agronomy

Article
Seed Physiological Potential of Capsicum annuum var.
glabriusculum Genotypes and Their Answers to
Pre-Germination Treatments
Juan Samuel Guadalupe Jesús Alcalá-Rico 1 , Alfonso López-Benítez 1, * ,
Mario Ernesto Vázquez-Badillo 1 , David Sánchez-Aspeytia 2 , Sergio Alfredo Rodríguez-Herrera 1 ,
Miguel Ángel Pérez-Rodríguez 3 and Francisca Ramírez-Godina 1
1 Department of Plant Breeding, Universidad Autónoma Agraria Antonio Narro, Calzada Antonio Narro 1923,
Buenavista, Saltillo 25315, Mexico; [email protected] (J.S.G.J.A.-R.);
[email protected] (M.E.V.-B.); [email protected] (S.A.R.-H.); [email protected] (F.R.-G.)
2 INIFAP-Experimental Field Saltillo, Carretera Saltillo–Zacatecas km. 342+119 # 9515 Hacienda de Buenavista,
Saltillo 25315, Mexico; [email protected]
3 Department of Botany, Universidad Autónoma Agraria Antonio Narro, Calzada Antonio Narro 1923,
Buenavista, Saltillo 25315, Mexico; [email protected]
* Correspondence: [email protected]; Tel.: +52-844-105-6151




Received: 8 May 2019; Accepted: 17 June 2019; Published: 20 June 2019


Abstract: Piquin pepper (Capsicum annuum var. glabriusculum) is an important species that supports
the economy of rural households; it is part of Mexican gastronomy and it is a highly valuable
phytogenetic resource. There has been recent interest in domesticating and exploiting piquin pepper
commercially, which has been limited until now due to the low germination rate, and this work had
the purpose of promoting germination and determining the physiological capacity of genotypes.
Ten piquin pepper genotypes from different geographical origins in Mexico were submitted to
11 pre-germination treatments. A completely randomized experimental design was carried out with
arrangement in split-plot. The large plot had the treatments and the small plot had the genotypes.
The results showed differences (p < 0.01) among treatments, genotypes, and treatment–genotype
interaction. On one hand, treatments gibberellic acid (GA) and mechanical scarification + gibberellic
acid (MSGA) increased the physiological potential of genotypes, reaching the highest values of
germination speed (GS), germination index (IG) and germination percentage (GP); as well as the
lowest values of dead seeds (DS) and hard Seeds (HS). In turn, the genotypes that presented the same
condition were G8, G7, and G10. Regarding the interaction, each variable had a different condition.
In conclusion, we can increase the physiological potential and solve the dormancy of piquin pepper
seed by applying gibberellic acid. Likewise, the best genotypes were G8 and G10.

Keywords: piquin pepper; dormancy; physiology; Treatment-Genotype interaction; biplot

1. Introduction
Piquin pepper (Capsicum annuum var. glabriusculum) has several names, including “chiltepín”,
“chile de monte”, and “chile silvestre” (wild pepper), among others [1]. The distribution of this
species reaches Colombia, Central America, Mexico, and the South of United States [2]. In Mexico, it is
widespread from Sonora to Chiapas and from Tamaulipas to Yucatan and Quintana Roo, showing
great environmental adaptability, probably resulting from its genetic diversity that allows it to adapt
itself to different environments [3,4]. The importance of piquin pepper lies in the fact that it is the
main source of income for many people and households in rural areas, who make their living from
picking of the fruit of wild populations. This hot pepper is preferred over Jalapeño and Serrano

Agronomy 2019, 9, 325; doi:10.3390/agronomy9060325 www.mdpi.com/journal/agronomy

Agronomy 2019, 9, 325 2 of 12

hot peppers in the market, although its value can be up to 40 times more expensive [5,6]. The great
acceptance of piquin pepper by consumers is due to its pleasant flavor, and even when it is very spicy
(50,000–100,000 SHU), that sensation disappears quickly from the mouth and does not irritate the
digestive system [7]. Furthermore, it is a highly valuable resource in breeding programs, since it is
considered the ancestor of all Capsicum annuum [8,9]. At present, there is great interest in domesticating
this species and establishing it as a commercial crop. It is known that piquin pepper seeds have a natural
dormancy mechanism that acts as a potential blockage to complete germination [3]. This mechanism
has helped the species to survive under adverse climate conditions and avoid competing for light,
water, and nutrients [10,11]. From an agronomic management standpoint, this is the main problem
of piquin pepper; the dormancy mechanism results in low and uneven germination at planting [7].
Therefore, the purpose of this research work was to determine the effect of pre-germination treatments
on the germination traits of different piquin pepper genotypes and to assess their physiological capacity.

2. Materials and Methods

2.1. Location of the Experimental Site

The experiment took place in the Seed Test Laboratory of the Training and Development Seed
Technology Center, “Centro de Capacitación y Desarrollo de Tecnología de Semillas” (CCDTS) from
the Plant Breeding Division of “Universidad Autónoma Agraria Antonio Narro” (UAAAN), located in
Buenavista, Saltillo, Coahuila, Mexico.

2.2. Plant Material

This research work included ten piquin pepper genotypes from different geographical origins
in Mexico provided by the National Institute of Forestry, Agricultural and Livestock Research,
“Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias” (INIFAP), “Las Huastecas”
Experimental field (Table 1). The mother plants of each genotype were kept in a controlled greenhouse
environment, so they had the same conditions. For the obtaining of the seed, ripe fruits were collected,
which were allowed to dry under ambient temperature conditions, were placed in paper bags with
their identification, and then stored for 3 months at a temperature of 17 ◦ C. Subsequently the seed of
the fruits was extracted, at the time it was required to use it.

Table 1. Identification and origin of 10 piquin pepper genotypes.

ID Locality Municipality State Geographical Coordinates

G1 Estación Álamo Villaldama Nuevo León 26◦ 230 5100 N, 100◦ 230 4200 W
G2 Ej. Potrero de Zamora Aramberri Nuevo León 24◦ 020 2000 N, 99◦ 550 1500 W
G3 Ej. Lázaro Cárdenas Burgos Tamaulipas 24◦ 560 5600 N, 98◦ 470 5900 W
G4 Barranco Azul San Carlos Tamaulipas 24◦ 240 0100 N, 99◦ 060 5000 W
G5 Palo Blanco Castaños Coahuila 26◦ 460 0200 N, 101◦ 300 0100 W
G6 Colatlán Ixhuatlán de Madero Veracruz 20◦ 490 0400 N, 98◦ 050 4500 W
G7 Tiopancahuatl Ixhuatlán de Madero Veracruz 20◦ 410 0600 N, 98◦ 000 4400 W
G8 Colatlán (Nursery) Ixhuatlán de Madero Veracruz 20◦ 490 0400 N, 98◦ 050 4500 W
G9 El Rincón Linares Nuevo León 24◦ 530 4000 N, 99◦ 280 1300 W
G10 La Laborcilla Rioverde San Luis Potosí 21◦ 510 3700 N, 99◦ 540 4000 W

2.3. Treatment of Seeds

Eleven pre-germination treatments showed in Table 2 were used in order to evaluate the
germination response in piquin pepper seeds. The treatments were selected according to previous
works, due to their effect on the seed [3,12–18]. The distilled water was used as a solvent to obtain the
desired concentration. In each treatment the seed was submerged for the indicated time, except for
the mechanical scarification, which consisted of sanding the seed. After submitting the seed to each
Agronomy 2019, 9, 325 3 of 12

treatment, a rinsing with distilled water was carried out. Three replicates were performed and 20 seeds
per replicate of each genotype were used.
The seeds were disinfected with sodium hypochlorite at 1% during 30 s, before rinsing them
with running water under the faucet, followed by a final rinse with distilled water. The seeds were
spread over absorbing paper and were left to dry for 15 min. The corresponding pre-germination
treatments were applied afterwards (Table 2). Petri dishes (100 mm × 15 mm) lined with sterilized
filter paper were used, moistened with a solution of distilled water and fungicide. Each week two
types of fungicides were rotated to prevent and control the development of pathogenic fungi, namely
Captan 50 (Captan) and Tecto 60 (Thiabendazole). Both at a concentration of 1 g L−1 , were applied by
spray at the time the filter paper required moisture. This was done throughout the duration of the
assay. The seeds were planted in the dishes by distributing them in circles, a small and a bigger circle,
to facilitate the evaluation of the variables. Afterwards, the Petri dishes were put into the germination
chamber LAB-LINE at 25 ◦ C, with 12 h of light and 12 h of darkness.

Table 2. Treatments used to evaluate the germination response of piquin pepper seeds.

ID Treatment Application Time

HP Hydrogen peroxide 3% 24 h
GA Gibberellic acid 5000 ppm 24 h
AV Agromil-V® 2% v/v 24 h
HC Hydrochloric acid 10% 30 min
KN Potassium nitrate 3% 168 h
HW Hot water At 83 ◦ C let cool naturally 24 h
MS Mechanical scarification Sanding softly with 1500 grit sandpaper 30 s
HWGA Hot water + Gibberellic acid 24 h per tratament
Hydrogen peroxide +
HPGA 24 h per tratament
Gibberellic acid
Hydrochloric acid + 1st tratament 30 min and
HCGA
Gibberellic acid 2nd tratament 24 h
Mechanical scarification +
MSGA 2nd tratament 24 h
Gibberellic acid
WIT Witness Direct seeding

2.4. Assessed Seed Germination Traits (Variables)

Germination percentage (GP): it was defined as the relationship among the number of germinated
seeds that had all their essential structures and the number of planted seeds, using the following
formula: GP = (NGS/TNS), where NGS is the number of germinated seeds and TNS is the total number
of planted seeds. Germination index (GI): is the time of germination based on the germination capacity,
GI = Σ(niti)/TNS, where ni, is the number of seeds germinated in day i and ti is the number of days
after planting. Germination speed (GS): is the number of seeds germinated in relation to the time of
germination: GS = Σ(ni)/t, where t is the germination time measured in days, from planting until the
germination of the last seed. Abnormal seedlings (AP): those seedlings that lacked one or more of their
essential seedling structures were counted. Dead seeds (DS): The counting included all those seeds
that were soft; they absorbed water, but they did not produce any seedlings. Hard seeds (HS): all those
seeds that remained waterproof at the end of the assay were counted, since they did not absorb water.
The results of these last three variables were expressed in percentages.

2.5. Statistical Analysis

A randomized experimental design was used with arrangement in split-plot. The large plot had
the treatments and the small plot had the genotypes. In order to increase the data normality before the
analysis, the percentages were converted into square root values.
The data was analyzed using a variance analysis in accordance with our design. When the
differences were significant, Tukey’s multiple comparison test was performed, and Biplot graphics
Agronomy 2019, 9, 325 4 of 12

were used for the interactions. Furthermore, the relationship among the variables was studied using
Spearman correlation coefficient and the genotypes similarity was determined by a dendrogram using
the UPGMA technique. All analyses were performed using the TukeyC, agricolae, and corrplot packages
of the statistical software R version 3.5 (R Foundation for Statistical Computing, Vienna, Austria).

3. Results
In the variance analysis of the physiological tests, highly significant differences (p < 0.01) were
found in the sources of variation treatments, genotypes and treatment–genotype interaction (Table 3).
This could be due to the different genetic constitution of the genotypes studied and the particular effect
of the treatments.

Table 3. Mean squares of the analysis of variance of physiological variables.

SV DF GS GI GP AP DS HS
Treat 11 1.285 ** 9.590 ** 0.129 ** 0.079 ** 0.045 ** 0.150 **
Error (a) 24 0.004 0.134 0.002 0.002 0.004 0.004
Gen 9 0.331 ** 5.149 ** 0.062 ** 0.018 ** 0.074 ** 0.029 **
Treat × Gen 99 0.030 ** 0.442 ** 0.007 ** 0.005 ** 0.007 ** 0.009 **
Error (b) 216 0.006 0.115 0.002 0.002 0.002 0.003
CV (a) 4.89 18.68 4.34 4.49 5.13 5.200
CV (b) 6.12 17.35 4.27 3.83 4.14 4.500
** Significant at probability levels ≤ 0.01, SV = Sources of Variation, DF = Degrees of Freedom,
GS = Germination speed, GI = Germination index, GP = Germination percentage, AP = Abnormal seedlings,
DS = Dead seeds, HS = Hard seeds, Treat = Treatments, Gen = Genotypes, % CV = Coefficient of Variation.

3.1. Effects of Pre-Germination Treatments

According to the mean comparison test (Table 4), the gibberellic acid treatment and its combination
with mechanical scarification (sanding) showed superior results, with increases in GS (77%), GI (52%),
GP (69%), and a decrease in DS (35%) and HS (74%), in comparison with the rest of the treatments.

Table 4. Comparison of Tukey’s means (p > 0.05) for pre-germinative treatments in physiological variables.

Treat GS GI GP AP DS HS
AV 0.41 c 3.35 cd 15.17 bc 9.83 cde 30.67 cde 44.33 abc
GA 1.52 a 6.19 a 40.00 a 26.67 ab 21.83 e 11.17 d
HC 0.46 c 4.54 bc 0.00 e 30.67 a 34.83 bcde 34.5 c
HCGA 1.22 b 5.79 ab 37.33 a 19.67 bc 26.5 de 16.5 d
HP 0.11 de 1.53 ef 5.17 de 4.00 de 39.83 bc 51.00 ab
HPGA 0.40 c 3.61 cd 13.83 bcd 11.83 cd 36.17 bcd 38.17 bc
HW 0.02 e 0.20 fg 1.00 e 0.33 e 53.67 a 45.00 abc
HWGA 0.00 e 0.00 g 0.00 e 0.00 e 45.00 ab 55.33 a
KN 0.18 d 2.48 de 8.67 cde 5.17 de 31.5 cde 54.67 a
MS 0.19 d 2.50 de 9.67 cde 4.33 de 34.33 bcde 51.67 ab
MSGA 1.46 a 6.01 ab 34.17 a 29.5 ab 24.67 de 11.67 d
WIT 0.38 c 5.19 ab 23.33 b 6.67 de 26.33 de 43.67 abc
Means with the same letter are not significantly different, Treat = Treatment, GS = Germination speed,
GI = Germination index, GP = Germination percentage, AP = Abnormal seedlings, DS = Dead seeds,
HS = Hard seeds, AV = Agromil-V® , GA = Gibberellic acid, HC = Hydrochloric acid, HCGA = Hydrochloric acid
+ Gibberellic acid, HP = Hydrogen peroxide, HPGA = Hydrogen peroxide + Gibberellic acid, HW = Hot water,
HWGA = Hot water + Gibberellic acid, KN = Potassium nitrate, MS = Mechanical scarification, MSGA = Mechanical
scarification + Gibberellic acid, WIT = Witness.

Hot water and its combined with gibberellic acid affected in a negative way the germination
variables, reducing GS by 97%, GI by 97%, and GP by 96%; on the other hand, it increased the
percentages of DS (27%) and HS (13%) as compared to the average of the rest of the treatments. It also
Agronomy 2019, 9, 325 5 of 12

showed low AP values, which, despite being a positive aspect, was influenced by low germination,
in this case.
Hydrogen peroxide and its combination with gibberellic acid, besides potassium nitrate,
showed lower results than the witness; obtaining 40, 51, and 61% less GS, GI, and GP respectively,
besides the increases of 5, 27 and 9% in AP, DS, and HS.
The treatments to break physiological dormancy presented better results than the treatments that
were used to break physical dormancy, which is an indication that there are inhibiting physiological
mechanisms that impair germination.

3.2. Physiological Response of Genotypes

With regards to the genotypes (Table 5), the results have shown that G8, G7, and G10 had the
ideal traits, by presenting increases in the germination variables (51% of GS, 53% of GI, and 66% of
GP), besides having the lowest values of DS and HS (48% and 11% less) as compared with the rest of
genotypes. Likewise, the variation of results might be due to the different environmental conditions to
which each genotype had to adapt and therefore the physiological needs were different.
G1 and G5 showed a deficient answer by giving very low values in the desirable traits and very
high values in the undesirable traits, with the exception of AP. This might be due to the conditions in
which they normally develop, causing different needs.

Table 5. Comparison of Tukey’s means (p > 0.05) for genotypes in physiological variables.

Genotype GS GI GP AP DS HS
G1 0.29 e 1.73 fg 5.14 de 9.72 c 38.06 abc 47.08 a
G2 0.59 cd 3.57 cd 18.19 b 11.67 bc 30.83 cde 39.03 abc
G3 0.54 d 3.34 cde 14.31 bc 13.19 bc 45.14 a 27.36 c
G4 0.50 d 3.01 def 12.36 bcd 13.06 bc 33.47 bcd 41.11 a
G5 0.28 e 2.19 efg 6.11 cde 10.42 c 44.72 ab 38.75 abc
G6 0.47 d 3.16 de 15.97 b 9.31 c 38.75 abc 35.97 abc
G7 0.67 c 4.63 bc 27.08 a 9.58 c 23.61 def 39.72 ab
G8 1.01 a 5.46 ab 27.64 a 22.36 a 21.11 ef 29.17 bc
G9 0.15 f 1.05 g 3.06 e 5.14 c 44.86 ab 46.94 a
G10 0.80 b 6.35 a 27.08 a 19.44 ab 17.22 f 36.25 abc
Means with the same letter are not significantly different, GS = Germination speed, GI = Germination index,
GP = Germination percentage, AP = Abnormal seedlings, DS = Dead seeds, HS = Hard seeds.

3.3. Specific Response of Treatment by Genotype

According to the main component analysis (Figure 1), the two first components contributed to
explaining from 53.8 to 72% out of the total variation. PC1 explained the greatest variation from 33.7 to
56.3%; followed by PC2, with a variation of 20.1 to 26.2%. A different distribution was observed
in the genotypes and in the treatments for each variable, which is indicative of a great genetic and
physiological diversity of seeds.
Figure 1 GS corresponds to the germination speed, determined that genotype G8 had the best
response despite being the least stable, followed by genotypes G2 and G10, that also had positive
answers. These results were observed when applying GA treatments and their combinations with MS
and HC. On the other hand, genotypes G5 and G9 had very different responses, influenced by negative
environments (the one that is not suitable for genotypes to express desirable characteristics, but on
the contrary), such as HW, HWGA, HP, and MS. Genotypes G4, G5, G6, and G7 were the most stable.
For this variable, genotypes G8 and G9 had the highest contribution of G and/or GE.
Regarding Figure 1 GI, shows that genotypes with the desirable trait were G10 and G8 in HPGA
and HC environments, the latter being the most discriminating. In turn, these environments presented
negative correlation with HW, HWGA, HCGA, GA, MSGA, MS, and KN. The genotypes with greater
ponse in HPGA (Figure 1 DS).
Regarding the percentage of hard seeds, Figure 1 HS shows that there are specific intera
ach genotype with every environment. The genotypes showing lower percentage of this trait
with applying HC,2019,as9, 325
Agronomy well as G9 applying HPGA and AV. On the other hand, 6 of 12AV and HW

most discriminating treatments. Furthermore, HW, HWGA, and KN interacted in a negativ

h most of thestability were G5, G6, and G7, since they were closer to the origin of PC2 associated to stability of
genotypes.
the genotypes.

Figure 1. Principal component analysis of physiological variables of interaction Treatment by Genotype,

Figure 1. Principal component
(GS) Germination analysis
speed, (GI) Germinationofindex,
physiological
(GP) Germinationvariables of Abnormal
percentage, (AP) interaction Treatmen
seedlings, (DS) Dead seeds, (HS) Hard seeds.
by Genotype, (GS) Germination speed, (GI) Germination index, (GP) Germination
percentage, (AP) Abnormal
Figure 1 GP, regardingseedlings,
the percentage(DS) Dead seeds,
of germination, shows (HS) Hard G2,
that genotypes seeds.
G7, and G8
obtained the highest values. The first one and the second one behaved well in MSGA, considering that
this was the most discriminating and most representative environment for this variable. Genotype
G8 showed a higher than average response in HCGA, AV, and WIT environments. It is worthwhile
mentioning that most of the treatments containing gibberellic acid were grouped and considered as
a favorable environment, with the exception of HWGA that was classified as a negative environment,
together with HW and HC. These last three treatments contributed to decrease the values of genotypes
G1, G5, and G9, considered among the most stable.
One of the desirable traits was having a low percentage of abnormal plants. Genotypes G1 and
G3 in MSGA environment and genotype G9 in WIT complied with this trait. The opposite happened
with genotypes G8 and G10 in HC, an environment that besides being the least ideal is the most
discriminating one, since it presented the longest vector (Figure 1 AP).
Agronomy 2019, 9, 325 7 of 12

As for the percentage of dead seeds, treatment HW showed a negative correlation with most
of the treatments; with the exception of HWGA and MSGA, since they had angles greater than 90◦
between their vectors. HW was the environment with the highest percentage of dead seeds and it
was the most discriminating environment. On the other hand, Genotype G6 showed good adaptation
in treatment MS, resulting in lower incidence of dead seeds. Genotype G7 also presented a positive
response in HPGA (Figure 1 DS).
Regarding the percentage of hard seeds, Figure 1 HS shows that there are specific interactions of
each genotype with every environment. The genotypes showing lower percentage of this trait were G3
with applying HC, as well as G9 applying HPGA and AV. On the other hand, AV and HW were the
most discriminating treatments. Furthermore, HW, HWGA, and KN interacted in a negative way with
most of 2019,
Agronomy the genotypes.
9, x FOR PEER REVIEW 7 of 11

3.4. Association
3.4. Associationbetween
betweenPhysiological
PhysiologicalVariables
Variables
Figure 22 shows
Figure shows thetheassociation
association among
among the
the test
testvariables.
variables. Germination
Germinationvariables
variables(GI,
(GI,GS,
GS,and
andGP)
GP)
were closely
were closelyrelated
relatedinin
a positive way,way,
a positive besides beingbeing
besides influenced by AP. These
influenced by AP. variables were negatively
These variables were
associated associated
negatively with DS and withHS.
DSThese data
and HS. allow
These dataus allow
to forecast
us to the behavior
forecast of piquin
the behavior of pepper seed in
piquin pepper
an indirect
seed way. way.
in an indirect

AP
1 0.66 0.65 0.19 − 0.38 − 0.57
0.8

0.6

GI
1 0.78 0.75 − 0.62 − 0.66

0.4

0.2
GS
1 0.82 − 0.56 − 0.76

GP
1 − 0.55 − 0.61
−0.2

−0.4

DS
1 − 0.04

−0.6

−0.8
HS
1

−1

Figure 2.
Figure Correlationofofphysiological
2. Correlation physiologicalvariables
variablesofofpiquin
piquinpepper,
pepper,(GS)
(GS)Germination
Germinationspeed,
speed,
(GI) Germination index, (GP) Germination percentage, (AP) Abnormal seedlings, seeds,
(GI) Germination index, (GP) Germination percentage, (AP) Abnormal seedlings, (DS) Dead (DS)
(HS) Hard seeds.
Dead seeds, (HS) Hard seeds.
3.5. Similarity of Genotypes
3.5 Similarity of Genotypes
AAcluster
clusteranalysis
analysisusing
using UPGMA,
UPGMA, gathered
gathered thethe 10 genotypes
10 genotypes in three
in three groups,
groups, at a distance
at a distance of 17
of 17 units. The first group included three genotypes with the highest seed physiology
units. The first group included three genotypes with the highest seed physiology potential. The potential.
The second
second group group had three
had three genotypes
genotypes with intermediate
with intermediate influence.
influence. The group
The third third group
was thewas the largest,
largest, with
withgenotypes
four four genotypes
of poor ofperformance.
poor performance. These results
These results show variability
show variability among genotypes
among genotypes and we
and we believe
believe
that mostthat most of
of these these were
groups groups were formed
formed due to
due to their their geographical
geographical closeness,
closeness, leading leading to similar
to similar needs,
needs, with the exception of genotype G6
with the exception of genotype G6 (Figure 3). (Figure 3).
25
20
t
 A cluster analysis using UPGMA, gathered the 10 genotypes in three groups, at a distance of 17
units. The first group included three genotypes with the highest seed physiology potential. The
second group had three genotypes with intermediate influence. The third group was the largest, with
four genotypes of poor performance. These results show variability among genotypes and we believe
that most of these groups were formed due to their geographical closeness, leading to similar needs,
Agronomy 2019, 9, 325 8 of 12
with the exception of genotype G6 (Figure 3).

25
20
Height

15

G3
G7
10

G5

G6
G10

G8
5

G1

G9

G2

G4
Figure 3. Dendrogram of physiological characteristics of 10 piquin pepper genotypes.
Figure 3. Dendrogram of physiological characteristics of 10 piquin pepper genotypes.
4. Discussion

4.1. Treatment Effect on Seed Physiology

Pre-germination treatments can present specific effects on piquin pepper seeds. Koornef & Bentsink [19]
mentioned that the use of gibberellic acid increased the seed germination rates of several species. In this
assay, the values were similar to the values reported by García et al. [16], where seed germination
and seedling vigor increased after applying this chemical to piquin pepper seeds under greenhouse
conditions. Likewise, Née et al. [20] mention that the dormancy-breaking is controlled by various
regulators, such as plant hormones and latent proteins. Hot water applications produced negative
effects in germination, coinciding with the results of García et al. [16], who reported that the hot water
treatment in piquin pepper seeds delays germination and seedling emergence. On the other hand,
we differed with the results reported by Cano et al. [3] who reported that hydrogen peroxide and
potassium nitrate applied to 16 collections of piquin pepper did not modify the germination percentage,
as compared to the witness (without any treatment).

4.2. Variability among Genotypes

Due to the diversity presented by genotypes and their great adaptability to different environments,
this species is an important phytogenetic resource for pepper crops’ breeding programs [21]. Differences
due to genetic diversity of genotypes can provide high phenotypic plasticity and variation of
morphological traits [22]. Therefore, the variation of the germination capacity among and within plant
species is an adaptation to the particular conditions of local and regional habitats [23]. Within the
same context, Hernández-Verdugo et al. [24] found great variation in the germination capacity
of wild pepper related populations. This variation allowed the detection of three genotypes with
physiological potential.

4.3. Interaction between Genotypes and Treatments

The interpretation of the interactions can be done by means of biplot graphics. Genotypes with
PC1 > 0 were identified as having positive value according to the variable studied and their opposites
were identified as PC1 < 0 [25,26]. Likewise, the genotypes located farther from the center contributed
the most to G and/or GE [27]. On the other hand, the environments with the longest vectors were the
most discriminating ones [28]. In turn, the angle formed between the location of genotypes and their
environments, has a correlation. The correlation is negative if the angle is obtuse, while an acute angle
depicts a positive relation [29,30]. On the other hand, PC2 is related to the stability of the genotypes,
being more stable than those genotypes that approached 0 [31]. Concerning the relationship between
the genotype and the environment, the closer were the genotypes from the vector, the greater their
adaptation to that specific environment [32].
Agronomy 2019, 9, 325 9 of 12

In this assay, three genotypes performed well in combination with GA that promote quick
germination [33]. Regarding the GI variable, the HC and HP treatments had a positive effect in two
genotypes, simulating the effect of the digestive tract of birds in breaking dormancy in piquin pepper’s
seeds [15]. In the case of GP, some genotypes required gibberellic acid to promote a positive effect
on germination, leading to pre-germination metabolic processes that reduced dormancy drastically.
However, in order to reach better results, it was necessary to combine the application of gibberellic
acid with treatments that control physical dormancy, such as mechanical scarification (sanding) and
hydrochloric acid applications, to improve water uptake and help the emergence of the primary root
through the seed coat [15,34]. Negative environments affected those genotypes that were close to the
vector, such as hot water, which decreased the GP variable value in three genotypes, and increased the
incidence of DS in three other genotypes. In previous works, the same condition has been observed
in comparison with other treatments, since temperature is an important factor that affects latency
and germination [35,36]. Abnormal seedlings are an undesirable condition that can be caused by
environmental factors. In this case, HC promoted an increase of this variable, specifically in two
genotypes, by interrupting the germination of the seeds, which could have altered the hormonal
metabolites and their regulators [37]. The opposite happened with direct planting (WIT) and seed
sanding, where the three genotypes drastically reduced the values of this variable. One of the
best-known mechanisms of dormancy is the waterproof seed coat (hence the name “hard seeds”),
where dormancy persists until part of the seed coat cracks and allows water to penetrate, giving
rise to the imbibition process [38]. The genotypes presented this germination-limiting condition
at different levels, but germination improved after the application of hormones like GA and AV.
These applications, combined with scarification treatments; either mechanical sanding (MS) or acid
applications (HC), can improve that impact. These results confirm that endogenous plant hormones like
GA play an important role in seed dormancy release. On one hand, ABA inhibits germination, while on
the other hand GA increases germination [39]. Also, the methods to soften artificially waterproof seeds
can cause positive changes in germination [40].

4.4. Relationship among Physiological Variables Related to Germination

Knowledge of the existing correlations among the traits of economic importance is a useful tool
for indirect selection [41]. The increase in desirable variables (GI, GS, and GP) led to a reduction of the
most undesirable variables, such as DS and HS. These results can be useful in future works, which will
require a smaller number of test traits.

5. Conclusions
Of the 11 treatments included in the present study, the one that increased the desirable
characteristics of physiological potential of the germination of genotypes was the gibberellic acid.
This showed that dormancy on piquin pepper seed is mostly influenced by physiological aspects.
Genotypes 8 and 10, originating in Colatlán (Nursery), Ixhuatlán de Madero, Veracruz and
La Laborcilla, Rioverde, and San Luis Potosí showed a greater germination capacity, as they were
statistically superior in 5 of 6 evaluated variables.
The analysis of the main components through a biplot graph helps to understand in a practical
way the interactions. The treatments had different effects on each genotype. G8, G2, and G10 genotypes
showed higher germination speed when applying gibberellic acid and its combination with sanding and
hydrochloric acid. Likewise, the interaction of the G8 and G10 genotypes with the Peroxide treatments
of hydrogen + gibberellic acid and hydrochloric acid showed superiority in the germination index.
In the case of the germination percentage, the combinations G2 and G7 with sanding + gibberellic acid
and G8 without treatment stood out. Abnormal seedlings were reduced especially in the genotypes G1
and G3 by applying sanding + gibberellic acid and G9 without any treatment. A significant reduction of
dead seeds in the G6 genotype was achieved by sanding and G7 with hydrogen peroxide + gibberellic
Agronomy 2019, 9, 325 10 of 12

acid. The combination of G3 with hydrochloric acid and G9 with hydrogen peroxide + gibberellic acid
and Agromil-V® greatly reduced hard seeds.
The relationship between the variables is an important factor that will allow us to predict a certain
feature in future work.
The association between genotypes made it possible to know their similarity and diversity
according to the characteristics studied.

Author Contributions: Conceptualization, J.S.G.J.A.-R., and A.L.-B.; methodology, J.S.G.J.A.-R., A.L.-B.,

and M.E.V.-B.; software, J.S.G.J.A.-R., and M.A.P.-R.; validation, D.S.-A., and F.R.-G.; formal analysis, J.S.G.J.A.-R.,
A.L.-B., S.A.R.-H., and M.E.V.-B.; investigation, J.S.G.J.A.-R., A.L.-B., and M.E.V.-B.; resources, J.S.G.J.A.-R.,
and A.L.-B.; data curation, J.S.G.J.A.-R. and S.A.R.-H.; writing—original draft preparation, J.S.G.J.A.-R. and
A.L.-B.; writing—review and editing, J.S.G.J.A.-R., A.L.-B., M.E.V.-B., D.S.-A., S.A.R.-H., M.A.P.-R., and F.R.-G.;
visualization, J.S.G.J.A.-R., A.L.-B., M.E.V.-B., D.S.-A., S.A.R.-H., M.A.P.-R., and F.R.-G.; supervision, J.S.G.J.A.-R.
and A.L.-B.; project administration, J.S.G.J.A.-R. and A.L.-B.; funding acquisition, J.S.G.J.A.-R. and A.L.-B.
All authors have read and approved the final manuscript.
Funding: This research was funded by Universidad Autónoma Agraria Antonio Narro (UAAAN), project 2134.
Acknowledgments: We thank to the National Council for Science and Technology (CONACYT) for a graduate
student’s scholarship.
Conflicts of Interest: The authors declare no conflict of interest.

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