2019 Volume 88 Issue 2 Pages 245-252
This study was conducted to evaluate the influence of fruit maturity and postharvest storage on the physiological qualities of chili seeds during development. Two types of chili were used (ancho and guajillo). Fruits were harvested at 40, 60, 80, 100, 120 and 140 days after anthesis (daa) and stored for 0, 7 and 14 days after harvest (dah). The moisture content of guajillo seeds throughout development decreased from 86 to 17%, while ancho maintained moisture at 47% at 80 dda. It was confirmed that precocious harvest (40 daa) was not beneficial to the physiological quality of chili seeds, even when it was associated with 14 dah. Germinability was correlated with electrical conductivity (R = −0.76): Ancho seeds germinated starting from 472.5 μS·cm−1·g−1 and guajillo seeds from 679.3 μS·cm−1·g−1. In fruits harvested 80 daa, 14 dah was essential to ensure the physiological quality of seeds in both types of chilies: mean germination was above 93%, and seed vigor was higher (germination after accelerated aging and mean germination speed were more than 90% and 5.6 radicle d−1, respectively). Seeds harvested 120 daa were of high quality, and post-harvest storage of fruits was not necessary. Expression of two late embryogenesis abundant (LEA) proteins was detected in both types of chilies: the first synthesis (65 kDa) occurred 80 daa, and the second (50 kDa) occurred 120 daa and this was directly related to the maximal physiological quality of chili seeds.
The genus Capsicum belongs to the Solanaceae family and comprises more than 30 species. C. annuum types are highly diverse; this species accounts for the greatest production and consumption worldwide. According to FAO data (2016), the world area planted with chilies is around 1.9 million hectares, producing 34.5 million tons. In Mexico, the best-known chili types are jalapeño, ancho, guajillo, pasilla, serrano, chili de árbol and piquín (Aguilar-Rincón et al., 2010).
Despite the importance of this crop, few studies on its seed development exist. Research on this area has focused on sweet peppers (Vidigal et al., 2009). Sweet pepper characteristics contrast with other pepper types. For example, the fruits and seeds develop in half the time (Ayala-Villegas et al., 2014; Vidigal et al., 2009). Thus, studies on the development of seed quality attributes in other types of chilies are needed.
Using high quality seeds is a basic requirement for profitable crop production. During seed development there is an optimal moment for harvesting to obtain seeds with the best attributes (Bino et al., 1998; Doijode, 2001). In orthodox seeds, the formation of structures and the acquisition of other characteristics is divided into three phases during seed development: i) histodifferentiation and cell expansion, ii) accumulation of reserves, and iii) acquisition of tolerance to desiccation (Otho et al., 2007).
Seed development initiates immediately after pollination when the ovule is activated to begin cell division and histodifferentiation to form embryo and endosperm tissues. Simultaneously, water flows expand the formed cells and transport compounds that permit reserve synthesis and storage. Seed maturity is reached with loss of water (tissue desiccation) and development of processes that allow the seed to survive with a low moisture content (Taiz et al., 2015). In agronomic crops, the physiological processes that occur in the final phases of seed maturation and drying have traditionally been considered unimportant for seed quality (Bewley et al., 2013). However, research has clearly demonstrated that seed vigor and potential for storage increase after physiological maturity and that the final phases of development and drying are important for maximizing quality (Bewley et al., 2013).
The final seed development stage, maturation, begins with a reduction of seed water content. This process is called acquisition of tolerance to desiccation (Amara et al., 2014). Tolerance to desiccation is associated with processes such as accumulation of sugars, biosynthesis of late embryogenesis abundant (LEA) and heat shock (HSP) proteins, activation of antioxidants and changes in the physical structure of the cell (Angelovici et al., 2010). Both LEA and HSP proteins appear during seed maturation and storage, and later disappear gradually during germination. This disappearance indicates that tolerance to desiccation is achieved and, later, lost (Kaur et al., 2016).
Moreover, LEA proteins are also associated with the response to water deficit in plant tissues, principally by preferential hydration of diverse molecules and replacement of water in dehydrated cells (Kalemba and Pukacka, 2007).
In some species, seed physiological maturity coincides with commercial maturity of the fruit (Bewley et al., 2013); for fleshy fruits such as peppers, the effect of post-harvest fruit storage on the seed quality needs to be studied. Some reports suggest that harvesting the fruit before seed maturity and after a period of postharvest storage decreases the period in which high quality seeds can be obtained (Ayala-Villegas et al., 2014; García-Ruiz et al., 2018). Finally, it is necessary to take into account that chili seed maturity and acquisition of tolerance to desiccation occurs in the fruit while its moisture content is very high.
For these reasons, the objective of this study was to describe the development kinetics of the physical and physiological quality parameters of the seeds of two chili types, ancho and guajillo, harvested at different stages of development and in different periods of postharvest fruit storage, as well as to identify the expression of LEA proteins during the process of seed maturation.
For the study, we used seeds from chili ancho (CP-1243) and guajillo (CP-1150) populations from the germplasm bank of the Colegio de Postgraduados, Mexico. One hundred seeds of each chili type were planted in polystyrene trays with 200 cavities in a sterile substrate (peat moss) and inside a greenhouse with polyethylene covering. Seeds were planted on April 29, 2016, in Texcoco, Mexico (19°27'32" N, 98°54'22" W, altitude 2248 m). Transplant was 56 days later to 30 × 30 cm black polyethylene bags. From transplant to onset of flowering, the plants were irrigated three times a week and fertilized twice with multipurpose Ultrasol® (N:P:K=18:18:18 + micronutrients; SQM, Jalisco, México) in irrigation water (2 g·L−1). At flowering onset NutriCalcio® (8.1% N, 12.75% Ca; 2 mL·L−1) was added.
Flowering was considered to have begun when 50% of the plants had at least two flowers in anthesis (more than 50% of the anthers had pollen). As of this moment, flowers in anthesis were labeled every day in the morning. Then, 90 fruits of each chili type were harvested 40, 60, 80, 100, 120, 140 d after anthesis (daa). Seeds were extracted from 30 of these fruits immediately after harvesting (0 d after harvest, dah). Seeds were also extracted from 30 fruits 7 dah and those from the remaining 30 were extracted 14 dah. The fruits were stored at 18 ± 1°C and 35 ± 4% relative humidity. Seed extraction was done manually. The seeds were washed with running water for 1 min and left to dry on paper toweling for 24 h at 20°C. They were then placed in paper envelopes and stored for 3 months at 18 ± 1°C.
Measured variablesMoisture content (%) was determined in fresh seeds (immediately after extraction) and dry seeds (stored for 2 months). This variable was determined after oven drying (mod. 31480; Thelco, Livonia, MI, USA) for 1 h at 130°C in three replications of 50 seeds. The percentage of moisture in the seeds was calculated with the formula of Bewley and Black (1994), and dry seeds were used to determine the rest of the variables.
To obtain the 1000-seed weight (g), we used the standard ISTA (2012) procedure; eight replications were done with 100 seeds. An Ohaus balance (PA2202; Ohaus, Pine Brook, NJ, USA) with precision of 0.001 g was used to weigh the samples.
A germination test was conducted. Seeds were placed on a layer of Whatman No. 2 filter paper moistened with 2% KNO3 in 11 cm × 11 cm × 3.5 cm plastic boxes, which were placed in a SD8900 germinator (Seedburo Equipment Co., Des Plaines, IL, USA) at 25 ± 1°C with constant light for 21 d, following the ISTA (2012) norms. We replicated the test four times with 50 seeds, and at the end calculated the germination percentage (%).
Seed vigor was evaluated with an adaptation of the accelerated aging test proposed by Delouche and Baskin (1973), which consisted of subjecting four replications of 25 seeds to 40 ± 0.5°C and 100% relative humidity for 72 h. The seeds were distributed evenly over a wire mesh suspended over 100 mL distilled water inside plastic boxes placed in a drying oven (mod. 31480; Thelco), which was programmed at the indicated temperature (AOSA, 1983). Later, in a germination test as described previously, speed of germination (radicles·d−1) was assessed. This was calculated with the Maguire formula (1962) with the count of emerged radicles every 24 h over 21 d.
Electrical conductivity of the imbibition solution (EC, μS·cm−1·g−1) was determined in four replications of 50 seeds, which were weighed and submerged in 50 mL deionized water for 24 h at 25°C (ISTA, 2012). EC was measured with a conductimeter (mod. 72729; Oakton Instruments, Vernon Hills, IL, USA).
Statistical analysisData were captured on an Excel® 2016 spreadsheet and graphs were constructed. For the analysis of variance, we used SAS (Version 9.1.3; SAS Institute, Cary, NC, USA) statistical software. Means were compared with Tukey’s test P = 0.05). Data of variables in percentages were transformed with the arc sine function
Fifty milligrams of frozen embryos were extracted from pepper seeds at 60, 80, 100, 120, and 140 days after anthesis (daa). Embryos were ground in 1 mL of high ionic strength extraction solution (50 mM Tris-HCl pH 8.5, 15 mM Beta-Mercaptoetanol, 1 mM, Dithitiothreitol) using refrigerated mortars and pestles. The extracts were put into 1500 μL Eppendorf tubes and centrifuged at 16000 × g for 15 min at 4°C. The supernatants were transferred to Eppendorf tubes and the precipitate was discarded. Prior to electrophoresis, the amount of proteins in the supernatant was quantified by the Bradford (1976) method. Volumes of supernatant were adjusted to 40 μg soluble protein in each sample. To these samples, 10 μL “Magic Mix” loading buffer solution were added (SDS 1%, B-ME 1%, EDTA 0.074%, glycerol 10%, bromophenol blue 0.05%, all in Tris-HCl 50 mM, pH 6.8). The soluble protein samples were denaturalized in a boiling water bath for 5 min, and the samples were loaded onto each track of a polyacrylamide-sodium dodecyl sulfate (SDS) gel (10 × 10 cm). A 12% acrylamide gel (Laemmli, 1970) was used and introduced into the electrophoresis chamber with a Tris glycine run buffer (Tris-HCl 0.1% pH 8.0, glycine 1.15%, SDS 0.8%, adjusted to 100 mL with distilled water). Electrophoresis was conducted at 120 V for 2 h. To determine the relative intensity of the proteins (separate bands), the gel was dyed for 2 h in a solution of Coomassie blue (Coomassie blue R-250 0.1%, methanol 40%, acetic acid 10%, adjusted to 100 mL with distilled water), and color was removed in a solution of acetic acid (10%), methanol (40%), water (50%) (Laemmli, 1970). Molecular weight (MW) of the separated proteins was calculated based on a curve formed by a kit of protein markers of known MW (Prestained SDS-PAGE Standards, Low range. Bio-Rad, Hercules, CA, USA).
Anthesis occurred 40 d after transplant in guajillo chilis and 45 d after transplant in ancho chilis. In both types of chilis, the extracted seeds dried for two months had an average moisture content of 6.4 ± 0.6%, but the seeds at 40 daa had 9.5 ± 1% moisture content. It is probable that because of the physical characteristics of the immature seeds they were able to conserve more moisture, since the seed moisture content continues to modify until it acquires dynamic equilibrium with the surrounding air (Rodríguez-Burgos et al., 2011). The moisture percentages observed are within the range for a live seed (Ayala-Villegas et al., 2014; Copeland and McDonald, 2001).
Recently extracted seeds (fresh seeds) from fruits 40 daa from both chili types were immature, mucilaginous, and thin with a moisture content above 75% (Fig. 1). Seeds from ancho chilis 40, 60 and 80 daa, regardless of postharvest fruit storage time, underwent rapid loss of moisture, which stopped at 47 ± 3%. This moisture content was constant from 80 daa until 140 daa (Fig. 1A). This indicates that the seeds inside the fruit reached a point of equilibrium at around 47% moisture. Pérez-Camacho et al. (2008) observed this same point of equilibrium in tomatillo seeds (Physalis ixocarpa Brot.), that reached 45% moisture. Other similar studies in tomatoes (Demir and Ellis, 1992a; Dias et al., 2006) and sweet peppers (Demir and Ellis, 1992b; Vidigal et al., 2009) found moisture contents of seeds inside the fruit that oscillated between 38 and 42% and remained high during the entire seed development period, reaching values of 17 ± 3% in the sample 140 daa for the three seed extraction times. Bewley et al. (2013) reported that in cherry tomatoes during seed ripening, a flow of water toward the placenta occurred; this flow may have been associated with a flow of soluble sugar entering the seed, allowing deposition and accumulation of reserves. In the case of ancho chilis, equilibrium in the moisture content of the seed was achieved when the seed surpassed its period of maximum growth and neared its maximum weight (Fig. 2A), which could be interpreted as the final period of deposition and accumulation of reserves. In guajillo chilis, although seed moisture content continued to decrease during the entire development cycle, seed weight did not continue to increase (Fig. 2B). It is postulated in this case that exit of water from the seed is not correlated with the entry of soluble sugars and biosynthesis of reserves.
Moisture content in recently extracted seeds from chili fruits, (A) ancho chili Y = 82 − 37e−231249819820.4x−6.4 (R2 = 0.89) and (B) guajillo chili Y = 259 − 48.2 Ln x (R2 = 0.93) harvested at six development stages and stored for three postharvest periods. dah = days after harvest.
Evolution of the 1000-seed weight of (A) ancho and (B) guajillo chilis harvested at six development stages and stored for three fruit postharvest periods. dah = days after harvest. Values with the same letter in the same sampling are statistically equal (Tukey’s test, P ≤ 0.05).
Thus, in terms of growth kinetics of 1000-seed weight (1000SW) in fruits not stored after harvest (0 dah) for both types of chilis (Fig. 2), we can distinguish two main phases. The first phase of rapid growth occurred 80 daa, while the second phase showed slower growth, which remained stable until the final sampling. Ayala-Villegas et al. (2014) obtained similar results with the same chili types.
Postharvest storage of the fruits of both chili types collected 40, 60 and 80 daa led to an increase in weight relative to the fruits with no postharvest storage (0 dah). This indicates that the seeds inside the fruit continue to receive photosynthates, so they could complete the phase of accumulation of reserves, even though the fruit had already been detached from the mother plant.
According to Singkaew et al. (2017), it is well-known that seeds act as a sink and accumulate food from the fruit (source) to complete physiological development during post-harvest ripening of berry fruits, including tomatoes.
Beginning and development of germinabilityThe source of variation (Table 1) of the interaction chili type with extraction date (C × E) was not significant for the variables germination (G) and speed of germination (SG) of aged seeds. This indicates that the combined effect of chili type and extraction date did not induce changes in any of these variables. Moreover, the effect of the factor chili type (C) on the electrical conductivity (EC) of the imbibition solution was not significant.
Degrees of freedom, mean squares and statistical significance in the analyses of variance of the variables of seed physiological quality.
Seed quality evolves during development until it reaches a maximal point, which is traditionally associated with physiological maturity (Bewley et al., 2013). In our study, the seeds in all the treatments extracted 40 daa and some extracted 60 daa did not germinate (Fig. 3). These results are similar to those found by Ayala-Villegas et al. (2014) with the same chili types. These seeds had not yet reached a level of morphological and physiological development to allow adequate germination. It is likely that they were in the phase of histodifferentiation, as the high electrical conductivity of the imbibition solution shows (EC; Fig. 4): the cell membranes had not yet formed, permitting greater flow of solutes of the forming cells. The EC test is used as an indicator of seed germinability and vigor (Rinaldi et al., 2017). When electrical conductivity of an imbibition solution is measured, the solutes released by seeds can be measured. EC is inversely proportional to seed germinability: a higher EC indicates that the organization or integrity of the membranes is deficient. In our study, the value of the negative correlation (R) between G and EC was −0.76 (P ≤ 0.01). Thus, the decrease in EC (Fig. 4) indicates improvement in the membrane organization during the process of maturation. Demir and Ellis (1992a) found that electrical conductivity of tomato seeds was higher 25 daa than 55 daa, the date that coincided with the maximum germination of the seeds. The magnitude of the EC values registered in our study is similar to that observed by Vidigal et al. (2009) in seeds of the sweet pepper Capsicum annuum L. in the maturation process and during different periods of postharvest fruit storage.
Evolution of seed germination of (A) ancho and (B) guajillo chilis harvested at six development stages and stored for three fruit postharvest periods. dah = days after harvest. Values with the same letter in the same sampling are statistically equal (Tukey’s test, P ≤ 0.05).
Kinetics of electrical conductivity of chili seeds, (A) ancho chili Y = 1499.2 − 1205.1e−44412517027.3x−4.2 (R2 = 0.71) and (B) guajillo chili Y = 1452.1 − 1201.6e−235034144.9x−4.8 (R2 = 0.8), harvested at six development stages and stored for three fruit postharvest periods. dah = days after harvest. Open symbols and arrow indicate values with zero seed germination.
In this study, the beginning of germination in ancho chili seeds was associated with EC values below 495.5 μS·cm−1·g−1 and that of guajillo chilis with EC of 679.3 μS·cm−1·g−1 (Fig. 4). Another variable that can be an indicator of the beginning of germinability in chili seeds is seed weight. In the first samples taken there was a low 1000SW (Fig. 2) and a high moisture content (Fig. 1). It seems that when 1000SW is above 8.2 g in ancho chilis (60 daa) and 5.3 g in guajillo chilis (14 daa), germination begins. This means that from 60 daa and 140 daa, a certain percentage of embryos was able to germinate, even before accumulation of maximum weight. However, it cannot be considered the optimal stage for harvesting the seeds because most of them had not reached maturity. It is at the end of the phase of reserve accumulation that the 1000SW stops increasing (Fig. 2A, B).
Germination of seeds from guajillo chili fruits harvested 60 daa and stored for 7 and 14 dah reached values of 21 and 47%, respectively, in contrast to seeds from fruits without post-harvest storage for which germination was 0%. Seeds from fruits harvested 80 daa and stored for 7 dah had significantly higher germination (G), reaching more than 75% of seeds of both chili types. When the fruits were stored for 14 dah, G improved significantly relative to those stored for 7 dah, reaching germination rates of 92% for ancho chili seeds and 96% for guajillo chili seeds (Fig. 3A, B).
Maximum germinability and vigorSeeds extracted from guajillo chili fruits with no postharvest storage (0 dah) developed more rapidly than seeds from ancho chili fruits (Fig. 3B). Guajillo chili seeds reached germination values close to the maximum 100 daa, while ancho chili seeds required 120 daa to reach that level. Postharvest fruit storage did not benefit seed germination at 100 daa for guajillo seeds and 120 daa for ancho seeds.
A decrease in germination percentage after this stage of development can be explained by longer exposure to environmental factors that cause the seeds to deteriorate. According to Rodríguez-Burgos et al. (2011) and Pichardo-González et al. (2014), quality attributes (germination, weight, vigor, etc.) increase as the seed develops and reaches its maximum expression in the phase of physiological maturity. From this moment, the irreversible process of loss of these attributes begins to deteriorate the seed and ends with its death.
Vigor is considered the last physiological quality parameter the seed acquires during its formation (Bewley et al., 2013) and the easiest to lose to deterioration. Speed of germination (SG) is a variable that is traditionally used to evaluate vigor (Ayala-Villegas et al., 2014; Pichardo-González et al., 2014). In general, during seed development in all the treatments, this variable followed a sigmoidal function characterized by a phase in which the growth curve was very low or did not exist, followed by exponential growth that concluded with a slower growth phase (Fig. 5). In fruits with no postharvest storage (0 dah), SG increased significantly in seeds from fruits of both types harvested 100 daa. In guajillo fruits harvested 60 daa and stored for 7 and 14 dah, there was a significant increase in SG, reaching 0.9 and 2.8 radicles·d−1, respectively. However, these values are considered too low to recommend these treatments. Samples of fruits of both chili types harvested 80 daa showed the greatest benefit from postharvest storage. By 14 dah, SG was established at 5.6 radicles·d−1 for ancho chilis and 6.1 radicles·d−1 for guajillo chilis. In both cases, the values represent between 80 and 85% of the maximum germination value achieved at 140 daa (Fig. 5).
Evolution of germination speed after aging accelerated in seeds of (A) ancho and (B) guajillo chilis harvested at six development stages and stored for three fruit postharvest periods. dah = days after harvest. Values with the same letter between samplings are statistically equal (Tukey’s test, P ≤ 0.05).
At the end of seed development, the process of acquisition of tolerance to drying occurs. This process is characterized by synthesis of late mRNA in the developing fresh seeds (Han et al., 1997), and then transcript code molecules such as LEA proteins. In the case of chili seeds, there are few studies of the presence and activity of this group of proteins during the desiccation phase, mainly the study of Vidigal et al. (2009); they identified LEAs in seeds of Capsicum annuum, a sweet pepper type. It is known that seeds of this type of chili have a development period of less than 70 daa, while ancho and guajillo chilis do not complete their cycle until 140 daa. The protocol of our study (modified from Vidigal et al., 2009) enabled us to separate a soluble protein profile from guajillo chili seeds (Fig. 6) that was very similar to that of ancho chili seeds. These separated proteins are in a molecular weight range (MW) between 15 and 65 kDa. The expression and degree of intensity shown in several of the separated bands is very similar among the different extraction dates after anthesis (60, 80, 100, 120, and 140 daa) and the different periods of fruit storage (0 and 14 dah). In the protein profile, the expression of two proteins P1 and P2 (Fig. 5) stands out. These proteins may be members of the family of late embryogenesis abundant proteins that are correlated with better seed germination and vigor. P1 had a molecular weight of approximately 65 kDa. It was absent up to 60 daa and it was found in the subsequent harvest dates (80 daa to 140 daa). P1 remained after 0 or 14 days of postharvest storage (Fig. 6). Another LEA protein expressed in the protein profile has a MW of 50 kDa and may be the P2 reported by Vidigal et al. (2009) in sweet pepper seeds. This P2 protein was evident in guajillo chili embryos at 100 daa and remained constant until 140 daa, just during the late maturation and desiccation of the seeds. Because this protein is found in three different types of Capsicum annuum, it may be considered a marker protein of the species. There is at least one more LEA protein in ancho and guajillo chilis (Fig. 6), with an MW of 30 kDa, reported in the literature for Capsicum annuum seeds and also reported by Vidigal et al. (2009). In our study, it was expressed from 60 daa to 140 daa. The expression of LEA proteins found in our study supports the physiological traits that have negative effects in terms of chili seed quality attributes.
Separation of proteins by PAGE-SDS of guajillo chili seed embryos from fruits harvested 60, 80, 100, 120 and 140 daa and stored 0 and 14 d postharvest (dah). Each track contains 40 μg soluble protein. MW: molecular weight (kDa).
In this study, we confirmed that 14 d postharvest storage improved germination and vigor characteristics of seeds extracted from both types of chili fruits harvested 80 daa, reaching high commercial quality germination rates (93% in ancho, and 96% in guajillo, chilis) and high vigor values (5.6 and 6.1 radicles·d−1 SG in ancho and guajillo chilies, respectively), maintaining the optimal 1000SW. These high physiological and physical quality values coincide with the expression of protein P1 (Fig. 6). At 100 daa in ancho chilis, germination in treatment 0 dah was significantly lower than in treatments 7 and 14 dah. Protein P2 was expressed more highly 120 daa in the seeds of both chilis and in all the postharvest treatments, coinciding with statistically superior vigor values (Fig. 5).
ConclusionsThe moisture content of seeds inside ancho chili fruits decreases from 40 daa to 80 daa, remaining at around 47% up to 140 daa. In guajillo chilis, it decreases during the entire period of seed development, diminishing to 17% 140 daa. Physical and physiological quality attributes increase as the seed develops. Germination of seeds from fruits of both chili types harvested 80 daa and stored for 14 d was on average 93%, and speed of germination of aged seeds was 5.6 radicle·d−1; both values are considered high. Germinability correlated with electrical conductivity and the thousand seed weight. Ancho chili seeds germinated at 472.5 μS·cm−1·g−1 EC and 8.2 g 1000SW, while guajillo chili seeds germinated at 679.3 μS·cm−1·g−1 EC and 5.3 g 1000SW. Seeds harvested from 120 daa onwards were of high quality and there was no need for postharvest storage of the fruits. We detected expression of two LEA proteins. The first, of 65 kDa, became at 80 daa, while the second protein, of 50 kDa, was evident 120 daa. These LEA proteins are directly related to the maximal physiological quality of ancho and guajillo chilis.
