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Introduction
The genus Agave is endemic to America and is the largest and most diverse of the Asparagaceae family with 210 species of angiosperm and monocotyledonous plants; of these, 159 are native to Mexico (García-Mendoza A, 2002). An example is Agave salmiana that is distributed mainly in the Mexican states of Tlaxcala, Puebla, Mexico, and Hidalgo (Molina-Vega et al., 2021). This species is used in the elaboration of traditional foods, fibers production, pulque and mezcal, saponins, fructans and in obtaining biofuels (Álvarez-Chávez et al., 2021). It is a semelparous species and takes 10 to 15 years to reproduce sexually. In comparison, asexual reproduction takes about five years with the formation of offshoots, being this form of propagation the most used for commercial purposes. However, this has led to the incidence of pests and diseases due to the decrease in genetic variability (Puente-Garza et al., 2015). Plant tissue culture can be a biotechnological tool of great help to preserve the genetic diversity of this species and take advantage of its diverse uses. In vitro propagation of Agave by the proliferation of axillary buds has been reported in different species between them A. salmiana subspecies crassispina (Nava-Cruza et al., 2015).
Somatic embryogenesis (SE) is an alternative to the mass culture of plants, which is characterized as a pathway for the regeneration of whole plants from a bipolar structure derived from somatic cells. This process is carried out through the induction of plant cells to dedifferentiate and enter a new biological development program and the subsequent expression and germination of the resulting embryos (Gutirrez-Mora et al., 2012), by using growth regulators such as auxins and cytokinins. Auxins being an essential factor for the initial cell polarization and asymmetrical first division (Asghar et al., 2023).
Some of the factors that have been studied to carry out the embryogenesis process in Agave are the type of explant used, the combination and hormonal concentration, osmotic conditions, concentration of amino acids and salts, etc. In the SE of Agave spp, different explants have been used: leaf segments, in A. victoria-reginae (Rodríguez-Garay et al., 1996), A. salmiana (Flores Benítez et al., 2007), A. vera-cruz (Tejavathi et al., 2007) and A. tequilana Weber, blue cultivar (Portillo et al., 2007; Rodríguez-Sahagún et al., 2011); stem tissues in A. victoria-reginae Moore (Martínez-Palacios et al., 2003) and A. fourcroydes Lem (Monja-Mio & Robert, 2016); tissue of bulbil apices in A. sisalana (Nikam et al., 2003) zygotic embryos in A. angustifolia Haw (Arzate-Fernández & Mejía-Franco, 2011; Reyes-Díaz et al., 2017) The combination of hormones is another of the parameters that has been manipulated to induce ES in different species of Agave, being 2,4-D auxin in combination with other hormones the common one of all. Such as the study carried out on Agave marmorata in which it was combined with BAP, using mature seeds as explants (Alvarez-Aragón et al., 2020).
Therefore, the use of somatic embryogenesis using zygotic embryos as explants of Agave salmiana Otto ex Salm-Dyck subsp. salmiana, a product of in-casa cross-pollination, could be a useful alternative in the production of plants, conservation of genetic diversity, and for the genetic improvement of this species.
Materials and methods
Plant material.In the April-June 2017 period, panicles and inflorescences of Agave salmiana Otto ex Salm-Dyck subsp. salmiana in the stage of sexual reproduction were collected in the municipalities El Arenal (N: 20º 10’ 39.1” W: 98º 53’ 56.1” 2217 m.s.n.m , N: 20º 10’ 36.8” W: 98º 53’ 54.5” 2239 m.s.n.m., N: 20º 10’ 37.1” W: 98º 53’ 51.5” 2246 m.s.n.m, N: 20º 10’ 38.4” W: 98º 53’ 53.1” 2267 m.s.n.m), Cardonal (N: 48º 78’ 94.0” W: 22º 77’ 97.3” 1961 m.s.n.m, N:
20º 36’ 03.8” W: 99º 06’ 40.0” 1953 m.s.n.m, N: 20º 36’ 03.8” W: 99º 06’ 40.0” 1953 m.s.n.m.), San Agustín Tlaxiaca (N: 51º 40’ 99” W: 22º 18’ 749” 2624 m.s.n.m, N: 50º 96’ 66” W: 22º 25’ 077” 2301 m.s.n.m) and Emiliano Zapata (N: 19º 39’ 20.2” W: 98º 33’ 10.4” 2502 m.s.n.m). The taxonomic classification was made according to dichotomous keys (Little & Gentry, 1983). The plant material was kept in the laboratory in containers with clean water (flower vases) at an average room temperature of 21-23 ºC.
Pollen viability tests. Mature flowers were emasculated, and anthers were collected and placed on a sheet of bond paper at room temperature, for three days, to carry out the pollen harvest and its subsequent viability test. The viability of pollen grains was observed through the formation and growth of the pollen tube. The pollen grains were placed in Petri dishes (100 mm x 15 mm) with 25 mL of culture medium containing 102.69 gl-1 sucrose, 0.02 g L-1 boric acid, 0.287 g L-1 calcium nitrate and 5 g L-1 agar, subsequently incubated for 30 minutes at 25 ºC (López Díaz & Rodríguez Garay, 2008).
In-casa cross-pollination. After checking the viability of pollen and when the flower was receptive with the presence of a pollen drop in the stigma, pollination was carried out as described by Rodríguez-Garay et al., (2018).
Callus formation. After 30 to 40 days post-pollination, the fruits were collected from the panicles and disinfected with 96% v/v alcohol and flamed within a laminar flow hood, then opened with a sterile scalpel and extracted the immature viable seeds (black color). The immature zygotic embryos were extracted with the help of a stereoscopic microscope, fine forceps, and a scalpel under aseptic conditions (Rodríguez-Garay et al., 2018). These immature zygotic embryos were used as explants. Embryos were placed in Petri dishes (100 mm x 15 mm) containing 25 mL of MS medium; supplemented with 30 g L-1 sucrose, L2 vitamins (Phillips & Collins, 1979), 8 g l-1 agar, 9.0 μM 2,4-D (Sigma-Aldrich D8407) and 1.3 μM of BAP (Sigma-Aldrich B3408) (Portillo et al., 2007). The pH was adjusted to 5.8 prior sterilization in an autoclave. Five explants (immature zygotic embryos) were seeded in each Petri dish and incubated at 27 °C ± 2 °C for 40 days under total darkness.
Experimental method
To increase the number of genotypes that could form somatic embryos, the explants that formed calluses were distributed in two experiments that differed by the use of plant growth regulators and their concentration.
Experiment 1: A 3 × 3 bifactorial experiment was designed: The first factor was 2,4-D in concentrations of 4.5, 9.0, and 13.5 μM, in combination with the second factor BAP in concentrations of 0.4, 1.3 and 2.2 μM (Portillo et al., 2007).
Experiment 2: A 3 × 3 bifactorial experiment was designed, using the auxin αnaphthaleneacetic acid (NAA) (Sigma-Aldrich N0640) in concentrations of 1.34, 2.7 and 4.06 μM, and the cytokinin BAP in concentrations of 2.5, 5.0 and 7.05 μM (Flores-Benítez et al., 2007).
Induction medium
For each of the experiments, MS medium (Murashige & Skoog, 1962), supplemented with 30 g L-1 sucrose, L2 vitamins (Phillips & Collins, 1979) and 8 g l-1 agar was used, with plant growth regulator, respectively.
Each experiment resulted in nine treatments, with eight and seven replications respectively for both experiments. Each experimental replication consisted of four calluses per Petri dish (nodular calluses, reliable and beige in color). They were incubated at 27ºC ± 2ºC for 40 days under total darkness. After this period, the number of calluses that could express somatic embryogenesis was quantified, as well as the number of somatic embryos formed in each callus before its maturation and germination.
Statistical analysis. A unifactorial analysis of variance (ANOVA) with a level of significance of 0.05 was performed, using the PAleontological STatistics program version 3.20 (Hammer & Harper, Norway).
The response surface methodology was used with the help of Design-expert software version 7.1.5 (Stat-Ease, Minneapolis), adjusting the data to a Miscellaneous 32 design with 4 points in the center to estimate the optimal conditions within the experimental area, (Table 1).
Table 1 Experimental range of the levels of the two independent variables in terms of real values in the experiments of induction of somatic embryogenesis in Agave salmiana Otto ex Salm-Dyck subsp. salmiana.
| Variables | Simbol | Coded variable levels -1 0 +1 | ||
|---|---|---|---|---|
| 6-bencilaminopurine (BAP) | X1 | 0.4 µM | 1.3 µM | 2.2 µM |
| 2,4-diclorofenoxiacetic acid (2,4-D) | X2 | 4.5 µM | 9.0 µM 13.5 µM | |
Maturation and germination of somatic embryos. The somatic embryos generated in both experiments were transferred to Petri dishes (100 x 15 mm) containing 25 mL of MS medium with the addition of 30 g L-1 sucrose, 500 mg L-1 glutamine, 250 mg L-1 casein hydrolysate and supplemented with L2 vitamins, and 6 g L-1 phytagel (Sigma P8169) (Santacruz-Ruvalcaba et al., 1998). For this purpose, eight embryos per Petri dish were incubated at 27ºC ± 2ºC with a photoperiod of 16 h light for 60 days, to quantify the percentage of germination.
Propagation. Two germinated plants without roots were placed in each bottle containing 100 mL of MS medium supplemented with 30 g L-1 sucrose, 44.3 μM BAP, 0.18 μM 2,4-D, and 8 g L-1 agar (Santacruz-Ruvalcaba et al., 1999). The pH was adjusted to 5.8 prior sterilization in an autoclave. The cultures were incubated at 27 ° C ± 2 ° C for 60 days with a photoperiod of 16 h light to generate the formation of multiple shoots.
For rooting, the same light and temperature conditions were used; however, the culture medium did not contain plant growth regulators and with a salts reduction of 50% of the MS medium. Finally, the rooted plants were transplanted to vessels made of expanded polystyrene (EPS) with an equivalent mixture of peat moss, vermiculite, and soil (1: 1: 1). The survival rate of plants was quantified 21 days after the initiation of incubation.
Results
Pollen viability tests in most of the collected parent plants indicated that 90% of the collected pollen could form a pollen tube. In Figure 1, the general process of the formation of zygotic embryos is depicted. 89 fruits were formed after 30 to 40 days as a product of the in-casa pollination. 25 out of 89 fruits were selected randomly, of which 1608 immature zygotic embryos were rescued. Seven of the rescued embryos had two cotyledons. 744 (46.3%) out of 1608 immature zygotic embryos could form calli, and 30 (1.8%) of them expressed somatic embryogenesis after 40 days of incubation in the induction medium; however, it was observed that only explant formed somatic embryos within 20 days.
Fig. 1 Formation of zygotic embryos by in-casa cross-pollination of Agave salmiana Otto ex Salm-Dyck subsp. salmiana. a) Flowers ready to be emasculated (Bar = 1cm). b) Mature flower ready to be pollinated (Bar = 1cm). c) Mature and open anthers for pollen harvest. d) Fruit ripening (Bar = 1cm). e) Viable (black color) and non-viable (white color) seeds (Bar = 5mm). f) Monocotyledon zygotic embryo (Bar = 1mm). g) Dicotyledon zygotic embryo (Bar = 0.5mm).
The analysis of variance (ANOVA) for the results of experiment 1 showed that there was a statistically significant difference between the treatments, after quantifying the number of calli that expressed somatic embryogenesis (p = 0.040) (Table 2). The response surface methodology predicted the number of calli expressed by somatic embryogenesis as a function of BAP and 2,4-D, and the results were adjusted to a second-order equation:
Table 2 Number of calli that expressed somatic embryogenesis of Agave salmiana Otto ex Salm-Dyck subsp. salmiana in nine treatments with different concentrations of growth regulators: 2,4-D / BAP.
| BAP / 2,4-D | 4.5 µM | 9.0 µM | 13.5 µM |
|---|---|---|---|
| 0.4 µM | 1.750 ± 0.250 a | 1.875 ± 0.479
ab
1.500 ± 0.189 ac |
1.125 ± 0.227
c 1.125 ± 0.227 c |
| 1.3 µM | 1.250 ± 0.250 c | ||
| 2.2 µM | 1.250 ± 0.313 c | 1.000 ± 0.267 bc | 0.500 ± 0.189 c |
*Number of calluses per Petri dish (Maximum value 4) ± standard error
*Values with the same letter do not have a statistically significant difference (p <0.05) using the LSD test.
The model predicted that the optimal concentration of plant growth regulators within the experimental area was 0.44 μM BAP and 7.39 μM 2,4-D with a response of 1.812 calli expressing SE. According to the model, the factor with the highest level of significance was auxin 2,4-D (p = 0.003). Figure 2 shows the response surface as a function of different concentrations of BAP and 2,4-D. It was found that there was no significant statistical difference between treatments (p = 0.456) when quantifying the number of somatic embryos formed.
Fig. 2 Contour plot of the surface for the number of embryogenic calluses (Z) using the interaction of the variables (X1) cytokinin BAP and (X2) auxin 2,4-D. Having as answer the number of calluses formed per Petri dish (The coding of the data is shown in Table 1).
In experiment 2, the ANOVA demonstrated that there was no statistically significant difference between treatments when quantifying the number of calli that expressed somatic embryogenesis (p = 0.315), as well as in the number of somatic embryos formed (p = 0.617). However, when comparing the results of both experiments, a statistically significant difference was observed in the number of genotypes (each zygotic embryo) that expressed somatic embryogenesis (p = 0.035), and in the number of somatic embryos formed (p = 0.000) (Table 3).
Table 3 Comparison of two experiments with different auxin in combination with BAP for the SE of Agave salmiana Otto ex Salm-Dyck subsp. salmiana.
| Experiment | Number of calluses per Petri dish | Number of somatic embryos per callus |
|---|---|---|
| (1) 2,4-D / BAP | 1.264 ± 0.137 | 42.419 ± 5.858 |
| (2) NAA / BAP | 0.873 ± 0.099 | 10.698 ± 1.925 |
Number of calluses per Petri dish have a Maximum value 4 ± standard error.
The germination percentage of the somatic embryos in experiments 1 and 2 was 97.11 ± 0.98% and 73.03 ± 5.43%, respectively. There was a 95% survival rate as a result of the acclimatization. Figure 3 shows some images of the process.
Fig. 3 In vitro propagation of Agave salmiana Otto ex Salm-Dyck subsp. salmiana by somatic embryogenesis. a) Dicotyledonar somatic embryo (heart) (Bar = 0.5 mm). b) Globular and torpedo structures seen under the stereo microscope on the surface of the embryogenic callus. (Bar = 0.5 mm). c) Germination of a somatic embryo (Bar = 5 mm). d) Bud formation (Bar = 1 cm). e) Rooted plant ready to be transplanted to soil (Bar = 1cm). f) Plant regenerated in substrate after 21 days (Bar = 1cm)
Discussion
In-casa cross-pollination could be a useful tool for the conservation of the genetic diversity of agave plants of the same species and variety. The previous procedure can be carried out in the laboratory under controlled conditions for the production of hybrids through sexual reproduction (Rodríguez-Garay et al., 2018), avoiding open pollination of unknown species/varieties. The results obtained in this proposal allowed the rescue of zygotic embryos, which were used as explants in the process of somatic embryogenesis, facilitating the selection of phenotypes and the production of cell lines with genetic diversity of Agave salmiana Otto ex Salm-Dyck subsp. salmiana for conservation and propagation purposes (Angeles Vázquez et al., 2018).
The genus Agave is composed of monocotyledonous plants, which are characterized by having only one cotyledon in their embryonic phase (Rodríguez-Garay, 2016). However, in this work, embryos with two cotyledons were found, similar to those reported for Agave tequilana with an incidence of 4% (Ayala-González et al., 2014). The presence of more than one cotyledon in this genus represents an opportunity to study the mechanisms that gave rise to its unique cotyledon since the phylogenetic groups of monocots and dicots are closely related (Ayala-González et al., 2014).
The embryogenic calli obtained in this work showed beige, yellow, and green colorations, with friable and compact textures, many of them nodular. These characteristics have been found in embryogenic calli of Agave victoria-reginae (Martínez-Palacios et al., 2003), Agave sisalana (Nikam et al., 2003), and Agave tequilana (Portillo et al., 2007), showing a specific morphology for embryogenic callus where they are generally friable and nodular of a beige coloration. After 20 days of incubation in the induction medium, somatic embryos were observed in some of the explants. This incubation period is shorter than that reported for other Agave species such as A. victoria-reginae (Rodríguez-Garay et al., 1996), A. tequilana (Rodríguez-Sahagún et al., 2011), A. fourcroydes (Monja-Mio & Robert, 2013), A. sisalana (Carneiro et al., 2014). This period is attributed to the type of explant used, in which the suspensor plays a significant role since several studies indicate that it is made up of totipotent cells that have the possibility of producing somatic embryos (Liu et al., 2015). In this work, it was observed that in most of the explants (zygotic embryos), the formation of embryogenic callus was initiated in the suspensor. As can be seen in Figure 3. At the end of 40 days of incubation, indirect embryogenesis occurred, which has been described in most Agave species (Arzate-Fernández & Mejía-Franco, 2011).
Recent studies have reported the use of zygotic embryos as explants to carry out the process of somatic embryogenesis in the Agave genus, rescued from seeds formed by open pollination, such as the one reported for A. angustifolia, A. cupreata and A. salmiana, the which were subjected to osmotic stress induced by the application of compounds such as polyethylene glycol (PEG) and abscisic acid (ABA), reaching percentages of embryogenic callus formation above 50% (Hernández-Solis et al., 2023). Other factors have also been explored, such as the concentration of vitamins in the induction medium, such as Thiamine, using the same type of explants (embryonic axes) in A. angustifolia, demonstrating that the higher the concentration of this compound, it is possible to obtain a greater number of embryos somatic (Reyes-Díaz et al., 2020.)
The addition of plant growth regulators in the in vitro culture indirectly causes local variations in the auxin concentration of the explant, possibly triggering the de novo synthesis and relocation of the endogenous auxin. Also, it has been demonstrated that the auxin analog 2,4- D can act directly as an inducer of genes related to stress (Fehér, 2015). Besides, it has been shown that it causes hypermethylation of DNA, inducing the expression of the MET1 gene that leads to the reprogramming of the genome and the acquisition of embryogenic competence (Leljak-Levanić et al., 2004).
The auxin 2,4-D is the most used analog in SE protocols in different species, including the genus Agave (Monja-Mio & Robert, 2016). Finally, the results obtained in this work show that the SE of Agave salmiana Otto ex Salm-Dyck subsp. salmiana is favored by relatively high concentrations of 2,4-D with the addition of low concentrations of the cytokinin BAP, as established for other Agave species (Delgado-Aceves et al., 2021).
Conclusions
The use of complete inflorescence of Agave salmiana Otto ex Salm-Dyck subsp. salmiana in-casa cross-pollination allowed obtaining viable zygotic embryos after 30 days post-pollination, which presented embryogenic capacity when using an MS medium supplemented with vitamins L2, 2,4-D and ANA in combination with the cytokinin BAP. The presence of 2,4-D auxin compared to ANA, in a 40-day incubation period in the dark, increased the number of somatic embryos formed and the number of embryogenic calli. The proposal to carry out in-casa crosspollination facilitates the conservation of diversity through the controlled pollination of a certain species or variety of Agave. Furthermore, it is possible to induce and express the embryogenic capacity of zygotic embryos of Agave salmiana Otto ex Salm-Dyck subsp. salmiana, this being an alternative to help the genetic conservation of Agave plants in Mexico.
