Vigor: essential element for seed quality

El vigor, elemento indispensable de la calidad de las semillas

Marlen Navarro,^I G. Febles,^II R. S. Herrera,^II

^IEstación Experimental "Indio Hatuey". Universidad de Matanzas "Camilo Cienfuegos". Central España Republicana. CP44280. Matanzas, Cuba.
^IIInstituto de Ciencia Animal. Mayabeque, Cuba.

ABSTRACT

In order to find methodologies with enough sensitivity to determine more precisely the degree of deterioration of seeds, several procedures have been developed under the conventional name of "vigor tests". There are profound debates and suppositions with regard to the concept and processes related to vigor. One idea is that vigor tests were developed in order to provide only additional information to that obtained with the germination test and they also allow to estimate the potential of emergency in the field in a wide range of environmental conditions. In this article, the authors offer a review on vigor, its concept and the most commonly used tests to evaluate it. At the same time, these researchers try to highlight that vigor is not a simple and isolated characteristic from the general performance of a seed feature, but it is the interaction of those biotic and abiotic properties, which influence on seeds and determine the activity level and their performance in time, such as the expressions of viability, dormancy, germination and emergence. Therefore, the term vigor is an integrated and dynamic concept, which cannot be separated because it is an essential part of seed quality.

Key words: vigor, quality, seeds.

RESUMEN

En la búsqueda de metodologías con sensibilidad suficiente para determinar con mayor precisión el grado de deterioro de las semillas, se han desarrollado diversos procedimientos con el nombre convencional de "pruebas de vigor". Con respecto al concepto y los procesos vinculados al vigor, se especula y se debate profusamente. Una de las ideas es que las pruebas de vigor se desarrollaron con la finalidad de ofrecer solo información complementaria a la obtenida por la prueba de germinación y que, a su vez, permiten estimar el potencial de emergencia en el campo en una amplia gama de condiciones ambientales. En este artículo, se ofrece una revisión sobre el vigor, su concepto y las pruebas más utilizadas para evaluarlo. Se intenta resaltar que el vigor no es una característica simple y aislada del comportamiento general de una simiente, sino que es la interacción de aquellas propiedades bióticas y abióticas, que influyen en las semillas y que determinan el nivel de actividad y el comportamiento de las mismas en el tiempo: expresión de la viabilidad, la dormancia, la germinación y la emergencia. Por ello, el término vigor es un concepto integrado y dinámico, que no se puede desvincular como parte esencial de la calidad de las semillas.

Palabras clave: vigor, calidad, semillas.

The seed is a living organism and, as such, is subjected to gradual degenerative processes that end with its death. The concept of vigor arises from the need to distinguish among seed lots with different potential, capable of producing normal, vigorous, and healthy seedlings and of establishing in the field under a wide range of environmental conditions (Heydecker 1972, Delouche 1976 and Perry 1984). Although there is no universally accepted definition of vigor, there is general consensus to consider it as the most important factor in seed quality.

From a biochemical point of view, vigor involves the ability of an organism for the biosynthesis of energy and metabolic compounds such as proteins, nucleic acids, carbohydrates and lipids, all associated with cell activity, integrity of cell membranes and transport or use of reserve substances (Bewley 1986 and Bewley and Black 1994). Meanwhile, the appearance of vigor in germination is demonstrated in the speed, consistency and intensity of this process, as well as the plantlet tolerance to unfavorable environmental conditions (Marcos Filho 2005).

Considering that vigor tests should estimate the quality of seeds with higher reliability than germination test, the evaluation, through integral studies, of any of the factors that antecede the lost of viability and are closely related to deterioration, may, theoretically, be used as tests for evaluating vigor.

VIGOR

The concept seed quality, besides being related to the germination response, also involves morphological, physiological and genetic aspects (Carvalho and Nakagawa, 2012). Therefore, germination test is not enough for expressing quality degree of seeds. On the other hand, Marcos Filho (2005) stated that this test has no ability to detect differences in quality among the lots of seeds with high percentage of germination, while the analyses of vigor show higher sensitivity to find such differences.

The Association of Official Seed Analysts (AOSA)  and the International Seed Testing Association (ISTA)offer their own concept of vigor:

- Vigor is considered as those seed properties that determine the fast and uniform emergence for the development of normal plantlets under a wide range of nursery or field conditions (AOSA 1983).

- Seed vigor is the sum of those properties that determine the level of activity and performance of seed lots with acceptable germination in a wide range of environments, and it is related to the rate and uniformity of germination and growth of plantlets, the ability for their emergence under unfavorable environmental conditions and seed performance after storage, mainly in the retention of the germination capacity (Hampton and Tekrony, 1995).

Likewise, the Brazilian Association of Seed Technologies (ABRATES) has a publication that offers vigor tests and concepts, which is edited by Krzyzanowski, Vieira and França Neto (1999).

Ferguson (1995), in an attempt to conceptualize seed vigor, stated that it is based on the physical and physiological performance of a seed lot, and includes: 1) changes on biochemical process; 2) rate and uniformity of germination and growth of plantlets; and 3) germination or emergence ability of seeds after being exposed to stress conditions. However, this author never developed any experimental procedure for integrating these three principles. 

Detection of seed deterioration through vigor tests may be understood as an important component on the evaluation of physiological quality and, according to Tekrony (2006), it contributes to the solution of problems from seed industry, such as storage, and there are other elements involved, like optimal moment for harvest, sweating and drying.

Researches, including those of biological nature, related to vigor and the creation of appropriate methodologies, focus, essentially, on horticultural  species from different environments, mainly form temperate areas. However, the work on this direction is generally scarce and fragmented as it would be seen later in tropical grass species and even more in shrub and tree legumes.

Therefore, Navarro (2009), after conceptualizing the term vigor, reached a new approach, as an integrated and dynamic concept that can be considered as the interaction of those biotic and abiotic properties, influencing on seeds and determining their activity level and performance during the time, such as the expressions of viability dormancy, germination and emergence. That is why vigor cannot be separated because it is an essential part of seed quality.

METHODS AND TESTS FOR EVALUATING VIGOR IN SEEDS

Vigor effects may remain and influence on growth of an adult plant, on harvest uniformity, and on yield of species. Therefore, several methods are used to characterize seed vigor (Tekrony 2003). Additionally, vigor may affect viability of the seed, germination speed, growth, plantlet sensitivity to external factors and storing capacity of different seed lots (Corbineau, 2012). 

Seed vigor tests are based on concepts such as resistance to stress, germination speed, integrity of membranes and plantlet development (Matthews et al., 2012). Based on the available technologies and recent physiological discoveries, evaluation methods are constantly improving (Kodde et al. 2012).

a) Plantlet growth test. Plantlet evaluation tests are based on the statements of Popinigis (1977) and Marcos Filho et al. (1987). Specifically, the analysis of plantlet growth as a vigor test implies the germination under controlled standard conditions and includes measurings of plantlet size and weight, or the classification of plantlets into vigor types (Krzyzanowski et al. 1999). It is important to point out that there are few research studies with this type of test in tree seeds.

b) Stress tests. The evaluation of vigor through stress tests requires the seed samples to germinate under stressing conditions or under the standard test of germination followed by a separated stress treatment (Lima and Marcos Filho, 2011).

Accelerated aging.One of the most used stress tests for evaluating vigor is the accelerated aging (AA), which is based on the increase of deterioration of seeds when they are exposed to adverse conditions of high temperature and high relative humidity (McDonald 1999). Under these conditions, low quality seeds deteriorate faster than the vigorous ones, so it is possible to establish differences among the evaluated samples. However, this requires a standard procedure for each species.

The need of a precise control over the environment and procedures, makes the AA to look more difficult than what it really is (Tekrony 1995). Bonner (1998), in a review on vigor tests, concluded that the results of biochemical studies on tree seeds during the aging treatments confirm that the fast use of energy reserves during the time of AA test is accompanied by the declining of germination and vigor. This supports the concept that seeds and trees react similarly when they are under AA conditions.

The key to these accelerate aging tests lies on the exposition period of seeds to the process and this is achieved through trials with several temperatures and amount of hours of seeds within the aging chamber (Tekrony 1995). One of those deficiencies is that, depending on the species, and at the same temperature, the increase of exposition period provides gains in the percentages of seed water (Marcos Filho 1999a). In order to counteract this factor, the use of salt saturated solutions (NaCl, KCl or NaBr) during the test is suggested, with the objective of reducing relative humidity within individual compartments, delaying water absorption by the seed.

This modification to the test is called Salt Saturated Accelerate Aging test (SSAA) and it was proposed by Jianhua and McDonald (1996). The inclusion of salts within the procedure for the accelerate aging test allows to discard that the increase of humidity content of seeds is the cause of seed deterioration, assuring that deterioration effects are a consequence of temperature and exposition period. Therefore, McDonald (1999) states that SSAA test facilitates the obtaining of more precise measurings of seed vigor.

Despite the diverse studies regarding the adequate exposition periods and temperature for AA test, there is a lack of information about several species of economic interest (Corte et al. 2010) and how to standardize the different parts of the method.

Controlled deterioration. For the evaluation of vigor, the test of controlled deterioration (Powell and Matthews 1984) may also be used. According to Krzyzanowski and Vieira (1999), this is an aging technique with similar bases to AA and includes a better control of humidity degree of seeds and temperature during the aging period.

 In the AA, seeds gain humidity during the initial period of the test due to the high temperature and different degrees of humidity, which results in different aging intensities during the same period of time (Matthews 1980). 

Its main advantage is that it is a stress method that does not require additional equipment, even though it is a test of simple implementation. Up to this moment, there are no reports of this test for evaluating vigor of seeds from tropical and subtropical shrubs and trees.

c) Biochemical tests. These tests evaluate indirectly the integrity of the cell membranes system, which is an extremely important aspect for guaranteeing the normal functioning of vital tissues of seeds (Marcos Filho 2011).

Tetrazolium salt test. I is most known biochemical test for evaluating vigor and it is frequently used as a fast test to estimate viability (França Neto and Krzyzanowski 2009), which is its main objective.

The theoretical supposition, in which this test is based, was stated by Harrington (1972), after assuming that maximum viability is associated with the maximum value of vigor and that both are closely related to the maximum point of physiological maturity (Tekrony and Egli 1997). It is known that the lost of viability is usually accompanied by the lost of respiration capacity, non-saturated fatty acids and lipids in the membrane, as well as reductions of energy from adenylate, enzymatic activity and content of messenger RNA (Smith and Berjak 1995).

The determination of viability through tetrazolium test do not detect the presence of pathogens or phytotoxical effects and its difficult the visual identification of abnormal plantlets (Miloševic, Vujakovic and Karagic, 2010).

Electric conductivity. The operating technique starts when seeds are soaked up in distilled or de-ionized water at a certain temperature and during a previously determined period and, later, a conductivity meter is used for the reading. The principles of this test establishes that less vigorous (more deteriorated) seeds show lower velocity of reestablishment of cell membrane integrity during imbibition and, as a consequence, release larger amounts of solutes (Vieira and Krzyzanowski 1999 and Carvalho et al. 2009). Results are expressed in μS/cm/g or μmho/cm/g. The lack of reference values to characterize the high or low vigor, the limiting aspects of the test of seeds with relatively small embryos and the expression of results in units that are less familiar to the user, have made difficult its use at a larger scale.

Protein electrophoresis. The main challenge of the studies on vigor tests is to identify indicators related to seed deterioration, which precede the lost of prortein capacity.  Within this concept, the technique of electrophoresis is important because it makes possible the detection of initial stages of deterioration through the activity of enzymes related to degradation and oxidation of reserve substances, as well as biosynthesis of new substances (Corte et al. 2010).

Priestley (1986) associated the loss of isoenzymes, such as peroxidase, acid phosphatase,  dehydrogenase, esterase and amino peptidase, with a severe aging of seeds within a group of species.

Chauhan et al. (1985), when studying the electrophoretic variation of proteins and enzymes of soy bean and barley in relation to seed quality, observed that the bands of proteins and enzymes (esterase, phosphatases and transaminases) work as molecular brands in the evaluation of quality. In addition, Vieira (1996) considered the electrophoretic variations of proteins and enzymes like glutamate dehydrogenase, malate dehydrogenase, acid phosphatase, malic enzyme, peroxidase and 6-phosphogluconate as promising indicators of the state of deterioration of cotton seeds.

d) Numeric variables based on results of germination and emergence tests. It is known that germination rate is positively related to the fast emergence in the field and to plantlet development of many species, including tree seeds. The most used method for tree seeds, for instance, was developed by Czabator (1962), who proposed the combination of germination rate with germination integrity in only one numeric index, which was known as germination value (GV). It was determined through the calculation of the maximum value (MV), which consists on the maximum value of germinated seeds in a day and daily mean germination (DMG) and it is calculated through the division of daily germination into the number of days since the beginning of test. Many studies have evaluated GV and MV as indicators of vigor in tree seeds (Poulsen et al. 1998).

Modified Weibull function. It was proposed by Brown and Mayer (1988), after analyzing a group of non-linear, linear fitted and logistic functions, as a model of data processing to compare vigor, based on frequency of accumulated germination. Previously, Scott et al. (1984) concluded that the parameters contained within it partially define the main characteristics of germination and emergence process.  From the study performed by Brown and Mayer (1988) to Weibull function, it began to be known as modified Weibull function, in the area of seeds. 

This non-linear function is regularly used to describe the dynamics of germination from the effect (in this indicator and in the emergence of plantlets) of the temperatures, stratification periods, pre-germination treatments, seminal characteristics and different especies, cultivar and hybrid, optionally through the analysis of their parameters (Huang and Yang 1995, Hernández and Paoloni 1998, and Cerabolini et al. 2004). 

In a review, Gardarin et al. (2011) detected that the three parameters of Weibull have been evaluated in 25 arvense species with different characteristics in seeds.

Other results obtained by Casco et al.(2008) show the usefulness of Weibull model for studies the seed bank of the soil, as well as the suitability of the function to describe distributions of size and number of seeds.

Navarro et al. (2013) also used Weibull function in order to estimate vigor of Albizia lebbeck (L.) Benth seeds, and perform comparisons when cropping is developed under three environmental conditions and with seeds of different storage times.

Germination speed.The first counting of germination test can be used as a vigor test, once the germination speed is related to evolution of seed deterioration. Likewise, samples presenting the highest germination values in the first counting can be considered as more vigorous. It is a simple test with an easy implementation, but generally shows low sensitivity because it does not detect small differences of vigor among lots (Marcos Filho 2011).

Germination percentage only includes the percentage of seeds that germinate during the test period, without taking into account whether the germination occurred during the first or last stage of the test. However, under field conditions (nursery), the fast germination is obviously an advantage for the establishment of plantlets. Therefore, germination speed takes part of vigor expression, and it is known that seeds with high vigor germinate faster than those of low vigor under any condition (van de Venter 2000).

Germination speed, known as “germination energy”, may be expressed in several ways, according to the daily germination records (Schmidt 2000):

•The percentage of seeds from a sample that germinate up to the moment of maximum germination

•The number of days required to reach the percentage of final germination

•Germination speed average in the total period of the test

Combination of biological variables and mathematical tools. The evaluation of vigor during germination, emergence and growth of plantlets consists of two phases: the expression of vigor as such, and the vigor reflected in time, through storage. In order to establish the importance and efficiency of the whole performance of biological variables per each time of storage, apart from the scarification method, Navarro et al. (2012) adjusted the statistical method for measuring the impact of innovation and technological transfer in the agricultural and livestock field, stated by Torres et al. (2008), in order to achieve an original and integral methodology for estimating seed vigor as an indicator of quality and later success of  the harvest. Therefore, it was decided that the statistics named by Torres et al. (2008) as “impact index” should change to “efficiency index”, and it is interpreted as the efficiency of evaluated variables in storage times, in their relation with vigor variability.

e) Computer analysis of seeds and plantlets. Silva et al. (2013) stated that integration of image analysis systems with vigor tests, considered as traditional, may contribute to the development of methodologies that will help on the standardization of the previously mentioned tests.

Automated evaluation of X-ray test. The X-ray test is recommended in the rules of ISTA (ISTA, 2004) and in the Rules of Seed Analysis (RAS) from Brazil (BRASIL, 2009). This technique, because it is a non-destructive method, favors the performance of physiological assays with seeds exposed to radiation, which allows the establishment of cause and effect relations (Cicero and Banzatto Junior, 2003) among the damages or changes observed in the interior of seeds and the prejudices caused to germination (Cicero et al., 1998).

A considerable amount of researches with X-rays in seeds relate the study of internal morphology with physiological potential of seeds from several species (Cicero, 2010). Gomes Junior (2010) provides an review of the most outstanding results, per species, for the evaluation of the internal morphology of seeds with the use of X-rays and their interpretation with the use of Tomato Analyzer and Image-Pro® software, which can determine the embryonic area or empty spaces inside the seed and establish relationships with vigor.

Analysis of plantlet images. Several systems of image analysis of plantlets have been proposed for the analysis of seed vigor. One of the first systems described in the literature was developed by Keys (1982) and improved by Keys et al. (1984).

Later, Geneve and Kester (2001) obtained promising results using image analysis to evaluate the size of plantlets as a vigor indicator. Meanwhile, Sako et al. (2001) proposed an automated method for evaluating vigor of lettuce seeds, by capturing images of plantlets and simultaneously determining the length of hypocotyl, primary root, the entire plantlet and root/hypocotyl relationship. The technique includes image processing with the use of "Seed Vigor Imaging System" (SVIS®) software, developed at Ohio State University.

According to Marcos Filho (2010), image processing of plantlets, or their parts, allows establish indexes of vigor, uniformity and growth of plantlets.

Despite the reliability of the available seed vigor tests, there are still opportunities to improve or develop new methods (Marcos Filho et al. 2009). In addition, there is a considerable interest in the development of methods and equipment that enable a rapid and automated evaluation of seed quality.

CONCLUSIONS

The detection of seed deterioration through vigor tests can be understood as an important component in the evaluation of quality and contributes to the solution of problems among them the storage and the characterization of the seed industry, such as storage. The main challenge for researches on vigor tests is to identify indicators related to seed deterioration, preceding the loss of germination capacity and quality.

In the last 50 years, seed specialists have proposed, studied and used different methods for evaluating vigor. Various methods have been studied in order to verify the possibility of their implementation and standardization for different species, in isolation or combined. Both, seed producer and the consumer need more information regarding the physiological quality of seeds than those provided by the germination test.

One of the symbols of the rapid progress of scientific researches in seed sector is the analysis of digital images of seeds and plantlets. This new generation of analysis has demonstrated high potential of use for several purposes of seed industry because the automatization of the analyses favors the fast obtaining of results, reduces human errors that occur during the interpretation of analysis based on visual estimates.

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Received: June 3, 2014 ]]> Accepted: December 8, 2015

Marlen Navarro, Estación Experimental "Indio Hatuey". Universidad de Matanzas "Camilo Cienfuegos". Central España Republicana. CP44280. Matanzas, Cuba. Email: boulandier@ihatuey.cu