American Journal of Plant Sciences, 2013, 4, 2227-2239
Published Online November 2013 (
Open Access AJPS
Seed Vigor Variation of Agave durangensis
Gentry (Agavaceae)
Gerardo Barriada-Bernal1, Norma Almaraz-Abarca1*, Tzahyri Gallardo-Velázquez2,
Isabel Torres-Morán3, Yolanda Herrera-Arrieta1, Socorro González-Elizondo1,
Eli Amanda Delgado-Alvarado1
1Interdisciplinary Research Center for the Integral Regional Development, Durango, National Polytechnical Institute (CIIDIR IPN
Durango), Durango, México; 2National School of Biological Sciences, National Polytechnical Institute (ENCB IPN), México DF,
México; 3University Center of Biological and Agropecuary Sciences, University of Guadalajara (CUCBA), Zapopan, México.
Email: *
Received September 27th, 2013; revised October 20th, 2013; accepted October 29th, 2013
Copyright © 2013 Gerardo Barriada-Bernal et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Agave durangensis propagates basically by seeds. This species is economically important because it supports a mescal
industry in Durango, Mexico. At present, its exploitation is overall by collecting plants from the wild populations. In
order to reduce the negative impact of the collection in the natural populations of A. durangensis and to improve the
mescal industry, it is necessary to establish plantations with selected seeds for a high vigor. In this paper, the variation
in morphological features, physiological behavior of germination, and biochemical indicators of seed vigor among three
natural populations of A. durangensis was assessed. Variation was found in the seed weight (0.68 to 1.15 mg/seed), seed
dimensions (3.51 × 5.29 to 4.62 × 5.92 mm), germinability reduction at 15˚C related to 25˚C (4% to 51%), germination
rate at 25˚C (44.0 to 48.5 seeds/day) and at 15˚C (5.17 to 6.77 seeds/day), development reduction at 15˚C related to
25˚C (86.66% to 91.99%), levels of seed accumulated phenols (71 to 85 µg/seed), antioxidant potential (42% to 50%
reduction of DPPH*), seed alcohol dehydrogenase activity (ADH) (180 to 1100 µmol NAD+/mg protein/min), highest
ADH activity after imbibitions at 25˚C (310.24 to 520.2 µmol NAD+/mg protein/min), and highest ADH activity after
imbibitions at 15˚C (170.74 to 440.71 µmol NAD+/mg protein/min). The variation in the seed vigor was revealed by a
principal component analysis (PCA) based on all the parameters evaluated. PCA clearly discriminated among the three
Keywords: Agave Durangensis; Seed Vigor; Germinability; Natural Variation
1. Introduction
Agave durangensis Gentry is the base of a mescal indus-
try in Durango, Mexico. At present, its exploitation is
carried out by collecting plants from the wild populations
[1], which is causing the reduction and fragmentation of
its natural distribution [2]. Agave durangensis propagates
basically by seeds, and under natural conditions it is in-
frequent to observe offshoots; this favors high levels of
genetic variability [2], which may represent a source of
worthy alleles to select for the establishment of commer-
cial plantations. Thus, it is relevant to carry out studies to
assess the variability in the seed vigor among its natural
populations. Natural variability in the seed vigor has
been found among varieties of cultivated species [3] as
well as among the natural populations of wild species
The definition of seed vigor given by the Association
of Official Seed Analyst’s Vigor Committee in 1979 was
mentioned by McDonald [5] as those seed properties
determine the potential for rapid, uniform emergence and
development of normal seedlings under a wide range of
field conditions. Seed vigor has genetic and environ-
mental determinants [3]. Most tests to evaluate the vigor
of seeds consider the germination behavior [6,7] and the
deterioration of that behavior under condition of stress
[8]. The correlation between the morphological, physio-
logical and biochemical indicators of seed vigor may
assist to broad the understanding of how the vigor is
*Corresponding author.
Seed Vigor Variation of Agave durangensis Gentry (Agavaceae)
expressed and facilitate the selection of high vigor seeds
for breeding programs. Among the biochemical charac-
ters used as indicators of seed vigor are the ADH activity
[3,9,10] and the levels of antioxidant phenols [11,12].
The aim of the present study was to evaluate the varia-
tion in the seed vigor among three natural populations of
A. durangensis as a way to select seeds to establish plan-
2. Materials and Methods
2.1. Seeds
Seeds of Agave durangensis were collected from three
natural populations in August 2009. The Table 1 shows
the geographical data of those populations. Voucher
specimens were collected from each population and de-
posited at the Herbarium MEXU. Seeds were aged for
nine months in paper bags at room temperature. Three
lots of 50 seeds of each population were prepared for the
2.2. Seed Morphological Features
Weight, length, and width were individually determined,
by using a vernier, for 150 seeds of each population.
2.3. Germination
The germination behavior, as a vigor indicator, was
evaluated at 25˚C ± 2˚C (temperature of reference) and at
15˚C ± 2˚C (stress temperature) for 360 h. Those tem-
peratures were selected because the seeds of Agave ger-
minate properly at 25˚C [13,14], because 15˚C has been
reported as a critical temperature for the germination of
the species of that genus [15], and because the tempera-
ture is the main factor regulating the germination in areas
with a marked thermal seasonality [16], as is the case
where A. durangensis grows. The relative humidity
ranged between 98% and 100%. Previous evaluations
made by the authors of the present study indicated that
the seeds of A. durangensis are indifferent to light to
germinate; that has been reported for the seeds of other
plant species [13,17,18].
Table 1. Collection sites for three natural populations of
Agave durangensis in Durango, Mexico.
Sample Number of
reference Latitude N Longitude WAltitude
Pino Suárez D05EPS001 23˚47'09.9" 104˚25'02.2"2054
La Parrilla D16EPA101 24˚00'53.5" 103˚56'43.6" 2050
Veracruz D22EVE205 24˚00'53.5" 103˚56'43.6"2050
2.4. Rate of Germination
The germination performance was evaluated at 24 h in-
tervals. The germination rate (V) was determined ac-
cording to Maguire [19] with the Equation (1).
Equation (1):
Totalofsproutsatday 1
Total ofsprouts atday n
where V is the rate germination and n is the number of
days after imbibition.
2.5. Germinability (% G)
Germinability was determined according to García and
Lasa [20], using the Equation (2).
Equation (2):
where ni is the number of seeds which germinated on the
day i and N is the number of seeds evaluated. Seeds with
visible radicle (length > 0.10 mm) were considered as
2.6. Growth Reduction of Plantets under Cold
Stress (15˚C)
Seed vigor was also evaluated by the percentage reduc-
tion of plantlet growth (representing the distance between
the tip of the primary leaf and the primary root tip) under
cold stress at 15˚C, compared with the growth at 25˚C,
after 360 h imbibition. This evaluation was made ac-
cording to Talai and Sen-Mandi [3], using the Equation
Equation (3):
% reduction of lengCth at 15
op et
where LESop is the plantlet length after 360 h, at 25˚C
and LESet is the plantlet length after 360 h, at 15˚C.
2.7. Extraction of Phenols
The seeds (5 g) of each sampled population were indi-
vidually grinded in liquid nitrogen. The extraction of
phenols was carried out according to Ardekani et al. [21],
by maceration in 20 mL of a solution containing water-
methanol-acetic acid-formic acid (20:40:39:1), at room
temperature, darkness, and shaking (120 rpm) for 24 h.
The extracts were centrifugated (8000 rpm, 25˚C, for
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Seed Vigor Variation of Agave durangensis Gentry (Agavaceae) 2229
10 min). The supernatants were decanted and concen-
trated to dryness. The dry extracts were disolved in 2 mL
of methanol. Aliquots were used to determine the total
phenol levels, the phenol profiles, and the antioxidant
activity. The extracts were prepared from three inde-
pendent seed lots of each population.
2.8. Total Phenols
The concentrations of total phenols were determined with
the Folin-Ciocalteu reagement, according to Lozoya-Sa-
ldaña et al. [22]. The phenol contents were expressed as
mg gallic acid equivalents (EAG)/g seeds, using a cali-
bration curve of gallic acid (Abs760nm = 0.0657 [gallic
acid] + 0.0159, correlation coefficient r = 0.9874). The
absorbances were registered at 760 nm (Jenway Genova
spectrophotometer), after 120 min in darkness. All sam-
ples were analyzed in triplicate.
2.9. Phenolic Profiles
The phenolic compositions of the extracts (aliquots of 20
µL) were determined by HPLC-DAD, according to a
modified gradient method from that of Campos and
Markham, [23], by using a Perkin Elmer Series 200
HPLC system and a Perkin Elmer Brownlee Validated
C18 (250 × 4.6 mm, 5 µm) column. Solvent A was water
acidified with phosphoric acid (pH = 4.0) and the solvent
B was acetonitrile. The gradient was: 0 to 24 min, 100%
A; 24 to 40 min, 91% A; 40 to 80 min, 68% A; 80 to 84
min, 67% A; 84 to 104 min, 57% A; 104 to 120 min,
57% A, and 120 to 125 min, 100% A. The flow was 0.8
mL/min. The chormatograms were registered at 260 and
340 nm. Spectral data for all the peaks were obtained
between 200 to 400 nm, using diode-array detection
(Perkin Elmer Series 200). Structural information was
obtained by direct comparisons of retention times and
UV spectra of resolved compounds with those of stan-
dards and according to Mabry et al. [24] and Campos and
Markham [23].
2.10. Antioxidant Activity
The antioxidant activities of the extracts were determined
by the free radical scavenging potential, using a freshly
solution of 2,2-diphenil-1-picrilhydracil (DPPH*), ac-
cording to Campos et al. [25]. One hundred microliters
of each extract were added to 900 µL of the solution of
DPPH* (57.5 µg/ml ethanol). The decrease in absorb-
ance at 523 nm (Jenway Genova spectrophotometer) af-
ter 30 min was registered. The percent of DPPH* scav-
enged by each sample was calculated by the equation (4).
Equation (4):
% DPPHscavengingactivitity =100AAA
where A0 = absorbance of DPPH* solution, and A1 =
absorbance of DPPH* solution/sample of extract after 30
min reaction. Ascorbic acid was evaluated in the same
manner as reference. Measurements were taken in tripli-
A lineal regression analysis was done to evaluate the
association between the seed phenol contents and the
scavenging activity by registering the A523 nm reduction
with increased volumes of seed extracts.
2.11. ADH Activity
The alcohol dehydrogenase (EC activity was
registered before seed imbibition and after seed imbibi-
tion at 6 h intervals until the radicle profusion, by the
reduction of NAD in the presence of ethanol according to
Rumpho and Kennedy [26], in extracts prepared from
1.25 mg of embryo and 1.5 mL of a solution containing
10 mM Tris-HCl (pH 7.5), 2 mM EDTA, and 20 mM
β-mercaptoethanol. Protein assay was performed by the
method of Lowry [27], expressed as mg bovine serum
albumin equivalents (EBSA equivalents)/mL of extract.
A solution of 1 mM NAD and a buffer of 50 mM
Tris-HCl were prepared. The reaction mixtures were
composed of 400 µL of embryo extract, 200 µL of Tris
buffer, and 50 µL of NAD solution. The reaction was
initiated by the addition of 50 µL of ethanol. The ab-
sorbance was registered at 340 nm (Jenway Genova
spectrophotometer) after 2 min of incubation at 25˚C and
at 15˚C. The ADH activity was expressed as μmol NAD+
reduced/min/mg protein. The value of molar extinction
coefficient of 6.22 × 103/M/cm was used to estimate the
concentration of NAD+. Measurements were taken in
2.12. Data Analysis
An ANOVA test was used to evaluate differences among
samples. Mean comparisons were made by using Statis-
tica 7.0. A principal component analysis (PCA) and a
correlation test, using Past 2.12 [28], were carried out
from a matrix constructed with the results of all the
evaluated parameters. The PCA analysis, grouping the
traits, was employed to evaluate the percentage contribu-
tion of each trait to the seed vigor variation.
3. Results
3.1. Seed Morphological Characterization
The seeds of the three evaluated populations of Agave
durangensis were lacrimiform, smooth, and black
(Figure 1), features that are common to all species of
genus Agave [29].
The weights and dimensions of the seeds of the
populations evaluated are showed in Tabl e 2. Significant
variations (p < 0.05) in the weight and width among the
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Seed Vigor Variation of Agave durangensis Gentry (Agavaceae)
Figure 1. Seeds of Agave durangensis from three popula-
tions of Durango, Mexico ((a) Pino Suárez, (b) La Parrilla,
(c) Veracruz).
Table 2. Weight and dimensions of the Agave durangensis
seeds of three natural populations. The values represent the
mean and standard deviation for 150 seeds. Different letters
mean significant differences (p < 0.05).
Population Weight/seed
Pino Suárez 0.68 ± 0.02a 3.99 ± 0.5b 5.56 ± 0.68
La Parrilla 0.99 ± 0.02b 4.62 ± 0.3c 5.92 ± 0.33
Veracruz 1.15 ± 0.01b 3.51 ± 0.52a 5.29 ± 0.60
three kinds of seed samples were observed. Weight
varied from 0.68 mg in the seeds of Pino Suárez (p) to
1.15 mg in the seeds of Veracruz (v). The biggest seeds
were those of La Parrilla (4.62 × 5.92 mm).
3.2. Germinability
The values of the germination percentage of the analyzed
seeds of A. durangensis are present in Tabl es 3 and 4. At
25˚C (Table 3) the seeds of the three populations began
the germination at 72 h. At that time, the highest values
of germination were for the seeds of Pino Suárez (17%),
whereas those of Veracruz showed the lowest percentage
(10%). The seeds of La Parrilla reached 100% of
germination at 120 h, those of Pino Suárez at 192 h, and
those of Veracruz reached 99% of germination at 216 h.
Variability in the proportion of seeds beginning the
germination and in the time reaching the highest
germinability was observed. However, non significant
differences were found after 240 h of imbibition in the
germinability values.
At 15˚C, 24% of the seeds from Veracruz (the first
ones starting the germination at that temperature) began
the germination at 264 h (Table 4). That means a gap of
192 h in the beginning of germination compared with the
one at 25˚C (Table 3); 95% of the seeds of that same
population germinated at 15˚C, which was the highest
value of germinability at the stress temperature. The
seeds of Pino Suárez and La Parrilla started the ger-
mination 24 h later, and reached values of 49 and 87%,
respectively, at the end of the experiment (360 h). The
variations found in the germinability at this temperature
were significant (p < 0.05).
3.3. Rate of Germination (V)
The Table 3 presents the rates of germination of the dif-
ferent lots of seeds of A. durangensis at 25˚C. Those
corresponding to 15˚C are present in Tabl e 4 . Significant
differences (p < 0.05) were found between the rates of
germination estimated at each condition of temperature,
and at 25˚C, significant interpopulation differences were
also found. At 25˚C, the seeds of Veracruz and Pino
Suárez showed the highest germination rates (48.5 and
47.6 seeds/day, respectively), and at 15˚C, the seeds of
Veracruz showed the highest rate (6.77 seeds/day).
3.4. Growth Reduction of Plantets under Cold
The plantlet growth of the three analyzed populations of
Agave durangensis was sensitive to temperature decrease
from 25˚C to 15˚C (Table 5). Significant differences (p <
0.05) in the growth reduction were observed among the
populations. The plantets of La Parrilla and Veracruz
were the most affected, showing a growth reduction of
91.45% and 91.99%, respectively, whereas the ones of
Pino Suárez were the least affected having reduced their
growth only 86.66%.
3.5. Total Phenols and Antioxidant Capacity
Significant interpopulation variability (p < 0.05) in the
phenol contents of the Agave durangensis seeds was
found (Table 6). The highest value corresponded to the
seeds of Pino Suárez, with 85 µg/g seeds; however, the
seeds of La Parrilla, with a lower phenol content (71
µg/g seeds), and with no significant differences with the
seeds of Veracruz, showed the highest antioxidant
potential (50.35%). The antioxidant potential of ascorbic
acid was significantly higher (65.28% ± 1.04%, p < 0.05)
than any of the extracts of seeds of A. durangensis.
A linear reduction of DPPH* concentration associated
to increasing of the phenol contents in the seed extracts
was observed: Pino Suárez seeds: A523nm = 0.0551 –
0.0003(extract volume), r = 0.9944; La Parrilla seeds:
A523nm = 0.0421 0.0002(extract volume), r = 0.9860;
and Veracruz seeds: A523nm = 0.0532 0.0002(extract
volume), r = 0.9894) (Figure 2).
3.6. Phenolic Profile of Seeds
Important differences in the phenol composition, deter-
mined by HPLC-DAD, were found in the seeds of the
three natural populations of Agave durangensis. The re-
tention times (RT) and the UV spectral data for the 14
compounds resolved are shown in the Table 7 . The seeds
of Pino Suárez accumulated four phenolic acids; the
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Seed Vigor Variation of Agave durangensis Gentry (Agavaceae)
Open Access AJPS
Table 3. Accumulated germination at 24 h intervals, germination percentage (% G), and rate of germination (V), at 25˚C, of
seeds of Agave durangensis from three natural populations. The values represent the mean and standard deviation for three
independent samples. Different letters mean significant differences (p < 0.05).
Population 72 h 96 h 120 h 144 h 168 h 192 h 216 h 240 h G (%) V (seeds/day)
Pino Suárez 0.17 ± 0.04 0.45 ± 0.07 0.92 ± 0.01 0.96 ± 0.040.99 ± 0.011 1 1 100 47.62 ± 1.78b
La Parrilla 0.15 ± 0.06 0.29 ± 0.01 1 1 1 1 1 1 100 48.5 ± 1.06b
Veracruz 0.10 ± 0.09 0.42 ± 0.01 0.75 ± 0.05 0.95 ± 0.050.98 ± 0.020.98 ± 0.020.99 ± 0.020.99 ± 0.02 99 ± 0.02 44.0 ± 0.84a
Table 4. Accumulated germination at 24 h intervals, germination percentage (% G), and rate of germination (V), at 15˚C, of
seeds of Agave durangensis from three natural populations. The values represent the mean and standard deviation for three
independent samples. Different letters mean significant differences (p < 0.05).
Population 168 h 192 h 216 h 240 h 264 h 288 h 312 h 336 h 360 h G (%) V (seeds/day)
Pino Suárez 0 0 0 0 0 0.26 ± 0.010.30 ± 0.010.44 ± 0.070.49 ± 0.08 49 ± 0.08a 5.17 ± 0.64
La Parrilla 0 0 0 0 0 0.15 ± 0.050.21 ± 0.020.67 ± 0.070.87 ± 0.07 87 ± 0.07b 6.10 ± 0.33
Veracruz 0 0 0 0 0.24 ± 0.01 0.48 ± 0.050.72 ± 0.060.78 ± 0.060.95 ± 0.01 95 ± 0.01c 6.77 ± 0.70
Table 5. Growth reduction of plantets of three natural populations of Agave durangensis, after 360 h, due to the temperature
reduction of germination. The values represent the mean and standard deviation for three independent samples. Different
letters mean significant differences (p < 0.05).
Population Plantets growth (cm) Growth reduction (%)
25˚C 15˚C
Pino Suárez 5.49 ± 0.31 0.73 ± 0.02 86.66 ± 3.10a
La Parrilla 5.85 ± 0.90 0.5 ± 0.07 91.45 ± 1.48b
Veracruz 5.01 ± 0.08 0.4 ± 0.05 91.99 ± 0.97b
Table 6. Total phenol contents and antioxidant potential of the seeds of three populations of de Agave durangensis. The values
represent the mean and standard deviation for three independent samples. Different letters mean significant differences (p <
Population Total phenol content (mg gallic acid equivalents/g seeds) DPPH* inhibition (%)
Pino Suárez 0.085 ± 0.00b 42.13 ± 3.27
La Parrilla 0.071 ± 0.008a 50.35 ± 6.99
Veracruz 0.078 ± 0.002a 49.39 ± 2.96
Figure 2. DPPH* disappear associated to the increasing in the phenol concentration of the seed extracts of three populations
of Agave durangensis.
Seed Vigor Variation of Agave durangensis Gentry (Agavaceae)
Table 7. Retention time and spectral data of the phenolic compounds found in the seeds of three populations of Agave du-
Location Number of Compound Type of phenolic compound λmax (nm) RT (min)
Pino Suárez 1 O-coumaric acid derivative 240 sh, 272, 302 sh 70.462
2 Phenolic acid 272 78.236
3 Phenolic acid 247 96.393
4 Phrnolic acid 282 sh, 295 116.956
La Parrilla 5 Phenolic acid 235 sh, 290, 320 sh 64.346
6 Dihydroflavonoid 280, 310 sh 68.556
7 Flavonol, possible herbacetin derivative 273, 296 sh, 317 sh 70.58
8 Phrnolic acid 230 sh, 277, 315 74.129
9 Phenolic acid 240, 305 sh 322 77.4
10 Flavone, possible scutellarein derivative 272, 330 83.926
11 Flavone, possible scutellarein derivative 272, 330 95.66
12 Flavone, possible scutellarein derivative 274, 330 97.405
Veracruz 13 Phenolic acid 290, 320 50.824
14 Dihydroflavonoid 290, 320 sh 61.432
7 Flavonol, possible herbacetin derivative 273, 296 sh, 317 sh 70.389
3 Phenolic acid 247 96.276
seeds of La Parrilla, three phenolic acids, one dihy-
droflavonoid, one flavonol, and three flavones; and the
seeds of Veracruz, two phenolic acids, one dihydrofla-
vonoid, and one flavonol. The compound 3, a phenolic
acid, was common to the seeds of Pino Suárez and those
of Veracruz, and the compound 7, one flavonol, possible
derivated of the flavonol herbacetin, was common to the
seeds of La Parrilla and to those of Veracruz. The re-
spective chromatograms are displayed in Figure 3.
3.7. ADH Activity
Significant variability (p < 0.05) in the ADH activity of
the seeds, before inbibition, among the three natural
populations of Agave durangensis was found (Figure 4),
being the highest for the seeds of Pino Suárez (1100
μmol NAD+/mg protein/min), and the lowest for the
seeds of La Parrilla (180 μmol NAD+/mg protein/min).
At 25˚C and in imbibitions conditions, after a con-
tinuous diminish for 6 h, the ADH activities of the seeds
of Veracruz and Pino Suárez, increased to reach a high
level (520.2 and 400.9 μmol NAD+/mg protein/min, re-
spectively) at 12 h after imbibition. The seeds of La
Parrilla reached latter (18 h after imbibition) their highest
level of ADH activity (310.24 μmol NAD+/mg pro-
tein/min) (Figure 4).
The highest level after imbibitions of the ADH activity
at 15˚C was observed in the seeds of Veracruz (440.71
μmol NAD+/mg protein/min). Lower activities were es-
timated for the seeds of Pino Suárez and La Parrilla
(170.74 and 185.62 μmol NAD+/mg protein/min, respec-
tively). The seeds of Veracruz reached the highest ADH
activity sooner (at the 12th day after imbibition) than the
seeds of La Parrilla and Pino Suárez (both at the 24th day
after imbibition) (Figure 5). The time taken for the
diminution of the ADH activity by the three populations
at this temperature was longer than at 25˚C (Figures 4
and 5), and this gap matched with the delay in the ger-
mination at 15˚C (Table 4).
3.8. PCA and Correlation Analysis
The results of a PCA, based on the genetic variability
revealed by the different indicators of seed vigor, are
showed in the Figure 6. The clear discrimination
between the three natural populations of Agave duran-
gensis can be observed.
Six principal components accounted for practically
100% of total variance, being the PC1 the mean one,
taking 78.9%; this same component had the highest
relative discriminating power (eigen value 190774) and
the PC6 had the lowest relative discriminating power
(eigen value 4.299) (data not shown). The PC1 was
mostly correlated with the germinability at 15˚C, rate
germination at 15˚C, weight, and growth reduction; PC2
with the ADH activity at both temperatures; PC3 with
the germinability at 25˚C; PC4 with the growth reduction;
PC5 with the DPPH scavenging; and PC6 with the ADH
activity at 25˚C.
The correlation coefficients for the pairs of the seed
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Seed Vigor Variation of Agave durangensis Gentry (Agavaceae) 2233
Figure 3. HPLC chromatograms of seed extracts of Agave durangensis from three populations of Durango, Mexico ((A) Pino
Suárez, (B) La Parrilla, (C) Veracruz). The numbers of compounds correspond to those in Table 7.
Figure 4. ADH activity at 25˚C in extracts of embryos of seeds of three natur al populations of Agave durangensis.
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Seed Vigor Variation of Agave durangensis Gentry (Agavaceae)
Figure 5. ADH activity at 15˚C in extracts of embryos of seeds of three natur al populations of Agave durangensis.
Figure 6. Results of a PCA based on the variation of the weight, and physiological, chemical and biochemical indicators of
seed vigor of three lots of three natural populations of Agave durangensis.
vigor indicators used to characterize each of natural population of A. durangensis were calculated. The corre-
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Seed Vigor Variation of Agave durangensis Gentry (Agavaceae) 2235
lation matrix is showed in Table 8.
4. Discussion
4.1 Seed Morphological Characterization
Seed weights of 9.50 mg have been reported for Agave
durangensis [15], a value around nine fold higher than
the values reported in the present paper for any of the
three natural populations evaluated. This suggests an
important intraspecific variation in the seed weight for
that species of Agave. Some authors have suggested than
the morphological variations in the seeds of plants of the
same species occurring in adjacent populations can result
from interpopulation, and in some cases, even from in-
terspecific hybridization processes [30], can be the result
of a processes of adaptation to store high levels of nutri-
ents [31,32], or can be associated to the seed dispersion,
the lighter ones favoring the conservation or extending of
the natural distribution area of the species with wind
dispersed seeds, as is the case of most species of Agave
[33]. The nutritional conditions of progenitors also can
determine the weight of seeds [32,34]. As well the seed
moisture content may strongly influence the weight.
4.2. Germinability
The seeds of Pino Suárez and La Parrilla, which had the
highest fast germination capacity at 25˚C, could have a
higher potential than those of Veracruz to take advantage
of the favorable temperature and humidity, which are
variable over seasons and years; the favorable season for
seedling growth is short (four months) in the geographi-
cal region of the natural distribution of A. durangensis.
The formation of seed banks has been reported as an
important strategy of species of unpredicted precipitation
environments [35], as those occurring in Durango, Mex-
ico; however, the fast and total germination showed by
the seeds of A. durangensis at 25˚C, happening in a short
time (5 to 9 days) suggests that the seeds of that species
do not form seed banks. That germination behavior could
represent, in natural conditions, a risk in years when the
precipitation distribution is irregular and scarce.
Ramírez-Tobías et al. [15] reported, for A. durangen-
sis, a germination value, at 25˚C, of 91%, reached at 150
h. Those results are different from those found in the
present study for the same species (Table 3); that could
be a consequence of the intraspecific variability con-
cerning the germination capability of A. durangensis.
The results of the present study indicated that the de-
crease of temperature caused a delay in the beginning of
the germination and a reduction of the germination po-
tential. Ramírez-Tobías et al. [15] reported that the ger-
mination of A. durangensis was inhibited in around 50%
at 15˚C. Our study revealed that between 49% and 95%
of the seeds of the natural populations of A. durangensis
can germinate at that temperature (Table 4), although
taking longer than at 25˚C.
The temperatures favorable to germination vary much
between different species of plants. The boundaries are
often narrow for seeds of species adapted to very specific
habitats and broader for seeds of species of wide distri-
bution [36]. Agave durangensis is a species of reduced
distribution [2]; according to the results of the present
study, its germination is compatible with a broad interval
of temperatures, indicating a high potential of germina-
tion response, what is related to its main propagation
mechanism, which is by seeds.
Table 8. Correlation coefficients of 11 seed vigor indicators used to characterize the natural populations of Agave durangen-
Rate of Germ
Rate of Germ
15˚C Weight
G25˚C 1 0.461 0.568 0.690 0.743 0.887 0.642 0.740 0.220 0.765 0.332
G15˚C 1 0.353 0.0009 0.012 0.033 0.022 0.0002 0.807 0.135 3.4 × 105
Rate of Germ
25˚C 1 0.132 0.703 0.505 0.800 0.878 0.067 0.023 0.175
Rate of Germ
15˚C 1 0.067 0.115 0.010 0.012 0.935 0.112 0.001
reduction 1 0.141 0.172 0.013 0.925 0.215 0.010
Phenol contents 1 0.090 0.0009 0.072 0.841 0.106
scavenging 1 0.042 0.573 0.459 0.079
Seed ADH 1 0.299 0.518 0.004
ADH 25˚C 1 0.012 0.706
ADH 15˚C 1 0.029
Weight 1
Open Access AJPS
Seed Vigor Variation of Agave durangensis Gentry (Agavaceae)
4.3. Rate of Germination (V)
The germination rate is a method to evaluate seed vigor;
it considers the number of normal plants that germinate
per day in preestablished conditions of germination. A
high value of germination rate is related to seeds with
high vigor [19].
The results of the present study indicate that the seeds
of Veracruz showed the best response of germination
under temperature stress conditions (Table 4). Several
reports about the use of germination rate evaluating the
seed vigor of monocotyledonous species [37-39] and of
dicotyledonous species [40-42] have been published.
Ramírez-Tobías et al. [15] reported that the germination
rate of Agave durangensis at 25˚C was around 0.5%
seeds/h. The rates found in the present study for any of
the three populations analyzed were higher (also at 25˚C),
between 3.66% seeds/h for the seeds of Veracruz to
4.04% seeds/h for those of La Parrilla (values estimated
from our raw data to compare with the results of
Ramírez-Tobías et al. [15]).
4.4. Growth Reduction of Plantets under Cold
According to Talai and Sen-Mandi [3] there is an inverse
relation between the growth reduction of plantets under
cold stress and the seed vigor. In this frame, the seeds of
A. durangensis of Veracruz (v), with a growth reduction
of 91.9% at 15˚C, would be the less vigorous, and those
of Pino Suárez (p), with a growth reduction of 86.6% at
15˚C compared with the growth at 24˚C, would be the
most vigorous (Table 5). To our knowledge, no reports
on the evaluation of this parameter in Agave have been
4.5. Phenol Composition and Antioxidant
The germination is related to the antioxidant potential of
seeds [43]. Some authors have suggested the use of that
potential as vigor indicator [44]; according to those au-
thors the seeds of La Parrilla, with the highest antioxi-
dant potential (50.35%, Table 6) would display the up-
permost vigor. The antioxidant potential, before and
during germination, is determined by the antioxidant
enzymatic activity of the embryo cells once these are
hydrated [44-46], and by the non-enzymatic antioxidant
compounds found in an active way in seeds, independ-
ently of the hydratation of seeds [47,48]. Phenolics are
among those compounds; actually they are the main re-
sponsible in maintaining the growth potential of the em-
bryo at storage conditions (pregerminatory stage) until
the imbibition allows the activation of the antioxidant
mechanism directed by enzymes [3]. A high correlation
between the phenol content and the antioxidant potential
in the seeds of Agave durangensis was found in the pre-
sent study (Figure 2), in accordance to the proposal of
Talai and Sen-Mandi [3]. The differences between the
free radical scavenging activities exhibited by the seeds
of each natural population of Agave durangensis can be
due to the variations in phenol contents (Table 6) and the
types of phenols accumulated, which were different in
the seeds of each population (Table 7). The particular
phenol profile is a relevant feature to determine the anti-
oxidant properties of any plant tissue or structure [49].
Seed phenols form complexes with carbohydrates and
lipids, enhancing the stability of those organic com-
pounds in the endosperm under oxidative stress condi-
tions, avoiding the beginning of a tandem oxidative deg-
radation caused by oxygen reactive species [50]. Seed
phenols also regulate the osmoconditioning in the first
stage of germination, in which the phenolic levels inside
seeds reduce, as a consequence of being moved out the
seed tissues [51].
4.6. ADH Activity
The ADH activity results suggest that the seeds of the
natural populations of Agave durangensis begin the ger-
mination sensu strict between 12 and 18 hrs after imbibi-
tions. At that stage the polypeptide synthesis is activated,
causing the metabolization of the endospermic tissues
and the development and growth of the vascular struc-
tures of the new plant [52]. The major anaerobiosis con-
ditions are present as a consequence of imbibition and
minimum oxygen exchange [53], and then the highest
metabolic activity in anaerobic conditions is displayed
[54]. All those conditions shoot the metabolic pathways
to synthesis of adenosin triphosphate (ATP), with ethanol
as a subproduct and using ADH like catalyzer [55].
At 25˚C, the seeds of Agave durangensis from Pino
Suárez and from Veracruz began a reduction of the en-
zymatic activity 12 hrs after imbibition, while the seeds
from La Parrilla began a reduction 18 hrs after imbibition.
At this temperature the seeds of Veracruz showed the
highest level of ADH activity (520.2 μmol NAD+/mg
protein/min) (Figure 4).
At 15˚C and after imbibitions, the seeds of Veracruz
showed the highest ADH levels (440.71 μmol NAD+/mg
EBSA/min) (Figure 5). The time taken by the three
populations of A. durangensis, for the diminution of
ADH activity, was longer at 15˚C than at 25˚C, because,
according to Labouriau [56], at 15˚C the seeds are closer
to the required thermodynamic limit to start germination.
The ADH activity decrease through the time, at both
15˚C and 25˚C, is due to a change in the permeability of
the seed membranes, increasing the oxygen exchange of
the embryo [9]. That decrease went on until the emer-
gence of the hypocotile and radicle, when the plantets
could carry out the aerobic respiration (Figures 4 and 5,
Open Access AJPS
Seed Vigor Variation of Agave durangensis Gentry (Agavaceae) 2237
Table 3).
Talai and Sen-Mandi [3] stated that the ADH activity
is essential during the anaerobic respiration in the pre-
germination and germination of seeds and for that reason
the ADH activity is a vigor indicator. According to that,
the seeds of A. durangensis of Veracruz (v), which
showed the highest ADH activity at either 15˚C or 25˚C,
represent the lots with the best vigor features.
4.7. PCA and Correlation Analysis
The seed vigor variation of A. durangensis has not been
well investigated; just a few reports have been published
about some issues of germination process [15]. To our
knowledge, the present study represents the first attempt
to investigate that kind of variation among natural popu-
lations of this species. The clear separation of the three
populations analyzed, with base on the variation of seed
weight and the evaluated physiological, chemical, and
biochemical indicators of seed vigor, suggests that wor-
thy alleles can be found from the natural populations of A.
durangensis to select seeds with high vigor to be used in
the establishment of plantations. Among the three popu-
lations of A. durangensis analyzed, that of Veracruz
showed high values in eight (weight, germinability at
25˚C and 15˚C, germination rate at 15˚C, phenol content,
scavenging activity, and ADH activity at 25˚C and 15˚C)
of the 11 seed vigor features evaluated.
The biochemical indicators (DPPH scavenging and
ADH activity) showed high associations with the phy-
siological indicators of seed vigor and with the phenol
contents in the seeds (Tab le 8 ). The results support those
previously reported by several authors about the signifi-
cance of the chemical and biochemical markers as seed
vigor indicators [3,44] and emphasize the importance of
the antioxidant phenols in the seeds to prevent oxidative
damage in the embryos and the essential participation of
alcohol deshydrogenase in the anaerobic respiration in
the germination of the seeds of A. durangensis.
5. Conclusion
Morphometric differences can be found among the seeds
of Agave durangensis of different natural populations.
Variation in some physiological, chemical and biochemi-
cal indicators of vigor was detected. According to the
results of germination potential, and to biochemical at-
tributes, like ADH activity, phenolic composition, and
antioxidant potential, the seeds of Veracruz have the
highest vigor. Each of the three natural populations of A.
durangensis could be typified by their morphological
attributes and by their physiological, chemical and bio-
chemical indicators of seed vigor. High correlations be-
tween chemical and biochemical markers and the germi-
nation markers were found, in such a way that the evalua-
tion of the former ones as indicators of seed vigor can
assist in the selection of seed lots with high germination
performance. The results of the present study suggest that
the variability of the natural populations of A. durangen-
sis is an important source of worthy alleles, which can
provide relevant support for the genetic improvement of
this species of Agave.
6. Acknowledgements
The authors thank the Comisión de Fomento a las Ac-
tividades Académicas del Instituto Politécnico Nacional
(COFAA IPN) for stimuli for research. Also, thanks are
given to Vicente Hernández Vargas (CIIDIR IPN Du-
rango) for the help in the collection of seed material and
Octavio Rosales for the important comments to the
[1] J. A. Ávila-Reyes, N. Almaraz-Abarca, E. A. Delgado-
Alvarado, L. S. González-Valdez, G. Valencia-del Toro
and E. Durán-Páramo, “Phenol Profile and Antioxidant
Capacity of Mescal Aged in Oak Wood Barrels,” Food
Research International, Vol. 43, No. 1, 2010, pp. 296-300.
[2] N. Almaraz-Abarca, V. Hernández-Vargas, I. Torres-
Morán, A. Delgado-Alvarado, G. Orea-Lara, A. Cifuentes-
Díaz de León, J. A. Ávila-Reyes, J. Herrera-Corral, N.
Uribe-Soto, R. Muñiz-Martínez and N. Naranjo-Jiménez,
Agave durangensis,” IPN-CONACYT, México DF, 2011.
[3] S. Talai and S. Sen-Mandi, “Seed Vigor-Related DNA
Marker in Rice Shows Homology with Acetyl CoA Car-
boxylase Gene,” Acta Physiologiae Plantarum, Vol. 32,
No. 1, 2010, pp. 153-167.
[4] M. Koornneef, L. Bentsink and H. Hilhorst, “Seed Dor-
mancy and Germination,” Current Opinion in Plant Bi-
ology, Vol. 5, No. 1, 2002, pp. 33-36.
[5] M. B. McDonald, “The History of Seed Vigor Testing,”
Journal of Seed Technology, Vol. 17, No. 2, 1994, pp.
[6] O. Chloupek, P. Hrstková and D. Jurecka, “Tolerance of
Barley Seed Germination to Cold- and Drought-Stress
Expressed as Seed Vigour,” Plant Breeding, Vol. 112, No.
3, 2003, pp. 199-203.
[7] M. Yamauchi, A. Aguilar, D. Vaughan and D. Seshu,
“Rice Germoplasm Suitable for Direct Sowing under
Flooded Soil Surface,” Euphytica, Vol. 67, No. 3, 1993,
pp. 177-184.
[8] D. J. Osborne, A. Dell’Aquila and R. H. Elder, “DNA
Repair in Plant Cells. An Essential Event of Early Em-
bryo Germination in Seeds,” Folia Biologica (Prague),
Vol. 30, Special Issue, 1984, pp. 155-169.
[9] S. Shimomura and H. Beevers, “Alchohol Dehydrogenase
Open Access AJPS
Seed Vigor Variation of Agave durangensis Gentry (Agavaceae)
and an Inactivator from Rice Seedlings,” Plant Physiol-
ogy, Vol. 71, No. 4, 1983, pp. 736-741.
[10] H. Kato-Noguchi, “Ethanol Sensitivity of Rice and Oat
Coleoptiles,” Physiologia Plantarum, Vol. 115, No. 1,
2002, pp. 119-124.
[11] C. Bailly, “Active Oxygen Species and Antioxidants in
Seed Biology,” Seed Science Research, Vol. 14, No. 2,
2004, pp. 93-107.
[12] B. A. Cevallos-Casals and L. Cisneros-Zevallos, “Impact
of Germination on Phenolic Content and Antioxidant Ac-
tivity of 13 Edible Seeds Species,” Food Chemistry, Vol.
119, No. 4, 2010, pp. 1485-1490.
[13] C. E. Freeman, “Germination Responses of a New Mex-
ico Population of Parry Agave (Agave parryi Engelm. var.
parryi) to Constant Temperature, Water Stress, and pH,”
The Southwestern Naturalist, Vol. 20, No. 1, 1975, pp.
[14] L. G. Orea, A. Cifuentes-Díaz de León, O. S. Gómez and
V. V Hernández, “Seed Germination (Agave durangensis)
at different temperatures and Effect of Fertilization on the
Plantet Development,” Vidsupra, Vol. 2, No. 1, 2009, pp.
[15] H. M. Ramírez-Tobías, C. B. Peña-Valdivia, J. R. R.
Aguirre, J. A. Reyes-Agüero, A. B. Sánchez-Urdaneta
and S. G. Valle, “Seed Germination Temperatures of
Eight Mexican Agave Species with Economic Impor-
tance,” Plant Species Biology, Vol. 27, No. 2, 2012, pp.
[16] G. Funes, S. Díaz and P. Venier, “Temperature as
Principal Determinative Factor of Germination in Species
of Chaco Seco, Argentina,” Ecología Austral, Vol. 19, No.
2, 2009, pp. 129-138.
[17] M. K. Kettenring, G. Gardner and S. M. Galatowitsch,
“Effects of Light on Seeds Germination of Eight Wetland
Carex Species,” Annals of Botany, Vol. 98, No. 4, 2006,
pp. 869-874.
[18] M. Sood and V. Thakur, “Effect of Light and Tempera-
ture on Germination Behavior of Aconitum deinorrhizum
Stapf,” International Journal of Farm Sciences, Vol. 1,
No. 2, 2011, pp. 83-87.
[19] J. D. Maguire, “Speed of Germination-Aid in Selection
and Evaluation for Seedling Emergence and Vigor,” Crop
Science, Vol. 2, No. 2, 1962, pp. 176-177.
[20] A. García and J. M. Lasa, “Test of Seed Vigor: Bi-
bliographical Review,” Boletin 14 de la Estación Ex-
perimental Aula Dei, Zaragoza, 1991.
[21] M. R. S. Ardekani, M. Khanavi, M. Hajimahmoodi, M.
Jahangiri and A. Hadjiakhoondi, “Comparison of Anti-
oxidant Activity and Total Phenol Contents of Some Date
Seed Varieties from Iran,” Iranian Journal of Pharma-
ceutical Research, Vol. 9, No. 2, 2010, pp. 141-146.
[22] H. Lozoya-Saldaña, R. Rivera-Hinojosa and M. T. Colinas-
León, “Phenols, Peroxidase and Phenylalanine Ammonia-
Lyase: Their Relationship to the Genetic Resisteance
Against Late Blight (Phytophthora infestans Mont. De
Bary) in Potato (Solanum tuberosum L.) Clones,” Agro-
ciencia, Vol. 41, No. 4, 2007, pp. 479-489.
[23] M. Campos and K. Markham, “Structure Information
from HPLC and On-Line Measured Absorption Spectra:
Flavones, Flavonols and Phenolic Acids,” Coimbra Uni-
versity Press, Coimbra, 2007.
[24] T. J. Mabry, K. R. Markham and M. B. Thomas, “The
Systematic Identification of Flavonoids,” Springer-Verlag,
New York, 1970.
[25] M. G. Campos, P. A. Da Cunha, M. C. Navarro and M. P.
Utrilla, “Free Radical Scavenger Activity of Bee Pollen,”
17th International Conference on Polyphenols, Palma de
Mallorca, 1994, pp. 415-416.
[26] M. E. Rumpho and R. A. Kennedy, “Anaerobic Metabo-
lism in Germinating Seeds of Echinochloa crus-galli
(Barnyard Grass),” Plant Physiology, Vol. 68, No. 1,
1981, pp. 165-168.
[27] M. A. Schuler and R. E. Zielinski, “Methods in Plant
Molecular Biology,” Academic Press, Millbrae, 1989.
[28] Ø. Hammer, D. A. T. Harper and P. D. Ryan, “PAST:
Paleontological Statistics Software Package for Education
and Data Analysis,” Paleontologia Electronica, Vol. 4,
No. 1, 2001, p. 9.
[29] H. Gentry, “Agaves of Continental North America,” The
University of Arizona Press, Tucson, 1982.
[30] M. G. Barbour, J. H. Burk, W. D. Pitts, F. S. Gilliam and
M. W. Schwartz, “Terrestrial Plant Ecology,” Benjamin/
Cummings, San Francisco, 1999.
[31] H. Baker, “Seed Weight in Relation to Environmental
Conditions,” Ecology, Vol. 53, No. 6, 1972, pp. 997-1010.
[32] N. Pesĕv, “Genetic Factors Affecting Maize Tolerance to
Low Temperatures at Emergence and Germination,”
Theoretical and Applied Genetics, Vol. 40, No. 12, 1970,
pp. 351-356.
[33] P. S. Nobel, “Environmental Biology of Agaves and
Cacti,” Cambridge University Press, New York, 2003.
[34] R. Austin and P. Longden, “The Effects of Nutritional
Treatments of Seed-Bearing Plants on the Performance of
Their Progeny,” Nature, Vol. 205, No. 4973, 1965, pp.
[35] T. Philippi, “Bet-Hedging Germination of Desert Annuals:
Beyond the First Year,” American Naturalist, Vol. 142,
No. 3, 1993, pp. 474-487.
[36] G. F. Pérez and L. J. B. Martínez, “Introducción a la
Fisiología Vegetal,” Mundi Prensa, Madrid, 1994.
[37] S. J. Martínez, J. V. Vargas, O. M. G. Peña and A. S.
Romero, “Speed of Emergence of Inbred Maize Lines,”
Revista Mexicana de Ciencias Agrícolas, Vol. 1, No. 3,
2010, pp. 289-304.
Open Access AJPS
Seed Vigor Variation of Agave durangensis Gentry (Agavaceae)
Open Access AJPS
[38] H. Zhang, L. J. Irving, C. McGill, C. Matthew, D. Zhou
and P. Kemp, “The Effects of Salinity and Osmotic Stress
on the Germination of Two Barley Varieties: Sodium as
an Osmotic Regulator,” Annals of Botany, Vol. 106, No.
6, 2010, pp. 1027-1035.
[39] M. Mercado and P. Fernández, “Enhancing Rice Seed
Germinability and Vigor Through Hydration-Dehydration
(HD) Technique,” Philippine Journal of Crop Science,
Vol. 27, No. 1, 2002, pp. 13-24.
[40] T. Mostarin, S. R. Saha and K. Khatun, “Seed Quality of
Bush Bean as Iinfluenced by Different Storage Containers
and Conditions,” Journal of Experimental Biosciences,
Vol. 3, No. 1, 2012, pp. 83-88.
[41] R. R. Sokht-Abandani and M. R. Ramezani, “The Phy-
siological Effects on Some Ttraits of Osmopriming Ger-
mination of Maize (Zea mays L.), Rice (Oryza sativa L.)
and Cucumber (Cucumis sativus L.),” International Jour-
nal of Biology, Vol. 4, No. 2, 2012, pp. 132-148.
[42] R. L. Hassell, R. J. Dufault and T. L. Phillips, “Influence
of Temperature Gradients on Triploid and Diploid Wa-
termelon Seed Germination,” HortTechnology, Vol. 11,
No. 4, 2001, pp. 570-574.
[43] C. Bailly, A. Benamar, F. Corbineau and D. Côme, “Free
Radical Scavenging as Affected by Accelerated Ageing
and Subsequent Priming in Sunflower Seeds,” Physiolo-
gia Plantarum, Vol. 104, No. 4, 1998, pp. 646-652.
[44] S. Balešević-T ubi ć, D. Malenčić, M. Tatić and J. Miladi-
nović, “Influence of Ageing Process on Biochemical
Changes in Sunflower Seed,” Helia, Vol. 28, No. 42,
2005, pp. 107-114.
[45] S. Nandi, S. Sen-Mandi and T. P. Sinha, “Active Oxygen
and Their Scavengers in Rice Seeds (Oryza sativa cv.
IET4094) Aged under Tropical Environmental Condi-
tions,” Seed Science Research, Vol. 7, No. 3, 1997, pp.
[46] U. M. N. Murthy, Y. Liang, P. P. Kumar and W. Sun,
“Non-Enzymatic Protein Modification by the Maillard
Reaction Reduces the Activities of Scavenging Enzymes
in Vigna radiate,” Physiologia Plantarum, Vol. 115, No.
2, 2002, pp. 213-220.
[47] L. Lepiniec, I. Debeaujon, J. M. Routaboul, A. Baudry, L.
Pourcel, N. Nesi and M. Caboche, “Genetics and Bio-
chemistry of Seed Flavonoids,” Annual Review of Plant
Physiology, Vol. 57, 2006, pp. 405-430.
[48] S. Pukacka and E. Ratajczak, “Age-Related Biochemical
Changes during Storage of Beech (Fagus sylvatica L.)
Seeds,” Seed Science Research, Vol. 17, No. 1, 2007, pp.
[49] L. G. Barriada-Bernal, N. Almaraz-Abarca, E. A. Del-
gado-Alvarado, T. Gallardo-Velázquez, J. A. Ávila-Reyes,
M. I. Torrres-Morán, M. S. González-Elizondo and Y.
Herrera-Arrieta, “Flavonoid Composition and Antioxi-
dant Capacity of the Edible Flowers of Agave durangen-
sis (Agavaceae),” CyTA-Journal of Food, Published on-
line: 14 Jun 2013.
[50] F. Abderrahim, E. Huanatico, R. Repo-Carrasco-Valencia,
S. M. Arribas, M. C. Gonzalez and L. Condezo-Hoyos,
“Effect of Germination on Total Phenolic Compounds,
Total Antioxidant Capacity, Maillard Reaction Products
and Oxidative Stress Markers in Canihua (Chenopodium
pallidicaule),” Journal of Cereal Science, Vol. 56, No. 2,
2012, pp. 410-417.
[51] Z. S. Siddiqui and M. A. Khan, “The Role of Seed Coat
Phenolics on Water Uptake and Early Protein Synthesis
during Germination of Dimorphic Seeds of Halopyrum
mucronatum (L.) Staph,” Pakistan Journal of Botany, Vol.
42, No. 1, 2010, pp. 227-238.
[52] J. Bewley and M. Black, “Seeds: Physiology of Devel-
opment and Germination,” Plenum Press, New York, 1994.
[53] D. Priestley, “Seed Ageing. Implications for Seed Storage
and Persistence in the Soil,” Cornell University Press,
Ithaca, 1986.
[54] M. C. Drew, “Oxygen Deficiency and Root Metabolism;
Injury and Acclimation under Hypoxia and Anoxia,” An-
nual Review of Plant Physiology and Plan Molecular Bi-
ology, Vol. 48, 1997, pp. 223-250.
[55] D. Schwartz, “An Example of Gene Fixation Resulting
from Selective Advantage in Suboptimal Conditions,”
The American Naturalist, Vol. 103, No. 933, 1969, pp.
[56] L. G. Labouriau, “Seed Germination as a Thermobiologi-
cal Problem,” Radiation and Environmental Biophysics,
Vol. 15, No. 4, 1978, pp. 345-366.