Exudate pH and primary root emergence tests in coriander seedsABSTRACT
The use of efficient, fast, and low-cost tests is essential in seed quality control programs. The objective of this study was to adapt exudate pH and primary root emergence tests for coriander (Coriandrum sativum) seeds. A completely randomized experimental design was used, with four replications of 50 seeds. Four coriander (C. sativum cv. Super Verdão) seed batches were evaluated for moisture content, first count, germination, emergence, emergence speed index, seedling total dry weight, and subjected to accelerated aging and tetrazolium tests. Exudate pH tests were conducted using soaking times of 30, 60, 90, and 120 minutes at 20, 25, and 30 °C. Primary root emergence tests were conducted at 20, 25, and 30 °C, starting observations 24 hours after the beginning of the test at 12-hour intervals and concluding after 120 hours. The exudate pH test at 20 °C for 30 minutes classified the batches similarly to the germination test, but differently from vigor tests. The primary root emergence test conducted at 20 °C, with evaluation after 60 hours, effectively classifies the vigor of coriander seed batches.
Key words:
Coriandrum sativum; rapid tests; viability; vigor
HIGHLIGHTS:
Exudate pH test classifies seed batches similarly to the germination test.
The primary root emergence test provides faster results than conventional vigor tests.
The vigor of seed batches can be classified using the primary root emergence test, after soaking for 60 hours at 20 °C.
RESUMO
A utilização de testes eficientes, rápidos e de baixo custo, é fundamental nos programas de controle da qualidade de sementes. No presente estudo, almeja-se adequar as metodologias dos testes de pH de exsudato e precocidade de emissão de raiz para sementes de coentro. O delineamento experimental foi o inteiramente casualizado com quatro repetições de 50 sementes. Quatro lotes de sementes da cv. Super Verdão foram avaliados em relação ao grau de umidade, primeira contagem, germinação, emergência, índice de velocidade de emergência, envelhecimento acelerado, massa seca total de plântulas e tetrazólio. No teste do pH de exsudato, os tempos de embebição foram de 30, 60, 90 e 120 minutos a 20, 25 e 30 °C. O teste de precocidade de emissão de raiz primária foi conduzido a 20, 25 e 30 °C, iniciando as avaliações com 24 horas após a instalação, com intervalos de 12 horas e finalizando com 120 horas. O teste de pH de exsudato a 20 °C por 30 minutos classificou os lotes de forma semelhante ao teste de germinação, no entanto, não foi eficiente nos testes de vigor. O teste de precocidade de emissão de raiz mostrou-se eficaz na classificação do vigor de lotes de sementes de coentro, sendo conduzido a 20 °C, com avaliação após 60 horas.
Palavras-chave:
Coriandrum sativum; testes rápidos; viabilidade; vigor
Introduction
Coriander (Coriandrum sativum L.) is an annual vegetable crop. Its leaves are rich in essential oils, flavonoids, phenolic compounds, and alkaloids and are commonly utilized in culinary practices worldwide (Mahleyuddin et al., 2022; Talebi et al., 2024). The germination test is routinely used in the laboratory and is officially recognized by the Brazilian Ministry of Agriculture to evaluate the physiological quality of seeds. However, this test lasts 21 days for coriander seeds (BRASIL, 2009), which is a significantly long time to make decisions.
Thus, rapid tests provide a quicker and reliable alternative for assessing seed batch quality and monitoring internal seed quality control programs of companies. However, rapid tests require appropriate methodologies for each species. The exudate pH test is based on the pH difference of the solution of viable and non-viable seeds subjected to soaking, whose viability assessment is linked to deterioration events, such as membrane permeability and solute leaching (Tillmann et al., 2019).
The primary root emergence test assumes that the reduced germination speed is an early expression of the seed’s physiological aging, which is the main cause of reduced vigor. High primary root emergence values at the beginning of the germination test indicate high seed vigor, while low values indicate lower vigor (Krzyzanowski et al., 2020). This test was effective in classifying the vigor of radish (Raphanus sativus L.; Mavi et al., 2016), eggplant (Solanum melongena L.; Ozden et al., 2018), chia (Salvia hispanica L.; Oliveira et al., 2019), and soybean (Glycine max; Rego et al., 2023) seeds. In this context, the objective of this study was to adapt exudate pH and primary root emergence tests to assess the physiological potential of coriander seeds.
Material and Methods
The experiment was conducted from May to September 2023, at the Seed Analysis Laboratory and in the greenhouse of the Universidade Federal Rural do Semi-Árido (UFERSA). Exudate pH and primary root emergence tests were conducted using a completely randomized experimental design, with four batches of coriander seeds and four replications of 50 seeds. Four batches of coriander seeds (A, B, C, and D) of the cultivar Super Verdão were purchased from local stores and kept in plastic bags in a controlled environment (17 °C; 50% relative air humidity) until the beginning of the experiment. The initial physiological potential of each batch was evaluated through various tests and assessments, as described below.
Seed moisture content: determined using the oven method at a temperature of 105 ± 3 °C for 24 hours (BRASIL, 2009), with two replications, each containing approximately 3.5 g. The results were expressed as a percentage (wet basis).
Germination test: conducted according to recommendations of the Rules for Seed Analysis (BRASIL, 2009), using four replications of 50 seeds sown on two blotting paper sheets moistened with distilled water equivalent to 2.5 times the dry paper weight and placed in transparent acrylic boxes (11 × 11 × 3.5 cm). The boxes were placed in a germination chamber at 20 °C with a photoperiod of 8 hours. The first count (at 7 days) and germination (at 21 days) were evaluated and the results were expressed as a percentage of normal seedlings.
Accelerated aging test: conducted in the same boxes used for the germination test, but with an aluminum mesh inside. The seeds (4 g) were evenly distributed on the mesh, and 40 mL of distilled water was added to the bottom of the box. The boxes were placed in a germination chamber for 48 hours at 41 °C (Radke et al., 2016). After this aging period, the batches were subjected to a germination test, using the same methodology described earlier, with normal seedlings assessed seven days after sowing. Seed moisture contents were determined after the accelerated aging period.
Emergence in greenhouse: conducted with four replications of 50 seeds, sown in aluminum trays containing washed and sterilized sand and placed in an oven at 105 °C for 2 hours (BRASIL, 2009). Daily counts were carried out from the sixth to the eighteenth day to evaluate the emergence speed index (ESI) and seedling emergence percentage in each batch. The formula proposed by Maguire (1962) was used to calculate the ESI.
Seedling total dry weight: determined in ten seedlings per repetition for each batch. Seedlings were randomly obtained from the germination test, placed in a Kraft paper bag, and dried in a forced air-circulation oven at 65 °C for approximately 48 hours, until a constant weight was achieved. The materials were then weighed on an analytical balance with a precision of 0.0001 g.
Tetrazolium test: conducted on seeds that had been previously immersed in distilled water for 16 hours at 20 °C. The seeds were then cut lengthwise with a knife, placed in disposable cups containing a 0.5% tetrazolium solution, and incubated for five hours at 40 °C in the dark (Silva et al., 2021).
Seed exudate pH test: conducted using phenolphthalein and sodium carbonate solutions. The phenolphthalein solution was prepared using one gram of the product dissolved in 100 mL of ethyl alcohol and 100 mL of distilled, boiled water (Souto et al., 2019). The sodium carbonate solution was prepared using three previously tested concentrations (0.8, 1.4, and 2.0 g L-1). The number of drops of the two solutions was adjusted; thus, when added to 2 mL of distilled water, the strong pink reference color was obtained (Tillmann et al., 2019). Pasteur pipettes were used to add the drops to each cell. In this sense, one drop of phenolphthalein and two drops of sodium carbonate were suitable.
After determining the concentration of the sodium carbonate solutions (0.8 g L-1) and the appropriate number of drops, the exudate pH test was implemented in 50-cell trays containing 2 mL of distilled water, with one coriander seed per cell and four replications of 50 seeds per batch. The trays were incubated in a germination chamber at temperatures of 20, 25, and 30 °C for 30, 60, 90, and 120 minutes.
After soaking, a drop of phenolphthalein solution and two drops of sodium carbonate were added, each with a separate Pasteur pipette, and the color of the exudate was immediately interpreted. A strong pink or light pink color indicated viable seeds, while a very light pink color indicated non-viable seeds (Souto et al., 2019). The results were expressed as a percentage of viable seeds.
Primary root emergence test: conducted using four replications of 50 seeds. The seeds were placed on two blotting paper sheets moistened with distilled water equivalent to 2.5 times the dry paper weight, and placed in transparent acrylic boxes. The boxes were placed in germination chambers at temperatures of 20, 25, and 30 °C. The evaluation criterion was at least 1 mm of root emergence, based on a preliminary test. Evaluations began 24 hours after the test was set up, at 12-hour intervals and continued until 120 hours had elapsed.
The results of the tests were subjected to analysis of variance using the F test, and means were compared using Tukey’s test at 0.05 probability level, using the statistical program SISVAR 5.3 (Ferreira, 2019).
Results and Discussion
The initial moisture contents of the batches were uniform, differing only 0.2 percentage points between batches B and C (Table 1). According to Marcos-Filho (2020), variation in moisture content between batches should not exceed 3.0 percentage points to prevent influencing the test results. The results for germination and seedling dry weight showed no differences between batches; however, the batches were stratified differently in the first count, emergence, emergence speed index, and accelerated aging tests (Table 1).
The germination test results showed all batches with germination above 80%, with no statistical difference among them. This result indicates that the coriander seed batches meet the minimum standards for commercialization, as required by the Brazilian Ministry of Agriculture and Livestock (Brasil, 2009). This result is usually found in routine analysis in seed testing laboratories, as germination tests are conducted under ideal conditions for each species. This reinforces the importance of using vigor tests, which estimate the development of seed batches after sowing under different environmental conditions.
However, most vigor tests indicated batch C with lower seed quality compared to the others (Table 1). This was demonstrated by the results of first count test, where batch C had 43% germination, while the others had more than 80%. This greater sensitivity of the first count in the germination test for detecting differences between coriander seed batches with similar germination was confirmed by Torres et al. (2015).
The classifications of coriander seed batches by emergence, emergence speed index, accelerated aging, and tetrazolium tests were similar to that of the first count test, with batches A, B, and D being more vigorous than batch C (Table 1). Decreases in the performance of less vigorous batches are commonly observed under less favorable environmental conditions, as is the case of the emergence and accelerated aging tests (Marcos-Filho, 2015). According to the results of the exudate pH test, the batches were distinguishable in most combinations of temperatures and soaking times (Table 2).
The mean percentage of viable seeds at 20 °C showed no statistical differences between the 30- and 120-minute soaking times (Table 2). The results for the 30-minute soaking time at this temperature were consistent with those of the germination test for all four batches, with mean seed viability above 80%. The viability percentage for the 120-minute soaking time was below 80%. Similar research on soybean (Glysine max L.) seeds demonstrated that the exudate pH test at 20 °C for a 30-minute soaking time effectively classified seed batches into different vigor levels (Theodoro et al., 2018). The percentage of viable seeds in batch B was lower at 60 and 90 minutes, differing from the classification obtained in the germination and vigor tests for all batches, and batch C exhibited inferior quality.
The seed batches exhibited no difference at 25 °C, regardless of the soaking time, with percentages of viable seeds below 80%, except for the 30-minute soaking time in batches C and D (Table 2). In contrast, similar research on rice seeds (Oryza sativa L.) demonstrated that the exudate pH test at 25 °C for 60 minutes effectively classified seed batches in a similar way to other initial quality tests (Santos et al., 2019). The seed batches also exhibited no difference at 30 °C, except for the 60- and 120-minute soaking times (Table 2). However, batch B had the lowest viability under these soaking times, differently from the results obtained in most initial quality tests, in which batch C exhibited the lowest physiological potential.
Increasing temperatures typically reduce the percentage of viable seeds during the exudate pH test, as observed in melon seeds (Costa et al., 2023). Higher temperatures accelerate water absorption, affecting the release of exudates with high buffering capacity (Tillmann et al., 2019). Furthermore, soaking rate is directly linked to water absorption time, which is influenced by species-specific seed characteristics, including size, moisture content, shape, internal reserves, and outer layer thickness. These attributes determine the speed and time required for water absorption and, consequently, the flow of soluble substances (Araújo et al., 2014).
The exudate pH test results should align with viability tests, such as germination or tetrazolium, to validate the effectiveness of the methodology applied to the seed batches. In the present study, the classifications of batches using certain combinations of the exudate pH test were similar to those obtained in the germination test. However, they differed from those of the tetrazolium and most vigor tests. In wheat seeds, batch classifications based on the exudate pH test differed from those obtained using initial quality tests (Grzybowski et al., 2021). In contrast, this test was effective in classifying melon batches (Costa et al., 2023).
Results of primary root emergence test at 20 °C showed a significant effect starting at 36 hours (Table 3). However, batches B and C did not differ from each other at 36 and 48 hours, in contrast to the initial vigor test results (Table 1), where only batch C was inferior to the others. Between 60 and 84 hours, the classification of batch was similar to that observed in the initial vigor tests (Table 3).
At 24 hours after the beginning of the test, seeds from batches A, B, and D exhibited higher percentages of root emergence at 25 °C compared to 20 °C. At 36 hours, batch C exhibited earlier root emergence at 25 °C than at 20 °C (Table 4). Overall, the temperature of 25 °C accelerated primary root emergence. However, seed batches were not stratified as effectively as in the initial tests, as batch A did not differ from batch C at 25 °C in most evaluations (Table 4). Seed enzymatic activities are typically faster under higher temperatures, accelerating the germination process (Silva et al., 2018).
The temperature of 30 °C did not favor primary root emergence in the four batches, nor did it significantly differentiate them at most evaluation times, except at 24 hours (Table 5). Nevertheless, the batch classification at 24 hours differed from the initial tests, indicating that this temperature is not suitable for the assessment. This inadequate performance may be due to species-specific requirements, as the Rules for Seed Analysis (BRASIL, 2009) recommend constant temperatures of 15 and 20 °C and alternating temperatures between 20 and 30 °C for optimum germination. For lentil (Lens culinares Medik.) seeds, root emergence was hindered at 30 °C, while temperatures of 20 and 25 °C favored the process (Borges et al., 2024).
Temperature has a direct impact on the percentage and speed of seed germination. An optimum temperature results in high germination in a short period of time, while temperatures above and below the optimum range are unfavorable to the germination process (Batlla & Arnold, 2015). Furthermore, more vigorous seeds usually exhibit higher germination rates across a broader temperature range; however, as they lose vigor become increasingly dependent on specific temperature conditions (Marcos Filho, 2015).
The temperature of 25 °C resulted in the highest primary root emergence speed index and was effective in stratifying the batches, with batch C exhibiting inferior quality compared to the others (Table 6).
Reduction in germination speed is an early indicator of the seed’s physiological aging, which is the main cause of reduced vigor. Germination speed can be accurately identified by a single count of primary root emergence at the beginning of the germination process, which is closely related to indicators that characterize germination speed. High primary root emergence speed indices at the beginning of the germination test indicate high seed vigor, while low indices indicate lower primary seed vigor (Krzyzanowski et al., 2020).
The methodology used for primary root emergence tests varies according to the species, particularly in terms of temperature and evaluation period. For eggplant seeds (Solanum melongena L.), Ozden et al. (2018) recommend temperatures of 20-30 °C or 25 °C, with substrate on paper, no light, and an evaluation time of 104 hours for obtaining at least 2 mm of primary root. For radish (Raphanus sativus), Mavi et al. (2016) recommend a temperature of 20 °C, with substrate on paper, and evaluation after 48 hours. For pumpkin (Cucurbita sp.), Souza et al. (2013) recommend a temperature of 25 °C, paper rolls, and root emergence evaluations after 72 or 96 hours.
The primary root emergence test can be used to identify the level of seed physiological deterioration, since germination speed is influenced by the level of seed deterioration (Krzyzanowski et al., 2020). In general, the seed batch with the lowest vigor according to the initial tests resulted in the lowest primary root emergence. Furthermore, temperatures also significantly influenced the results. Therefore, the primary root emergence test proved effective and can be used in ranking coriander seed batches, with results consistent with those of the other initial vigor tests.
Conclusions
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The classification of coriander seed batches by the exudate pH test at 20 °C, with a soaking time of 30 minutes, is consistent with the classification by the germination test. The use of the exudate pH test under the conditions tested is not as efficient as the vigor test for coriander seeds.
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Primary root emergence test is effective in classifying the vigor of coriander seed batches and can be performed at 20 °C with evaluation 60 hours after the beginning of the test.
Acknowledgments
The authors thank the entire team of the Seed Analysis Laboratory at the Universidade Federal Rural do Semi-Árido for their support.
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