Efficiency of seed vigor tests in estimating Melissa officinalis L. seedling emergence in soil and the effects of iron oxide nanoparticles on the seedling’s physiological properties
محورهای موضوعی : Stress Physiology
Hamidreza Eisvand
1
,
Zeinab Farajollahi
2
,
Dariush Goodarzi
3
,
Elham Jahangirinia
4
,
Donald L. Smith
5
1 - Department of Agrotechnology, Lorestan University
2 - Department of Plant Production and Genetic Engineering, Faculty of Agriculture, Lorestan University, Khorramabad, Iran.
3 - Department of Plant Production and Genetic Engineering, Faculty of Agriculture, Lorestan University, Khorramabad, Iran.
4 - Department of Plant Production and Genetic Engineering, Faculty of Agriculture, Lorestan University, Khorramabad, Iran
5 - Department of Plant Science, McGill University
کلید واژه: Hiltner test, Nano fertilizer, Plant nutrition, Seed vigor, Soil texture,
چکیده مقاله :
This study was conducted on Melissa officinalis (lemon balm) to compare the effects of seed vigor tests, under laboratory conditions, on seedling emergence from soil (greenhouse) and to study the effects of foliar application of iron oxide nanoparticles (ION) on growth variables and physiological performance of Melissa officinalis seedlings. Seed vigor tests were conducted on seeds of three sizes (large, medium, and small) in the laboratory. These tests included standard germination, accelerated aging, cold, Hiltner, and electrical conductivity (EC). Seeds of three sizes were planted in two different soil types, namely loam and clay loam, in a greenhouse. At the two-node stage, foliar applications of iron oxide nanoparticles (control, 15, and 30 ppm) were also made. The Hiltner test best predicted seedling emergence form the soil, whereas the other tests showed no significant predictive power. The findings indicated that the combined treatment of large seeds plus clay loam soil plus application of 30 ppm ION produced the highest seedling height, chlorophyll a and chlorophyll b levels, soluble sugars content, essential oil percentage, and gas exchange, while the combined treatment of small seed + loam soil + no foliar ION application resulted in the lowest levels of these variables. Loam soil with small seeds and no foliar ION spray increased proline concentration and antioxidant enzyme activity. Results of soil texture analysis and Hiltner seed vigor test were found crucial for farmers who cultivate Melissa officinalis L. Finally, ION foliar spraying is suggested for better physiological performance and yield of this plant.
This study was conducted on Melissa officinalis (lemon balm) to compare the effects of seed vigor tests, under laboratory conditions, on seedling emergence from soil (greenhouse) and to study the effects of foliar application of iron oxide nanoparticles (ION) on growth variables and physiological performance of Melissa officinalis seedlings. Seed vigor tests were conducted on seeds of three sizes (large, medium, and small) in the laboratory. These tests included standard germination, accelerated aging, cold, Hiltner, and electrical conductivity (EC). Seeds of three sizes were planted in two different soil types, namely loam and clay loam, in a greenhouse. At the two-node stage, foliar applications of iron oxide nanoparticles (control, 15, and 30 ppm) were also made. The Hiltner test best predicted seedling emergence form the soil, whereas the other tests showed no significant predictive power. The findings indicated that the combined treatment of large seeds plus clay loam soil plus application of 30 ppm ION produced the highest seedling height, chlorophyll a and chlorophyll b levels, soluble sugars content, essential oil percentage, and gas exchange, while the combined treatment of small seed + loam soil + no foliar ION application resulted in the lowest levels of these variables. Loam soil with small seeds and no foliar ION spray increased proline concentration and antioxidant enzyme activity. Results of soil texture analysis and Hiltner seed vigor test were found crucial for farmers who cultivate Melissa officinalis L. Finally, ION foliar spraying is suggested for better physiological performance and yield of this plant.
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1405
Hamid Reza Eisvand1*, Zeinab Farajollahi1, Dariush Goodarzi1, Elham Jahangirinia1, Donald L. Smith2
1. Department of Plant Production and Genetic Engineering, Faculty of Agriculture, Lorestan University, Khorramabad, Iran
2. Department of Plant Science, McGill University, Canada
________________________________________________________________________________
Abstract
This study was conducted on Melissa officinalis (lemon balm) to compare the effects of seed vigor tests, under laboratory conditions, on seedling emergence from soil (greenhouse) and to study the effects of foliar application of iron oxide nanoparticles (ION) on growth variables and physiological performance of Melissa officinalis seedlings. Seed vigor tests were conducted on seeds of three sizes (large, medium, and small) in the laboratory. These tests included standard germination, accelerated aging, cold, Hiltner, and electrical conductivity (EC). Seeds of three sizes were planted in two different soil types, namely loam and clay loam, in a greenhouse. At the two-node stage, foliar applications of iron oxide nanoparticles (control, 15, and 30 ppm) were also made. The Hiltner test best predicted seedling emergence form the soil, whereas the other tests showed no significant predictive power. The findings indicated that the combined treatment of large seeds plus clay loam soil plus application of 30 ppm ION produced the highest seedling height, chlorophyll a and chlorophyll b levels, soluble sugars content, essential oil percentage, and gas exchange, while the combined treatment of small seed + loam soil + no foliar ION application resulted in the lowest levels of these variables. Loam soil with small seeds and no foliar ION spray increased proline concentration and antioxidant enzyme activity. Results of soil texture analysis and Hiltner seed vigor test were found crucial for farmers who cultivate Melissa officinalis L. Finally, ION foliar spraying is suggested for better physiological performance and yield of this plant.
Keywords: Hiltner test, nano fertilizer, plant nutrition, seed vigor, soil texture
Eisvand, H.R., Z. Farajollahi, D. Goodarzi, E. Jahangirinia, D. L. Smith. 2024. 'Efficiency of seed vigor tests in estimating Melissa officinalis L. seedling emergence in soil and the effects of iron oxide nanoparticles on the seedling’s physiological quality'. Iranian Journal of Plant Physiology, 14(2): 4911-4923.
Medicinal plants are those that have at least one therapeutically useful active component in one or more of their organs. These components serve as basis for production of plant-based chemical-pharmaceuticals. Due to their wide biological and pharmacological activities, higher safety margins, and cheaper production costs, medicines produced from these plants are in demand as primary health care components in both developed and developing nations )Yudharaj et al., 2016(. Agriculture is the most significant economic activity in rural areas of Iran. The strategy of planting and producing medicinal plants and processing the resulting crops is a technique to lessen the impact on natural vegetation, diversify source of income, and potentially double earnings in villages. To accomplish this goal, it may be crucial to allocate rainfed fields, low-yield lands, or national lands for the cultivation of medicinal plants near villages. Reduction of agricultural water use, preservation of water and soil, creation of jobs, reduction of migration to cities, stimulation of the rural economy, provision of medicinal plants required by pharmaceutical companies, provision of trade, increase in non-oil exports, development of the rural ecotourism sector, and protection of the genetic resources of these plants are some advantages of these measures (Tarhani, 2015).
Melissa officinalis L., sometimes known as lemon balm is a dicotyledon that is a member of the mint family (Lamiaceae). The Mediterranean and northern parts of Iran have the highest abundance of this herbaceous, perennial plant. Its dried leaves include 11.8% polyphenolic constituents and 0.134% citral, along with 11.34% cinnaminic chemicals. Chemical screening also revealed 2.2% methyl heptenone, 14.4% citronellal, 2.7% linalool, 47.5% geranial, 6-7% isogeraniol, geranyl acetate 10%, and 11% caryophyllene oxide along with alpha-pinene, cispara-Meth2-en-7-ol, nerol acetate, IR-alpha-pinene, carane, verbenol, verbenone, menthol, germanicol, longifolene, himachala 2, 4 -diene, and andropholide. Lemon balm is well known for having a variety of medical benefits for ailments like digestive disturbance, oxidative stress, and neurological diseases )Awad et al., 2009(.
Most plants are propagated from seed, which makes seed quality assurance a top priority in contemporary seed science and a requirement for producing good yields across all crop plant species. High quality seeds must be used in order to provide a healthy crop yield because seed germination is one of the most crucial plant phenological stages that is influenced by seed vigor. The effects of seed production, harvesting, and storage conditions combine to affect seed quality. Considering that seeds with high germination ability in the laboratory do not necessarily have sufficient germination in the field and produce fewer seedlings, determining seed vigor by appropriate and specific tests for a plant and cultivation conditions is crucial (Hassan and Hassan, 2018(.
Seed quality has many aspects including seed germination, seed health, seed purity, seed size, genetic characteristics, seed vigor, and storability. Normally, physiological activity of seeds is determined by a standard germination test. But the standard germination test often estimates seed germination percentage to be greater than is seen under field conditions, because in the standard germination test, natural seedling production is measured under favorable conditions (Zhang and Ghanem, 2018). Determining seed vigor through appropriate and specific tests for a plant and set if cultivation conditions is of particular importance because seeds with high germination ability in the laboratory may not necessarily have sufficient germination in the field and produce fewer seedlings (Hassan and Hassan, 2018(. Seed germination, health, purity, size, genetic features, vigor, and storage ability are just a few of the numerous factors that make up seed quality. Typically, the standard germination test is used to assess the physiological quality of seeds. However, because natural seedling production is assessed in a controlled environment, the common germination test frequently overestimates the seed germination percentage compared to field circumstances (Zhang and Ghanem, 2018).
There are numerous seed vigor tests available, but only a small number of them are accepted by seed experts and seed testing organizations. Several studies on seed vigor tests have been published, including standard germination tests for Vicia sativa (Tao et al., 2022), accelerated aging test for rice (Wang et al., 2022( and Salvia officinalis (Eisvand and Farajollahi, 2017), electrical conductivity test for endemic species (Marin et al., 2018), and cold test for maize (Jha and Mohamed, 2023(. Contrary to tests, which are often carried out in a laboratory environment in petri dishes, seed germination takes place in the soil matrix of a farm, where a range of stresses are often present. Under field conditions, seedling emergence and establishment occur in a range of climatic conditions that are often undesirable. For this reason, seed vigor tests have been developed to provide an appropriate prediction of emergence and seedling deployment for planting under different climatic conditions. Moisture and soil density are important factors affecting germination percentage, germination rate and plant establishment at the field level. Rapid germination is important because it enables the seedling to emerge from the soil before specific conditions lead to the formation of a crust following rainfall or irrigation. In addition to moisture, soil physical resistance also affects germination and initial seedling establishment. As soil physical resistance increases, seed germination decreases. Physical resistance of soil is affected by seed planting depth and soil compaction )Chimungu et al., 2015.( Soil density and texture affect porosity, air permeability, rooting ability, nutrient flow and biological activity of soil (Descalzi et al., 2018(.
Developing crops can often be iron deficient. Despite the typically high soil iron content, much of this element is fixed as Fe3+, especially at high pH levels, and cannot be absorbed. It is thought that iron shortage in agricultural soils can be remedied by adding ION to the soil via fertilizer or spray applications. In most cases, leaf applied nutrients serve as a supplement to soil fertilizer in order to meet the plant's nutritional needs )Mimmo et al., 2014(. Researchers found that foliar application of iron in nano form has positive and significant effects on vegetative and reproductive traits for Vicia faba when compared to iron chelate in sand soil conditions in their study of the effects of foliar application of different sources of iron improve morpho-physiological traits and nutritional quality of plants such as lemon balm )Mahmoud et al., 2022).
The aim of this study was to obtain answers to the following questions. First, can the use of seed vigor testing techniques and determination of convergence of laboratory and greenhouse results be effective in identifying the quality of seeds of lemon balm for the farmer? Second, whether seed quality, soil texture, and foliar application of iron nano-oxide have different effects on growth and physiological and antioxidant traits. The final question was whether the difference in seed size and micronutrient leaf nutrition can modulate morphophysiological indices of Melissa officinalis under various soil texture conditions.
Materials and Methods
This study was carried out in 2022 to evaluate the seed vigor of the medicinal plant Melissa officinalis in laboratory and the greenhouse conditions of Plant Production Engineering and Genetics Department, Faculty of Agriculture, Lorestan University, Iran. Medicinal Plants Research Center in Khorramabad provided 90% viable and 98% pure lemon balm seeds.
Laboratory survey
In the experimental phase, seeds were split into three groups according to their appearance and 1000-seed weight, namely large (1000-seed weight = 0.8 g), medium (1000-seed weight = 0.5 g), and small (1000-seed weight = 0.3 g). A scale with a 0.001-gram precision was utilized for these groupings. The moisture contents of seed samples were then randomly evaluated to prevent the impact of moisture on seed weight. Seeds were subjected to a variety of vigor tests, including normal germination, accelerated aging, cold test, Hiltner, and electrical conductivity tests. The association between the outcomes of each test and the rate of emergence in pot soil was assessed using the T-test. Also, Pearson correlation was used to examine the connection between the electrical conductivity test and the seedling emergence percentage in potted soil.
Vigor tests
Standard germination test
Petri dishes of 9 cm diameter and 1.5 cm height were disinfected with 15% sodium hypochlorite for the standard germination test. Eight replications (400 seeds) were used in total, with 50 seeds sown on filter paper in each Petri dish. Seeds were kept in a germinator at a constant temperature of 25 ± 2 ℃, 8 hours of darkness, 16 hours of light, and 70% relative humidity. According to ISTA guidelines (ISTA, 2009), normal seedlings with their usual roots, shoots, colors, and sizes as well as abnormally germinated seeds were counted.
Fig. I. Results of seed vigor tests; A: Accelerated aging test, B: Standard germination test, C: Cold test, and D: Hiltner test.
|
Eight (8) replications of 50 seeds were utilized for this test. Seeds were exposed to a temperature of 40 ± 1 ℃ and 100% relative humidity for 24 h (Hampton and Tekrony, 1995). After the seeds underwent accelerated age, they were kept in a germinator for 10 days at a constant temperature of 25 ± 2 ℃, 8 h of darkness, 16 h of light, and 70% relative humidity. Following the criteria of the ISTA (ISTA, 2009) criteria for abnormally germinated seeds, natural seedling count with roots, shoots, and natural color and size was carried out (Fig. I).
Cold test
Eight of the 50-seed replications were put in plastic containers with wet paper for seven days at 8 ℃. The percentage of germination and seed vigor were then assessed after being placed in a germinator at 25 ± 2 ℃ (Fig. I).
Hiltner test
Brick grit weighing 1100 grams and having diameters between 2 and 3 mm was autoclave sterilized; then, 250 ml of distilled water was poured to the brick grits to moisten them. Next, a three-cm layer of brick grits was spread out on the bottom of each container, and the seeds were then covered with a one-cm-thick layer of brick grits (Fig. I). A total of 400 seeds were used in 8 replications (50 seeds in each replication) for this test. The containers holding seeds were put in a germinator at 20 ± 2 ℃ for 14 days, at which timey, normal and abnormal seedlings were counted (Hampton and Tekrony, 1995).
Electrical conductivity test
First, 250 mL distilled water was poured in a 500 mL container and stored at 20 ℃ for 24 h to achieve equilibrium temperature. Then, 8 samples of 50 seeds from each seed mass were carefully weighed and placed in each container containing distilled water. Each container was covered with an aluminum cap and placed at 20 ± 1 ℃ for 24 h. Afterwards, the seeds were removed and the solution was slowly shaken for about 5 minutes and the electrical conductivity of the solutions was determined with an EC meter. The electrical conductivity of the distilled water was measured at 20 ℃, and the electrical conductivity per gram of seed weight of each experimental unit was calculated from the following equation (Hampton and Tekrony, 1995).
EC=ECs/W
EC: electrical conductivity, ECs: EC given by the EC meter, W: seed weight in grams.
Greenhouse experiment
Table 1 Physico-chemical analysis of soils
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Seedling emergence percentage
The pots were assessed daily and number of emerged seedlings were counted. Seedling emergence percentage and speed of seedling emergence were calculated using the obtained daily data.
Gas exchange and photosynthesis
Leaf gas exchanges was monitored using a portable infrared gas analyzer (IRGA—LC pro+; ADC Bio Scientific, the UK) five days following ION spraying. The stomatal conductance (gs) and net photosynthesis rate (PN) were measured on a sunny day, between 9:00 and 10:00 a.m., using the central leaflet of the first leaf completely expanded from the apex with a photosynthetic flux density (PPFD) of 1200 (μ mol m−2 s−1, ambient CO2 concentration (~ 400 μ mol mol−1), ambient air temperature 22 ℃, and an air flow rate 0.3 mol m−2 s−1).
Antioxidant enzyme
Antioxidant enzymes and soluble proteins were extracted from leaf tissues according to the method of )Badawi et al., 2004(. The extraction mixture was prepared by homogenizing 500 mg of fresh plant material in 5 mL of extraction buffer, which consisted of 50 mM phosphate buffer (pH 7.6), 1.0 mM ascorbate, and 1.0 mM EDTA. Samples were centrifuged at (14,000 g for 4 mins. at 3 ℃), and the supernatant was collected. Quantification of the total soluble protein levels was performed using the method described by Bradford (1976). Absorbance was measured at 595 nm, using bovine albumin as a standard.
Catalase (CAT) assay
To assay CAT (EC 1.11.1.6), 0.2 mL of the supernatant and 1.8 mL of a reaction mixture containing 50 mM phosphate buffer (pH 7.0) and 12.5 mM hydrogen peroxide were mixed, and the absorbance was measured at 240 nm )Havir and McHale, 1987). The CAT activity was expressed in units mg–1 protein.
Proline and soluble sugars content
Fig. II. Effects of soil type and seed size on seedling emergence of M. officinalis L; bars indicate standard error. Means with at least one common letter are not significantly different from each other according to LSD Test (p ≤0.05).
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Photosynthetic pigment assay (chlorophyll a and b)
After extraction of leaves (0.2 g) in 0.5 mL acetone (3% v/v) and then centrifuging (10000 rpm, 10 min), the absorptions of the supernatants were recorded at 645 nm (Chl b) and 663 nm (Chl a) by UV–Vis spectrophotometry (UV-1800 Shimadzu, Japan). The obtained absorptions were converted to the pigment values (Lichtenthaler, 1987).
Essential oil content
Branches were harvested and air dried in the shade. Then, 10 g of the dry branches were crushed and ground into powder to determine the amount of essential oil content by water distillation using a Clevenger device (model EMO500/C). The amount of essential oil (in percent) was also calculated after desiccating the water with dry sodium sulfate (Kapoor et al., 2002).
Data Analysis
Minitab 16 was used to conduct T-tests and drawing graphs. SAS software (Version 9:00 TS Level 00M0) was used for ANOVA. The means were compared by LSD test at 5% probability level.
Results
Seedling emergence percentage
The highest percentage of emergence per pot was observed for clay loam soil and the use of large size seeds (83.07%); the lowest percentage of emergence (77.17%) was observed for loamy soil and the use of small seeds (Fig. II).
Plant height
Based on the results of the comparison of the average of triple effects, the highest plant height was obtained in the clay loam soil substrate with the application of 30 ppm iron nano-oxide (50.43 cm) in planting conditions. Minimum plant height was observed for loamy soil and using smaller seeds, and control nutrient conditions (no spraying) (Table 2).
Chlorophyll a
Based on comparisons of the means of triple effects, the highest chlorophyll a content was obtained for clay loam soil, using large size seeds and application of 30 ppm iron nano-oxide (3.91 μg.g FW-1). Also, the lowest chlorophyll a content was observed for loamy soil culture conditions, using smaller seeds, and under control nutrient conditions (no foliar application of iron nano-oxide) (Table 2).
Chlorophyll b
Based on the results of the comparison of the means, the highest chlorophyll b content was obtained for clay loam soil, using large size seeds and application of 30 ppm iron nano-oxide (2.25 μg g FW-1). Also, the lowest chlorophyll b content was for loamy soil culture conditions, using smaller seeds, and under control nutrient conditions (no foliar application of iron nano-oxide) (Table 2).
Stomatal conductance (gs)
Table 2. Effect of seed size and ION foliar on plant height and some physiological traits M. officinalis L. under clay loam and loam soil conditions.
* Means in each column with at least one same letter are not significantly different from each other according to LSD Test (p ≤ 0.05). Values described corresponding to means from three replications ± standard errors (FS: foliar spraying) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Net photosynthesis rate (PN)
Based on the comparison of the means, the highest net photosynthesis rate in pots was occurred for clay loam soil, the use of large seeds, and application of 30 ppm iron nano-oxide (7.70 μmol CO2 m−2 s−1). Also, the lowest net photosynthesis rate was the result of culture of smaller seeds in loam soil and under control nutritional conditions (Table 2).
Table 3. T-test probability level of vigor test results with seedling emergence in soil.
ns, *, and ** indicate non-significant, significant at 0.05, andsignficant at 0.001 probability levels, respectively.
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Based on comparison of the mean effects of the three factors of the study, the lowest proline content was obtained in clay loam soil, using large seeds, and application of 30 ppm iron nano-oxide solution of 0.77 (molg-1 FW). Also, the highest amount of proline resulted from cultivation in loamy soil, using smaller seeds, and under control nutrient conditions (no spray application) (Table 2).
Soluble sugars
Based on comparison of the average effect of three factors, the highest soluble sugars content was 2.71 mg g-1 FW in clay loam soil, using large seeds, and application of 30 ppm iron nano-oxide solution. On the other hand, the lowest soluble sugars content was the result of culture in loamy soil, using smaller seeds, and under control nutritional condition (no spraying) (Table 2).
Essential oil
Based on comparison of the mean effects of the three factors of the study, the highest percentage of essential oil (8.91%) resulted from culture in clay loam soil, using large seeds, and with application of 30 ppm iron nano-oxide. The lowest essential oil content occurred with culture in loamy soil, using smaller seeds and under control nutritional conditions (no spraying) (Table 2).
Catalase (CAT)
Based on the results of the comparison of the average of the three factors of the lowest catalase enzyme level occurred for clay loamy soil, the use of large size seeds, and application of 30 ppm of iron Nano-oxide (3.82 U mg–1 protein) was obtained. Also, the highest amount of catalase enzyme resulted from the loam soil, using seeds with other small and control nutrient conditions (no spraying) factor combination (Table 2).
Vigor tests and seedling emergence percentage in pot soil
The relationship between the results of vigor tests as germination percentage and seedling emergence percent from pot soil was investigated using a T-test (Fig. III). Also, the relationship between electrical conductivity measurement as dS m-1 and seedling emergence in pot soil was evaluated by Pearson correlation coefficient. The results showed that among the vigor tests used in this study, only the Hiltner test could accurately and significantly estimate seedling emergence in soil (Table III). As the figure shows, applying a cold test with lower temperature or duration and an accelerated aging test with longer time may make it possible to estimate the emergence of M. officinalis L. seedlings in soil, although more research is required for this. Furthermore, there was a negative, albeit non-significant, relationship between EC test and seedling emergence in soil (r=-0.29, p=0.38).
Discussion
To make economical use of medicinal plants in the agricultural production fields and to use the potential of such natural resources, proper research on the cultivation of these plants is essential. Considered in the present study were the effects of seed size and soil type on germination and emergence of M. officinalis and the effect of these factors on some physiological characteristics of seedlings. In addition, the best test for seedling emergence in soil was determined amongst several common seed vigor tests.
Fig. III. Box plot of the relationship between vigor tests results and seedling emergence percentage in soil (greenhouse) according to T-test. AA-test: accelerated aging test. |
In the present study, decrease in the seed size affected plant quality in two ways. First, the percentage of seedlings appearing in pots decreased. Poor seedling emergence from small seeds, which have lower growth than normal seedlings, use less environmental facilities such as light, moisture, and soil nutrients, and are more sensitive to adverse conditions. Therefore, the optimal quality of lemon balm seedlings grown from smaller seeds was significantly lower than those produced from larger seeds.
One of the goals of foliar application of iron oxide nanoparticles in this experiment was to improve the robustness of weak seedlings produced from smaller seeds. Foliar feeding of iron oxide nanoparticles to seedlings from smaller seeds improved physiological quality of M. officinalis seedlings to some extent, but its effect on medium size seeds was greater than smaller seeds. The results showed that with increasing iron content, most of the growth characteristics increased compared to the control. Considering that the soil is mostly calcareous in the agricultural and garden lands of Iran, the use of nano-iron fertilizer, due to its light and small particles and high responsiveness, can be considered as an appropriate solution across a range of environments. Due to the inactivation of high levels of Fe in some calcareous soils and the reduction of iron absorption in other soils, iron deficiency symptoms such as calcium-induced chlorosis are observed. There are various methods to eliminate iron deficiency such as the use of acidic modified materials, iron chelates, organic compounds, and foliar application of elements (Morales et al., 1998.( Foliar feeding (foliar application) and iron chelators are two general methods of application. Iron compounds are the best solution to remove iron chlorosis in all soils, especially in alkaline soils, and can cure the most severe iron-related nutritional problems in plants (Rodríguez‐Lucena et al., 2010.( Foliar spray addition of elements such as iron, boron, manganese, and copper is more suitable than their application through soil because when nutrients are added to the soil, they adsorb to soil particles and then have less access to the root environment. By the foliar application method, the elements are directly available to the shoots of the plant (Wissuwa et al., 2008(. The role of iron in photosynthesis is due to its participation in oxidation and reduction reactions in chloroplast, in which iron participates in electron donor-receptor groups. By increasing the amount of photosynthesis in order to prevent over-accumulation of photosynthetic materials in chloroplasts, the transfer of these substances to active growth tissues is increased and this stimulates overall plant growth (Marschner et al., 2011(.
The effect of foliar application of levels of iron oxide nanoparticles on physiological characteristics and growth of Lallemantia iberica showed that iron oxide nanoparticles significantly increased physiological characteristics such as chlorophyll and carotenoid contents, and growth variables except leaf number. However, there was no significant effect on proline levels (Javanmard et al., 2022.( Mitigating the effect of environmental stresses by micronutrients can be attributed to the activity of antioxidant enzymes such as superoxide dismutase, catalase, and peroxidase in plant cells and reduction of toxicity of reactive oxygen species ROS, which modulates stress conditions (Lipiec et al., 2013(. Research on the effect of drought stress and foliar application of iron oxide nanoparticles on quantitative and qualitative traits of sesame seeds suggested that foliar application of iron oxide nanoparticles, especially at 15% concentration, is recommended to reduce the harmful effects of drought stress and improve quantitative and qualitative traits of sesame (Karamian Hasan Abadi et al., 2021).
Emergence is the first and most sensitive stage of plant growth and development because the early stages of plant growth including germination, growth and seedling establishment play an important role in the subsequent development of plants. The main point of seed planting is to place seeds at a certain distance apart and depth in the seedbed. Smooth and uniform planting depth provides a better cultivation area for each seed, this is crucial for the timing of the uniformity of emergence, germination rate, and later for crop development and productivity (Batlla and Benech-Arnold, 2007). A major problem for seedlings is the strength of soil resistance, which changes continuously depending on the physical and mechanical properties of the soil. Soil texture, moisture content, compactness, strength, and other physical properties of soil, which are the sources of variability in seed position at planting time (Gonzalez‐Andujar et al., 2016). For this reason, comparative evaluation of M. officinalis seeding conditions in two different soil textures showed that germination and establishment of healthy plants in clay loam soils were greater than loamy soils. In explaining this phenomenon, it can be stated that in clay loam soil, the clay content in the heavier soil texture was higher than loamy. Heavier soils have higher electrical conductivity due to higher ratios of clay and silt. Such soils have higher water contents and more favorable conditions for rapid seed germination (ISTA, 2009); the distribution of aggregate size in seedbed affects germination, development, and productivity of crops (Baskin and Baskin, 2004).
Poor soil porosity, low oxygen permeability, and poor seed cover cause hypoxic conditions in the soil matrix around the seeds. Such hypoxia leads to an increase in fermentation-related volatile metabolites such as acetaldehyde, methanol, and acetone, which play known toxic roles in the seed germination process )Holm, 1972(. Germination tests are used as a qualitative index in evaluation of seeds and seedlings obtained from them. But it cannot be considered as a major indicator of seed quality in the field; the seed is enclosed by cultivation in soil in special environmental conditions that differ significantly from ideal laboratory conditions. It is important to know in such conditions which seed vigor test has the greatest relationship to field conditions. In this case, by identifying the correct test, it is possible to do more accurate testing prior to planting to select seeds that have better capabilities regarding emergence and establishment on the farm. Therefore, in this study, in addition to evaluating and grading the potency and vigor of lemon balm seeds using seed vigor assay tests, the relationship between these tests with the rate of seedling emergence and green cover under pot conditions was investigated in order to predict seedling status, in pots. The results of this study showed that the Hiltner test was able to predict the percentage and rate of emergence of lemon balm seedlings. The cold test was somewhat consistent with pot results, but standard germination, accelerated aging, and electrical conductivity tests lacked this capability. Therefore, we can suggest the Hiltner test to predict the emergence status of M. officinalis in pots.
Acknowledgment
This work was supported by an approved grant (No. 1401-6-02-6-10-1403) from the Lorestan University.
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