Extraction and benefit of the seed.

The seed was extracted by macerating the fruits with hands and latex gloves, the macerate was placed in drinking water and the seeds were allowed to settle, then dried at room temperature. Half of the seed was conditioned and the other half was only extracted, dried by sun exposure and stored in a conventional way in a Ziploc brand polyethylene bag and at the ambient temperature of the seed laboratory of the CDDTS. The conditioning consisted of drying the seed with forced air, until reaching a humidity of 9%, for which a South Dakota brand blower was used, it was homogenized by weight and size and it was stored in Uline brand plastic bags of 10 × 20 cm, hermetically sealed and at a temperature of 15 °C (^{Aguirre and Peske, 1998}; ^{Reveles-Hernández et al., 2013}).

Germination tests

Four ages of the seed, 0, 2, 4 and 12 months after extraction were evaluated, seeds benefited and not benefited and seeds preconditioned with gibberellic acid at 5 000 mgL^-1 and seeds without preconditioning (dry seeds) each treatment with three repetitions and each repetition with 50 seeds. The experimental design was factorial A × B × C, where: A= age, B= benefit and C= conditioning (^{Zar, 1996}). The seeds were germinated in Petri dishes on Whatman No. 1 filter paper in a Lab-line branching chamber at 25 ±1 °C, with 8 hours of light and 16 hours of darkness. Germinated seeds, ungerminated seeds and abnormal plants were recorded (^{AOSA, 1993}).

Imbibition test of the seed

This test was performed additionally to determine if the seed was impervious to water. The 50 seeds of each age were weighed with four repetitions on an Ohaus brand precision balance, model Adventurer Pro, then placed in a 13 × 100 mm test tube, 10 cc of distilled water was added with an Eppendorf brand micropipette and by difference of the volume, the water absorbed was quantified, the excess water was removed and the hydrated seed was weighed again, to determine the weight increase by the water absorption. This test was performed at 12, 24, 36, 48 and 60 h.

Viability test with tetrazolium

To determine the viability of the seed, the 2, 3, 5-Triphenyl test, Tetrazolium chloride (TZ), Sigma-Aldrich brand (^{ISTA, 2004}) was carried out. It started with an osmoconditioning of the seed, which consists of placing the seeds in distilled water for 16 hours. Once the seed was hydrated, longitudinal cuts were made with a scalpel, to expose the cotyledons. The sections were placed in a test tube of 13 × 100 mm, adding the solution of 2, 3, 5 Tetrazolium Trifenil chloride 0.1% to cover the seed, then the test tubes were wrapped with aluminum foil to avoid light and they were placed in a Lab-Line brand incubation chamber at 30-35 °C for 90 min. Subsequently, in a Carl Zeiss brand stereoscope, seed staining was observed.

Seedling production

In this test, three different ages of the seed, 2, 4 and 12 months were evaluated (the seeds were removed recently harvested, because there was no germination in that test), seeds benefited and not benefited, seeds preconditioned with gibberellic acid at 5 000 mgL^-1 and seeds without preconditioning (dry seeds); each treatment with three repetitions and each repetition with 50 seeds. The experimental design was factorial A × B × C where, A= age, B= benefit and C= conditioning (^{Zar, 1996}). The seeds were planted in polystyrene trays of 200 cavities and as a substrate moss peat moss brand Sunshine was used. At 48 days after sowing, percentage of seedlings suitable for transplanting, height and diameter of the seedlings and number of true leaves were evaluated.

Germination tests

The results show that age and benefit, are the factors that have the most effect on germination, the conditioning with gibberellic acid at 5 000 mgL^-1, was also significant, but to a lesser degree. The highest germination percentages of piquín or wild pepper were obtained with the seeds of 2 and 4 months of age, which were benefited and conditioned before sowing with gibberellic acid (Table 1). The piquín or wild pepper seed that is currently used for research, backyard production or small scale, comes from collections that are made directly in the field, fruits left behind vendors or nuts that retain fans of this crop or that are purchased in stores.

The form of storage is also very varied and goes from the conservation in nuts or the extraction and conservation in containers, between the most common, polyethylene bags, paper and glass jars. The management of the seed, from the production or collection, form of extraction, selection, conditioning, drying and storage, as a whole is called profit (^{Aguirre and Peske, 1988}). This benefit has a high impact on the germination and viability of the seed and no evidence was found to be made on piquin pepper seeds. This may be a cause of the low and variable germination rate reported from 5 to 80% (^{Ramírez-Meraz, 2001}; ^{Rodríguez et al., 2003}; ^{Cano-Vázquez et al., 2015}).

Among other possible causes, are cited from myths such as the fact that the seed of wild pepper, require passing through the digestive system of birds to germinate (^{Ariza-Lizarde et al., 2011}). The investigations to determine the causes of the latency, have basically started from two hypotheses; One is that the seed has physiological latency, for this purpose germination tests have been carried out with hydrogen peroxide (^{Flores et al., 2008}), potassium nitrate and gibberellic acid (^{García-Federico et al., 2010}; ^{Araiza-Lizarde et al., 2011}), another hypothesis is that the seed presents physical latency, attributed to the impermeability of the testa or seminal cover and to the low permeability of the endosperm (^{Bañuelos et al., 2008}; ^{Araiza et al., 2011}), for this studies of scarification, stratification and previous hydration of the seed are reported. Therefore, in a complementary way, this study included a conditioning test with gibberellic acid and another one of imbibition that are cited as the most common tests to check whether there is physical or physiological latency.

Imbibition test

The results show that the piquín pepper seed absorbed a greater volume of water during the first 12 hours, while in the subsequent 48 h the absorption was minimal. The above shows that the seed is not impervious to water. The impermeability of the testa or seminal cover is reported as the most probable cause of the physical latency of the piquín pepper seed (^{Ramírez-Meraz, 2001}, ^{Rodríguez-Del Bosque et al., 2004}). However, the imbibition tests show that the seed is hydrated during the first 12 hours of exposure to water (Figure 1), and therefore the latency is not associated with the impermeability of seed coat or cover (^{García-Federico et al., 2010}; ^{Prado-Urbina et al., 2015}).

Figure 1 Absorption of water by the piquín pepper seed at different times of imbibition. 

This information is important, because in practice, performing osmoconditioning prior to sowing, commonly known as seed soaking, makes this activity difficult. If the seeding is manual, the seed adheres to the hands and if it is mechanical, the seeders are designed to plant dry seeds, which is the way to plant commercial pepper seeds, such as serrano peppers, jalapeños, morrones, habaneros, etc.

Tetrazolium stain test

The tetrazolium staining test (2, 3, 5 triphenyl tetrazolium chloride) is a technique used to determine if the seeds have physiological activity. It is based on the reduction of tetrazolium by the hydrogen that is released by the activity of the hydrogenase enzymes, which are activated as a result of respiration, once the seeds have begun their germination process. Because of the reduction of tetrazolium, which is colorless, a red color is formed, which is indicative that the seed has begun its germination (^{ISTA, 2007}, ^{SNICS, 2017}). The red coloration can appear in any part of the seed that has respiratory activity, so it is necessary to identify if this red color is present in the embryo, because in this way it can be more certain that the seed is able to germinate.

The results of this test are presented with photographs, where graphically it is shown that the seeds extracted from fresh red fruits (Figure 2a) do not present staining in the embryo, only in the endosperm of the seed, which indicates that there is activity Respiratory, not necessarily as part of the germination process, but rather as an activity of the reserve storage process. In Figures 2b and 2c corresponding to the seeds of 2 and 4 months of age, the red color is observed in the embryo, which indicates respiratory activity in this tissue, when correlating these results with the germination tests, it is concluded that respiratory activity is the result of the germination process of the seed. Finally, when looking at Figure 2d, which corresponds to the seeds of 12 months of age without benefit, no staining is observed, which indicates that there is no respiratory activity in any tissue; that is, dead seeds.

Figure 2 Seed staining by tetrazolium 

The seeds of 12 months and that were benefited, presented a similar staining to the seeds of 4 months of age, so that the benefit and adequate storage of the seed reduces the deterioration conserving the seed for more than a year, as it happens with seeds of commercial peppers.

Seedling production

In this test, the analysis of variance showed that the percentage of seedlings is affected by age, benefit and seed conditioning with gibberellic acid, but once the seedlings emerged no effect on their growth and development was observed and these results coincide with those obtained in the germination test.

Also, these results show that it is possible to obtain up to 96% of seedlings suitable for transplant if the seed benefits, later it is stored properly for 4 months and before planting it is conditioned with a solution of 5 000 mgL^-1 of gibberellic acid before sowing (Table 2 and Figure 3). It was also observed that if the seed benefits and stores properly, it can maintain its viability for more than 1 year.

Figure 2 Piquín pepper seedling with seeds of 4 months old, benefited and conditioned with 5 000 mg L^-1 of gibberellic acid. 

The response of the piquín pepper seed to the benefit and conditioning is very similar to that of other types of commercial pepper, given that commercial pepper seeds carry a period of rest after being extracted by the dynamics of their preparation for sale. For example, broad-leaved, free-pollinated peppers seeds or varieties, in the northern region of Guanajuato, Zacatecas, Aguascalientes, are harvested during the months of september to october, and later planted in the months of november to january. Which implies that the seeds are at rest at least 2 months before planting. In the case of hybrids, harvest, profit, packaging and distribution for sale, it is estimated that it lasts more than two months.