a) Workspace

The experiment was conducted during December 2014 and January 2015 in the microbiology laboratory and nursery of the Multidisciplinary Academic Division of Ríos, located at km 1 of the Tenosique road - Waterfalls Reforma, Mexico Tenosique Tabasco.

b) Plant material

A collection of fruits amashito pepper healthy plant in the mountains of Tenosique region was performed between parallels 17° 28' north latitude and 91° 25' west longitude. The fruits were stored in Petri dishes for 3 days at room temperature (24 ± 2 °C), then the fruit ripened orange-red colour is selected, then extracted the seeds were extracted by friction of the fruit. In Figure 1 (green and ripe) fruit and seed amashito pepper shown.

c) Treatment design

A completely randomized design was used with 5 treatments and three replications, 20 seeds per replicate. After removing the seeds from the fruits, these were treated with 1.2% Cupravit^® for an exposure time of 10 min, then were given 3 rinses with sterile water. Then the seeds were distributed uniformly in a Petri dish, then were grouped into five equal parts into a cake, then took each of the divisions so random for each treatment. Seeds were emptied quickly to immersion in a solution with sterile water and Biozyme TF^® (1.6% v/v) for 0, 12, 18 and 24 h, and a control consisting of only hydration with sterile water for 24 h. It is important to mention that the seeds were not dried in the sun as is done in a common way with other species; this in order not to favour the hardening of the testa, while simulating the natural process, where birds eat the ripe berries orange-red colour, and after 8-24 h pass through the digestive tract and are dispersed with feces. The dependent variable was the percentage of germination, while observing the 50 + 1% of sprouts. Table 1 describes the five treatments.

Table 1 Treatments for evaluating Biozyme TF^® on germinating seeds of amashito (Capsicum annuum L. var. Glabriusculum). 

The seeds from each treatment were planted in trays of transparent polyethylene 40 x 10 cm. The Cosmopeat^® (grade horticultural substrate) previously sterilized in autoclaved All American^®, at 121 °C for 15 min, the substrate was maintained at field capacity.

Statistical analysis

An analysis of variance of a completely randomized design with equal numbers of replicates per treatment and comparison tests Tukey (p≤ 0.01) was performed. Data were analysed using the statistical package ^{Olivares (1994)}.

The results were highly significant (α= 0.01); that is, the time of immersion of seeds in Biozyme TF^® had a positive effect on the germination percentage and time. According to technical information phytohormonal balanced composition of Biozyme TF^®, gibberellic acid can induce in pepper seeds amashito hydrolysis of starch and sucrose to glucose and fructose, and promote the release of energy and becomes negative potential allowing water ingress of water and increasing cell wall plasticity and cell growth leading to tissue. Indole acetic acid, meanwhile could interfere with the translation of the message on the amino-ATP activates link to join the messenger RNA. For its size, zeatin could act with specific receptor proteins, initiating a translation path of the signal. Thus Biozyme TF^® assets can penetrate through hard epicuticular waxy outer layer, and disable the possible natural inhibitors, responsible for a lower germination of 5% as indicated by the ^{INIFAP (2002)} and ^{INIFAP (2004)}. Biozyme TF^® stimulate germination of seeds expressing their full genetic potential. In Figure 2 we can see that on the eighth day treatment 2, 3 and 4 had over 50 + 1% of the sprouts.

Figure 2 Appearance of seed germination of amashito (Capsicum annuum L. var. Glabriusculum) at 8 days of immersion in Biozyme TF^® (1.5%) for 18 h. 

As seen in the Table 2, the treatment 2 with 12 h of immersion with 1.5% Biozyme TF^® 61.5% germination, was obtained while in treatment 3 and 4 with longer immersion at 18 and 24 h increased to 83.3 and 86.6%, comparing mean there is no difference between these two treatments; however, when comparing treatment 1, 2 and 5 if there is a highly significant difference. The results are clear of the effect of immersion time of seeds with Biozyme TF^®, for the treatment 5 which only consisted only hydrate the seeds in water for 24 h germination percentage was 43% similar as Only the seeds are exposed for a moment with the product, obtaining 40%. It is noteworthy that the maximum potential germination treatments was 12 days, after which time no longer germinated seeds was evident.

Table 2 Results of the percentage and time of amashito (Capsicum annuum L. var. Glabriusculum) germination. 

* Letras distintas indican diferencias significativas, según la prueba de Tukey (p≤ 0.01).

These results are significant, compared to those reported by ^{INIFAP (2004)} used gibberellic acid at a concentration of 5 000 ppm of the commercial products Biogib, ProGibb plus or Activol immersed for 24 h at 25 to 30 °C; and germination was between 60 to 80% at 15 and 20 days. Another study reported by ^{Araiza et al., (2011)} found that treating seeds with pepper chiltepín AG 400 ppm at 35 °C and a period of 10 h light at 16 days was 65% germination and 47% control to eight weeks. In Figure 3, amashito pepper seedlings with 6 leaves on average at 25 days after germination is observed, and transplanted into styrofoam cups ½ liter in commercial substrate.

Figure 3 a) Seedlings of amashito (Capsicum annuum L. var Glabriusculum) at 25 days of age; and b) seedlings with a height of 6 cm and 6 leaves on average. 

This technology enables a high germination percentage pepper amashito as an alternative production under intensive management, to improve the income of many families in rural areas; Additional conservation of biodiversity of species in their natural habitat, decreasing the pressure of field collection in favour. In a study conducted by ^{Sandoval-Rangel et al. (2011)} described that pepper “piquín” (ecotype amashito) the overall average growth of pepper plants grown in open field with padding and fertigation was 44.90 cm and a diameter of 50.78 cm coverage to 98 days after transplantation, and an average yield of 130.91 g per plant in two cuts with antioxidants trolox equivalent of 8.58 mg^-1 and capsaicin content of 672.80 mg kg^-1 (100 Units Escoville 92.03). Organoleptic characteristics that make according to ^{Rodriguez-Del Bosque (2005)} the preference level is very similar to “jalapeño”.

Conclusions

BiozymeTF^® due to the content of phytohormones: gibberellic acid (31 ppm), indole acetic acid (31 ppm) and zeatin (83 ppm) at 1.6% (v/v) has a positive effect on increasing the germination rate of pepper amashito to 86.6%. Therefore, it cannot be considered a limiting low germination for intensive cultivation of this species for commercial purposes factor. In addition, the results can be used as a strategy to repopulate devastated areas, because the plants obtained by seeds are a good source of genes leading to genetic variability and ecosystem balance. On the other hand, it is important to evaluate the potential of this species and propose a model of production, management and conservation with a focus on sustainability; as the wild species are an important source of genes for plant breeding through genetic engineering.