4or POD-1after pretreatment by5950V for10s and20s, respectively. Expression of APX isoenzymes were all changed after different dose treatment.
Summarily, atmospheric pressure air plasma seed pretreatment have follow-up effects on seedling vigor. After seed pretreatment by5950V for10s, there were more rapid seedling emergence and strong seedlings, more accumulation of protein in seedlings, acceleration of seedling growth, promotion of CAT activity and its expression, less lipid peroxidation degree of seedling cells. Therefore, this air plasma dose is promising. As for dose of3400V for20s, the effects of increasing of seedling height, fresh weight and protein of seedling, and CAT expression were observed; however, the seedling emergence was retarded, and less strong seedlings were obtained, which means not an ideal dose.
5. Effects of air plasma seed pretreatment on plant growth and yield of A. paniculata:
Cultivation test was repeated. After planted for40d in1st cultivation test, plant growth were measured, but there were no significant difference between treated groups and ctrl group. However, after planted for30d in2nd cultivation test, data of seedling height, node number, and leaf number of treated groups were all smaller than ctrl. But, the3rd leaf areas of group3,4, and5(the treating dose were5100V for10s,5950V for10s, and3400V for20s) were larger than ctrl. Different results of these two cultivation tests were presumed to be the influence of the different development stage.
As for the yield, there were increasing trend of fresh weight, dry weight, and ratio of dry weight with fresh weight, along with the increasing treating voltage when treating time was10s; especially, the data of group4(treated by5950V for10s) were significantly higher than ctrl.
All in all, after air plasma seed pretreatment, germination and seedling emergence may be enhanced by effect of seed coat etching follow-up penetrability increase. The active species, charge particles in air plasma should play a key role in seed vigor change, lead to variation of seedling and plant growth. However, more direct evidence is required to explain the mechanism. In this study, A. paniculata seed treated by air plasma excitated at5950V for10s actually gained seed invigoration.
1.穿心莲种子质量检验规程的制订：
按照《农作物种子检验规程》及《<规程>实施指南》的指导性原则制订了穿心莲种子的真实性鉴定、净度分析、重量测定、水分测定、生活力测定及发芽试验的具体检验规程。扦样种子批重量<50kg,送检样品最少重量>50g,净度分析样品≥5.0g；以百粒法进行重量测定；以130℃高恒温烘150min测定种子水分；发芽试验规程为：种子以室温水浸种18-20h,以纸上法(TP)、27.5℃进行光照培养；初次及末次计数时间分别为置床后第2d,第7d。
2.穿心莲种子质量分级标准的制订：
按照《农作物种子检验规程》及《<规程>实施指南》的指导性原则及本文制订的具体规程对我国穿心莲主产区的种子进行了质量检测。对国内常用的4大质量指标(发芽率、水分、千粒重及净度)检测结果进行K-means聚类,围绕聚类中心值,结合生产实际和可操作性制订穿心莲种子质量分级标准,把穿心莲种子分为3级。其中1级种子要求发芽率≥85%,水分≤14.0%,千粒重≥1.2g,净度≥85%,为优质种子；2级种子发芽率65.0-85.0%,水分≤14.0%,千粒重1.1-1.2g,净度80.0-85.0%,为合格种子；任何一项低于2级标准为不合格种子。
不同等级穿心莲种子重复育苗试验表明：质量不合格种子出苗率差,基本无种用价值。进行GAP生产应选择1级的优质种子进行育苗种植。
3.空气等离子体对穿心莲种皮透性、种皮结构及荫发率的改变：
发芽试验表明：5950V激发的空气等离子体处理穿心莲种子60s虽然对萌发率的影响与对照组比较未见显著性差异；然而,由于初生根长度显著小于对照组,从而引起萌发活力指数(vigor index)显著低于对照组；可见,较长时间等离子体处理有一定抑制萌发的作用。推测是由于种子表面受到等离子体再沉积(re-deposition)作用(扫描电镜观察)的影响,阻碍了吸胀的进程,使得发芽受到抑制。而较低的激发电压或较短的处理时间(30s)穿心莲种子萌发参数改变不明显。
后续的试验缩短了处理时间,处理剂量分别为3400V、4250V、5100V、5950V处理l0s以及20s。
4250V激发的空气等离子体处理10s以及3400V和5950V处理20s后,种子的发芽势及活力指数显著高于对照组；可见,较低剂量的空气等离子体处理能够促进穿心莲种子萌发。
种子浸出液电导率测定表明：空气等离子体确实能改变穿心莲种皮的透性,其中5950V处理10s种皮透性明显高于对照组,推测是等离子体刻蚀作用使得种子表面颗粒状的突起上产生多数细小的孔洞(扫描电镜观察)引起透性增强并促进萌发。3400V处理20s种皮被刻蚀的孔洞较少,透性明显低于对照组；推测是再沉积作用占优势。
4.空气等离子体预处理对穿心莲种子出苗率、幼苗生长量、蛋白质积累、抗氧化酶类活性及其同工酶表达的影响：
育苗试验表明：空气等离子体处理改变了穿心莲种子的出苗速率；不同剂量的效应不一致。5950V电压激发的空气等离子体处理种子10s能够提高出苗速率,使得穿心莲提前出苗,并提高壮苗率。5100V处理20s有相似的效应,但与对照组比较的差异不显著。而5100V处理l0s及3400处理20s对后期出苗有抑制作用。
空气等离子体预处理穿心莲种子对苗期株高及真叶面积具有后续效应。5950V处b理10s及3400V处理20s显著促进了株高的增长；3400V处理10s及4250V处理20s抑制了株高的增长。5950V处理20s对真叶面积增加有明显促进作用,然而对幼苗干重却无正面的影响；而3400V处理10s,4250V处理10s及4250V处理20s显著抑制了真叶面积的增长。处理时间为10s随着电压升高,对株高及叶面积的增长都有促进的作用；处理时间为20s,随着电压的升高,对幼苗干重的增加有抑制作用。综合看来,5100V处理10s、5950V处理10s及3400V处理20s促进幼苗生长效果较好。
通过测定穿心莲幼苗叶片SOD、POD、CAT活性,MDA含量以及可溶性蛋白含量表明：空气等离子体预处理种子改变了穿心莲50日龄幼苗抗氧化酶活性和细胞脂质过氧化程度；同时,对于苗期蛋白质的积累有促进作用。5950V电压处理种子10s能激发穿心莲幼苗的CAT酶活性,降低MDA含量,表明环境胁迫下细胞过氧化损伤程度减轻；换而言之,就是抵御外界环境的胁迫能力增强。
采用聚丙烯酰胺凝胶电泳分析表明：空气等离子体预处理穿心莲种子对幼苗的CAT、SOD、POD及APX同工酶的表达都有影响。其中5950s处理10s及3400V处理20s明显促进了幼苗期CAT-1表达。3400V,4250V,5100V处理10s和20s促进了SOD-2的表达。5950V处理10s和20s则分别(比对照组)各少了1条窄的酶条带POD-4和POD-1。等离子对各组APX的表达都有一定影响。
综合以上结果,大气压空气等离子体预处理穿心莲种子对幼苗活力具有后续影响。其中,5950V处理l0s能够促进穿心莲种子提前出苗,增加壮苗；对于苗期的株高及幼苗鲜重增长效应明显；促进苗期初生代谢产物(蛋白质)的积累；促进过氧化氢酶(CAT)同工酶的表达,提高过氧化氢酶的活性,并且细胞脂质过氧化程度轻。因此,5950V处理10s是较为理想的处理剂量。
3400V处理20s有促进幼苗株高增长,鲜重增加,蛋白质积累增加,以及促进CAT同工酶的表达的效应；然而,其出苗速度受到抑制,壮苗少,并不是理想的处理剂量。
5.空气等离子体预处理种子对穿心莲植株生长及药材产量的影响：
第1次栽培试验,种植40天后,各处理组的植株生长量与对照组比较总体差异不大。第2次栽培试验,种植30天后,处理组的株高、茎节数、叶片数均低于对照组；然而,第3、4、5组的第3对真叶面积大于对照组。两次栽培试验生长量结果的不尽一致可能是由于植株处在不同生长时期(不同植株年龄)引起的。
从最终药材产量看来,空气等离子体预处理种子10s,随着处理电压的升高,各组药材的鲜重、干重及折干率有逐渐升高的趋势,5950V处理10s与对照组比较达到显著性差异。
综上所述,空气等离子体预处理种子确实可以通过刻蚀种皮改善通透性等作用促进种子萌发、出苗及幼苗生长。其中5950V处理10s对于穿心莲种子活力的强化有显著的作用,是较好的处理剂量。空气等离子体所含的活性分子、带电粒子等是影响种子活力,继发使得萌发后幼苗及植株的生长发生改变的重要因素；然而其作用机理有待深入研究。
Seed vigor is the total sum of those properties of the seed which determine the potential level of activity and performance of the seed or seed lot during germination and seedling emergence. It reflects the potential activities of germination, seedling emergence, plant growth and production. That means seeds with high vigor level germinate rapidly and orderly, develope into strong seedlings that can resist environment stress, result in healthy plant growth and stable and high production. However, the seed vigor of Andrographis paniculata decline because of germ-plasm degeneration and other reasons, lead to variation of seed quality. This dissertation focuses on the following issues:establish the seed testing rules and seed quality grade standard for A. paniculata which mean testing method and quality evaluation. One qualified seed lot with high seed purity level was selected for pretreatment using atmospheric pressure dielectric barrier discharge air plasma with different dose. Seed invigoration effects and an optimal plasma treating dose which can increase seed vigor of A. paniculata, then accelerate the germination and seedling emergence, promote plant growth, finally increase yield, were investigated. Methods and results were as following:
1. Establishment of seed testing rules for A. paniculata:
Based on the Rules for agricultural seed testing and its implementation guide, testing rules of seed trueness, purity analysis, weight determination, moisture content determination, viability test by TTC method, and germination test were established. Weight of sampling seed lot, seed testing sample, and seed sample for purity analysis were prescribed as less than50kg, more than50g, and no less than5.0g, respectively;100seeds method was proposed for seed weight determination; high-temperature (130℃) baking method for moisture content determination. Germination test procedure was established as: seeds soaked in water at R.T. for18-20h, then cultured at27.5℃by TP method (top of paper) under light; germinated seed should be counted on the2nd and7th day after sowing for the first and last time, respectively.
2. Establishment of seed quality grade standard for A. paniculata:
Seed lots from main producing area in China were tested according to the testing rules for A. paniculata and based on the guideline of Rules for agricultural seed testing and its implementation guide. Data of germination percentage, moisture content,1000seeds weight, and seed purity were classified into3grades by K-means cluster analysis. Then, the seed quality grading standard was preliminarily established by cluster center in combination with actual institution, agricultural production needs and maneuverability. The germination percentage, moisture content,1000seeds weight, and seed purity of1st grade high quality seed were prescribed as no less than85%, no more than14.0%, no less than1.2g, and no less than85%, respectively; and2nd grade seeds prescribed as65.0-85.0%, no more than14.0%,1.1-1.2g, and80.0-85.0%, respectively; otherwise, the seed lot would be disqualified.
Different grade seeds lots were used to conduct seedling emergence test, results showed that seedling emergence percentage of unqualified seeds (3rd grade) was poor, and had no cultivated value. In good agricultural practice, the1st grade seed lot is suggested to be chosen for cultivation.
3. Effects of air plasma on germination, penetrability, ultra-microscopy surface characteristic of A. paniculata seeds:
Germination test showed that germination percentage of seeds pretreated by air plasma excitated in5950V for60s showed no significant difference compared with the Ctrl (untreated). However, the primary root length of the treated seeds was significantly shorter than that of the Ctrl, resulted in lower germination vigor index. That means long time air plasma treatment can lead to germination suppression. The seed coat re-deposition effect of plasma observed by SEM was presumed to be the reason of suppression. Germination of the other treated seeds by lower voltage or shorter time had no significant change.
The plasma exposure time in follow-up investigation was decreased as3400V.4250V.5100V,5950V treated for10s (group1-group4), and3400V,4250V,5100V,5950V for20s (group5-group8).
Results showed that the germination energy of group1,2,3,4,5,6and8were higher than Ctrl; and the germination vigor index of group2,5,8were both significantly higher than Ctrl. It indicates that lower air plasma treatment could promote the germination.
Electric conductivity (EC) determination of soaked seeds leakage showed that air plasma could change the permeability of A. paniculata seeds. The EC of seeds treated by5950V for10s was significant higher than that of Ctrl, which was explained as the etching effect of air plasma produced tiny holes on the seed coat observable by SEM, resulted in imbibition and germination acceleration. On the other side, EC of seeds treated by3400V for20s was significant lower than ctrl; and the presumable reason was predominant re-position effect by air plasma.
4. Effects of air plasma seed pretreatment on emergence, growth, and protein accumulation, antioxidant enzyme activities, antioxidant isoenzyme expression of A. paniculata seedling:
Results showed that seedling emergences were changed after air plasma seed pretreatment, effects varied in different dose. Seeds pretreated by air plasma excitated in5950V for10s achieved emergence acceleration, resulted in more amounts of strong seedlings. Seeds pretreated by5100V for20s had the similar effect, but had no significance compared with Ctrl. On the other side, pretreatment by5100V for10s and3400for20s presented inhibition effects.
There were follow-up effects on seedling height and leaf area after A. paniculata seeds pretreated by air plasma. The seedling heights were significantly increased after air plasma pretreating excitated by5950V for10s and3400V for20s, but decreased by3400V for10s and4250V for20s. Leaf areas of seedlings pretreated by5950V for20s were significantly increased, but no significant change was shown in dry weight of the seedlings. Leaf areas of seedlings pretreated by3400V for10s,4250V for10s, and4250V for20s were significantly decreased. There were also rise trend of the seedling height and leaf area when the voltage rising with10s of treating time; on the other hand, falling trend of seedling dry weight presented when voltage rising by20s of treating time. In summary, the seedling growth promotion effects of air plasma seed pretreatment by5100V for10s,5950V for10s, and3400V for20s were shown beneficial for growth of the herb.
After extraction of protein from50day seedlings, activities of SOD, POD, CAT, and content of MDA and protein were determined. Results showed that activities of antioxidant enzymes and cellular lipid peroxidation degree were change variously; protein accumulation was promoted by air plasma seed pretreatment. After seed pretreatment with5950V for10s, the CAT activity was increased and MDA content decreased, which meant alleviation of lipid peroxidation and enhancement of environment stress-resistant ability of the seedlings.
SDS-PAGE analysis of seedlings'protein appeared no significant change in protein expression. However, native-PAGE analyses showed that isoenzymes expression of CAT, SOD, POD, and APX were changed variously after plasma seed pretreatment. There was an additional clear band of CAT-1after air plasma pretreatment by5950V for10s and3400V for20s, compared with ctrl. There was one more band of SOD-2after air plasma pretreatment by3400V,4250V,5100V for10s and20s. However, there was one narrow band less of POD-4or POD-1after pretreatment by5950V for10s and20s, respectively. Expression of APX isoenzymes were all changed after different dose treatment.
Summarily, atmospheric pressure air plasma seed pretreatment have follow-up effects on seedling vigor. After seed pretreatment by5950V for10s, there were more rapid seedling emergence and strong seedlings, more accumulation of protein in seedlings, acceleration of seedling growth, promotion of CAT activity and its expression, less lipid peroxidation degree of seedling cells. Therefore, this air plasma dose is promising. As for dose of3400V for20s, the effects of increasing of seedling height, fresh weight and protein of seedling, and CAT expression were observed; however, the seedling emergence was retarded, and less strong seedlings were obtained, which means not an ideal dose.
5. Effects of air plasma seed pretreatment on plant growth and yield of A. paniculata:
Cultivation test was repeated. After planted for40d in1st cultivation test, plant growth were measured, but there were no significant difference between treated groups and ctrl group. However, after planted for30d in2nd cultivation test, data of seedling height, node number, and leaf number of treated groups were all smaller than ctrl. But, the3rd leaf areas of group3,4, and5(the treating dose were5100V for10s,5950V for10s, and3400V for20s) were larger than ctrl. Different results of these two cultivation tests were presumed to be the influence of the different development stage.
As for the yield, there were increasing trend of fresh weight, dry weight, and ratio of dry weight with fresh weight, along with the increasing treating voltage when treating time was10s; especially, the data of group4(treated by5950V for10s) were significantly higher than ctrl.
All in all, after air plasma seed pretreatment, germination and seedling emergence may be enhanced by effect of seed coat etching follow-up penetrability increase. The active species, charge particles in air plasma should play a key role in seed vigor change, lead to variation of seedling and plant growth. However, more direct evidence is required to explain the mechanism. In this study, A. paniculata seed treated by air plasma excitated at5950V for10s actually gained seed invigoration.
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