COMPARISON BETWEEN BIOMETRIC AND ELECTROLYTE LEAKAGE TESTS AS INDICATORS OF MAIZE SEED VIGOR

– The objective of this work was to compare the accuracy of seedling tests based on electrolyte release to distinguish different maize seed vigor levels with the accuracy provided by biometric tests for seedling length and biomass. For this purpose, seeds with different vigor levels of the Garra Viptera hybrid had their physiological potential evaluated through tests of germination, accelerated aging, seed electrical conductivity, field emergence, seedling length, seedling dry biomass, seedling electrical conductivity, potassium leakage and membrane injury index. Completely randomized design was used, with four replications, except for the field emergence variable, when the randomized complete block design was adopted. The obtained means were compared by Fisher’s LSD test, at 5% probability, and then Pearson’s correlation coefficients were calculated between the tests based on seedling performance and the physiological quality results obtained with the seeds. In addition to their lower sensitivity to distinguish the different vigor levels, the biometric tests (length and biomass) showed values of correlation with field emergence considerably lower than those obtained with the membrane permeability tests (seedling electrical conductivity, potassium leakage and membrane injury index). Among the tests based on electrolyte extravasation, the membrane injury index was the most accurate to differentiate maize seed lots in different vigor levels, as well as to estimate the seed emergence in the field.


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The use of high quality maize seeds is a determining factor for maize crop establishment and, consequently, for a successful crop yield.
Therefore, an important stage in this process is the prior selection, through lab tests, of seed lots with high physiological performance (Marcos Filho, 2015a). Among those tests, the most relevant are the biometric analyses of length and dry biomass of seedlings, as well as the methods based on electrolyte release to the medium (Miguel & Marcos Filho, 2002;Marcos Filho, 2015b).
Seedling length and biomass are probably the most applied seedling performance tests for seed quality control, as they are based on physical quantities that minimize subjectivities in data interpretation and, at the same time, allow repeatability in relatively simple conditions (Marcos Filho, 2015a), though with controversial results, such as those reported by Boligon et al. (2011) and Marcos Filho (2015b), as to the differentiation of seed lots with close levels of conservation.
With regard to electrical conductivity or potassium leakage, Miguel and Marcos Filho (2002), Pereira et al. (2019), Sharma et al. (2011), and Zucareli et al. (2013), among other authors, report high degrees of correlation between those tests, when applied to seeds, and the subsequent performance observed at field condition.
Basically, such tests associate the plasma membrane integrity loss, which is pointed out as the first event in the seed deterioration process, with the concentration of electrolytes released to the medium during the imbibition process (Marcos Filho, 2015b). The membrane injury index is another test based on electrolyte leakage that has showed promising results for identification of the biotic and abiotic stress level that a plant or its vegetal tissue was submitted to (Demidchik et al., 2014). However, its application as a tool to assess the seed physiological condition is still a little-studied aspect.
In this context, the present work aims to investigate the accuracy of tests based on electrolyte release by maize seedlings to distinguish vigor levels of the original seed lot in comparison with the accuracy provided by the biometric tests for length and biomass.

Material and Methods
The experiment was carried out in the Seed vigor, respectively.
The main cause of vigor differences between seed lots is their aging stage. In the deterioration process, one of the first signs of seed viability loss is the coagulation of membrane proteins (Marcos Filho, 2015a). Therefore, the purpose of the abovementioned procedure was to simulate the natural aging of seeds, a process that results in vigor reduction and culminates in their loss of viability. Although this aging process is currently more used as a vigor parameter, its original purpose is to estimate the storage potential of a seed lot by combining the most critical environmental factors during storage: high temperatures and relative humidity (Marcos Filho, 2015b). Similar procedure for obtaining seed lots with different vigor levels has already been adopted in other studies, as in Kranner et al. (2011).
Once the seed lots of each vigor level were obtained, they all had their physiological potential analyzed by tests based on the performance of seedlings and seeds.

Seed Analysis
Germination test: it was performed for each vigor level, with eight replications of 50 seeds. To germinate, the seeds were placed on two sheets of Germitest ® paper, moistened with distilled water in the proportion of 2.5 times the paper dry weight. Subsequently, a third sheet, which was also moistened as previously described, was positioned over the seeds so as to make paper rolls. The rolls were then taken to a germinator that was regulated to maintain a constant temperature of 25ºC. Evaluation was carried out seven days after the installation by calculating the percentage of normal seedlings (International Seed Testing Association, 2017). At the end of this period, the reading of electrical conductivity in the imbibing solution was performed with the use of a benchtop conductivity meter. Before the reading, the imbibing solution was slightly stirred with the use of a glass rod.
The electrode of the equipment was washed with demineralized water and dried with paper towel before each measurement. The value indicated by the equipment was written down and divided by the weight obtained from each replication.
Thereby, the result obtained was expressed in μS cm -1 g -1 .
Field emergence: it was performed with four replications of 50 seeds. The seeds were distributed at 1.5 cm depth, in furrows of 1.0 m length, spaced 20 cm apart. The normal emerged seedlings were counted 15 days after sowing (Nakagawa, 1999 Where VI = result of the germination on the 5 th day (%) x measured length (cm).

Seedling dry biomass: for each vigor level,
four subsamples of 20 seeds were distributed in Germitest ® paper rolls, moistened with distilled water in the proportion of 2.5 times the paper dry weight. The seeds were maintained in a germinator at 25 ± 2 ºC, for seven days. After that, the seedlings were dried in a forced air circulation oven at 65 o C until a constant weight was achieved (Nakagawa, 1999). The average dry biomass of the seedlings was obtained through weighing process using a 0.0001 g precision analytical balance.
Electrical conductivity of seedlings, potassium leakage and membrane injury index: maize seedlings were obtained as described in the seedling length test.

Results and Discussion
Regardless of the test applied, the seeds with initial high and low physiological quality were grouped into different vigor classes with the application of mean test (Table 1). Leakage of ions, amino acids and sugars is regarded as an indirect measure of the membrane permeability level and, consequently, of its deterioration condition (Marcos Filho, 2015b Table 1. Means of germination (G), accelerated aging (AA), electrical conductivity of seeds (EC), field emergence (EM), electrical conductivity in seedlings (ECs), membrane injury index (MII), potassium leakage (LK), total seedling length (TL), aerial part length (APL), main root axis length (RL), vigor index based on total seedling length (VI TL ), vigor index based on aerial part length (VI APL ), vigor index based on main root axis length (VI RL ), and seedling dry biomass (BIO) of five lots with different vigor levels of the Garra Viptera maize hybrid.
Vigor levels G (%)  Table 2. Pearson's correlation (p<0.05) between variables based on seedlings (electrical conductivity in seedlings -ECs, potassium leakage -LK, membrane injury index -MII, total seedling length -TL, aerial part length -APL, main root axis length -RL, seedling dry biomass -BIO, vigor index based on total seedling length -VI TL , vigor index based on aerial part length -VI APL, and vigor index based on main root axis length -VI RL ) and variables based on seed analysis (germination -G, accelerated aging -AA, electrical conductivity of seeds -EC, and field emergence -EM) of five lots with different vigor levels of the Garra Viptera maize hybrid. Simultaneously to a lower sensitivity to distinguish vigor levels (Table 1) can be attributed to the nature of the tests, whose theoretical basis suggests that the activation of membrane protection and repair mechanisms, during the imbibition process, occurs more slowly in deteriorated seed lots, which results in the uncontrolled water uptake and, consequently, the release of larger quantities of intracellular substances to the medium.
As the deterioration advances, enzymes are inactivated, alterations in DNA, RNA and protein synthesis become more intense, and the free radical content is incremented with the consumption of reserves (Kaewnaree et al., 2011). However, as it is the structure responsible for the cell-medium interaction, the membrane permeability has long been regarded as the first phenomenon of the aging process (Kaewnaree et al., 2011;Marcos Filho, 2015b). In the present work, the MII was the electrolyte test that presented correlation values closer to -1 (Table   2). This greater accuracy may be related to the nature of its methodology, which allows the collection of exudates released by plant cells of internal and external tissues, while the others primarily collect those released by the external tegument. Although demanding more time to provide a result, the MII presents an advantage in relation to the other tests based on electrolytes (electrical conductivity and potassium leakage) which is to break the membrane resistance to the exudate release, thus neutralizing the genotypic variations associated with the tegument (Demidchik et al., 2014).

Conclusion
The methodologies based on electrolyte leakage of maize seedlings, especially the membrane injury index, were more accurate than the biometric tests to identify the vigor level of the maize seed lots used.