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Equipment to assess vigor in soybean seeds using CO2 produced during respiration1 1 Research developed at Laboratório de Fisiologia de Sementes, and Laboratório de Agrotecnologia, Universidade Federal de Pelotas, Pelotas, RS, Brazil

Equipamento para avaliar vigor em sementes de soja através do CO2 produzido durante a respiração

ABSTRACT

The adoption of quick and reliable laboratory techniques and equipment to choose the best seed lots for marketing will influence the production of soybeans with superior physiological quality, among other areas in the sector. Therefore, the objective of this study was to evaluate the CO2 concentrations produced by water-soaked soybean seeds and to verify the effectiveness of new equipment to help choose lots with different vigor levels. To evaluate the physical and physiological quality of the seeds, eight soybean lots were evaluated with the following tests: water content, weight of thousand seeds, first germination count, germination, electrical conductivity, emergence, and respiration evaluated by the Pettenkoffer apparatus and with equipment designed to measure CO2 in seeds. The results were subjected to analysis of variance with means compared by Tukey’s test at p ≤ 0.05. Conventional methods showed significant differences in vigor and viability in soybean seeds. The equipment designed was efficient in detecting CO2 produced by seeds soaked in water for 8 hours. The CO2 readings with the equipment presented satisfactory results to predict the vigor in soybean seeds through respiration.

Key words:
Glycine max L.; MG811 sensor; quick tests; carbon dioxide

RESUMO

A adoção de técnicas e equipamentos laboratoriais rápidos e confiáveis para a escolha dos melhores lotes de sementes para comercialização influenciará a produção de soja com qualidade fisiológica superior, entre outras áreas do setor. Diante disto, objetivou-se avaliar as concentrações de CO2 produzido por sementes de soja embebidas em água e comprovar a eficácia de um novo equipamento que auxilie na seleção de lotes com diferentes níveis de vigor. Para avaliação da qualidade física e fisiológica das sementes, oito lotes de soja foram avaliados com os seguintes testes: teor de água, peso de mil sementes, primeira contagem de germinação, germinação, condutividade elétrica, emergência e respiração avaliadas pelo aparelho Pettenkoffer e com equipamento projetado para medir CO2 em sementes. Os resultados foram submetidos à análise de variância com médias comparadas pelo teste de Tukey a p ≤ 0,05. Os métodos convencionais evidenciaram diferenças significativas do vigor e viabilidade em sementes de soja. O equipamento desenvolvido se mostrou eficiente na detecção de CO2 produzido pelas sementes embebidas em água durante 8 horas. As leituras de CO2 com o equipamento apresentaram resultados satisfatórios para predizer o vigor em sementes de soja por meio da respiração.

Palavras-chave:
Glycine max L.; MG811 sensor; testes rápidos; dióxido de carbono

Introduction

Seed vigor is the set of properties that determines the physiological potential for rapid and uniform emergence and development of normal seedlings under a wide range of environmental conditions (Rocha et al., 2017Rocha, G. C.; Rubio Neto, A.; Cruz, S. J. S.; Campos, G. W. B.; Castro, A. C. de O.; Simon, G. A. Qualidade fisiológica de sementes de soja tratadas e armazenadas. Revista Científic@, v.5, p.50-65, 2017. ). The ambient temperature, water content, and chemical composition of the seeds influence the preservation of physiological quality by altering biological activities and accelerating the respiratory one (Tillmann et al., 2019Tillmann, M. A. A.; Tunes, L. M. de; Almeida, A. da S. Análise de semente. In: Peske, S. T.; Villela, F. A.; Meneghello, G. E. Sementes: fundamentos científicos e tecnológicos. 4.ed. Pelotas: Becker & Peske, 2019, p.147-258. ).

During this process, the stored glucose is used as an energy source, consuming oxygen, and producing CO2 with subsequent release of water and energy (Silva et al., 2017Silva, J. L. da; Costa, F. B. da; Nascimento, A. M. do; Santiago, M. de M.; Gadelha, T. M. Cinética de respiração de frutos de juazeiro (Ziziphus joazeiro Mart.) sob diferentes concentrações de NaOH. Revista Verde de Agroecologia e Desenvolvimento Sustentável, v.12, p.798-801, 2017. https://doi.org/10.18378/rvads.v12i4.4741
https://doi.org/10.18378/rvads.v12i4.474...
). The released CO2 comes from the complete oxidation of carbohydrates and lipids, which are consumed during the degradation of these reserves in the germination process (Marcos Filho, 2015Marcos Filho, J. Fisiologia de sementes de plantas cultivadas. 2. ed., Londrina: ABRATES, 2015. 660p.; Wendt et al., 2017Wendt, L.; Malavasi, M. M.; Dranski, J. A. L.; Malavasi, U. C.; Gomes Junior, F. G. Relações entre testes de vigor com a emergência a campo em sementes de soja. Revista Brasileira de Ciências Agrárias , v.12, p.166-171, 2017. https://doi.org/10.5039/agraria.v12i2a5435
https://doi.org/10.5039/agraria.v12i2a54...
).

Depending on the temperature, humidity, and gas concentration to which the seeds are exposed, these metabolic reactions can vary significantly. Therefore, researchers have opted for reliable and economically viable checks, which help in deciding the fate of certain seed lots through vigor tests to identify less advanced stages of deterioration, according to Wendt et al. (2017Wendt, L.; Malavasi, M. M.; Dranski, J. A. L.; Malavasi, U. C.; Gomes Junior, F. G. Relações entre testes de vigor com a emergência a campo em sementes de soja. Revista Brasileira de Ciências Agrárias , v.12, p.166-171, 2017. https://doi.org/10.5039/agraria.v12i2a5435
https://doi.org/10.5039/agraria.v12i2a54...
).

Promising results were obtained by Aumonde et al. (2012Aumonde, T. Z.; Marini, P.; Moraes, D. M. de; Maia, M. de S.; Pedó, T.; Tillmann, M. A.; Villela, F. A. Classificação do vigor de sementes de feijão-miúdo pela atividade respiratória. Interciencia, v.37, p.55-58, 2012. ) in kidney bean seeds, and by Mendes et al. (2009Mendes, C. R.; Moraes, D. M. de; Lima, M. da G. S.; Lopes, N. F. Respiratory activity for the differentiation of vigor on soybean seeds lots. Revista Brasileira de Sementes , v.31, p.171-176, 2009. https://doi.org/10.1590/S0101-31222009000200020
https://doi.org/10.1590/S0101-3122200900...
) and Dode et al. (2013Dode, J. S.; Meneghello, G. E.; Timm, C.; Moraes, D. M.; Peske, S. T. Teste de respiração em sementes de soja para avaliação da qualidade fisiológica. Ciência Rural, v.43, p.193-198, 2013. https://doi.org/10.1590/S0103-84782013000200001
https://doi.org/10.1590/S0103-8478201300...
) in soybean seeds using the Pettenkoffer apparatus. However, Oliveira et al. (2015Oliveira, L. M. de; Cavalheiro, V. B. D.; Moraes, D. M. de; Tilmann, M. A. A.; Schuch, L. O. B. Medição do CO2 como método alternativo para a diferenciação do vigor de lotes de sementes de melancia. Ciência Rural, v.45, p.606-611, 2015. https://doi.org/10.1590/0103-8478cr20130594
https://doi.org/10.1590/0103-8478cr20130...
), while evaluating the respiratory activity in watermelon seeds, observed that there was no positive and significant correlation between respiration and other vigor tests, corroborating with Dranski et al. (2019Dranski, J. A. L.; Malavasi, M. M.; Malavasi, U. C. Methodology proposal for evaluation of vigor in soybean seeds by respiratory activity. International Journal of Agriculture and Biological Sciences, v.3, p.43-52, 2019. ).

In this context, the objective of this study was to evaluate the CO2 concentrations produced by soybean seeds soaked in water and to prove the effectiveness of new equipment that helps in choosing lots with different levels of vigor.

Material and Methods

The experiment was performed from January to June 2019, in the Laboratory of Seed Physiology, and the Laboratory of Agrotechnology, at the Federal University of Pelotas. Eight soybean seed lots benefited and had statistically similar germination and greater than 80 percent were used, according to IN nº 45 of MAPA (Brasil, 2013Brasil. Legislação Brasileira sobre Sementes e Mudas; Lei n° 10.711, de 05 de agosto de 2003, Instrução Normativa nº 45, de 17 de setembro de 2013. Available on: <Available on: http://www.agricultura.pr.gov.br/arquivos/File/PDF/padroes_milho.pdf >. Accessed on: Ago. 2018.
http://www.agricultura.pr.gov.br/arquivo...
). Owing to the low initial moisture (9.5 ± 0.5%), seeds were preconditioned until 11.8 ± 1.2% moisture (w.b.).

For this, they were evenly distributed on braided wire screens and placed in Gerbox® boxes containing 40 mL of distilled water under ambient conditions (25 ± 5 °C). The duration for which the seeds were in the boxes was based on preliminary tests (18 to 24 hours). After preconditioning, physical and physiological quality analyses were performed using the following tests:

Weight of a thousand seeds (PMS) was evaluated by weighing on analytical scale (0.1 mg) of 8 sub-samples of 100 seeds, from the portion of pure seeds in each lot (Brasil, 2009Brasil - Ministério da Agricultura e Reforma Agrária. Regras para análise de sementes. Brasília: MAPA, 2009. 399p.). The results were expressed in grams.

In conducting the germination test, five replicates of 50 seeds were used, distributed over two sheets of Germitest® paper, moistened with 2.5 times the dry weight of the paper, packed with Biochemical Oxygen Demand (BOD) type germinators at 25 °C with a 12- hour photoperiod, whose counts were performed on the 5th and 8th days, according to Brasil (2009Brasil - Ministério da Agricultura e Reforma Agrária. Regras para análise de sementes. Brasília: MAPA, 2009. 399p.). The results are expressed as percentages.

First germination count (PCG): obtained from the quantification of the number of seedlings accumulated on the first counting day, performed on the fifth day of the germination test. The results are expressed as percentages.

Electrical conductivity test (EC): 250 seeds were used, distributed in five replicates of 50 seeds, weighed, and placed in plastic cups containing 75 mL of deionized water for 24 hours in an air-conditioned room (25 °C). Then, the quality of seeds was evaluated, with the aid of a conductivity meter (Carvalho et al., 2009Carvalho, L. F. de; Sediyama, C. S.; Reis, M. S.; Dias, D. C. F. S.; Moreira, M. A. Influência da temperatura de embebição da semente de soja no teste de condutividade elétrica para avaliação da qualidade fisiológica. Revista Brasileira de Sementes, v.31, p.9-17, 2009. https://doi.org/10.1590/S0101-31222009000100001
https://doi.org/10.1590/S0101-3122200900...
). The results are expressed in μS cm-1 g-1 of seed according to Eq. 1.

C E = reading solution with seeds reading water mass dry seeds (1)

Accelerated aging (EA) was conducted with five replicates of 50 seeds, arranged in a single layer on a steel screen inserted into plastic boxes (Gerbox) containing 40 mL of distilled water (Krzyzanowski, 1991Krzyzanowski, F. C. Teste de comprimento de raiz de plântula de soja. Informativo ABRATES, v.2, p.11-14, 1991.). Subsequently, the boxes were transferred to a germination chamber type BOD at 41 °C for 48 hours. The seeds were then subjected to the germination test, as previously described. The evaluation was carried out on the fifth day after sowing, and the seedlings were considered normal, according to Brasil (2009Brasil - Ministério da Agricultura e Reforma Agrária. Regras para análise de sementes. Brasília: MAPA, 2009. 399p.). The results are expressed as the percentage of normal seedlings.

Emergence (E%): 5 replicates of 50 seeds from each lot were used. The evaluation was based on the final count of the total seedlings that emerged for each lot, carried out 16 days after sowing when the percentage of seedlings remained constant. The results were expressed as percentages. Soil moisture was maintained close to field capacity through daily irrigation with the aid of a watering can.

The assessment of the CO2 produced by the seeds during respiration was performed using equipment that uses MG811 sensor, and with the aid of the Pettenkoffer device, according to Mendes et al. (2009Mendes, C. R.; Moraes, D. M. de; Lima, M. da G. S.; Lopes, N. F. Respiratory activity for the differentiation of vigor on soybean seeds lots. Revista Brasileira de Sementes , v.31, p.171-176, 2009. https://doi.org/10.1590/S0101-31222009000200020
https://doi.org/10.1590/S0101-3122200900...
), using 5 replicates of 10 g of seeds, weighed on an analytical balance (0.1 mg). Respiratory activity was calculated based on the equation proposed by Müller (1964Müller, L. E. Manual de laboratório de fisiologia vegetal. Turrialba, Costa Rica: Instituto Interamericano de Ciencias Agricolas de la O.E.A., 1964. 165p.).

The equipment subject to patent (BR 10 2019 023534 9) transmits the data collected by the MG811 sensor and through electronic boards and circuits, expresses the values on an LCD display (16 x 2, 16-pin). To capture CO2, the sensor remained tightly coupled to a glass container (200 mL) with the seeds inside.

Under laboratory conditions (25 ± 2 °C) for 8 days, the data were obtained every 30 min, for 8 hours, using three replicates of 100 seeds immersed in a volume of 25 mL of distilled water in an airtight container containing the coupled sensor. The period and volume of water were established based on preliminary tests. The values were expressed in millivolts (mV). The analysis of the breathing data mentioned here was performed using descriptive statistics (mean, minimum, median, maximum, and standard deviation).

With the aid of the statistical program Rbio 1.0, the results were tested for normality (Shapiro-Wilk) and homoscedasticity (Brown-Forsythe) and, if complied with ANOVA assumptions, they were subjected to analysis of variance with means compared by the Tukey test at p ≤ 0.05.

Data on the mass of a thousand seeds, first germination count, accelerated aging, and respiration using the Pettenkoffer method showed normal distribution, eliminating the need for data transformation. However, the water content, germination, and emergence variables were transformed to arcsin √(x/100) and, for log (x) to the electrical conductivity variable.

Results and Discussion

The results for the water content variable show differences between the tested lots, classifying lots 3, 4, 5, and 7, with means statistically similar and superior to the others (Table 1). According to Tunes et al. (2012Tunes, L. M. de; Tavares, L. C.; Barros, A. C. S. A. Envelhecimento acelerado como teste de vigor para sementes de arroz. Revista Ciências Agrárias, v.35, p.120-127, 2012. ), when the water content of the seeds is relatively low, greater reliability is allowed for the results obtained in the physiological quality tests, because, within certain limits, the more humid seeds are, the more susceptible they are to deterioration.

Table 1
Mean values for the variables water content (TA), germination (G), electrical conductivity (EC), and field emergence (E), first germination count (PCG%), accelerated aging (EA%), respiration by the Pettenkoffer, and weight of a thousand seeds (PMS) in eight lots of soybean seeds

However, according to Barbosa et al. (2012Barbosa, R. M.; Silva, C. B.; Medeiros, M. A.; Centurion, M. A. P. C.; Vieira, R. D. Condutividade elétrica em função do teor de água inicial de sementes de amendoim. Ciência Rural, v.42, p.45-51, 2012. https://doi.org/10.1590/S0103-84782012000100008
https://doi.org/10.1590/S0103-8478201200...
) and Paixão et al. (2017Paixão, C. S.; Chrispin, C. P.; Silva, R. P. da; Girio, L. A.; Voltarelli, M. A. Physical and physiological quality of soybean seeds at three speeds of the harvester. Revista Brasileira de Engenharia Agrícola e Ambiental, v.21, p.214-218, 2017. https://doi.org/10.1590/1807-1929/agriambi.v21n3p214-218
https://doi.org/10.1590/1807-1929/agriam...
), the water content of the seeds directly influences the integrity of the membranes, as assessed by the electrical conductivity test, and can influence other analyses of physiological quality.

The evaluations of the first germination count referred to by Martins et al. (2017Martins, A. B. N.; Xavier, F. da M.; Dias, L. W.; Meneguzzo, M. R. R.; Vera, M. J. G.; Moraes, D. M. de. Qualidade fisiológica de lotes de sementes de amaranto. Revista CONGREGA URCAMP, v.14, p.2433-2440, 2017.) as an indication of vigor, showed significant differences between lots (Table 1), with the lowest means observed in lots 1 and 8. Eventually, this variable better expresses the differences in germination speed between seed lots (Martins et al., 2017Martins, A. B. N.; Xavier, F. da M.; Dias, L. W.; Meneguzzo, M. R. R.; Vera, M. J. G.; Moraes, D. M. de. Qualidade fisiológica de lotes de sementes de amaranto. Revista CONGREGA URCAMP, v.14, p.2433-2440, 2017.), which makes it liable to fail, indicating the performance of a population in totally favorable conditions, and to benefit many lots of medium vigor.

The germination evaluation (Table 1) allowed the classification of the lots at three levels of viability (high, medium, and low). Despite the statistical differences observed, all lots showed germination higher than the minimum recommended for commercialization (80%), according to IN nº 45, September 17, 2013 (Brasil, 2013Brasil. Legislação Brasileira sobre Sementes e Mudas; Lei n° 10.711, de 05 de agosto de 2003, Instrução Normativa nº 45, de 17 de setembro de 2013. Available on: <Available on: http://www.agricultura.pr.gov.br/arquivos/File/PDF/padroes_milho.pdf >. Accessed on: Ago. 2018.
http://www.agricultura.pr.gov.br/arquivo...
), which is preferable for physiological quality analyses.

The chemical instability of lipids is one of the main factors in reducing the germination speed of several species (José et al., 2010José, S. C. B. R.; Salomão, A. N.; Costa, T. da S. A.; Silva, J. T. T. T. da; Curi, C. C. da S. Armazenamento de sementes de girassol em temperaturas subzero: aspectos fisiológicos e bioquímicos. Revista Brasileira de Sementes . v.32, p.29-38, 2010. https://doi.org/10.1590/S0101-31222010000400004
https://doi.org/10.1590/S0101-3122201000...
); therefore, lipid peroxidation and oxidative stress cause the deterioration of seeds during their aging.

Through the accelerated aging test (Table 1), it was possible to classify lots 4 and 5 as high vigor (71% and 68%, respectively) and suggest that the other lots showed low vigor, with low tolerance to adverse conditions, since the results of the emergence in the sowing bed were superior to those of accelerated aging, showing little or no stress conditions during the emergence.

Greater water absorption during accelerated aging can promote further deterioration and, consequently, prevent the seed lot from expressing its maximum physiological potential (Tunes et al., 2012Tunes, L. M. de; Tavares, L. C.; Barros, A. C. S. A. Envelhecimento acelerado como teste de vigor para sementes de arroz. Revista Ciências Agrárias, v.35, p.120-127, 2012. ). However, the use of saline solutions promotes the reduction of relative humidity and water absorption by the seeds, thereby reducing the deterioration and favoring the expression of the vigor of the lot (Tunes et al., 2012Tunes, L. M. de; Tavares, L. C.; Barros, A. C. S. A. Envelhecimento acelerado como teste de vigor para sementes de arroz. Revista Ciências Agrárias, v.35, p.120-127, 2012. ).

Cellular damage assessed by the electrical conductivity of seeds in lots 1, 3, and 8 (Table 1) allowed them to be classified as having low vigor, as well as in EA and EC. Injuries caused by beneficiation or even during imbibition may have caused irreversible damage, causing the rupture of membranes and the release of exudates, showing more severe effects in lots with lower water content (Barbosa et al., 2012Barbosa, R. M.; Silva, C. B.; Medeiros, M. A.; Centurion, M. A. P. C.; Vieira, R. D. Condutividade elétrica em função do teor de água inicial de sementes de amendoim. Ciência Rural, v.42, p.45-51, 2012. https://doi.org/10.1590/S0103-84782012000100008
https://doi.org/10.1590/S0103-8478201200...
; Gordin et al., 2015Gordin, C. R. B.; Scalon, S. P. Q.; Masetto, T. E. Disponibilidade hídrica do substrato e teor de água da semente na germinação de niger. Pesquisa Agropecuária Tropical, v.45, p.312-318, 2015. https://doi.org/10.1590/1983-40632015v4535337
https://doi.org/10.1590/1983-40632015v45...
; Paixão et al., 2017Paixão, C. S.; Chrispin, C. P.; Silva, R. P. da; Girio, L. A.; Voltarelli, M. A. Physical and physiological quality of soybean seeds at three speeds of the harvester. Revista Brasileira de Engenharia Agrícola e Ambiental, v.21, p.214-218, 2017. https://doi.org/10.1590/1807-1929/agriambi.v21n3p214-218
https://doi.org/10.1590/1807-1929/agriam...
).

The differences between the lots when evaluating the weight of a thousand seeds (Table 1) are not associated with vigor, considering that some lots with higher PMS (Lots 1, 3, 4, 5, and 8) presented medium or low vigor. As reported by Conrad et al. (2017Conrad, V. A. D.; Radke, A. K.; Villela, F. A. Atributos físicos e fisiológicos em sementes de soja no beneficiamento. Magistra, v.29, p.56-63, 2017. ), the weight of a thousand seeds is influenced by the size of the sieve in which they are classified during beneficiation.

Hampton et al. (2013Hampton, J. G.; Boelt, B.; Rolston, M. P.; Chastain, T. G. Effects of elevated CO2 and temperature on seed quality. Journal of Agricultural Science, v.151, p.154-162, 2013. https://doi.org/10.1017/S0021859612000263
https://doi.org/10.1017/S002185961200026...
) also stated that there is no relationship between seed mass and vigor and germination in seed lots. However, the C/N ratio may result in a reduction in the capacity of seeds to meet the requirement of amino acids for the synthesis of proteins necessary for embryo growth and seed germination.

According to Table 1, seed respiration evaluations by the Pettenkoffer apparatus were not efficient in classifying the lots at different levels of vigor, since the observed values did not differ statistically by the Tukey test (p > 0.05). These results do not corroborate those obtained by Mendes et al. (2009Mendes, C. R.; Moraes, D. M. de; Lima, M. da G. S.; Lopes, N. F. Respiratory activity for the differentiation of vigor on soybean seeds lots. Revista Brasileira de Sementes , v.31, p.171-176, 2009. https://doi.org/10.1590/S0101-31222009000200020
https://doi.org/10.1590/S0101-3122200900...
), Aumonde et al. (2012Aumonde, T. Z.; Marini, P.; Moraes, D. M. de; Maia, M. de S.; Pedó, T.; Tillmann, M. A.; Villela, F. A. Classificação do vigor de sementes de feijão-miúdo pela atividade respiratória. Interciencia, v.37, p.55-58, 2012. ), and Leite et al. (2018Leite, M.S.; Torres, S.B.; Freitas, R.M.O.; Nogueira, N.W.; Leite, T.S.; Paiva, E.P. Vigor determination of okra seeds by respiratory activity. Bioscience Journal, v.34, p.1551-1554, 2018. https://doi.org/10.14393/BJ-v34n6a2018-39647
https://doi.org/10.14393/BJ-v34n6a2018-3...
), when assessing respiration in soybean, kidney beans, and okra seeds, respectively.

According to Lamarca & Barbedo (2012Lamarca, E. V.; Barbedo, C. J. Short storability of Caesalpinea echinata Lam. seeds as a consequence of oxidative process. Hoehnea, v.39, p.577-586, 2012. https://doi.org/10.1590/S2236-89062012000400006
https://doi.org/10.1590/S2236-8906201200...
), the water content and temperature influence the respiratory metabolism of the seeds, so that the increase in O2 consumption without the corresponding release of CO2 suggests the inefficiency of antioxidant systems in the seed. Besides, not all carbon in the respiratory route will become CO2, with intermediates that will be used in the synthesis of amino acids, lipids, and other compounds (Chen & Arora, 2011Chen, K.; Arora, R. Dynamics of the antioxidant system during seed osmopriming, post-priming germination, and seedling establishment in Spinach (Spinacia oleracea). Plant Science, v.180, p.212-220, 2011. https://doi.org/10.1016/j.plantsci.2010.08.007
https://doi.org/10.1016/j.plantsci.2010....
).

The difference observed between the consumption of O2 and the production of CO2 by the seeds, according to Lamarca & Barbedo (2012Lamarca, E. V.; Barbedo, C. J. Short storability of Caesalpinea echinata Lam. seeds as a consequence of oxidative process. Hoehnea, v.39, p.577-586, 2012. https://doi.org/10.1590/S2236-89062012000400006
https://doi.org/10.1590/S2236-8906201200...
), may also be related to the use of fatty acids as an initial substrate for respiration, producing less CO2 per mole of oxygen. During the incubation of Caesalphinea echinata seeds, Lamarca & Barbedo (2012Lamarca, E. V.; Barbedo, C. J. Short storability of Caesalpinea echinata Lam. seeds as a consequence of oxidative process. Hoehnea, v.39, p.577-586, 2012. https://doi.org/10.1590/S2236-89062012000400006
https://doi.org/10.1590/S2236-8906201200...
) measured the levels of O2 consumed and CO2 produced and found that seeds with a water content of 37% produced 60 µmol g-1 of dry mass day-1, and that seeds with 6% water content and O2 consumption were three times greater than CO2 release.

Regarding seed respiration evaluated with the equipment subject to the patent (Figure 1), in the first 2 hours there was no relevance in the data owing to the volume of water in the bottle and the need for gas stabilization, which would eliminate the interference of CO2 from the environment. In addition, an initial situation of hypoxia could occur in the early stages of seed imbibition (Marinho et al., 2019Marinho, J. de L.; Costa, D. S. da; Carvalho, D. U. de; Cruz, M. A. da; Zucareli, C. Evaluation of vigor and tolerance of sweet corn seeds under hypoxia. Journal of Seed Science, v.41, p.180-186, 2019. https://doi.org/10.1590/2317-1545v41n2209568
https://doi.org/10.1590/2317-1545v41n220...
).

Figure 1
Respiration in soybean seeds in function of time after moistening by means of readings using the equipment subject to patent

Thus, according to Figure 1, the respiration of lots 1 and 4 remained stable during the evaluations, although lot 1 had higher values, proving the hypothesis that less vigorous lots have greater respiration and, consequently, greater deterioration (Tillmann et al., 2019Tillmann, M. A. A.; Tunes, L. M. de; Almeida, A. da S. Análise de semente. In: Peske, S. T.; Villela, F. A.; Meneghello, G. E. Sementes: fundamentos científicos e tecnológicos. 4.ed. Pelotas: Becker & Peske, 2019, p.147-258. ), as proven by the emergence and accelerated aging, as shown in Table 2. This was also observed for lots 2 and 3, whose increase in respiration in lot 2 was noticed from two and a half hours to the five-hour evaluation period. During this period, enzymatic activation, and metabolites necessary for germination occur, characterized by phase 1 of imbibition (Carvalho et al., 2012Carvalho, T. C.; Grzybowsky, C. R. S.; Ohlson, O. C.; Panobianco, M. Comparação da qualidade fisiológica de sementes de soja convencional e de sua derivada transgênica. Revista Brasileira de Sementes , v.34, p.164-170, 2012. https://doi.org/10.1590/S0101-31222012000100020
https://doi.org/10.1590/S0101-3122201200...
).

Table 2
Means and complementary descriptive statistics of respiration data evaluated for 8 hours by the equipment

According to Carvalho et al. (2012Carvalho, T. C.; Grzybowsky, C. R. S.; Ohlson, O. C.; Panobianco, M. Comparação da qualidade fisiológica de sementes de soja convencional e de sua derivada transgênica. Revista Brasileira de Sementes , v.34, p.164-170, 2012. https://doi.org/10.1590/S0101-31222012000100020
https://doi.org/10.1590/S0101-3122201200...
), soybean seeds present radicle protrusion 38 hours after installing the germination test, a fact confirmed by the imbibition curve presented by the authors. The germination phases presented in that work were: phase 1 until 10 hours; phase 2 until 25 hours; and phase 3 until 38 hours.

According to Tillmann et al. (2019Tillmann, M. A. A.; Tunes, L. M. de; Almeida, A. da S. Análise de semente. In: Peske, S. T.; Villela, F. A.; Meneghello, G. E. Sementes: fundamentos científicos e tecnológicos. 4.ed. Pelotas: Becker & Peske, 2019, p.147-258. ), the expression of vigor in seeds under less favorable conditions can be better quantified owing to the degradation of reserves and the consequent release of CO2. Therefore, in high vigor seeds, the CO2 levels will be lower than those observed in low-quality lots. However, there is disagreement regarding the association of vigor and the concentration of CO2 produced by the seeds (Dranski et al., 2019Dranski, J. A. L.; Malavasi, M. M.; Malavasi, U. C. Methodology proposal for evaluation of vigor in soybean seeds by respiratory activity. International Journal of Agriculture and Biological Sciences, v.3, p.43-52, 2019. ).

The values obtained for seed lot 5 show a slight increase in breathing after the first hour of evaluation, whose values indicate levels of vigor lower than in lot 6. However, lot 7 expressed oscillation and high values in the first 3 hours of evaluation (Figure 1), which was also confirmed by the values presented in Table 2.

The increase in respiration after 3 hours of evaluation is a consequence of cellular reorganization and a lower volume of water after imbibition and exposure to O2 in the container. According to Metivier & Paulilo (1980Metivier, J.; Paulilo, M. T. The utilization of cotiledonary reserves in Phaseolus vulgaris, L. cv. carioca I. Journal of Experimental Botany, v.31, p.1271-1282, 1980. https://doi.org/10.1093/jxb/31.5.1271
https://doi.org/10.1093/jxb/31.5.1271...
) and Marcos Filho (2015Marcos Filho, J. Fisiologia de sementes de plantas cultivadas. 2. ed., Londrina: ABRATES, 2015. 660p.), the respiratory rate in beans and soybean seeds, respectively, is higher in the first 6 hours of imbibition.

In the respiration method used by Metivier & Paulilo (1980Metivier, J.; Paulilo, M. T. The utilization of cotiledonary reserves in Phaseolus vulgaris, L. cv. carioca I. Journal of Experimental Botany, v.31, p.1271-1282, 1980. https://doi.org/10.1093/jxb/31.5.1271
https://doi.org/10.1093/jxb/31.5.1271...
), bean seeds were subjected to 12 hours of soaking in distilled water and used a Warburg respirometer at 30 °C, with sucrose and KOH solution, as well as filter paper and thermometer. However, the time the containers remained open may have influenced the CO2 levels fixed on the filter paper strip.

Concerning seed lot 8, in the period from 1 hour 30 min to 5 h hours 30 min of evaluation, there was an increase in CO2 levels because of imbibition, consumption of O2, and beginning of the germination process (Carvalho et al., 2012Carvalho, T. C.; Grzybowsky, C. R. S.; Ohlson, O. C.; Panobianco, M. Comparação da qualidade fisiológica de sementes de soja convencional e de sua derivada transgênica. Revista Brasileira de Sementes , v.34, p.164-170, 2012. https://doi.org/10.1590/S0101-31222012000100020
https://doi.org/10.1590/S0101-3122201200...
). This seed lot had medium vigor levels, possibly owing to the low water content, which compromised the evaluation of PCG and germination (Table 1).

According to Lamarca & Barbedo (2012Lamarca, E. V.; Barbedo, C. J. Short storability of Caesalpinea echinata Lam. seeds as a consequence of oxidative process. Hoehnea, v.39, p.577-586, 2012. https://doi.org/10.1590/S2236-89062012000400006
https://doi.org/10.1590/S2236-8906201200...
), the physiological processes involved in the loss of viability of some seeds may be related to seed respiration or oxidative reactions, such as lipid peroxidation, significantly influencing the reorganization of membranes and resumption of the germination process, resulting in low vigor seeds.

Seeds with advanced levels of deterioration tend to have greater cellular damage, among which the activity of mitochondria can also be compromised, resulting in reduced breathing and generation of adenosine triphosphate (ATP) (Taiz et al., 2017Taiz, L.; Zeiger, E.; Møller, I.M.; Murphy, A. Fisiologia vegetal. 6.ed. Porto Alegre: Artmed, 2017. 888p. ). However, it is likely that during the process of cellular reorganization, the greater metabolic and respiratory activity will occur, according to Dode et al. (2013Dode, J. S.; Meneghello, G. E.; Timm, C.; Moraes, D. M.; Peske, S. T. Teste de respiração em sementes de soja para avaliação da qualidade fisiológica. Ciência Rural, v.43, p.193-198, 2013. https://doi.org/10.1590/S0103-84782013000200001
https://doi.org/10.1590/S0103-8478201300...
) and Venske et al. (2014Venske, E.; Abreu Júnior, J. de S.; Sousa, A. de M. de; Martins, L. F.; Moraes, D. M. de. Atividade respiratória como teste de vigor em sementes de algodão. Revista Brasileira de Ciências Agrárias, v.9, p.174-179, 2014. https://doi.org/10.5039/agraria.v9i2a3518
https://doi.org/10.5039/agraria.v9i2a351...
).

Therefore, when comparing the results between the two methods to assess respiration and vigor in soybean seeds, the new equipment using a CO2 sensor demonstrated differences between the lots evaluated at all time intervals (Figure 1 and Table 2), whereas, by the Pettenkoffer apparatus, the evaluations did not result in a significant effect.

Conclusions

  1. Conventional methods showed significant differences in vigor in different lots of soybean seeds.

  2. The equipment subject to the patent proved efficient in the detection of CO2 produced by soybean seeds soaked in water for eight hours, allowing the estimation of the vigor in soybean seeds through respiration.

Acknowledgements

This study was carried out with support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo a Pesquisa do Rio Grande do Sul (FAPERGS) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil.

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  • 1
    Research developed at Laboratório de Fisiologia de Sementes, and Laboratório de Agrotecnologia, Universidade Federal de Pelotas, Pelotas, RS, Brazil

Highlights:

  • There is a need for new seed vigor tests based on the respiration process.
  • The equipment should be designed to reduce uncertainty in vigor tests.
  • A vigor test based on the CO2 produced by the seeds’ respiration is an excellent method to check the physiologic quality of seed lots.
  • Edited by: Walter Esfrain Pereira

Publication Dates

  • Publication in this collection
    26 Mar 2021
  • Date of issue
    May 2021

History

  • Received
    13 May 2020
  • Accepted
    03 Feb 2021
  • Published
    05 Mar 2021
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