Pepper is one of the most important vegetable crops in Mexico; is preferably consumed fresh (^{Valadez, 2001}). From the economic viewpoint C. annuum is the most cultivated species in Latin America and worldwide, followed by C. chinense. Most hybrid seed production of this crop is produced in countries like China, India and Thailand with cheap and skilled labour (^{Berke, 2008}). There are so expensive genotypes that the price of the seed on the market ranges from 1 000 to $1 200 per thousand seeds.

Production and hybrid seed yield depends on several factors, including, emasculation technique, stigma receptivity, right time of pollination, pollen viability, pollinations number necessary for seed production number of fruit per plant (^{Kivadasannavar, 2008}). The flower opening occurs most often in the early hours of the day and remains open for a variable period, on average 24 h. Anthers open after the opening of the flowers, varying from 1 to 8 h at C. annum. Stigma can be receptive stage button on the start of anthesis, or 2-3 h after flower opening (protogyny) (^{Vallejo et al., 2004}). The technique used in the cultivation of pepper is the manual emasculation; however, it is a laborious and unprofitable procedure (^{Bosland et al., 2012}).

Some current alternatives emasculation used in removing pollen are: by heat, cold or ethanol, without emasculation and pollination gene-cytoplasmic male sterility (^{Kato, 1989}; ^{Chávez, 1993}; ^{Aloni, 1999}). ^{Hirose and Fujime (1973)} studied the influence of Dalapon^® to induce male sterility in pepper and reported that 6 applications per week in low concentrations (up to 300 ppm) suppressed anther dehiscence.

The aim of this study was to evaluate two techniques of emasculation in two genotypes of pepper (Euforia and Invicto), and two of pepper Bell (Canon and Dársena) and, compare with ethanol emasculation and manual emasculation and seed quality considering the state of ripening for removal.

The work was conducted under greenhouse conditions in the region of Celaya, Guanajuato, at an elevation of 1765 meters. The materials tested were two genotypes pepper (Euforia and Invicto) and two of pepper Bell (Canon and Dársena). The germination of seedlings of four genotypes was carried out in trays. Steiner nutrient solution in irrigation water daily was applied (^{Castañeda, 2001}). When the seedlings reached a height of 15-20 cm, these were transplanted in the greenhouse; four blocks were established, with rows of 3 m length; each block was constituted by 10 rows at a distance of 1 m between them. Each groove had nine plants at a distance of 40 cm, double row with a density of 2.2 plants m².

Two methods were evaluated, ethanol and manual emasculation. For the first one, parted buttons were selected in the presence of immature anthers. 47.5% ethanol (V/V) was evaluated for 5, 10, 30 and 45 s; after immersion, the buttons are covered with thin paper to prevent contamination five flower buds per treatment with three replications.

Variables assessed: performed quantitatively and qualitatively. Presence of pollen (PP), if the anther observed after application of ethanol had dehiscence and pollen release by visual observation. Fall Flower (CF), the number of the flowers that fell after application of the method is recorded. Percentage of aborted of the flowers (PFA), the number of the flowers that had no fruit set was recorded. Percentage of mooring and fallen fruit (PA), (PFC). Mooring percentage was measured by counting the number of fertilized of the flowers and fruit set. The number of fallen fruits, evaluated as total fruit was counted. Pollen viability. In Bell pepper genotypes, emasculations made with ethanol and of the flowers were left on the ground one day.

Subsequently, the anthers were cut to remove pollen and feasibility testing. For this we employed: sucrose 20% solution, 100 ppm of H₃BO₃, 300 ppm Ca(NO₃)₂, 200 ppm MgS0₄, 100 ppm of KNO₃ and 0.4% Tween 80, pH 5.5. Two drops of medium were placed on a slide, then samples of pollen from of the flowers subjected to treatment with ethanol were placed. These were kept at a temperature of 20 °C for 2 h. The observation of pollen was performed using a stereoscope microscope Leica Zoom 2000^®. Grains of viable pollen (GPV). Pollen grains that had presence of pollen tube length larger than or equal to the diameter of pollen (µm) were considered viable (^{González et al., 2002}).

The manual emasculation is performed between 08:00 and 10:00 am, because the pollinated requires an average temperature of 20 °C, for the anther to present the dehiscence and suitable pollen viability. Direct and reciprocal crosses were made; labelled and covered with tissue paper the flower. The evaluation was performed by using the quantitative characteristics as female parent and then as a male with 10 repetitions per treatment, giving a total of 20 experimental units; evaluation was done descriptively, estimating the mean percentage of mooring (number of fruits achieved). The fruits are harvested when they were red or red streaking, which is an indicator of ripeness of fruit and seed. To evaluate the most appropriate maturing phase for extracting seed and ensure the same quality were considered three degrees of maturity, at 30, 45 and 60 days with 8 replicates.

Seed Extraction time: immediately, the seed is removed at the time of harvest; late, the seed is removed after 14 days of harvested fruit. Extraction was performed by fruit and weighed individually; washed with sterile distilled water and allowed to dry for 14 days; stored in airtight bags. Physiological seed quality. The standard germination test was performed on germinator paper. 25 seeds were placed on paper previously wetted; spraying “captan” 1g L^-1. The paper was rolled to form the "tacos" and placed in plastic bags and subsequently entered the chamber Conviron^® germination at 25 °C. The first seven days and the second at 14 days, two counts were performed.

Emasculation with 47.5% ethanol (v/v); in genotypes of pepper Euforia and Invicto for 10 s, damaged in intermediate grade 80% of the flowers. Immersion for 30 s affected 60% of the flowers with intermediate damage. Treatment of 45 s caused a severe 100% of the flowers damage. Treatment for 10 s caused more damage due to the effect of surface tension generated by the morphology of the flower bud and the exposure time by not allowing to drip the solution. Treatment of 30 s was sufficient for the solution to accumulate and fall by gravity. As for the fall of the flowers, treatments of 10 and 30 s showed 10% of detachment. 45 s treatment resulted in 100% release of the flowers.

The immersion of floral button of Euforia and Invicto in 50% ethanol (v/v) for 10 s, showed that 40% had no visible damage to the stigma. Treatment for 5 s found that 40% had an intermediate damage, 20% slight damage and 40% had no damage. This immersion time seems right for emasculation in these varieties. During the dive of 5 s in 50% ethanol (v/v) showed that 40% of the stigmata had a yellow tint, 40% one light brown colour and 20% dark brown coloration. Immersion for 10 s caused 60% take a yellow colour, light brown 20% and 20% a brown coloration. Immersion in ethanol 52% (v/v) for 5 s, Euforia and Invicto, 60% showed no visible damage of the flowers on stigma and 40% with intermediate damage. The immersion time of 10 s showed 40% of the flowers without damage, 40% intermediate and 20% damage with severe damage to the stigma.

At this concentration of 5-10 s of exposure time 20% caused severe damage. The application of 52% ethanol (v/v) for 5 s and damage caused an intermediate light brown coloration in the stigma. According to the results of the application of ethanol at 50 and 52% (v/v) for immersion times of 5 and 10 s, show that treatment of 50% ethanol (v/v) for 5 s had higher stigma damage, compared to 52% immersion. This we believe is due to the arrangement of floral organs. In this connection it may be noted that when the stigmas are longer than the anthers, the damage is more severe. In accelerated aging tests of hybrid seed maize ^{Santipracha et al. (1997)} found that treatment of 43 °C for 96 h causes a decrease in the percentage of germination up to 26.5%, while treatment of 44 °C for 96 h in 67.5% reduces, in one of their hybrids. Physiologically, little difference in concentration or temperature can have marked effects.

Damage caused by immersing the flower bud in the concentration of ethanol 50% (v/v) for 5 to 10 s, in genotypes of pepper Bell, Canon and Dársena, treatment of 5 s, provided 48% of the flowers without visible damage to the stigma. In the treatment for 10 s, it was observed that 68% showed no visible damage. The damage on the stigma was more severe when the stamens were longer than the pistil (short-styled of the flowers). Immersion flower bud on Bell pepper Canon and Dársena in 52% ethanol for 5 s, caused no visible damage to 68% of the flowers. Treatment for 10 s had no visible damage 60% of the flowers. At this stage the more of the flowers you did not file damage immersion times of 5 to 10 s was observed. The type of floral morphology helps prevent damage to the stigma (styled flower).

In all the treatments with ethanol 47.5% (v/v) dehiscence of anthers and pollen shedding had normal coloration. 45 s treating damage caused burns similar anthers. The method of emasculation with ethanol did not interrupt the anther dehiscence at all.

Statistical analysis of the treatments with ethanol 50% and 52 during immersion times of 5 and 10 s, detected no statistically significant differences. In the trial for pepper was observed that the successful implementation of the method of emasculation with ethanol depends on the arrangement of floral organs, which coincides with the results found in the genotypes of pepper Bell.

In the Figure 1, the percentage of aborted of the flowers was observed; this result agrees with ^{Luna (2010)} who notes that non-pollinated of the flowers emerge (^{Jaimez, 2010}). An important factor fruit set is the position of the flowers on the plant. The FAO (2002) noted that in pepper, each plant produces hundreds of the flowers that can curdle at 100% when the first stage of flowering and are on the main stem; this percentage drops to 80% for subsequent stem and of the flowers of the same reduced up to 20-30% and even 10% for of the flowers of lateral branches. 80% of the flowers tied with hand pollination, while the ethanol treatment was only 20%.

Figure 1 Percentage of aborted flowers in treatments with ethanol 50 and 52% during immersion times of 5 and 10 s. 

^{García (2011)} working with Capsicum flexuosum found that in assessing greenhouse plants in different populations, fruit length was influenced by the source of pollination; as many small fruits was found in random pollination and hand pollination showed intermediate values and greater variability.

Treatments at 30, 45 and 60 days of ripening obtained that 30 days showed a mean of 5.5 ± 0.51 cm, 45 days had 7.7 ± 0.73 cm and 60 days, the average was 8.0 ± 0.68 cm. In pepper Bell, shows that after 45 days the fruit had reached the diameter of 60 days. In the period of 30-60 days, the product diameter increased 69%.

Treatment with 50% ethanol for 5 s 235 seeds were obtained per fruit; with 10 s of treatment, 304 seeds and 52% ethanol for 5 s 374 seeds were harvested; 10 s 296 seeds per fruit were obtained.

The number of seeds per fruit of pepper Bell, emasculated manually, treatments maturation at 30, 45 and 60 days was observed at 60 days an average of 170 seeds were obtained at 45 days, 238 seeds and 192 seeds 30 days. In researches on habanero pepper (^{Puente et al., 1991}) found that when advancing the status of fruit maturity there was an increase in the percentage of germination. In the work done in pepper Bell (^{Sánchez et al., 1993}) reported that, the seeds extracted from fruits of 30 days did not germinate, while the highest percentage of germination was found in the treatments of 50 and 60 days. This result overlaps with ^{Edwards et al. (1987)} who evaluated the effect of fruit maturity and harvest time and postmaturation germination on tabasco pepper.

Obtaining germination rates of 81% to be harvested berries; addition, the germination percentage rose to 86% after a period of after-ripening.

ANOVA (Table 1) variables fruit ripening at 30, 45 and 60 days and immediate and delayed extraction seed. There were highly significant differences between harvest times for the variable length of stem and root. For extraction time, it showed only significant differences in the variable length stalk; this indicates that at least one treatment is different.

Table 1 Mean squares, statistical significance and coefficients of variation of the analysis of variance of harvested fruit maturity and extraction of seed for physiological quality variables. 

FV= fuente de variación; GL= grados de libertad; Rep. Repeticiones; C= cosecha; E= extracción; CV= coeficiente de variación; *, ** indican significancia estadística al nivel 0.05 y 0.01 de probabilidad, respectivamente.

According to this, the comparing means (Figure 2) shows that, the treatment of fruit maturity harvest at 60 days was higher than at 45 days in treatment variables root and stem length; This result was expected because as time passes the length of root and stem increases because the plant has not reached maturity; results agree with ^{Carrillo et al. (2009)}, mentioning that among the factors that can have an effect on seed quality are the degree of maturity and ripening time of it.

Figure 2 Percentage of the flowers treatments moored in ethanol at 50 and 52% for immersion times of 5 and 10 s. 

Table 2 Comparison of means of the treatment ripening of harvest fruits for physiological quality variables. 

Medias con la misma literal son estadísticamente iguales (Tukey p< 0.05).

By comparing averages of seed extraction time, extraction late treatment was higher in the root length variable; there were no statistical differences in the variable length of stem. This result overlaps with ^{Randle et al. (1981)} who stated that, the seed of pepper completes its physiological maturity in a rest period ranging from one to six weeks after the fruits were harvested, depending on the type of pepper.

The Table 3 shows the analysis of covariance of the ripening of fruit to 30, 45 and 60 days and extraction immediate and late seed. There were statistically significant in both variables, indicating that at least one of the treatments is different.

Table 3 Mean squares, statistical significance and coefficients of variation of the analysis of variance covariance of harvested fruit maturity and extraction of seed for physiological quality variables. 

FV= fuente de variación; GL= grados de libertad; Rep. repeticiones; CoCE= covariancia de cosecha y extracción; C.V.= coeficiente de variación; ** indica significancia estadística al nivel 0.01 de probabilidad.

The Table 4 presents the comparison of means of covariance of harvested fruit maturity and seed extraction. The treatment of 60 days in both extraction times was over 45 days, for both variables.

Table 4 Comparison of means of covariance of the maturity of harvested fruit and removing seed for physiological quality variables. 

Medias con la misma literal son estadísticamente iguales (Tukey p< 0.05).