Seed Vigor Variation of <i>Agave durangensis</i> Gentry (Agavaceae)

doi:10.4236/ajps.2013.411276

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American Journal of Plant Sciences, 2013, 4, 2227-2239

Published Online November 2013 (http://www.scirp.org/journal/ajps)

http://dx.doi.org/10.4236/ajps.2013.411276

Open Access AJPS

2227

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: *nalmaraz@ipn.mx

Received September 27th, 2013; revised October 20th, 2013; accepted October 29th, 2013

License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

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

populations.

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

[4].

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)

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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-

tations.

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

analysis.

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

(m)

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):











00

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

germinated.

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

(3).

Equation (3):



% reduction of lengCth at 15

100

op et

LES LES

LES

















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):



010

% 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-

cate.

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 1.1.1.1) 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

triplicate.

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)

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Figure 1. Seeds of Agave durangensis from three popula-

tions of Durango, Mexico ((a) Pino Suárez, (b) La Parrilla,

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

(mg)

Width/seed

(mm)

Length/seed

(mm)

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

Stress

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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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 <

0.05).

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)

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Table 7. Retention time and spectral data of the phenolic compounds found in the seeds of three populations of Agave du-

rangensis.

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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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-

sis.

25˚C

15˚C

Rate of Germ

25˚C

Rate of Germ

15˚C

Growth

reduction

Phenol

content

DPPH

scavenging

Seed

ADH

25˚C

ADH

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 × 10−5

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

Growth

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

DPPH

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)

2236

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

Stress

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

published.

4.5. Phenol Composition and Antioxidant

Capacity

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

manuscript.

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