Agave Durangensis Research Articles

Characterization of the complete chloroplast genome sequence of Agave durangensis (Asparagales: Asparagaceae: Agavoideae)

Agave durangensis commonly known as agave cenizo, is an endemic Agave species in Mexico used for mescal production, yet its taxonomic delimitation is still controversial. This study aimed to enhance taxonomic clarity by characterizing its chloroplast genome. Chloroplast DNA was isolated from 2-year-old A. durangensis leaves. The complete chloroplast genome size was 156,441 bp, comprising a large single-copy region (LSC), a pair of inverted repeat regions (IR), and a small single-copy region (SSC). Annotation revealed 87 protein-coding genes, 38 tRNAs, and 8 rRNAs, with notable gene inversions. Phylogenetic analysis suggests, A. durangensis forms a separate lineage within the Agave genus.

Diversity of Culturable Yeasts Associated with the Technification Level in the Process of Mezcal Production in the State of Durango

Durango State has the denomination of origin for the production of mezcal, which is made from Agave durangensis, mainly in an artisanal way; therefore, differences in the fermentation process affect the quality of the final product. The main objective of the present study was to evaluate the diversity of culturable yeasts involved in the artisanal and semi-technified process of mezcal production in the State of Durango. Three distilleries with different production processes were monitored at different fermentation stages (beginning, mid-fermentation, and end of fermentation) in the spring and summer seasons. A greater diversity was found in the distillery of Nombre de Dios in both the spring and summer production seasons (H’ = 1.464 and 1.332, respectively), since it maintains an artisanal production process. In contrast, the distillery of Durango, where a Saccharomyces cerevisiae commercial inoculum is used to start fermentation, presented low diversity indexes (H’ = 0.7903 and 0.6442) and only S. cerevisiae, Kluyveromyces marxianus, and, sporadically, Pichia manshurica were found. Results suggest that the yeast microbiota involved in mezcal fermentation during the different seasons is affected by the type of inoculum; changes include the presence of some species that were only identified during a specific season in alcoholic fermentation, such as Torulaspora delbrueckii and Pichia kluyveri.

Variation of the chemical and biochemical responses to salinity during germination and early growth of seedlings of two populations of Agave durangensis Gentry

Agave durangensis (Asparagaceae) sustains a mescal industry in Mexico. The main reproductive strategy of the species is by seeds. The increased demand for agave-based beverages encourages producers to seek new cultivation areas. However, more than half of the territory of the country includes arid and semiarid zones, which are highly affected by salinity. The aim of the current study was to determine if salinity triggers different seed germination potential and variable biochemical and chemical responses in seedlings of two populations of A. durangensis, that might confer different tolerance to salinity. Seeds from each population were irrigated with four salinity treatments. Germination potential, as well as growth parameters and biochemical and chemical attributes of seedlings, were determined. Although with reduced germinability and germination speed, seeds of the two populations were able to germinate even under the strongest NaCl concentration (100 mM) evaluated. Effects in the growth parameters were registered; however, the seedlings of both populations survived throughout the experiments, increasing chlorophyll content and cell viability in most saline treatments. The enzymatic defense mechanism and the accumulation of proline were activated in a salt-dependent manner, which did not occur with the phenolic compounds; however, monomeric anthocyanin accumulation was outstanding under the two strongest NaCl concentrations evaluated. Important interpopulation differences were registered in each type of response, which were differently regulated under variable NaCl concentrations, some of them being more important than others under a particular saline condition. Seeds from the population Durango were more sensitive to salinity.

Extraction of agavins from Agave durangensis leaves: Structural, thermal and techno-functional characterization

This research aimed to optimize the extraction process of the agavins from Agave durangensis leaves and to carry out their structural, thermal, and techno-functional characterization. From the optimization methodology, a theoretical yield of 41.40 g agavins/g d.s. and a real yield of 40.31 g agavins/100 g d.s. were obtained. In addition, a characteristic carbohydrate profile by TLC and the functional groups by FT-IR were found. DSC analysis showed an endothermic peak between 65.43 and 210 °C, while the glass transition temperature (Tg) ranged between 34.92 and 52.66 °C. The particle size showed two principal distributions with good polydispersity. The techno-functional properties of moisture, wettability and bulk density were similar to those of commercial Agave tequilana agavins, while color, hygroscopicity, and water activity were statistically different. This work is a basis for using Agave durangensis leaves agavins in food applications and future scientific research.

Comparative study of four extraction methods of fructans (agavins) from Agave durangensis: Heat treatment, ultrasound, microwave and simultaneous ultrasound-microwave.

Agavins (fructans from the agave plant) are used for their technological and prebiotic properties in developing functional foods. In this study, four extraction methods were evaluated: ultrasound (U), microwave (M), simultaneous ultrasound-microwave (UM), and heat treatment (HT). Isomers with a degree of polymerization (DP)>10, as well as a significant concentration of fructooligosaccharides (FOS) were identified. The yield obtained by UM (72%) was similar to the other methods; however, the extraction time was shorter (5min). In U, M, and HT the yields were 86, 76, and 85% in 35, 30, and 180min, respectively. In addition, FTIR spectra showed evidence of regions corresponding to fatty acids and carbohydrates. Therefore, using UM to obtain agavins is a more ecological and faster process.

Biomass Estimation of Agave durangensis Gentry Using High-Resolution Images in Nombre de Dios, Durango

The high demand for distilled agave products reduces wild populations. The use of geospatial technologies such as unmanned aerial vehicles (UAVs) offer enormous benefits in spatial and temporal resolution and lower costs than traditional direct field observation techniques for natural resource monitoring. The objective was to estimate the green biomass (Wt) of Agave durangensis Gentry using high-resolution images obtained by a UAV in Nombre de Dios, Durango. Random sampling was performed in the agave area. A Pearson correlation analysis was performed, followed by a regression analysis. The results showed that NDVI was the most correlated (r = 0.65). The regression analysis showed that the model obtained explains 59% (RMSE = 32.06 kg) of the total variability in the estimation of green biomass (Wt) of agave using images derived from the UAV. The best estimate was achieved with B1, B2, NDVI, GNDVI, EVI2, and SAVI as predictor variables. High-resolution images were shown to be a tool for estimating Wt of Agave durangensis Gentry.

Improving the Physical, Mechanical and Energetic Characteristics of Pine Sawdust by the Addition of up to 40% Agave durangensis Gentry Pellets

Agave durangensis Gentry biomass, as a residue from the mezcal production process, may be an interesting bioenergy alternative; however, its high ash content limits its application. In this study, pellets were generated with agave fiber mixed with Pinus species sawdust in the following six proportions (%): 100–0 (control), 80–20, 60–40, 40–60, 20–80 and 0–100 (control). The physical, chemical and energetic properties of the pellets were evaluated according to the UNE-EN ISO 17225-6, UNE EN ISO 17827-2, UNE-EN ISO 17828, UNE-EN ISO 18122, UNE-EN ISO 18123, UNE-EN ISO 18125, and UNE-EN ISO 18134-1 standards. The results showed significant statistical differences (p < 0.05) among the treatments tested. The percentage of volatile material and fixed carbon ranged from 86.53 to 89.96% and 4.17 to 8.16%, respectively; the ash content ranged from 0.27 to 10.06%, and the calorific value ranged from 17.33 to 18.03 MJ/kg. Bulk density ranged from 725.76 to 737.37 kg/m3 and the impact-strength index was in the range of 69.33 to 126.66. The mechanical hardness and compressive strength were found to be in the ranges of 50.5 to 68.4% and 0.90 to 36.65 N/mm, respectively. Pellets generated with Agave residue mixture ≤ 40% were identified as promising biobased resources for the sustainable production of renewable energy.

Use of Agave durangensis Bagasse Fibers in the Production of Wood-Based Medium Density Fiberboard (MDF)

There is an increasing interest in using non-wood lignocellulosic materials for the production of wood-based medium density fiberboard (MDF). Agave durangensis Gentry bagasse is a waste product produced in large quantities in the mezcal industry. This study evaluated the incorporation of A. durangensis bagasse fibers (ADBF) to elaborate MDF wood-based panels. Three types of panels with different ratios (wood fibers: bagasse fibers) were investigated. The ratios evaluated were 100:0, 90:10, and 70:30. The density profiles, water absorption, and thickness swell of the panels were determined, as well as the modulus of elasticity (MOE), modulus of rupture (MOR), and internal bond (IB), according to the ASTM D1037-06a standard. The results were compared to the ANSI A208.2-2016 standard. The effect of the addition of ADBF on the properties of the panels was analyzed. Density profiles were comparable among the three types of panels, while water absorption, thickness swelling, MOE, MOR, and IB were similar between panels with ratios of 100:0 and 90:10. Panels with 10% and 30% of ADBF meet the minimum ANSI requirements for quality grade 115. It is feasible to use up to 30% of ADBF in the manufacture of wood-based MDF panels.

Agave durangensis vinasse as a biocide for forest pest control

In forestry practice, the prevention and fight against insect pests and diseases is a priority to preserve the health of these ecosystems. To combat it, insecticides of chemical and biological origin have been used. However, there are alternatives that have not yet been investigated, e.g., the use of agro-industrial waste. In the mezcal distillation process, polluting liquid residues called vinasses are generated, and these can be considered for pest control. In this project, the effect of vinasse from Agave durangensis subjected to different treatments was studied to evaluate its effect on forest phytopathogenic fungi. The mezcal vinasse was characterized physicochemically and by its metabolites. Furthermore, the percentage of inhibition in vitro of phytopathogenic (causing root wilt) fungi isolated from Pinus cooperi seedlings was studied. The fungi inhibition was related to the vinasse concentration. The lower pH and sterile raw vinasse showed a better inhibition effect. Four phytopathogenic strains of Pinus cooperi were isolated and identified, which corresponded to the genera Fusarium, Aspergillus, and Penicillium. None of the isolated were able to grow in potato dextrose-mezcal vinasse medium (PDMVM). Therefore, the mezcal vinasse showed fungicide activity in vitro against all strains.

Utilization of Agave durangensis leaves by Bacillus cereus 4N for polyhydroxybutyrate (PHB) biosynthesis

Lignocellulosic wastes may provide a means to economize polyhydroxybutyrate (PHB) production. This study has proposed the use of Agave durangensis leaves obtained from the artisanal mezcal industry as a novel substrate for this aim. Results revealed an increase in PHB biosynthesis (0.32 g/L) and improvement in %PHB (16.79–19.51%) by Bacillus cereus 4N when A. durangensis leaves used as carbon source were physically pre-treated by ultrasound for 30 min (ADL + US30’) and thermally pre-treated (ADL + Q). Chemical analyses and SEM studies revealed compositional and morphological changes when A. durangensis leaves were physically pre-treated. Also, elemental analysis of growth media showed that carbon/nitrogen ratios of 14–21, and low nitrogen, hydrogen, and protein content were well-suited for PHB biosynthesis. Confocal microscopy revealed morphological changes in the bacterial cell and carbonosome structure under the influence of different substrates. Finally, Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) analyses showed that homopolymeric PHB with a high thermal-resistance (271.94–272.89 °C) was produced. Therefore, the present study demonstrates the potential use of physically pre-treated A. durangensis leaves to produce PHB. These results promote the development of a circular economy in Mexico, where lignocellulosic wastes can be employed to produce value-added biotechnological products.

Chemical composition of Luffa aegyptiaca Mill., Agave durangensis Gentry and Pennisetum sp.

The particleboard industry faces problems of wood shortage, which has led to the use of non-wood lignocellulosic materials. Furthermore, there is also interest in looking for materials that improve their physical and mechanical properties. The species Luffa aegyptiaca Mill. (fruit), Agave durangensis Gentry (bagasse) and Pennisetum sp. (plant, leaves and stem) could be used in the elaboration of wood-based particleboards. The aim of this study is to determine the feasibility of using these materials to produce particleboards in accordance with their chemical composition. Five materials were studied, A. durangensis (bagasse), L. aegyptiaca (fruit) and Pennisetum sp. (whole plant, leaves and stem). Extractives, holocellulose, Runkel lignin and ash content was determined. The pH of the fibers was also measured and a microanalysis of the ash was performed. ANOVA and Kruskal-Wallis tests were carried out, in addition Tukey and Dunn tests for group comparison were performed. Pennisetum sp. leaves presented the highest total extractives and ash content, while L. aegyptiaca fruit and A. durangensis bagasse had the highest both content of holocellulose and Runkel lignin respectively. The lowest pH was presented by the L. aegyptiaca fruit, while the highest was from the Pennisetum sp. stem. The element with the greatest presence in the five materials was potassium, except in A. durangensis bagasse showing calcium. L. aegyptiaca fruit has better characteristics to be used in particleboards with greater mechanical resistance because of its higher holocellulose content. However, Pennisetum sp. (plant, leaves and stem) could be used to make particleboards with high resistance to water absorption.

Potential production of 2-phenylethanol and 2-phenylethylacetate by non-Saccharomyces yeasts from Agave durangensis

The participation of non-Saccharomyces yeasts in fermentation processes is of great importance due to their participation in the formation of esters and superior alcohols, which confer characteristic aromas to beverages such as wine and mescal. The aim of this study was identify and evaluate the potential aroma production of yeast native of Agave fermentation by the mescal production in Durango, Mexico. Isolated yeasts were molecularly identified by 5.8s ribosomal gene; the potential production of aromas was carried out in fermentations with the addition of l-phenylalanine and evaluated after 24 h of fermentation. Extraction and quantification of aromatic compounds by headspace solid-phase micro-extraction (HS-SPME) and gas chromatograph mass spectrometry (GC-MS). The isolated non-Saccharomyces yeasts could be classified into six different genera Saccharomyces cerevisiae, Clavispora lusitaniae, Torulaspora delbrueckii, Kluyveromyces dobzhanskii, Kluyveromyces marxianus, and Kluyveromyces sp. All probed strains presented a potential aroma production (ethyl acetate, isoamyl acetate, isoamyl alcohol, benzaldehyde, 2-phenylethyl butyrate, and phenylethyl propionate), particularly 2-phenylethanol and 2-phenylethylacetate; the levels found in the Kluyveromyces marxianus ITD0211 yeast have the highest 2-phenylethylacetate production at 203 mg/L and Kluyveromyces marxianus ITD0090 with a production of 2-phenylethanol at 1024 mg/L. Non-Saccharomyces yeasts were isolated from the mescal fermentation in Durango; the Kluyveromyces genus is the most predominant. For the production of aromas, highlighting two strains of Kluyveromyces marxianus produces competitive quantities of compounds of great biotechnological interest such as 2-phenylethanol and 2-phenylethylacetate, without resorting to the genetic modification of yeasts or the optimization of the culture medium.

Calidad de sitio para Agave durangensis Gentry en la Sierra de Registrillo, Durango

El Agave durangensis Gentry, es un recurso natural que ha sido aprovechado de manera empírica por los pobladores de la zona semidesértica del estado de Durango. No se cuenta con elementos científicos y normativos que regulen u optimicen la cosecha de este producto; por lo tanto, contar con el presente, proporciona los instrumentos necesarios para el correcto aprovechamiento del agave. En la presente investigación se probó la factibilidad de cinco modelos matemáticos para determinar la calidad de sitio del A. durangensis en la zona semidesértica de la Sierra de Registrillo, municipio de Durango, México. La base de datos utilizada consistió en 204 pares de agaves dominantes en altura-edad, seleccionados de 180 sitios de muestreo en 1 000 m2 cada uno, tomados en el área de estudio a través de un muestreo sistemático. El ajuste de los modelos matemáticos se realizó por métodos de regresión no lineal ponderada para corregir el problema de heterocedasticidad de varianza observada en la relación altura dominante-edad. Los resultados del ajuste de las ecuaciones indican que el modelo de Chapman-Richards fue el que mejor determina la calidad de sitio. A partir del análisis realizado, se concluye que el uso de modelos de índice de sitio es factible para clasificar la productividad de los sitios de A. durangensis en el área de estudio. El modelo desarrollado proporciona ecuaciones compatibles de índice de sitio y crecimiento en altura dominante.

Enzymatic Potential of Native Fungal Strains of Agave Residues

Twelve strains of fungi from residues of Agave durangensis were isolated and identified by molecular techniques for evaluation of their hydrolytic enzyme production capability. A proportion (50%) of the fungal strains belonged to the Aspergillus genus and the other strains used belonged to Alternaria, Neurospora crassa, Mucor sp, Rhizopus sp., Botryosphaeria sp., and Scytalidium sp. The isolated strains were evaluated for their potential to produce extracellular enzymes using different substrates (cellulose, xylan, inulin, Agave fructans, starch, and tannic acid). It was observed that most of the tested strains were capable of simultaneously secreting cellulases, xylanases, inulinases, fructanases, and laccases. Botryosphaeria sp. ITD-G6 was selected for its evaluation in the production of inulinase, using different substrates. Showing high inulinase activities (5.22 U / mL for Agave waste, 4.37 U/ mL for inulin and 5.00 U / mL for Agave fructans).

Effect of ultrasound pre-treatment on the physicochemical composition of Agave durangensis leaves and potential enzyme production

Approximately 1 million tons of agave plants are processed annually by the Mexican tequila and mezcal industry, generating vast amounts of agroindustrial solid waste. This type of lignocellulosic biomass is considered to be agroindustrial residue, which can be used to produce enzymes, giving it added value. However, the structure of lignocellulosic biomass makes it highly recalcitrant, and results in relatively low yield when used in its native form. The aim of this study was to investigate an effective pre-treatment method for the production of commercially important hydrolytic enzymes. In this work, the physical and chemical modification of Agave durangensis leaves was analysed using ultrasound and high temperature as pre-treatments, and production of enzymes was evaluated. The pre-treatments resulted in modification of the lignocellulosic structure and composition; the ultrasound pre-treatment improved the production of inulinase by 4 U/mg and cellulase by 0.297 U/mg, and thermal pre-treatment improved β-fructofuranosidase by 30 U/mg.

Effect of steroidal saponins from Agave on the polysaccharide cell wall composition of Saccharomyces cerevisiae and Kluyveromyces marxianus

In the fermentation process of distilled spirits from Agave sugars, saponins inhibit the growth of yeasts during this procedure. This type of compound is present in the must, and the mechanisms of the cell growth inhibition are poorly understood. The fundamental objective of this work was to study the effect of saponins on the cell walls of the fermentative yeasts Saccharomyces cerevisiae and Kluyveromyces marxianus. This study was performed in batch and continuous cultures, adding pulses of saponins extracted from the leaves of Agave durangensis and Agave salmiana ssp. crassispina. The saponin extract from A. durangensis exhibited a stronger growth inhibitory effect in both yeast strains than A. salmiana saponin extract due to the structure of the saponins. However, in K. marxianus growth recovery was observed, because these strain showed saponinase activity that performed the saponin hydrolysis. Finally, microscopic observations confirmed the changes in the wall cell of S. cerevisiae and K. marxianus.

English

Agave plants are members of the Agavaceae family and utilize crassulacean acid metabolism (CAM) for CO2 fixation. Fructans are the main photosynthetic products produced by Agave plants, and are their principal source of storage carbohydrates. The aim of this work was to determine the chemical and molecular characterization of fructans from Agave durangensis. Fructans were extracted from 10 year old A. durangensis plants. Trimethylsilyl derivatization was employed to determine the monomer composition. The linkage types in these carbohydrates were determined by methylation followed by reduction and O-acetylation, and finally analysis by gas chromatography-mass spectrometry (GC-MS). Samples were shown to contain t-β-D-Fruf, t-α-D-Glup, i-α-D-6-Glup and 1,6-di-β-D-Fruf linkages. The analysis of the degree of polymerization (DP) was confirmed by MALDI-TOF-MS, showing a wide DP ranging from 2 to 29 units. The analyses performed revealed that fructans from A. durangensis are formed of 97.11% fructose and 2.89% glucose, and are a complex mixture of fructooligosaccharides of the neo-fructan type containing principally β(2-1) and β(2-6) linkages, with branch moieties. Key words: Degree of polymerization (DP), GC-MS, MALDI-TOF-MS.

Genetic Variability in Natural Populations ofAgave durangensis(Agavaceae) Revealed by Morphological and Molecular Traits

Agave durangensis is an endemic species that occurs in a reduced area in southern Durango and northern Zacatecas, Mexico. We analyzed the genetic variability of three populations of the species using two pairs of Inverse-Sequence-Tagged-Repeat primers and nine morphological characters to determine the species of the morphologically variable populations. The primers generated characteristic amplification profiles, which distinguished between populations of A. durangensis and revealed interspecific variability when compared with individuals of A. asperrima. Some incongruence between morphological and molecular variability was detected. High genetic variability was indicated by an elevated level of polymorphism (24.18–61.50%) among 91 genetic loci amplified and by the level of gene diversity (0.0807–0.2337) among populations. Genetic differentiation (0.0328–0.4857), gene flow (0.5294–14.7511), genetic distance (0.0200–0.3019), and genetic identity (0.7653–0.9802) among populations suggested that one of the three populations of A. durangensis is in a present advanced diversification process but may still maintain some level of gene flow with the other two populations. Urgent conservation efforts, including protection from harvest, should be addressed for maintaining the genetic variability of the natural populations of A. durangensis.Agave durangensis es una especie endémica que crece en un área reducida del sur de Durango y norte de Zacatecas, México. Analizamos la variabilidad genética de tres poblaciones de esa especie usando dos pares de iniciadores de los marcadores de Repetición de Secuencias Inversas Marcadas (ISTR por sus siglas en inglés) y nueve atributos morfológicos para determinar las especies de las poblaciones morfológicamente muy variables. Los iniciadores generaron perfiles de amplificación característicos, mostrando su capacidad para distinguir entre poblaciones de A. durangensis y para revelar variabilidad intereespecífica cuando los perfiles se compararon con los de A. asperrima. Incongruencia entre la variabilidad morfológica y la molecular fue detectada. Alta variabilidad genética fue encontrada, indicada por los elevados niveles de polimorfismo (24.18–61.50 %) entre 91 loci genéticos amplificados y por los valores de diversidad génica (0.0807–0.2337) entre poblaciones. Diferenciación genética interpoblacional (0.0328–0.4857), flujo genético (0.5294–14.7511), distancia genética (0.0200–0.3019) y la identidad genética (0.7653–0.9802) sugieren que una de las tres poblaciones de A. durangensis se encuentra en un proceso actual avanzado de diversificación, pero que aún existe flujo genético con las otras dos poblaciones. Esfuerzos urgentes de conservación incluyendo la protección de recolección se deberían de contemplar para mantener la variabilidad genética de las poblaciones naturales de A. durangensis.

Flavonoid composition and antioxidant capacity of the edible flowers of Agave durangensis (Agavaceae)

The antioxidant properties and the flavonoid composition of extracts of different hydrophobicity prepared from the entire edible flowers of Agave durangensis were evaluated. Separately, total extracts of tepals and anthers–pollen were analyzed in the same manner. The high performance liquid chromatography with photodiode array detection (HPLC–DAD) analysis revealed a total of eight flavonols (five quercetin glycosides and three kaempferol glycosides), varying in number and concentration in the different extracts. The total extracts of the entire flowers showed the highest flavonoid content (1210.4 µg/g dry extract) and the most complex flavonoid profile (eight compounds). All the extracts showed important antioxidant activity, which was not evidently associated with their flavonoid content. The total extracts of tepals showed the highest antioxidant properties (total antioxidant capacity, free radical scavenging activity, and iron reducing capacity: 30.2 mg ascorbic acid equivalents, EC50 = 0.074 µg/mL, and IC50 = 43.28 µg/mL, respectively). The flowers of A. durangensis represent an important source of antioxidant flavonols.

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

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.