闫银发,韩守强,周盛祥,宋占华,李法德,张春庆,张晓辉,王 敬
极低频高压脉冲电场提高陈年棉种活力的参数优化
闫银发1,2,韩守强1,周盛祥3,宋占华1※,李法德1,2,张春庆4,张晓辉1,王 敬1
(1. 山东农业大学机械与电子工程学院,泰安 271018;2. 山东省园艺机械与装备重点实验室,泰安 271018; 3. 中天科技集团,南通 226009;4. 山东农业大学农学院,泰安 271018)
为研究极低频脉冲电场对陈年棉种活力的影响,该文利用高压脉冲电源和弧形电极-平板组成的电场系统在极间距离为50 mm条件下,以16 kV、10 Hz为中心点,利用Design-expert软件设计正交试验,高压脉冲电场处理棉种时间为40 s。并利用响应面分析法,对发芽势、发芽率、发芽指数、活力指数等4个指标进行参数优化,得出最优处理电压为16.25 kV,最优频率为10.90 Hz。在此基础上,对优化条件进行了试验验证。结果表明:陈年棉种在脉冲电压16.25 kV、脉冲频率10.90 Hz处理条件下,与对照相比,发芽势提高了44.2%,发芽率提高了56.8%,发芽指数提高了64.3%,活力指数提高了81.8%,各指标都达到极显著差异(<0.01)。陈年棉种的电场生物学效应对电场电压和频率都具有选择性,在电压为16.25 kV、频率为10.90 Hz的脉冲电场作用下,陈年棉种的电场生物学效应最明显,研究结果为后续作物种子高压电场处理的参数优化提供参考。
电场;种子;优化;弧形电极;种子活力
农作物是人类赖以生存的基础,随着农业科技与时俱进的发展,越来越多的新兴技术被应用于农业领域,为了调控和干预作物种子萌发及其生长发育,高压电场技术成功应用于种子处理[1]。到目前为止,高压电场种子处理主要采用交变电场和高压静电场,极少涉及高压脉冲电场[2-5]。
在高压电场处理种子方面已有试验证明,合理的电场强度和处理时间可以提高种子的发芽指标[6-10]、提升种子酶活性、促进细胞膜修复、降低细胞膜对干旱胁迫的敏感性并能有效增强种子的活力[11-12]。高压静电场能对种子细胞膜产生强烈的刺激作用,同时在细胞内部会产生感应电流,对整个生物系统产生影响[13]。由于细胞膜内外两侧存在电位差,当电位差达到一定数值时便能够使细胞膜产生可逆穿孔,这种效应改变了细胞膜的通透性[14]。因此,得益于高压脉冲电场的剧烈作用,使其生物学效应更为显著。
极低频高压脉冲电场是指频率在0~300 Hz范围内的高压脉冲电场,由于生物体的频率也处于这一频率范围,因此极低频高压脉冲电场能够与生物体电磁场产生耦合共振,进而产生非热生物效应[15]。早有研究证明,生命体电位波动能够影响呼吸代谢、光合作用、水分吸收和气孔导度等核心生理过程[16-18]。采用极低频率的高压脉冲电场来促进种子的生长发育,由此产生一种干预和调控种子萌发的极低频脉冲电场种子处理技术[19-21]。
本文从种子萌发及生长活力方面进行了探索,研究了低频高压脉冲电场对种子发芽势、发芽率、发芽指数和活力指数等活力指标的影响,试验设计了陈年棉种的最优电场处理剂量,并进行试验验证,为后续研究低频高压脉冲电场生物学效应提供参考。
试验所用陈年棉种是山东农业大学农学院提供的邯郸885,2013年生产,产地为山东夏津,初始含水率为7%±0.5%,挑选大小一致,外观饱满的陈年棉种进行发芽试验,未经电场处理的初始发芽率在40%左右。陈年棉种的物理特性如表1所示。
表1 棉种的物理特性
1.2.1 高压脉冲电源
本试验所用脉冲电源由山东农业大学机电学院自行研制,该脉冲电源在10~20 kV输出幅值范围内连续可调,输出频率在1~50 Hz范围内连续可调,脉宽为10~500 ms,占空比为50%。
1.2.2 电场极板间距可调的种子处理装置
图1为电场极板间距可调的种子处理装置,种子处理装置主要由滑动导轨、丝杠套、丝杠、上支撑板、芒刺板、下支撑板、支撑底座和提升法兰等部分组成,该装置使弧形芒刺电极片始终处于张紧状态而不发生弯曲,还能使电极片之间距离均匀且保持平行。当需要调节电极尖端与下极板距离时,可手动转动调节手轮,电极尖端与下极板间距离可调范围为0~300 mm。
1.滑动导轨 2.丝杠套 3.丝杠 4.上支撑板 5.芒刺板 6.下支撑板
弧形电极片为厚度0.6 mm的不锈钢金属片,每片电极片有24个弧形半圆顶端,半圆半径为4.5 mm,安装时将10片弧形电极片通过两端的圆孔与试验台固定,设置金属片之间的间距为24 mm,弧形电极阵列具体形状见文献[22-23]。
用高压脉冲电场处理系统[24]进行种子处理试验时,弧形电极尖端距离不锈钢下极板(接地)的距离调整为50 mm。在下极板上均匀平铺单层300粒左右的陈年棉种,调整高压脉冲电源输出的电压和频率,通过高压探头(美国泰克公司Tektronix P6015A)和示波器(美国泰克公司Tektronix TDS1012B-SC)实时读取电源电压、波形和频率信号。
1.3.1 高压脉冲电场处理
陈年棉种在恒定温度为25 ℃,空气相对湿度为30%的环境下进行电场处理,根据前期在弧形电极条件下电源电压和频率对陈年棉种活力影响的初步探索,选择电源电压12~20 kV、脉冲频率5~15 Hz,电场场强方向竖直向下,棉种处理时间为40 s[20,24],以16 kV、10 Hz为中心点[24],利用Design-expert软件设计正交试验并分析,因素水平表如表2所示。同时设置CK对照组,CK不做电场处理,将电场处理后的陈年棉种及时放入密封袋保存,在全部种子处理完成后(4 h以内)进行萌发试验。
表2 脉冲电场正交试验因素水平表
1.3.2 发芽试验
在置床前将每个密封袋中待发芽棉种放入质量分数为0.2% HgCl2溶液中进行杀菌处理,然后从每个密封袋随机选取50粒种子,清洗后用砂床法置床,所有置床种子在山东农业大学作物生物学国家重点实验室种子幼苗培养室进行萌发试验,培养室按照国家标准为:25 ℃恒温、持续光照、相对湿度为100%恒湿[25]。每个电场处理条件做4盒试验,为4次重复,每项指标取平均值计算。
棉种发芽期间要定时补水,保证幼苗发育所需水分。在此期间,若发现棉种霉变应及时进行清理,以免影响其他幼苗生长。在第3天,部分幼苗高度接近发芽盒盒盖,应及时去除盒盖,防止影响幼苗生长,从第3天开始每天记录每盒的发芽数,直到第12天进行洗苗,测量相关数据[26]。数据统计标准根据《种子检验学》进行界定,测量每盒所有棉花幼苗[26]。然后计算每个处理条件下棉种的发芽率、发芽势、发芽指数和活力指数,计算公式为(1)-(4)所示[27]。
式中GE为发芽势,%;GP为发芽率,%;4为棉花种子在第4天的发芽数;12为第12天洗苗后的正常苗数;为发芽天数,d;为与相对应的每天种子发芽数;GI为发芽指数;为幼苗长度,cm;VI为活力指数。
1.3.3 幼苗指标测量及数据处理方法
用流水将幼苗进行冲洗,去除砂子,用吸水纸吸干幼苗表面的水分,用数显式游标卡尺(世达工具(上海)有限公司生产,型号:91513,精度:±0.03 mm)测量苗的长度,每盒测量25株棉花幼苗,计算每盒幼苗的平均长度,然后取每个重复的平均值作为一次电场处理的幼苗长度,与CK对照组进行对比,用SPSS软件进行方差分析。
正交试验共13个处理,中心值点处有4个重复,试验结果如表3所示。
表3 脉冲电场正交试验结果
注:表中‘*’为<0.05显著 ‘**’为<0.01极显著。
Note: ‘*’ in the table represents significance as<0.05, ‘**’ in the table represents extremely significance as<0.01.
由表3可知,脉冲电场处理后陈年棉种的活力指数、发芽率、发芽势和发芽指数显著高于CK,当电源频率为10 Hz,电源电压在16 kV时,所有指标与CK相比都有极显著差异(<0.01)。
利用Design Expert软件对表3试验数据进行多元回归拟合,建立棉种活力指数、发芽势、发芽率、发芽指数与电压、频率之间的二次多项回归模型。对各项进行方差分析,分析结果如表4所示。
表4 响应面方差分析结果
注:表中‘*’为<0.05显著;‘**’为<0.01极显著;为脉冲电压,kV;为电源频率,Hz。
Note: ‘*’ in the table represents significance as<0.05; ‘**’ in the table represents extremely significance as<0.01;is pulsed voltage, kV;is power frequency, Hz.
由表3可知,脉冲电场处理后陈年棉种的活力指数显著高于CK,大部分有极显著差异(<0.01)。图2为脉冲电场与弧形电极对棉种处理后活力指数的响应面分析,从图2可以看出,不同频率下,提高电压对陈年棉种活力指数的影响趋势是相似的;不同电压下,提高频率对陈年棉种活力指数的影响趋势也是相似的;综合来看,弧形电极脉冲电场处理后的棉种活力指数随着电压和频率的升高都呈现出先升高后下降的趋势。而且脉冲频率的变化对棉种活力指数的影响较为明显。建立棉种活力指数与影响因素实际值之间的二次多项回归模型如式(5)所示。
式中VI为脉冲电场与弧形电极处理棉种活力指数,%;V为脉冲电压,kV;f为电源频率,Hz。
从表4可知,活力指数的值为8.07,值为0.008,说明建立的活力指数与试验因素的关系模型极显著(<0.01),关系模型的信噪比为8.378,说明模型较优,试验的准确度和可信度较高。从表4中数据可以看出,频率对活力指数的影响极显著,电压和频率的交互作用对活力指数的影响不显著。
由表3可知,弧形电极脉冲电场处理后陈年棉种的发芽势高于CK,有显著或极显著差异(<0.01或<0.05)。图3为脉冲电场与弧形电极对棉种发芽势的响应面分析,从图3可以看出,弧形电极脉冲电场处理后的棉种发芽势随着电压和频率的升高都呈现出先升高后下降的趋势,也就是说存在一个最优处理条件,使棉种经脉冲电场处理后发芽势达到最高。通过响应面分析,建立陈年棉种发芽势与影响因素实际值之间的二次多项回归模型如式(6)所示。
式中GE为脉冲电场与弧形电极处理棉种发芽势,%。
从表4可知,发芽势的值为12.72,值为0.002 1,说明建立的发芽势与试验因素之间的表达式是极显著的(<0.01),关系模型的信噪比为9.348,说明模型较优,试验的准确度和可信度较高。从表4中数据可以看出,电压和频率的平方对发芽势的影响极显著,电压、频率以及电压和频率的交互作用对发芽势的影响不显著。
图3 脉冲电场与弧形电极对棉种发芽势的响应面分析
由表3可知,弧形电极脉冲电场处理后陈年棉种的发芽率显著高于CK,大部分有极显著差异(<0.01)。个别处理达到显著差异(<0.05)。图4为脉冲电场与弧形电极对棉种处理后发芽率的响应面分析,从图4可以看出,不同频率下,提高电压对陈年棉种发芽率的影响趋势是相似的;不同电压下,提高频率对陈年棉种发芽率的影响趋势也是相似的;综合来看,弧形电极脉冲电场处理后的棉种发芽率随着电压和频率的升高都呈现出先升高后下降的趋势。通过响应面分析,建立陈年棉种发芽率与影响因素实际值之间的二次多项回归模型如式(7)所示。
式中GP为脉冲电场与弧形电极处理棉种发芽率,%。
从表4可知,发芽率的值为8.74,值为0.006 4,说明建立的发芽率与试验因素之间的表达式是极显著的(<0.01),关系模型的信噪比为6.872,说明模型是较优的,试验的准确度和可信度较高。从表4中数据可以看出,电压和频率的平方对发芽率的影响极显著,电压、频率以及电压和频率的交互作用对发芽率的影响不显著。
由表3可知,弧形电极脉冲电场处理后陈年棉种的发芽指数高于CK,有显著或极显著差异(<0.01或<0.05)。图5为脉冲电场与弧形电极对棉种发芽指数的响应面分析,从图5可以看出,弧形电极脉冲电场处理后的棉种发芽指数随着电压和频率的升高也都呈现出先升高后下降的趋势。通过响应面分析,建立陈年棉种发芽指数与影响因素实际值之间的二次多项回归模型如式(8)所示。
式中GI为脉冲电场与弧形电极处理棉种发芽指数。
从表4可知,发芽指数的值为8.35,值为0.007 3,说明建立的发芽指数与试验因素之间的表达式是极显著的(<0.01),关系模型的信噪比为6.137,说明模型是较优的,试验的准确度和可信度较高。从表4中数据可以看出,电压和频率的平方对发芽指数的影响极显著,电压、频率以及电压和频率的交互作用对发芽指数的影响不显著。
以陈年棉种发芽势、发芽率、发芽指数和活力指数的最大值为优化目标,发芽势、发芽率、发芽指数和活力指数的权重均为1,利用响应面法对二项回归数学模型式(5)-(8)进行数学优化,得到弧形电极脉冲电场处理陈年棉种的电压、频率最优组合是:电压16.25 kV,频率10.90 Hz。在此最优条件下,陈年棉种发芽势、发芽率、发芽指数和活力指数的期望值分别为60.06%、67.99%、44.45和266.84,都显著高于CK(<0.01)。
依据参数优化的结果,取脉冲电压16.25 kV、频率10.90 Hz为处理条件对陈年棉种进行电场处理,陈年棉种的发芽势、发芽率、发芽指数和活力指数分别为62%、69%、46和269,其发芽指标与CK的发芽势、发芽率、发芽指数和活力指数相比,发芽势提高了44.2%,发芽率提高了56.8%,发芽指数提高了64.3%,活力指数提高了81.8%,各指标都达到极显著差异(<0.01),符合参数优化的结果。
电场作为自然界存在物质,能够不同程度影响环境中生物的生长。高压脉冲电场的生物效应是由电场引起生物体内的物理和化学原发反应,从而形成一个综合的后效应[28-29]。适当电场剂量(电场剂量=电场强度×作用时间)的处理,对种子萌发起着积极的促进作用[30-33],从试验结果分析来看,陈年棉种的发芽势、发芽率、发芽指数、活力指数随电压和频率的升高呈现先升高后下降的趋势,在极板间距为50 mm,电压为16.25 kV,频率为10.90 Hz,处理时间40 s时,陈年棉种发芽的各项指标均达到最大值。因此电场剂量不仅与电场强度、作用时间有关,还与高压电场的频率有关,当电场频率大于或小于10.90 Hz时,陈年棉种的发芽指标都有所降低。
从试验结果来看,陈年棉种的电场生物学效应对电场电压和频率都具有选择性,在合适的电场电压和频率下,生物体的电场生物学效应最明显。根据“种子萌发的能量刺激假说”[34],陈年棉种经电压为16.25 kV,频率为10.90 Hz的电场处理后,种子内部经过复杂的物理和化学变化,为陈年棉种萌发提供了恰当的能量刺激,显著提高了陈年棉种的发芽势、发芽率、发芽指数、活力指数。
1)用弧形电极,脉冲电场对陈年棉种处理,进行正交试验,通过分析得出处理后的棉种发芽势、发芽率、发芽指数、活力指数随电压和频率的升高呈现先升高后下降的趋势,陈年棉种的电场生物学效应对电场电压和频率都具有选择性。
2)在弧形电极条件下,极板间距为50 mm,处理时间为40 s,脉冲电场的较优处理条件为16.25 kV、10.90 Hz,在此电场剂量条件下处理的棉种与CK相比,发芽势提高了44.2%,发芽率提高了56.8%,发芽指数提高了64.3%,活力指数提高了81.8%,各指标与对照(CK)相比都达到极显著差异(<0.01)。
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Parameter optimization on improving aged cotton seeds vigor by extremely low frequency high voltage pulsed electric field
Yan Yinfa1,2, Han Shouqiang1, Zhou Shengxiang3, Song Zhanhua1※, Li Fade1,2, Zhang Chunqing4, Zhang Xiaohui1, Wang Jing1
(1.,271018,; 2.,271018,; 3.,226009,;4.,’271018,)
In order to explore the effect of extremely low frequency high-voltage pulsed electric field on the aged cotton seed vigor, high voltage pulse power supply and arc electrode were used to treat aged cotton seeds with different voltages and frequencies. The treatment time was 40 s. The experiment environment temperature was set as 25 ℃, the relative humidity was set as 30%, and the temperature and humidity were remained relativelystable during the experiment. The effect of high voltage and extremely low frequency on germination ability of aged cotton seeds was studied. The voltage of vertically downward electric field was 12-20 kV, the pulse frequency was 5-15 Hz, the electrode was arc, and the distance between electrode plates was 50 mm. The point of 16 kV and 10 Hz was selected as the center point, and the orthogonal test was designed by Design-Expert software. The CK stood for the aged cotton seeds that had not been treated by electric field. The aged cotton seeds treated by electric field were taken into the sealed bag in time, and all the seeds were germinated within 4 h after treatment. The aged cotton seeds in sealed bag were put separately into the HgCl2solution with mass fraction of 0.2% for sterilization before seeds germination, and then 50 seeds were selected randomly from each seed sterilization group. Finally, the selected seeds were cleaned and put on the germination bed with sand bed method. The seed germination experiments were carried out at the National Key Laboratory of Crop Biology, Shandong Agricultural University, which were in accordance with the national standard: 25 ℃ constant temperature, continuous light, and relative humidity of 100%. Four boxes of seeds were treated under each electric field for 40 s, which were 4 repetitions, and then the average of each index was calculated.During the cotton seeds germination, the moldy cotton seeds should be removed timely, in order not to affect other seeds growth. According to the response surface analysis of the pulsed electric field and the arc electrode to the dynamic index of the aged cotton seed, and the single factor analysis of the pulsed electric field and the arc electrode to the cotton vigor index, the effect of increasing the voltage on the aged cotton seed vigor index was similar. At different voltages, the influence of increasing frequency on the cotton seed vigor index was similar. On the whole, the cotton seed vigor index after arc electrode electric field treatment showed a tendency of rising firstly and then falling with the increasing of voltage and frequency. And the influence of pulse frequency on cotton vigor index was obvious. Using the Design-Expert software, the pulse voltage and frequency were optimized, and the optimal condition was that the pulse voltage was 16.25 kV and the pulse frequency was 10.90 Hz. Under the optimal condition, the germination potential was increased by 44.2%, the germination rate was increased by 56.8%, the germination index was increased by 64.3%, and the vigor index was increased by 81.8%, and the indices were all significantly different (<0.01) from that of the CK.
electric field; seeds; optimization; arc electrode; seeds vigor
10.11975/j.issn.1002-6819.2017.22.039
S335.2
A
1002-6819(2017)-22-0301-07
2017-07-26
2017-11-07
2015年山东省农业重大应用技术创新课题;山东省现代农业产业技术体系棉花创新团队项目(SDAIT0309)
闫银发,汉族,山东曹县,副教授,主要从事高压静电技术在现代农业中的应用研究。Email:sd28@163.com
宋占华,山东临清市,博士,副教授,主要从事现代农业机械装备设计与开发。Email:songzh6688@163.com