Fruit collection

The first week of September 2019, Brosimum alicastrum fruits were collected in four locations in northern Campeche (Figure 1). The collection provenances are characterized by a coverage of medium sub-evergreen forest and high evergreen forest, with clayey soils, rainfall from 600 to 4 000 mm, with dry seasons of three to seven months, average annual temperature of 18 to 27 ºC and altitudes from 20 to 1 000 m (^{Inegi, 2021}). This region has been previously described by ^{Santillán-Fernández et al. (2021b)} as an area with a high natural distribution of B. alicastrum, homogeneous in its edaphoclimatic parameters.

Figure 1 Spatial location of the provenances for collecting fruits of Brosimum alicastrum Sw. in the state of Campeche. 

Fruit collection in X-Mabén and Hopelchén was carried out in areas disturbed by anthropic activities, unlike Sahcabchén and Calakmul whose collection was in less disturbed areas and even in the biosphere reserve, in the case of Calakmul. The collection of the fruits was made based on the methodology described by ^{Vallejos et al. (2010)}; 10 trees were selected per provenance with the best phenotypic characteristics (dasometric: greater height, greater dbh and straight stem), with a minimum distance between selected trees of 100 m; an average of 50 fruits was obtained from each individual, which were stored in sterilized plastic bags for transfer to the facilities of the Colegio de Postgraduados (Graduate Studies College), Campeche campus (Champotón, Campeche, Mexico). The fruit was pulped, exposing the seed, which was washed with water to remove pericarp residues.

Morphometry of B. alicastrum seeds

For the morphometric analysis of the seeds, those that were broken, vain, or with symptoms of damage by pests and diseases were removed. Four samples of 30 seeds per provenance (480 seeds in total) were randomly selected. The quantified variables were: length (cm), width (cm) and the length/width ratio, measured with a Vernier caliper (Mitutoyo 530-312CAL^TM); weight (g), calculated using an analytical balance (Wellish ^TM ); volume (cm³) and density (g cm^-3), with the water displacement method (^{Rashidi et al., 2007}) which consists of placing each seed inside a glass cylinder (Cylinder-250mL-2PK-Glass^TM) with a known volume of water (100 mL) and record the displaced volume.

The statistical differences between the morphometric variables by place of origin were determined with the R software (^{Venables and Smith, 2023}) through a means test by Tukey, with a 95 % reliability (α=0.05).

Seed viability analysis

Seed viability was determined with the tetrazolium chloride technique, which is used to differentiate metabolically active from inactive tissues (^{Berridge, 2005}). For this, a completely randomized experimental design was used, with four replicates of 30 seeds for each collection site (480 seeds). Since no previous works were found for B. alicastrum in which the viability of its seeds with tetrazolium is analyzed, the preparation of the solution and the evaluation of the staining patterns were made considering what has been cited for similar species.

A 1 % solution of 2,3,5-triphenyltetrazolium chloride (CTT) (Sigma T8877-10G) in sterile tridistilled water was used (^{Espitia-Camacho et al., 2017}). The seeds were cut following the equatorial axis and placed in 20 mL of CTT solution in a Petri dish; then, they were incubated (Labnet ^® modelo 222DS) in the dark at 28 °C for 4 h. The incubation time was previously determined from a 24-h staining kinetics with 4-h sampling intervals.

According to the methodology described by ^{Rodríguez et al. (2008)} and ^{Orantes-García et al. (2013)}, when the living cells of the seed embryo actively respire (they have a higher probability of germination), the CTT test stains them red, and in the case of dead cells, they keep the original color of the embryo. For this analysis, three categories were considered, according to the ^{Pérez-Mendoza (2018)} classification system: fully stained seeds (viable and vigorous embryos), partially stained seeds (viable embryos with medium vigor) and unstained seeds (non-viable embryos); in all cases they were counted and expressed as a percentage per replication and provenance. The statistical differences of the proportion of seeds that presented a category of staining by provenance were determined with the R software (^{Venables and Smith, 2023}) through a means test by Tukey with a 95 % (α=0.05) reliability.

Effect of storage time and temperature on seed viability

To analyze the effect of storage time and temperature on the viability of B. alicastrum seeds, a completely randomized experimental design was established with 24 replicates of five seeds for each storage temperature. The temperature gradients were defined based on preliminary studies for tropical trees such as B. alicastrum; in this regard, the temperature of 4 °C is one of the most evaluated in studies on the effect of storage temperature, while that of 28 °C is considered an ambient temperature within the range of 23 to 28 °C (^{Campbell-Martínez et al., 2022}; ^{Park et al., 2022}). For this experiment, the seeds from the place of origin that obtained the lowest percentages in the CTT test were selected, in order to study the effect in the worst scenarios.

The seeds were stored at 4 °C (Samsung side by side ^® refrigerator) and 28 °C (culture room) for a period of 92 days. Every 15 days, four replicates were selected randomly and without replacement by temperature gradient, to which the CTT test was applied to determine the percentage of fully stained seeds, partially stained seeds, and unstained seeds. Due to the number of replicates (24) and analysis period (15 days), the CTT test was performed six times. In addition, a germination test was carried out on 80 seeds (from the provenance that had the lowest percentages in the CTT test) stored for 30 days at 4 °C and 28 °C, four replicates of 10 seeds were made for each gradient of temperature, in each one the percentage of germination was obtained. The statistical differences of the proportion of seeds that presented a category of staining by analysis period, and the percentage of germination by temperature gradient were determined in the R software (^{Venables and Smith, 2023}) by means of a test of means by Tukey with a reliability of 95 % (α=0.05).

Morphometry of B. alicastrum seeds

An exploratory analysis of the collected seeds showed that they have a spherical or flattened shape, covered with a whitish-brown papyraceous testa that detaches itself when it dries, with a greenish seed with two asymmetric cotyledons (^{Brechú-Franco et al., 2021}) (Figure 2). When analyzing the morphometry of the seeds of the four provenances, the highest averages for length (cm) and width (cm) corresponded to the Hopelchén material and the lowest to Calakmul (Table 1). However, the Calakmul seeds had the best averages in volume (cm³) and weight (g).

A) Outer testa of brown-grey color that covers the seed, B) Seed without testa showing the endocarp, cotyledons and radicle of the embryo, and C) Seed with germination induced by high relative humidity. Endospermo = Endosperm; Cotiledón = Cotyledon; Radícula = Radicle.

Figure 2 Morphology of Brosimum alicastrum Sw. seeds. Seeds with different degrees of radicle development are illustrated. 

The Length (L)/ Width (A) ratio is one of the factors that define the shape of a seed: L/A<1 corresponds to a flattened seed, L/A=1 to a round one, and L/A>1 to an oblong one (^{Bezerra et al., 2015}). In the present study, although the statistical analyzes showed significant differences between the provenances, all the values of L/A are close to 1 (Table 1), that is, they correspond to round seeds, as previously cited (^{Brechú-Franco et al., 2021}).

The seeds with the highest volume were those from Hopelchén and Calakmul, which also registered the highest weights and densities (Table 1). These morphometric data are slightly different from those recorded by ^{Morales and Herrera (2009)}, who evaluated seeds of ramón trees located in Yucatán, with lengths less than 1.4 cm, widths greater than 1.8 cm, and average weights of 2.8 g. The difference in seed sizes may be due to genetic and environmental factors inherent to the regions where the populations of the species are located (^{Vergara et al., 2014}).

Tetrazolium chloride (CTT) staining test

Fully stained embryos and partially stained embryos were observed (Figure 3). According to ^{Mancipe-Murillo et al. (2018)}, staining intensity indicates a higher probability of germination. However, it should be considered that due to the recalcitrant property of B. alicastrum seeds, they are more likely to lose viability in the first 30 days (^{Magnitskiy and Plaza, 2007}).

Figure 3 Staining of Brosimum alicastrum Sw. seeds by the 2,3,5-triphenyltetrazolium chloride (CTT) test. 

Tukey’s means test by (α=0.05) showed that the X-Mabén and Hopelchén provenances had the seeds with the highest percentages of complete staining and were statistically different from those from Sahcabchén and Calakmul (Figure 4). The collection in X-Mabén and Hopelchén was carried out in areas disturbed by anthropogenic activities. According to ^{Romero-Saritama (2018)}, the viability of a seed is affected by various factors, including the age of the tree and the physiographic conditions of the collection area. However, forest species tend to develop greater resilience under unfavorable conditions, which helps explain why higher percentages of viability were recorded in disturbed sites.

Figure 4 Statistical differences in the staining percentage of Brosimum alicastrum Sw. seeds by provenance. Means with the same letter per column are not statistically different (Tukey, α=0.05). 

Effect of storage time and temperature on seed viability

Under room temperature conditions (28 °C) around 30 days, the percentage of partially stained seeds was higher than that of fully stained seeds, so it is considered that after 30 days the viability of the seeds is considerably reduced. In the case of seeds stored at 4 °C, the percentage of partially stained seeds exceeded the value of fully stained seeds after 70 days (Figure 5).

Figure 5 Effect of temperature and storage time on the viability of ramón (Brosimum alicastrum Sw.) seeds. 

From day 77, the seeds stored at different temperatures registered the lowest staining percentages. Due to the recalcitrant property of ramón seeds, a gradual increase in the percentages of partially stained seeds and non-stained seeds was expected for both storage temperatures throughout the evaluation period.

The effect of temperature and storage time on the viability of recalcitrant seeds has been described by ^{Magnitskiy and Plaza (2007)}, who postulated that temperature is the factor that most rapidly affects the increase in the viability of recalcitrant seeds. In this regard, ^{Valverde-Rodríguez et al. (2019)} pointed out that the longevity in the viability of recalcitrant seeds is greater with storage temperatures below 10 °C. However, given the null forest management applied to B. alicastrum, there is little information on the effect of temperature on the viability of its seeds (^{Vergara et al., 2014}).

Finally, the germination test of seeds stored for 30 days at 4 °C and 28 °C revealed 90 % germination in seeds stored at 4 °C, and 60 % for seeds stored at 28 °C (Table 2). The root system of the seeds stored at 4 °C was greater than that of the seeds at 28 °C (Figure 6). These results coincide with those obtained by ^{Del Amo et al. (2002)} who documented germination percentages higher than 75 % for the species. Although, unlike the study here described (controlled environment), this work was carried out in a nursery, with seeds without a prior storage period.

Figure 6 Brosimum alicastrum Sw. seeds germinated after 30 days of storage under controlled temperatures: 4 °C and 28 °C.