Introduction

The tomatillo (Physalis ixocarpa Brot. ex Horm.), also known as the Mexican husk tomato, is a vegetable that has been consumed in Mexico since pre-Columbian times (^{Santiaguillo-Hernández et al., 2012}). As an olericultural species, it has great economic and cultural importance for the Mexican population. It is currently ranked the sixth horticultural crop in terms of planted area, after chili pepper (Capsicum annuum L.), squash (Cucurbita argyosperma), potato (Solanum tuberosum L.), tomato (Solanum lycopersicum L.) and onion (Allium cepa L.). It is planted in 30 states of the Mexican Republic, and its cultivated area is 42,464 ha, with an average yield of 19.9 t·ha^-1 and a production value of $3,006,099.93 MXN (^{Sistema de Información Agroalimentaria y Pesquera [SIAP], 2021}).

One of the most important inputs in an efficient production system is the seed, which is the starting point to have a favorable response to commercial growing conditions, produce vigorous plants and, consequently, achieve higher fruit production. Seed represents a strategic input par excellence, which allows sustaining agricultural activities, and contributes significantly to improving production in terms of quality and profitability (^{Coronado-González, Peña-Lomelí, Magaña-Lira, Sahagún-Castellanos, & Ybarra-Moncada, 2019}).

Seed quality is a multi-faceted concept comprising several components, including physical, genetic, sanitary and physiological ones (^{Pérez-Camacho, González-Hernández, Molina-Moreno, Ayala-Garay, & Peña-Lomelí, 2008b}; ^{Pérez-Camacho et al., 2012}). Physiological attributes are those related to seed metabolism, i.e., the expression of the maximum developmental potential of the seed (^{Pichardo-González et al., 2010}), such as viability, germination capacity and vigor. On the other hand, deterioration means the loss of some of the key physiological functions, which ultimately leads to the loss of essential attributes for seed quality such as vigor and germination capacity (^{Food and Agriculture Organization of the United Nations [FAO], 2011}).

Seed deterioration is associated with changes in its metabolism. The factors that in interaction can lead to deterioration and loss of vigor and viability, total or partial, are temperature, moisture, oxygen pressure, bacteria, fungi, insects and rodents (^{Doria, 2010}). The rate of deterioration depends on the environmental conditions during storage and the storage time of the seeds.

The objective of storage is to reduce the speed and effects of deterioration to keep the seeds viable and in good physical and physiological condition until sowing, where they are expected to have satisfactory germination and adequate vigor in seedling emergence (^{Jara, 1997}). In tomatillo, as in many other species, it is common to observe that stored seed quickly loses viability with storage temperatures above 30 °C, a condition that frequently occurs in agricultural field warehouses (^{Pérez-Camacho et al., 2012}).

Little research has been conducted on the quality of tomatillo seed and its deterioration. Detailed knowledge of the morphological and physiological characteristics of the seeds of this species could help to identify the factors involved in their deterioration. This aspect is important in breeding and germplasm conservation programs (^{Pérez-Camacho et al., 2008b}; ^{Coronado-González et al., 2019}). Therefore, the aim of this research was to determine the impact of storage conditions and time on the germination and vigor of seeds of four tomatillo varieties.

Materials and methods

The research was carried out at the National Plant Germplasm Bank and the Ecology and Seed Analysis Laboratories of the Plant Science Department of the Universidad Autónoma Chapingo (UACh) (19° 30’ North latitude and 98° 59’ West longitude, at 2,250 m a. s. l.). This work is part of the Tomatillo Breeding Program.

The biological material used in the experiments was produced in the spring-summer 2013 cycle in the experimental field of UACh (Gema, Tecozautla 04 and Diamante varieties) and the Colegio de Postgraduados, Campus Montecillo, State of Mexico (Manzano Tepetlixpa variety). The varieties analyzed are registered in the National Catalogue of Plant Varieties (^{Servicio Nacional de Inspección y Certificación de Semillas [SNICS], 2021}).

Crop management was carried out as described by ^{Martínez-Solís, Peña-Lomelí, and Montalvo-Hernández (2004)} for the production of tomatillo seed. Seedlings were transplanted into the open field in the first week of April 2013, at a density of 30,000 plants·ha^-1. The fruits were harvested in the second week of June, when they were physiologically mature and had a light green to yellow color, in the case of the Gema, Tecozautla 04 and Diamante varieties, and a yellow color in Manzano Tepetlixpa. Seed extraction of each variety was performed immediately after harvest with a pulping machine suitable for tomatillo (^{Coronado-González et al., 2019}).

The seed was sun-dried on racks (^{Martínez-Solís et al., 2004}) and subsequently processed in October 2013 with an air screen cleaner (model LA-LS/B, Seedburo®, USA), with a 1 HP motor. With the use of 1.5, 2.0 and 3.0 mm round sieves, three seed sizes were obtained: small, standard and large, respectively. The impurities were separated and the empty seed was removed. At the end of the drying and conditioning process, the seed was stored with a moisture content of 5 % in Gema, 6 % in Tecozautla 04, 6.4 % in Diamante and 7.8 % in Manzano Tepetlixpa (^{Coronado-González et al., 2019}). For the study, a 1 kg sample of standard size (2 mm) seed was taken from each variety, which is the one generally marketed. Each sample was divided into two parts of 500 g each. Of the eight subsamples, four were stored in the seed laboratory and the other four in the UACh germplasm bank as described below.

One study factor was the storage condition, which had two levels and was established in February 2014. The first level was the environmental conditions of the Seed Laboratory, with no temperature or relative humidity (RH) control. The annual average temperature was 23.43 ± 0.64 °C; average RH, 23.55 ± 0.06 %; dew point temperature, 1.46 ± 0.56 °C, and absolute humidity, 4.95 ± 0.18 g·m^-3. Of each variety, 500 g of conditioned seed were placed in an aluminized plastic bag with a 30 x 22 cm "Ziploc"-type hermetic seal, which resembles the container in which tomatillo seed is marketed. The second level was the National Germplasm Bank, where the seeds were kept at -20 °C and 13 % RH. Of each variety, 500 g of conditioned seed were placed, together with a plastic bag with silica gel (3 x 3 cm), in translucent glass jars of 500 mL volume with a hermetic seal.

The second factor evaluated was the variety, which corresponded to four tomatillo genotypes produced in the spring-summer 2013 cycle. The Gema, Tecozautla 04 and Diamante varieties were produced at the Experimental Agricultural Field of the Plant Science Department, and the Manzano Tepetlixpa variety at the Colegio de Postgraduados.

The third study factor was seed storage time, which had four levels. Germination was evaluated, in the four varieties, in October 2013 (time 1, zero months of storage), October 2014 (time 2, one year of storage), October 2015 (time 3, two years of storage) and October 2016 (time 4, three years of storage). Vigor was evaluated in the four varieties and the two storage conditions at 6 (time 1), 12 (time 2), 24 (time 3) and 36 (time 4) months of storage. To evaluate germination and vigor, a sample of 5 g of seeds per variety was taken from both environments.

In order to determine physiological quality in relation to germination percentage, a 2 x 4 x 4 incomplete factorial treatment design was used, in which the levels of storage condition (2), variety (4) and storage time (4) were combined. Since at time 1 the seed was not yet stored, there were only four combinations (four varieties and one storage condition). At times 2, 3 and 4 there were eight combinations (two storage conditions and four varieties), giving a total of 28 treatments.

The experimental design was completely randomized, with four replications at time 1 (16 experimental units) and six replications at times 2, 3 and 4, where the four varieties of the two storage environments (48 experimental units per year) were evaluated. Each experimental unit consisted of a transparent plastic Petri dish 95 mm in diameter by 10 mm deep, with 100 seeds evenly distributed on medium-pore filter paper moistened with 5 mL of distilled water at the beginning, and that necessary to maintain moisture throughout the test. The experimental units were placed in a germination chamber (model D- 7140, Seedburo®, USA) at 30 ± 1 °C and 90 % RH, conditions proposed by ^{Martínez-Solís, Mendoza, Rodríguez-Pérez, Peña-Lomelí, and Peña (2006)}, for 21 days according to the standards of the International Seed Testing Association (^{ISTA, 2004}), and following the methodology used by ^{Coronado-González et al. (2019)}. Germination percentage (GP) was evaluated based on the number of seeds sown and the number of seeds with radicle emission in normal seedlings at the end of the test.

To determine physiological quality in relation to seed vigor with respect to storage time, a 2 x 4 x 4 full factorial treatment design was used, combining the levels of storage condition, variety and storage time, respectively, which resulted in 32 treatments. The experimental design was completely randomized with six replications at all times, where the four varieties were evaluated in the two storage environments, resulting in 48 experimental units per time. In this case, each experimental unit consisted of a plastic Petri dish 135 mm in diameter by 22 mm deep, with 50 seeds evenly distributed on medium-pore filter paper permanently moistened with 10 mL of distilled water. The experimental units were placed in a germination chamber (Precision 818, Thermo Scientific™, USA) at 30 ± 1 °C (^{Martínez-Solís et al., 2006}; ^{Coronado-González et al., 2019}), with 24-h lighting during the 14 days of the test. Germinated seedling counts were made every 48 h until the end of the test. Seven days after the start of the test, 5 % Captan® was applied to prevent fungal damage.

The vigor-related traits evaluated are described below. 1) Germination speed index (GSI): germinated seeds were counted every 48 h, considering as such the emergence of the radicle, and the GSI was calculated according to the formula proposed by ^{Maguire (1962)}; 2) average seedling length (SL): a sample of 10 seedlings per experimental unit was taken, and the length (cm) of each seedling was measured from the tip of the primary root to the tip of the cotyledonary leaves and the average length was calculated; 3) average seedling dry weight (SDW): once the seedlings were measured, they were dried to constant weight for 72 h at 72 °C in a constant climate oven (Memmert HPP110, Wisconsin Oven, USA), then weighed (mg) on an analytical balance (Pioneer, Oahus®, USA) and the average was calculated; 4) percentage of normal seedlings (NS): it was obtained as a function of the number of normal seedlings counted at the end of the test. Normal plants were those that showed all their essential structures, according to ^{ISTA (2004)} standards.

The data for germination percentage and percentage of normal seedlings were transformed before the analysis of variance using the arcosine y/100 formula (^{Sokal & Rohlf, 1995}), in order to correct their deviation from a normal distribution. In addition, an analysis of variance was performed on the data for each character evaluated and, subsequently, Tukey's comparisons of means (P ≤ 0.05) were made. For these analyses, the SAS version 9.3 statistical software package (^{SAS Institute Inc., 2011}) was used.

Results and discussion

The results of the analysis of variance for the germination percentage (Table 1) show a significant effect (P ≤ 0.01) of varieties and storage time, but not for storage environment. Only the Variety x Storage time interaction was significant. The low coefficient of variation indicates that the results are reliable.

The two storage environments evaluated were equally efficient in maintaining seed germination. This can be explained by the fact that storage occurred, in both cases, at temperatures below 30 °C, which is the point at which deterioration accelerates in tomatillo (^{Pérez-Camacho et al., 2008b}; ^{Pichardo-González et al., 2010}). In addition, the seed was stored in an airtight container, so its moisture was presumably kept low in both environments.

The varieties with the highest germination were Diamante and Tecozautla 04, which outperformed Gema and Manzano Tepetlixpa (Table 2), the latter with the lowest germination value. These results can be attributed to the genetic condition of the seeds, since the varieties come from different breeds, so they probably have different earliness and chemical composition, which is reflected in different seed sizes, in the degree of physiological maturity and, consequently, in different germination percentages (^{Pichardo-González et al., 2010}; ^{Pérez-Camacho et al., 2012}; ^{Peña-Lomelí, Ponce-Valerio, Sánchez-del Castillo, & Magaña-Lira, 2014}).

In the results of the comparison of storage time means, no significant differences were found between the samples obtained in the first year (time 2) of evaluation. From the second year (time 3), a descending behavior in germination was observed, although not significant with the 2015 (time 3) and 2016 (time 4) tests. In the three years of sampling, a 4.13 % decrease in germination was recorded, with the largest decrease from year two to year three (2.16 %). This dynamic coincides with that reported by ^{Pérez-Camacho et al. (2008a)} on the germination of tomatillo seeds stored for 1, 2, 3 and 5 years, where germination decreased linearly. However, the annual decrease (8.7 %) reported by these authors was much higher than that obtained in the present work. This may be due to the origin of the seed, since in the aforementioned study each variety was produced in a different year, and in the present work the seed was produced specifically for the study, in the same year and under the same growing conditions.

It is clear that there is a loss of germination due to the effect of deterioration, which begins at physiological maturity and can lead to the total loss of germination capacity (^{Delouche, 2002}), due to the consumption of seed reserves during storage (^{Copeland & McDonald, 2001}) or to an increase in the physiological processes of the seeds (such as respiration or metabolic activation) (^{Pichardo-González et al., 2010}).

The lowest germination recorded during the storage time was 91.5 %, a value that exceeds the standard required for certification, which is at least 85 % (^{FAO, 2011}). This indicates that, even after three years, the seed lots would be viable for commercial use.

The results of the analysis of variance of the vigor variables (Table 3) presented significant (P ≤ 0.05) effects in the variety, environment and storage time factors in all variables. Likewise, the Variety x Environment interaction was significant for NS; Variety x Storage time, for GSI and NS, and Variety x Environment x Storage time, for SL.

Unlike the germination percentage, in the vigor evaluation, the effect of the different storage conditions on the three variables can be observed. This is due to the methodology of both tests, since in the case of germination it was sufficient to have the emergence of the radicle at the end of the test, while in the vigor test the GSI, SL and SDW were recorded. It may also be due to deterioration affecting vigor, before reducing germination.

The results of the comparison of means for vigor (Table 4) show that the variety with the highest GSI was Diamante (P ≤ 0.05), which surpassed Tecozautla 04 by 8 %, Gema by 9.8 % and Manzano Tepetlixpa by 12 %. Diamante presented, statistically, a shorter SL compared to the other varieties by 8 %, and Manzano Tepetlixpa presented a lower percentage of NS by 6 %.

The first stage in germination is the rehydration of the seed with water by imbibition (^{Doria, 2010}), where the amount of water absorbed during this stage depends on factors such as seed size, which does not exceed two to three times its dry weight (^{Coronado-González et al., 2019}; ^{Jara, 1996}; ^{Méndez-Natera, Merazo-Pinto, & Montaño-Mata, 2008}). In this context, the average seed weight of Diamante (1.38 mg) is lower than that of the other three varieties, whose value fluctuates from 1.46 to 1.48 mg (^{Coronado-González et al., 2019}). This situation makes it evident that Diamante, being smaller, imbibes faster and, consequently, its GSI is higher than that of the other varieties. Additionally, having fewer reserves, Diamante’s SL was shorter than that of the other varieties, and its SDW was also the lowest, only significantly different from Tecozautla 04.

The seed conserved in the Germplasm Bank generated higher GSI and NS values than those obtained in seed stored in the Seed Laboratory by 3.3 and 2.8 %, respectively. These results are due to a lower deterioration generated by conservation at low temperature (-20 ° C) and low RH (13%) prevailing in the Germplasm Bank. In addition, the moisture content of the seeds (5 to 7 %) at the time of storage may have caused them to reach a glassy state (which is produced by the accumulation of sucrose, possibly in conjunction with oligosaccharides such as raffinose), which helps to preserve the membrane and protein structure (^{Bradford, 2004}). Under these circumstances, the diffusion of molecules in the cytoplasm is minimal and, consequently, seed deterioration is reduced (^{Carrillo-Salazar, Pichardo-González, Ayala-Garay, González-Hernández, & Peña-Lomelí, 2011}), which consequently minimizes the negative effect on germination and vigor.

The highest SL value was obtained in seeds stored without environmental control in the Seed Laboratory, exceeding by 3 % the results of seeds stored in the Germplasm Bank (P = 0.05). This behavior contrasts with what has been previously reported in the sense that the greatest expression of vigor must have come from the seeds in the best storage conditions. In this regard, ^{Popinigis (1985)} argues that seedling growth is highly variable, and strongly influenced by genetic and environmental factors during establishment, which may explain the discrepancy in the result discussed.

The results of mean comparisons to assess the effect of storage time on the four vigor variables (Table 4) show significant differences in all variables. In GSI, SDW, SL and NS, a trend of reduced vigor with a longer storage time is observed by 9.9, 29.9, 15 and 28.5 %, respectively, which implies that over the 36 months of evaluation, deterioration reflects a negative effect on vigor.

Under these circumstances, it is considered that the storage conditions in the laboratory (23 °C and 23 % RH) and in the Germplasm Bank (-20 °C and 13 % RH) allowed, to a large extent, maintaining seed vigor during the 36 months of evaluation, since it is proven that the reduction of temperature and RH during storage retards deterioration. In the first case, the decrease in temperature prevents an increase in the respiration rate (^{FAO, 2011}), while, in the second, the low relative humidity prevents the moisture content in the seed from rising (^{Navarro, Febles, & Torres, 2012}). The latter prevents increased metabolic activity that could lead to energy release and heating (^{Carrillo-Salazar et al., 2011}; ^{Pérez-Camacho et al., 2012}), which leads to the loss of seed viability, germination and vigor. On the other hand, having dried the seeds (5 to 7 % moisture), prior to their storage, also had an important effect, since it possibly reduced the respiration rate and, consequently, enzyme activity, which slowed deterioration (^{Pérez-Camacho et al., 2008b}; ^{Carrillo-Salazar et al., 2011}).

The Variety x Storage time interaction is reflected in germination, since as shown in Figure 1a, the Diamante, Tecozautla 04 and Gema varieties showed a decrease in germination of 7, 6.2 and 6 %, respectively. The Manzano Tepetlixpa variety showed a stable behavior, around 90 % germination. As previously stated, this behavior can be attributed to the genetic condition of the seeds due to the fact that the varieties come from different races (^{Coronado-González et al., 2019}).

Figure 1 Comparisons of means of combinations of germination and vigor variables in tomatillo (Physalis ixocarpa Brot. ex Horm.) seeds of four varieties stored for 36 months: a) germination percentage in four storage times, b) germination speed index in four storage times, c) percentage of normal seedlings in four storage times and d) percentage of normal seedlings in two storage conditions. Means with the same letter in each figure do not differ statistically (Tukey, P ≤ 0.05). 

The interaction also affected vigor in the GSI and NS variables. As can be seen in Figure 1b, at six months all varieties had similar GSI values. With 12 months of storage, the Gema variety remained stable, and Diamante had an increase in its GSI, while the Manzano Tepetlixpa and Tecozautla 04 varieties reduced their GSI by 12 and 13 %, respectively. The GSI of Manzano Tepetlixpa and Diamante remained unchanged from 12 to 24 months, while Gema and Tecozautla 04 showed a slight decrease. From 24 to 36 months of storage, all varieties reduced their GSI. In this period, Diamante was significantly superior to the other three varieties. Regarding NS (Figure 1c), Manzano Tepetlixpa was observed to have lower percentages, compared to the other varieties, by 4, 6 and 8 % during 6, 12 and 24 months of storage, respectively. However, at 36 months it is observed that the percentage of NS decreases severely in the four varieties, with no significant differences among them.

With these results, the loss of vigor and aging of the seed is evident, which increases over time (^{Pérez-Camacho et al., 2012}). During natural seed aging, the total content of reserves such as carbohydrates and proteins is reduced, causing important effects on the growth and development of the new seedling (^{Basavarajappa, Shetty, & Prakash, 1991}; ^{Pichardo-González et al., 2010}).

The Variety x Environment interaction was significant for NS, as shown in Figure 1d. In the Gema, Manzano Tepetlixpa and Diamante varieties, no significant differences were observed between storage conditions, while Tecozautla had a significantly higher percentage of NS in seeds stored in the Germplasm Bank compared to those stored in the Laboratory.

Conclusions

Storage for three years slightly impacted the germination of the Gema, Tecozautla 04 and Diamante varieties, while the Manzano Tepetlixpa variety maintained constant germination during the same period.

All four varieties studied maintained germination above 90 %, indicating that even after three years of storage, the seed lots would be viable for commercial use.

Both storage environments were favorable for maintaining germination, but not for vigor. Seeds stored in the Germplasm Bank had a higher germination speed and percentage of normal seedlings.

Storage time negatively affected the vigor of the seeds, which is an indicator of their deterioration.

In the vigor test, the Diamante variety presented the highest germination speed index values.