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Using the experimental design with an orthogonal matrix with four ultrasonic factors, the effect of ultrasonic treatment on seed germination and seedling growth of cane fescue (Festuca arundinacea) and Russian rye (Psathyrostaehys juncea Nevski) was determined. Multivariate analysis of variance revealed significant differences and association effects for paired factors. The activity of superoxide dismutase (SOD) and peroxidase (POD) and the content of malonic dialdehyde (MDA) influenced. Sonication had a positive effect on germination rate (GP) and seedling growth (GS) of old seeds, which provides the main evidence for the use of sonication for pre-treatment of old grass seeds. For the four ultrasonic factors, after binary quadratic regression analysis, the optimal conditions were that the ultrasonic time was 36.7 min, the ultrasonic temperature was 35 °C, the power output was 367 W, and the seed soaking time was 4.1 hours. Sonication can improve seedling growth. In addition, the life span of seeds of cane fescue and Russian rye was approximately 9.5 and 11.5 years, respectively, under natural storage conditions. Physiological mechanisms that may contribute to the improvement of GP and HS are discussed.
Seeds are needed to restore crop productivity, preserve germplasm, and increase species diversity. However, seed aging or spoilage during storage can lead to significant loss of genetic diversity, failure to emerge, abnormal emergence and ultimately reduced field colonization1. Studies have shown that seed aging is associated with major changes in cellular metabolism and biochemistry, including enzyme inactivation, lipid peroxidation, membrane integrity disruption, and damage to deoxyribonucleic acid (DNA) 2 . Although the exact mechanism leading to seed senescence remains unclear, one of the main causes of seed spoilage is considered to be the accumulation of reactive oxygen species (ROS).
As a rule, dried seeds can be stored for a longer period of time, but eventually the deterioration or aging of the seeds may become irreversible4. In addition, pre-storage conditions (i.e. seed temperature and moisture) and genotype effects play an important role in influencing seed longevity characteristics. Grasses such as cane fescue (Festuca arundinacea Schreb.) and Russian wild rye (Psathyrostachys juncea Nevski) have higher seed lipid content, which can accelerate seed spoilage compared to other forage or agricultural seeds. Thus, extending the shelf life of stored seeds and improving germination from both a biophysical and an economic point of view has become a subject of intense study.
Priming is a pre-planting treatment widely used to stimulate germination and improve seed quality under stressful conditions or after long periods of storage6,7. The positive effect of priming (including halogen, water, osmotic and thermal priming, etc.) on seed productivity has been demonstrated in many species. However, these treatments are relatively time-consuming and labor-intensive9. In contrast, the use of ultrasonic (US) treatment, which is easier to operate and saves time, can have multiple effects in a short period of time, including thermal, mechanical and chemical effects on seeds9. In addition, although it has generally not been well tested, US processing has shown promising results in millet9 and many other crops, such as hybrids of calantha, bean,10 corn,11 barley,12 fern spores,13 alfalfa, and broccoli,14 chickpeas, wheat, watermelon, and sugar delicacies15,16. Thus, methods to increase the germination of older cereal seeds and stimulate seedling growth may be effective, but there is no evidence of the effect of ultrasonication or studies of its optimal conditions on mature seeds of cane fescue and Russian ryegrass.
Reed fescue (Festuca arundinacea Schreb.) is primarily a cool grass found in many livestock pastures. It is widely cultivated in temperate regions of the world17,18 and is one of the most important forage species, which is also used for soil conservation. This grass thrives in grazing environments where multiple simultaneous stresses are common19. Tall fescue (Tf) is also a cold season lawn grass widely used in residential and commercial landscapes20, with excellent shade tolerance and dark green winter color, this grass outperforms warm season lawn grasses such as Bermuda grass and Zoysia.
Russian wild rye (Psathyrostachys juncea Nevski) is a perennial herbaceous plant that is fast growing, drought tolerant, calcium carbonate (CaCO3) tolerant and has low fertility requirements21,22,23. Russian wild rye is a cool fodder variety well adapted to semi-arid climates24,25, a highly competitive, high-yielding grass and an important source of livestock and wildlife in semi-arid pastures in Eurasia and northwestern China26 an excellent forage source22,23,24,27 ,28,29. Therefore, this study examined the effect of US treatments on increasing the emergence of old seeds and improving the vigor and growth of seedlings of Tf and wild Russian ryegrass (Rw) collected in 2006, 2010 and 2014. 2006 and 2009 respectively. In addition, by evaluating changes in physiological parameters associated with age, we aimed to test whether US treatment has a positive effect on repairing damage caused by the seed aging process.
In 2006, the Tf group of nesting experiments collected, namely Tf (2006), 2006 and 2003. harvest Rw (Rw 2006 and Rw 2003), the seeds did not germinate. Germination also did not occur with treatments of groups 7, 8 and 9 of cane fescue collected in 2014 and 2010 (Tf 2014 and Tf 2010) and Rw (2009), which were sonicated at 65°C (Figure 1A).-C ) The highest germination rate (GP) with Rw (2009) 4 seed treatment was 89.3% (Figure 1C) with sonication time of 15 minutes, sonication temperature of 45 °C, power output of 350 W, and seed soaking time of 1 hour. The lowest GP was at 5 sonication and sonication conditions were 35 minutes sonication time, 45°C sonication temperature, 500 W power output, and 9 hours seed soaking time (Fig. 1A-C). With the same treatment time (15 min) or the same temperature (45°C) as treatment 4, the highest GP Tf values (2014 and 2010) for treatments 1 and 6 were 78.7% and 79. 0%, respectively (Fig. 1A, B). However, the lowest GP of the three groups was also at 45°C on treatment 5 (Figure 1A-C). An analysis of a range of orthogonally designed experiments showed that sonication temperature was the most important factor among the four design factors.
Combined effects of ultrasonic temperature, ultrasonic time, and ultrasonic output power on seed germination of cane fescue (2014(A) and 2010(B)) and Russian wild rye (C). Numbers in circles indicate treatment, Ck – control. The highest and lowest values are shown, except for treatments 7, 8 and 9.
GP and Germination Index (GI) were significantly positively correlated with shoot and root length (Table S1). Shoot and root length was negatively correlated with malondialdehyde (MDA) content, but positively correlated with superoxide dismutase (SOD) activity.
方差分析表明，超声处理时间、超声处理温度、输出功率和浸种时间对GP、GI和SVi以及茎和根长度具有显着性（p < 0.01），且两者均存在显着的耦合效应。 Analysis of variance showed that ultrasound treatment time, ultrasound treatment temperature, output power, and seed immersion time were significant (p < 0.01) for GP, GI, and SVi, as well as stem and root length, and there was a significant coupling effect between the two. Дисперсионный анализ показал, что время обработки ультразвуком, температура обработки ультразвуком, выходная мощность и время замачивания семян оказывали значительное влияние на GP, GI и SVi, а также на длину стебля и корня (p <0,01), и оба имели значительные эффекты связи. Analysis of variance showed that sonication time, sonication temperature, output power, and seed soaking time had significant effects on GP, GI, and SVi, as well as stem and root length (p < 0.01), and both had significant interaction effects. . -Wise, among the four factors, except for the factors X3 and X4 and X1*X2 interaction length root (Table 1).
In the Rw group (2009), seed germination was significantly higher in all variants than in the control (Fig. 2C). Sonication has significantly and consistently increased the GP of Russian wild rye seeds since 2009 (Fig. 2C). Of the three groups, treatment 5 (T5) had the lowest GP (Figure 2A-C), while 2009 Rw seed treatment 4 (T4) had the highest GP (Figure 2C). For new (2014, Fig. 2A) cane fescue seeds, the VP of seeds of variant 1 (T1) was higher than that of the control (T7), and the VP of variant 3 (T3) was lower than that of the control (T3). control (Fig. 2A), these results differ from the results of the old seed GP (2010, Fig. 2B). The basic penetration percentage statistics are presented in Figure S1.
Model curves showing germination during seed treatment of cane fescue (2014 (A) and 2010 (B)) and Russian wild rye (C). T1 to T6 is treatment and T7 is control (black). All of the models were significant at P < 0.05. All of the models were significant at P < 0.05. Все модели были значимы при P <0,05. All models were significant at P<0.05.所有模型在P < 0.05 时均显着。所有模型在P < 0.05 时均显着。 Все модели значимы при P <0,05. All patterns are significant at P<0.05.
In the control, the MDA content and POD activity in Rw (2009) seedlings were significantly higher than in Tf 2014 or 2010 seedlings (Fig. 3A, C). POD activity was significantly higher among all treatments in Rw (2009) (Figure 3C). In the Tf (2010) group, MDA content was significantly higher and SOD activity was lowest in treatment 1, but opposite results were found in treatment 3 (Figure 3A,B). In addition, the trends in MDA content and SOD activity were opposite to the changes observed with Tf(2010) in the 2009 Rw seedling group and in variant 1 (Fig. 3A, B). Based on the analysis of variance, the effects of the four factors were significant for the MDA content and for the SOD and POD activities (p < 0.01) and there were significant coupling effects both pair-wise and among the four factors, with the exception of factors X2 and X4 and interaction X3*X4 on the activity of POD (Table 2). Based on the analysis of variance, the effects of the four factors were significant for the MDA content and for the SOD and POD activities (p < 0.01) and there were significant coupling effects both pair-wise and among the four factors, with the exception of factors X2 and X4 and interaction X3*X4 on the activity of POD (Table 2). На основании дисперсионного анализа влияние четырех факторов было значимым для содержания МДА и активности СОД и ПОД (p < 0,01), а также наблюдались значимые эффекты сцепления как попарно, так и между четырьмя факторами, за исключением факторов Х2 и Х4 и взаимодействия Х3*Х4 на активность ПОД (табл. 2). Based on the analysis of variance, the influence of four factors was significant for the content of MDA and the activity of SOD and POD (p < 0.01), and significant linkage effects were observed both in pairs and between four factors, with the exception of factors X2 and X4 and interaction X3* X4 for POD activity (Table 2).基于方差分析，四个因素对MDA含量和SOD和POD活性的影响显着（p < 0.01），并且在成对和四个因素之间存在显着的耦合效应，除了因子X2 和X4 以及交互作用X3*X4 对POD 活性的影响（表2）。基于 方差 分析 ， 四 因素 对 对 含量 含量 和 和 pod 活性 的 影响 显着 （（p <0.01） ， 并且 成 对 和 四 个 之间 显着 的 耦合 ， 除了 因子 x2 和 x4 以及 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用 作用X3*X4 На основании дисперсионного анализа влияние четырех факторов на содержание МДА и активность СОД и ПОД было значительным (p < 0,01), и наблюдались значительные эффекты связи между парными и четырьмя факторами, за исключением факторов X2 и X4 и эффектов взаимодействия Эффекты X3* X4 на активность POD (таблица 2). Based on analysis of variance, the effect of four factors on MDA content and activity of SOD and POD was significant (p < 0.01), and there were significant association effects between paired and four factors, with the exception of factors X2 and X4 and interaction effects Effects of X3* X4 on POD activity (Table 2).
MDA content (A), SOD (B) and POD (C) activity in seedlings of cane fescue (2014 and 2010) and Russian rye (2009). Letters denote differences in treatments only between years. FW – fresh weight.
MANOVA revealed significant differences between factors at p < 0.0001 (Table 3).
In option 1, the logarithmic ratios of GI, SVi, and shoot length were significantly higher in the 2009 Rw seedling group than in the control, while these values were lower in the Tf (2010) group than in the control (Fig. 4A). – C) In the year Rw (2009), the length of the shoots increased significantly, and the length of the roots was lower than in the control group in variants 1, 2 and 3 (Fig. 4C, D). In variant 5, shoot and root length was significantly longer in the Tf (2010) group than in the control group, but the results were reversed in the 2014 group (Fig. 4C,D). Gis and SVis were significantly higher in the Tf (2010) group. three years lower than in the control group (Fig. 4A, B).
Germination index (GI, A), seed vigor index (SVi, B) and logarithmic ratio of shoot length (C) and root length (D). * Indicates the difference between the treatment and the control was significant at p < 0.05. * Indicates the difference between the treatment and the control was significant at p < 0.05. * Указывает, что разница между лечением и контролем была значимой при p < 0,05. * Indicates that the difference between treatment and control was significant at p < 0.05. * 表示治疗组和对照组之间的差异在p < 0.05 时显着。 * 表示治疗组和对照组之间的差异在p < 0.05 时显着。 * Указывает, что разница между группой лечения и контрольной группой значима при p < 0,05. * Indicates that the difference between the treatment group and the control group is significant at p < 0.05.
112 quadratic models were selected for shoot length, root length, shoot-to-root ratio, shoot length, GP, Gi and SVi variables, which were sonication pair factor regression models. The critical values of the stationary point are plotted in pairs (Figs. 5A–F). The sample sizes for the four critical factor values were 59, 57, 51, and 56, as determined by model analysis. The value mode of the four factors is calculated and used as the optimal value. Thus, the optimal values of the four factors were 36.7 min for sonication time, 35 °C for sonication temperature, 367 W for power output, and 4.1 h for seed soaking time.
Scatterplots showing sonication temperature versus sonication time (A), power output versus sonication time (B) and sonication temperature (C), and seed soaking time versus sonication time (D), sonication temperature ( E) and power output (F)).
Ultrasonic technology (USA) is widely used in medicine, biology30 and to improve the technical properties of food products such as emulsifying power, solubility and texture, as well as in areas such as homogenization, extraction, viscosity modification, crystallization, drying and defoaming31, 32, 33, 34. Through a combination of pressure or heat treatment, the synergistic effect of ultrasound can be very effective in inactivating microorganisms and enzymes such as peroxidase and lipoxygenase during food storage. To explain the role of US treatment in seed priming, a number of biochemical and physiological mechanisms have been proposed, including repair of age-related cellular damage and accelerated metabolic uptake that activates seed-driven germ protrusion. 12. Cavitation activity. In this study, we investigated the effects of different levels of sonication output, time and temperature on Tf and Rw seeds to determine the optimal conditions to promote germination and seedling growth. GP and rhizome length were significantly higher in each treatment than in controls, especially in long-term storage seeds (GP: 79.0% vs. 54%, Tf, 2010; 89.3% vs. 36% Rw, 2009) (Fig. 1B ,C). ); one result may be that sonication increases seed porosity through acoustic cavitation, thereby facilitating oxygen access and moisture uptake, which are necessary to initiate the first stage of seed germination. Several other interesting studies have shown that ultrasound can effectively accelerate starch metabolism by activating an enzyme (alpha-amylase) that occurs during seed germination12,36. Similarly, the germination rate of sonicated barley seeds increased by about 1.042–1.065 times compared to the control, and the germination period was significantly reduced by 30–45% compared to the control. The results of this study (Figure 2C) for Rw (2009) seeds are consistent with this study. Seedling growth values, described as (Gi, Svi, root and shoot length), were significantly higher in variant 4 of both species than in the control group (Figs. 4A–D). Additionally, although the germination models were significant at p < 0.05, the comparably variations indicated the effects of US treatments9 (Fig. 2, Table S2 and eq. 3). Additionally, although the germination models were significant at p < 0.05, the comparably variations indicated the effects of US treatments9 (Fig. 2, Table S2 and eq. 3). Кроме того, хотя модели прорастания были значимыми при p <0,05, сопоставимые вариации указывали на влияние обработки в США9 (рис. 2, таблица S2 и уравнение 3). In addition, although germination patterns were significant at p < 0.05, comparable variations indicated the effect of US treatment9 (Fig. 2, Table S2 and Equation 3).此外，虽然发芽模型在p < 0.05 时显着，但可比较的变化表明美国处理的影响（图2、表S2 和eq.此外，虽然发芽模型在p < 0.05 3）。 Кроме того, хотя модель прорастания была значимой при p <0,05, сопоставимые изменения указывали на эффект обработки в США (рис. 2, таблица S2 и уравнение 3). In addition, although the germination pattern was significant at p < 0.05, comparable changes indicated a treatment effect in the US (Fig. 2, Table S2 and Equation 3). The physiological effects of ultrasonic treatments may be that they play a catalytic role in improving seedling productivity, especially for those seeds that are naturally aged, whose protective enzymatic systems are severely compromised by reactive oxygen species (ROS).
The use of orthogonal plans to optimize conditions in multifactor interaction processes is efficient and reliable9,37. According to the results of this study, optimization of the four factors resulted in a sonication time of 36.7 min, a sonication temperature of 35°C, a power output of 367 W, and a seed soaking time of 4.1 hours. For the critical values, the factor sample sizes were 59, 57, 51 and 56, respectively, which were treated as large sample statistics (n > 30) because of the diverse results under complex effects 38,39. For the critical values, the factor sample sizes were 59, 57, 51 and 56, respectively, which were treated as large sample statistics (n > 30) because of the diverse results under complex effects38,39. Для критических значений размер факторной выборки составлял 59, 57, 51 и 56 соответственно, что рассматривалось как статистика большой выборки (n > 30) из-за различных результатов при сложных воздействиях 38,39. For critical values, factor sample sizes were 59, 57, 51, and 56, respectively, which were considered as large sample statistics (n > 30) due to different outcomes for complex interventions 38,39.对于临界值，因子样本量分别为59、57、51 和56，由于复杂效应下的不同结果38,39，它们被视为大样本统计量(n > 30)。对于 临界值 ， 因子样本量 为 为 59、57、51 和 56 ， 由于 复杂 下 的 不同 结果 38,39 ， 它们 被 大样本 统计量 统计量 统计量 (n> 30)。。 Для критических значений размеры факторной выборки составляли 59, 57, 51 и 56, которые рассматривались как статистика большой выборки (n > 30) из-за разных результатов при комплексных воздействиях38,39. For critical values, factor sample sizes were 59, 57, 51, and 56, which were considered as large sample statistics (n > 30) due to different outcomes under complex interventions38,39. During sonication in an aquatic environment, the collapse of cavitation bubbles created by a series of compression and thinning creates shear forces that act mechanically and chemically on the submerged seeds. Under optimal US processing conditions in this study, mature seeds could be properly stimulated and seeds sufficient to initiate germination and emergence were prepared in a physicochemical state. A wide range of optimal conditions for sonication of various plants has been determined: 60 W, 22 °C and 2 minutes for spruce, 460 W, 30 °C and 15 minutes for barley 12, 135 W for Calanthe Hybrid 15, less than 7 minutes; chickpeas 45 minutes 16. Reasons for these differences may be related to different types of species or individual seed characteristics (eg, seed coat thickness, size, infective microbes and dormancy). Under appropriate sonication conditions, mating is relatively easy and inexpensive and can minimize damage from seed senescence, especially seeds of rare plant species. Secondly, the principle of complete energy balance can explain the different results of ultrasonic performance. Lower sonication temperature (35°C vs. 39.7°C previously), power output (267W vs. 348W) and longer sonication time (36.7 min vs. 22.5 min) and seed soaking compared to the previous study Time (4.1 hours compared to .0 hours) required. In addition, in this experiment, sonication was successful, namely for GP Tf (2010) and Rw (2009) (Fig. 1B,C); the highest GP value of the first group was at sonication time of 55 min and power output of 200 W (Figure 1B, C, Figure 1B), while the last group was 15 min and 350 W (Figure 1C). In addition, in our study, both germination ability and seedling growth of aged seeds were increased by US treatment, while treated immature seeds did not show a significant improvement in germination, despite the fact that the effect was observed in the model (equation 3, table S2 and Figure 2), which may indicate a redistribution of intracellular water3, disruption of the ultrastructure of embryonic cells, changes in cellular pH and redox states involved in natural aging7 obtained under the action of ultrasound priming, relative to reduced ones.
Interestingly, the critical death temperature for sonication was at or below 65°C, as shown in treatments 7, 8, and 9 (Fig. 1A-C). In general, with increasing sonication time, sonication temperature, or sonication output power, physical damage occurs due to the very rapid local pressure and temperature changes caused by sonication, resulting in shear failure, intense cavitation, thinning of cell membranes, and localized heating. . chemical damage and the formation of free radicals,34 which can be lethal to seeds immersed in the bath. Ultrasound treatment for more than 5 minutes negatively affected the germination rate of pepper seeds. Strongly damaged areas were observed on the surface of ginger granules when using ultrasound for 200 minutes. In addition, reactions such as protein denaturation, molecular degradation and starch gelatinization can occur during high temperature treatment (perhaps 65°C in the study), which can significantly impair seed viability during hydration. In addition, the asymmetric collapse of cavitation bubbles near the seed surface can generate microjets that affect mass transfer. Because of the significant association effects between these factors (Tables 1, 2, 3), further study of more complex mechanisms is required to explain damage caused by ultrasound treatment.
The accumulation of reactive oxygen species (ROS) is considered to be the main factor leading to seed aging during storage. Proteins and nucleic acids of mature seeds are damaged by excess ROS, which can accelerate lipid peroxidation reactions and accelerate membrane disassembly2,44. MDA, the end product of lipid peroxidation, gradually accumulated in the seeds as they deteriorated during storage, which is consistent with the results of this study. In the Rw (2009) group, MDA content was significantly reduced in all treatments (treatments 1–4 and 6) compared to controls (Fig. 3A), suggesting that restoration of membrane integrity was effective in treated older seeds. The large drop can also be largely due to the fact that the application of ultrasound of a certain intensity can effectively inactivate enzymes such as lipoxygenase in mature seeds. Studies have shown that during food storage, enzymes such as peroxidase and lipoxygenase can be successfully inactivated using a combination of heat and ultrasound. Thus, the cytoprotective ability to neutralize seed aging caused by ROS accumulation was evaluated. SOD activity directly regulates the amount of ROS37. In options 1 and 3 of the present study, complementary trends in MDA and SOD were observed in Tf (2010) and Rw (2009) (Fig. 3A,B), the same was observed for GP and UVI in both groups of cereal results. (Fig. 4A, B). However, the trend changed and became opposite for the length of shoots and roots (Fig. 4C,D). Changes in SOD activity under abiotic stress may reflect the same trend in changes in the production of superoxide radicals, which may be the result of electron leakage from the electron transport chain to molecular oxygen. An increase in SOD activity may be associated with the temporal regulation of specific isoenzymes and the induction of new isoforms. Further studies of SOD isoenzymes of the sonicated herb are required. Antioxidant enzymes such as POD in plant cells can remove reactive oxygen species47 caused by plant stress, thus effectively preventing their accumulation. Disruption of these antioxidant enzymes or a decrease in their activity can accelerate seed aging. In the present study, POD activity in the Rw (2009) group was significantly higher than in the other two older groups (Figure 3C). Similarly, optimal use of ultrasonic extraction (EAE) to extract phenolic compounds from hawthorn leaves enhances potent antioxidant activity to protect against H2O2-induced cell death48. This discrepancy can be partly explained by different plant species. Shoot and root lengths, as well as Gi and SVi values, were significantly different from controls in the three groups (Figures 5A–D), consistent with previous studies. Length is a large sample statistic (n = 30)38,39 and sonication significantly affected GP and seedling growth.
In addition, in the present study, neither the treatment group nor the control grass seeds (cane fescue in 2006 and Russian rye in 2006 and 2003) did or did not germinate under any of the nine treatment options during the experimental period. which indicates that the seeds of cane fescue and Russian rye have a life expectancy of approximately 9–10 years and 10–12 years, respectively, under natural storage conditions (room temperature, humidity ~13%). The longevity of seeds depends not only on the genetic characteristics of the seeds, but also on the physiological state, seed moisture, collection temperature and storage method, etc. 49 . The classification described by Ewart 50 suggests that most naturally stored grass species have a lifespan of 3 to 15 years, as grass seeds quickly lose their ability to germinate after completing a dormant period for a short period of time51. In addition, wheatgrass (Agropyron cristatum) had a CV of 33.0% stored for 8 years and then dropped to zero in the 9th year. Similarly, HR of brome and Siberian rye (Elymus sibiricus) were 45% and 64%, respectively, at 9 years of storage, and then dropped to zero 52 after 11 and 10 years, respectively. All of the above data supports the assumption that most grass seeds have a lifespan of about 10 years under natural conditions.
Eight seedling growth and germination variables formed 112 binary quadratic models based on balanced, separate, or unmixed orthogonal designs with paired independent variables in design factors. Thus, the optimized results are mathematically justified, since a large sample of 223 critical values of stationary points is used, obtained from 112 models (Fig. 5) and design modes.
Ultrasonic treatment had a positive effect on germination and seedling growth of aged seeds, providing the main evidence for the further use of sonication for pre-treatment of aged grass seeds. The optimal conditions for the four factors were ultrasonic time 36.7 min, ultrasonic temperature 35℃, power output 367W, and seed soaking time 4.1 hours. In addition, this method is simple, inexpensive, and time-saving, and sonication can improve seedling growth. In addition, the present study indirectly shows that the lifespan of Russian cane fescue and wild rye seeds under natural storage conditions is approximately 9.5 and 11.5 years, however, determining the exact duration of the natural aging process requires more in-depth research in the future. .
Seeds of cane fescue (Festuca arundinacea Schreb.) and Russian wild rye (Psathyrostachys juncea Nevski) were obtained from the Grassland Research Station of China Agricultural University in Hexi Corridor, Jiuquan City, Gansu Province, China. This study was performed at the Laboratory of the Grassland Science Department, Northwest A&F University, Shaanxi Province, China. This study was performed at the Laboratory of the Grassland Science Department, Northwest A&F University, Shaanxi Province, China. Это исследование было проведено в лаборатории отдела науки о пастбищах Северо-Западного университета A&F, провинция Шэньси, Китай. This study was conducted in the Grassland Science Department Laboratory of Northwestern A&F University, Shaanxi Province, China. Это исследование было проведено в лаборатории Департамента науки о пастбищах Северо-Западного университета A&F, провинция Шэньси, Китай. This study was conducted in the laboratory of the Department of Grassland Science, Northwestern A&F University, Shaanxi Province, China. The seeds of these herbs are stored at room temperature in the laboratory for 1 to 12 years. Six seed groups of two grasses made up the nesting experiments (Table S3). Each group was fixed in an orthogonal matrix design [L9(34)] for sonication.
Four factors were investigated, namely sonication time (factor A), sonication temperature (B), sonication output (C), and seed soaking time (D). Based on the orthogonal design, each of the four ultrasound exposure factors was assigned three levels (Table 4), and nine treatment combinations were established for the various factors. In addition, a control without sonication and immersion was included (Table S4). All treatments were carried out in triplicate.
Ultrasonic irradiation was performed using an ultrasonic generator (KQ-500DE; Kunshan Ultrasonic Instrument Co., Ltd., China) with a fixed frequency of 40 kHz, adjustable temperature (from 10 to 80°C), and ultrasound power. varied from 200 to 500 watts. It consisted of stainless steel. The container (capacity 22.5 l) consists of a piezoelectric element connected to a water inlet and outlet valve. The water in the circulating ultrasonic bath is heated to the desired temperature level before treatment, and the water is isolated to minimize heat transfer through the environment. Three specific volumetric power levels (0.013 W/mL; 0.029 W/mL and 0.033 W/mL) were recorded in the respective experimental condition groups (sonication time, temperature, power output), respectively. During the experiment, each group of seeds (pre-soaked), covered with gauze (in orthogonal design [L9(34)]), was immersed in distilled water (13 L, desired temperature) in a tray, and then immersed in appropriate conditions Turn on the equipment (as shown in Table 4 and Table S4) by monitoring the temperature of the water, requiring periodic checks to ensure that the temperature remains constant during each treatment.
In addition, an electric thermostatic oven (DHG-9140A; Shanghai Yiheng Instrument Co., Ltd., China), a plant incubator (ZPW-400; Harbin Dongtou SG-Tech Development Co., Ltd.), and an electronic analytical balance (YP1200; Shanghai Science and Industry Co., Ltd., China).
Seeds were surface sterilized in 0.1% (w/v) sodium hypochlorite solution for 15 min, washed 5 times with distilled water, and then germinated in sterile Petri dishes 100 mm in diameter lined with two layers of Whatman No.1 filter paper moistened with distilled water. or Solution Handling. The germination test was repeated 3 times with 50 seeds per treatment. The Petri dishes were wrapped in clear plastic wrap to keep the water from evaporating. The water level in the germination tank was maintained by adding distilled water daily. Petri dishes were placed in a germination machine (LRH-250-GS II; China) set to alternating day and night conditions: 16 hours light, 5500 lux at 25°C and 8 hours dark at 20°C for 14 days , according to Lu54 research evaluation. This temperature state was chosen to represent the mid-spring temperature, which corresponds to the time of year when grass seeds germinate. Germinating seeds were counted daily for 14 days. The length of the shoots and roots was measured on the fourteenth day after sowing and measured 10 seedlings from each Petri dish in triplicate. The sample size was 30 (3 × 10). When the total number of seedlings is less than 10, all seedlings are measured according to the rules of ISTA (International Seed Testing Association). Seed germination was determined by elongation of coleoptiles up to 0.2 cm. Germination rate (VP) was calculated. Germination index (Gi) was calculated using the following formula:
where Gt is the number of seeds germinated per day, Dt are the corresponding days of germination, t is the total number of germination periods (14 days).
The activity of SOD and POD and the content of MDA in the treatments were determined. SOD activity was determined by the method of Zhang55. For the antioxidant enzyme extract, each group of seedlings (0.5 g) was homogenized in 5 ml of 50 mM sodium phosphate buffer (pH 7.8 for SOD and POD containing 1% (w/v) PVP and 0.1 mM Na2EDTA). The homogenate was then filtered through two layers of filter paper at 10,000 g for 15 min at 4°C, after which an aliquot of the supernatant was analyzed for enzymatic activity (SOD and POD) at 25°C. SOD activity was measured spectrophotometrically as described by Beyer and Fridovich56, and one unit of SOD activity was defined as the amount of enzyme inhibiting 50% of HCT57 photoreduction. POD activity was measured by the method described by Fu using guaiacol (1-hydroxy-2-methoxybenzene, C7H8O2) as a substrate. The reaction mixture contained 50 μl of 20 mM guaiacol, 2.8 ml of 10 mM phosphate buffer (pH 7.8) and 0.1 ml of enzyme extract. The reaction was started with 20 µl of 40 mM H2O2. One unit of POD activity is defined as the amount of enzyme that increases absorbance by one unit of absorbance at 470 nm per minute56. Malondialdehyde (MDA) content The reaction with thiobarbituric acid (TBA) was carried out according to the method of Madhava and Sresti58. To determine the MDA content, each group of samples containing 0.1 g of seedling homogenate was mixed with 5 ml of TCA (0.5%) and centrifuged at 10,000×g for 25 min. The mixture was then heated at 95° C. for 30 minutes, then the tubes were rapidly cooled with an ice bath. After centrifugation of the tube at 10,000 g for 10 min, the MDA content was determined by the absorption of the supernatant. Subtract the A600 nonspecific absorbance value from the A532 value. The concentration of MDA was calculated using the extinction coefficient of MDA at 155 mM-1 cm-157.
The data were analyzed for analysis of variance (ANOVA) using the statistical package SAS (version 8.2)59. Differences between the means were tested with Student-Newman-Keuls tests and values of p < 0.05 were significantly different.38,39,60 Differences between the means were tested with Student-Newman-Keuls tests and values of p < 0.05 were significantly different.38,39,60 Различия между средними значениями были проверены с помощью тестов Стьюдента-Ньюмена-Кеулса, и значения p < 0,05 значительно различались.38,39,60 Differences between means were tested using Student-Newman-Keuls tests, and p values < 0.05 were significantly different.38,39,60用Student-Newman-Keuls 检验检验平均值之间的差异，p < 0.05 的值有显着差异。用Student-Newman-Keuls 检验检验平均值之间的差异，p < 0.05 Различия между средними были проверены с помощью теста Стьюдента-Ньюмена-Кеулса, и значения при p < 0,05 значительно различались. Differences between means were tested using the Student-Newman-Keuls test, and the values at p < 0.05 were significantly different. 38,39,60
where c, a and b are constants (Table S2) and X is the number of days until germination. The models were significant at Pr < 0.05. The models were significant at Pr < 0.05. Модели были значимы при Pr < 0,05. The patterns were significant at Pr < 0.05.模型在Pr < 0.05 时显着。模型在Pr < 0.05 时显着。 Модели значимы при Pr < 0,05. Models are significant at Pr < 0.05. The model curve is shown in Figure 2 and the main data are listed in Table S5.
For the overall results, the variables (factors A, B, C, and D) are labeled X1 through X4. Dependent variables GP, Gi, SVi, stem length, root length, stem/root ratio, shoot length (stem + root), MDA content, SOD and POD activities were designated Y1–Y10, respectively. These treatment group variables were treated and analyzed separately using quadratic pairwise regression models (X1 and X2, X1 and X3, X2 and X3, X1 and X4, X2 and X4, X3 and X4)38,39,59.
where β is a constant. The critical values for the stationary points (X1, X2, X3 and X4) were obtained from a significant quadratic model. The thickest value is used as mode 39. Calculate the mode of the value using the following formula:
Where L is the lower bound of the array on which the pattern is located, U is the upper bound of the pattern on which the pattern is located, ∆1 is the frequency distance between the lower neighboring array and the pattern array, ∆2 is the frequency distance between the upper neighboring array and the pattern array, d — distance between arrays. These analytical and graphical procedures were performed using the statistical package SAS (v8.2)59.
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