Seed quality tests

For each plant, 50 black seeds were selected in triplicate. Germination (^{Al-Ansari and Ksiksi, 2016}), imbibition, viability and moisture content were determined. For the weight of 1 000 seeds, 10 groups of 100 seeds of each species were formed. These tests were conducted in accordance with international rules for seed analysis (^{ISTA, 2016}).

Mineral analysis of seeds and soil

From each plant, seeds were ground to obtain a duplicate 0.5 g sample for wet digestion. A mixture of nitric acid and perchloric acid in a 2:1 ratio was used; once digested, the sample was made up to a volume of 50 ml with distilled water. The determination of the minerals Cu, Zn, Mn, Fe, Mg, Ca, Na and K in seeds was carried out by atomic absorption spectrophotometry (Thermo Scientific®, Model iCE 3000 Series) based on official methods of analysis (^{AOAC, 1990}). The content of S and P was quantified with an ultraviolet-visible spectrophotometer (GBC©, Cintra Model). The soil samples were analyzed based on the Official Mexican Standard (^{NOM-021-RECNAT-2000, 2002}). Texture, pH, electrical conductivity and organic matter content were determined. The quantified elements were Cu, Zn, Mn, Fe, Mg, Ca, Na, K, P and S.

Morphometric analysis

Photographs of 20 seeds for each species were taken; in each image, reference points were detected with the Makefan8 program, the TPSutil 1.44-2009 software, and the TPSdig2 2.32-2021 software and 10 landmarks were placed on each, which were analyzed with the MorphoJ software, version 1.08.02.

Statistical analysis

A test of means (Duncan, p ≤ 0.05) was performed with SAS Studio®, with species as treatments. Likewise, principal component analyses were performed with all variables.

Germination

A. potatorum and A. salmiana presented the highest values (65.5% and 58.3%, respectively). A. americana and A. nussaviorum had lower percentages (43.8% and 41.6%, respectively); no significant differences were detected between species (p > 0.05). Fernandez et al. (²⁰¹⁹) reported a high germination percentage for A. potatorum (>90%) without any pre-germination treatment, and they considered the time of seed harvest as a determining factor; that is, the shorter the storage time, the higher the germination percentage. The same germination pattern associated with storage time was observed by Martínez-Rodríguez et al. (²⁰²²) in A. marmorata, who reported germination of 90% and 77% for seeds with 12 and 18 months of storage, respectively. For their part, Ramírez-Tobias et al. (²⁰¹²) also reported a high value in the germination of seeds of A. salmiana (> 60%) and associated it with the temperature of the water used in the imbibition, less than 15 °C (Figure 1).

Figure 1 Percentage of germination, viability, imbibition, weight of one thousand seeds and moisture content in seeds of five Agave species. Different letters indicate statistically significant differences between species (p ≤ 0.05). 

Weight and moisture content

A. americana showed the highest weight of 1 000 seeds (11.2 g) significantly and A. nussaviorum had the lowest weight (4.8 g). This is because the first seeds are larger in size (length: 10.4 mm x width: 7.3 mm x thickness: 0.78 mm) and the second are smaller (length: 6.7 mm x width: 4.6 mm x thickness: 0.38 mm) (Figure 1). In their study, Hernández et al. (²⁰²³) reported a weight of 4.2 and 4.6 g for the same number of seeds of A. cupreata and 10.9 g for A. angustifolia; they also demonstrated a close association between seed weight and the amount of reserve substances, which could be reflected as an increase in germination capacity. The moisture content was less than 5% of the weight of each batch in the five species. A. salmiana and A. karwinskii showed significantly higher values, whereas A. potatorum presented the lowest value (Figure 1). The moisture percentage differs from that reported by Bejarano-León et al. (²⁰¹¹) for A. victoria-reginae (9%).

Imbibition and viability

A. nussaviorum presented the highest percentage of weight gain (389.4%), significantly higher than A. salmiana, which showed the lowest water absorption level (190.4%). This same behavior was observed in seeds of Opuntia sp. by Monroy-Vázquez et al. (²⁰¹⁷) (Figure 1). The highest significant viability value was in A. potatorum (72.8%). Authors such as Gutiérrez-Hernández et al. (²⁰²⁰) classify A. potatorum seeds as recalcitrant due to the loss of lipid content and the decrease in the percentage of germination due to the time of collection (Lechuga-Campuzano et al., ²⁰²⁵) (Figure 1).

Mineral analysis in seeds

It was observed that K was at a higher concentration in A. salmiana, A. potatorum and A. nussaviorum and that Ca was at a higher concentration in A. karwinskii and Mg in A. americana. S was in second-to-last place only in A. potatorum, and in the other species, it ranked last (Table 1). Micronutrients were found in the following decreasing order: Zn> Fe> Na> Mn> Cu, in A. salmiana, A. potatorum, A. nussaviorum, and A. americana. In A. karwinskii, the decreasing order was Zn> Na> Fe> Cu> Mn (Table 1). Gutiérrez-Hernández et al. (²⁰²⁰) relate the chemical composition of agave seeds to germination capacity and reproductive viability.

Mineral analysis and soil properties

The soils of Ocotlán de Morelos (ODM) and San Martín Mexicapam (SMM) had alkaline pH values (8.2 and 8.3), and Santa Cruz Mixtepec site 2 (SCM 2) registered a moderately acidic pH value. Electrical conductivity (EC) was higher in SMM and lower in SCM 2 and San Vicente Nuñú (SVN) (0.2-0.3 dS m^-1). Organic matter ranged between 1.4 and 4%. In texture, sandy-clay-loam soils predominated. Ca was found in concentrations similar to those reported by Ávila-Uribe et al. (²⁰²⁵) in soils of the district of Miahuatlán, Oaxaca. Calcium predominated in soils, followed by phosphorus and, to a lesser extent, potassium (Table 2). Ávila-Uribe et al. (²⁰²⁵) mention calcium as a predominant element in soils used for agave planting. Elements in optimal conditions provide an appropriate chemical environment for production (Echeverría et al., ²⁰²³). No relationship was found between the nutrient content of the soil and the nutrient content in the agave seeds (Figure 2).

Figure 2 Macro and microelements in seeds of five agave species and in the substrate associated with each species. 

Morphometry

The morphometric analysis presented 61.1% of accumulated variation with the first two components (PC1 38.7% and PC2 22.4%) in the defined space. A scattered grouping was presented for each species with overlapping ellipses (Figure 3). Points far from the central zone were observed, which suggests the existence of differences in shape, but with insufficient loading to separate each species. This coincides with what was found by Hernández-Castro et al. (²⁰²¹) and Villanueva-Castillo et al. (²⁰²¹) in morphometric studies, where they indicate variation in agave seeds.

Figure 3 Principal components of the seed morphometry of five Agave species. 

Principal component analysis

With the principal component analysis (PCA) applied to the quality variables, mineral content in seed and minerals in soil, it was demonstrated that the first two components reflected 71.4% of the total variance, and with the third component 89% was reached (PC1: 40.5%, PC2: 30.9%, PC3: 17.6%). PC1 was made up of the variables: copper in the substrate, sand, weight of 1 000 seeds, EC and iron content in the substrate; it separates A. karwinskii from the rest of the species, as it is mostly influenced by Cu and sand in the soil; this species was the one that presented a high concentration of this element (22.7 mg kg^-1); in the positive quadrants, the species of agaves collected in the Central Valleys region were concentrated, and in the negative quadrants, the two species from the Mixteca region. PC2 is composed of potassium in substrate, moisture percentage in seed, copper in seed and sodium in substrate. A. americana and A. potatorum grouped in the quadrant with negative values of component 2, since they were the species with the lowest moisture content and intermediate Cu concentration in the soil (Figure 4).

Figure 4 Dispersion of the species based on principal components 1 and 2, considering all quality variables, minerals in substrate and seed and physicochemical properties of the soil.