¹Laboratorio de Biotecnología Vegetal. Tecnológico Nacional de México, Campus Tuxtla Gutiérrez. México.

²Campo Experimental Huimanguillo.Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias. México.

Sideroxylon capiri (Tempisque) is a tropical tree of the Sapotaceae family, considered of great importance from its biological role, traditional use and high content of secondary metabolites of interest. However, both natural and anthropogenic factors have led to its classification as a threatened species in Mexico according to the NOM-059-SEMARNAT-2010 regulation. Therefore, the development of an in vitro propagation protocol for the conservation of this species is crucial. Within this context, the effect of the growth regulators 6-Benzylaminopurine (BAP) and Picloram on the formation, proliferation and development of embryogenic callus and/or pro-embryogenic structures, as well as the effect of L-Cysteine on the reduction of oxidation in callus which were exposed to UV-B irradiation, was evaluated. Results show a positive response to the application of the combination of BAP and Picloram at concentrations of 4 and 2 mg L^-1 respectively, on growth callus. Additionally, the generation of pro-embryogenic callus was observed at concentrations of 0.5 and 1 mg L^-1 of BAP and 1.5 and 2 mg L^-1 of Picloram. Furthermore, a culture medium was standardized that reduced callus oxidation and browning by using 100 to 200 mg L^-1 of L-Cysteine.

Key words: Somatic embryogenesis, total phenols, L-Cysteine, recalcitrance, regeneration, Tempisque.

Sideroxylon capiri (tempisque) es un árbol tropical de la familia Sapotaceae, considerado de suma importancia debido a su papel biológico, uso tradicional y alto contenido de metabolitos secundarios de interés. No obstante, debido a factores tanto naturales como antropogénicos, en México se ha catalogado como una especie amenazada de acuerdo con la NOM-059-SEMARNAT-2010. Por lo anterior, es crucial el desarrollo de un protocolo de propagación in vitro para su conservación. En este contexto, se evaluó el efecto de los reguladores de crecimiento 6-Bencilaminopurina (BAP) y Picloram en la formación, proliferación y desarrollo de callo embriogénico y estructuras proembriogénicas, así como el efecto de la L-Cisteína en la reducción de la oxidación en callos, los cuales fueron expuestos a irradiación UV-B. Los resultados mostraron una respuesta positiva ante la aplicación de la combinación de BAP y Picloram en concentraciones de 4 y 2 mg L^-1,respectivamente sobre el crecimiento de los callos. Además, se observó la generación de callos proembriogénicos en concentraciones de 0.5 y 1 mg L^-1 de BAP y 1.5 y 2 mg L^-1de Picloram. Adicionalmente, se logró estandarizar un medio de cultivo que disminuyó los procesos de oxidación y pardeamiento en los callos mediante el uso de L-Cisteína en concentraciones de 100 a 200 mg L^-1.

Palabra clave: Embriogénesis somática, fenoles totales, L-Cisteína, recalcitrancia, regeneración, tempisque.

Sideroxylon capiri (A. DC.) Pittier, better known by its common name as “tempisque”, is a timber tree that belongs to the Sapotaceae family. Its distinctive morphological characteristics include a cylindrical stem and elongated green leaves; the fruits of tempisque are ovoid in shape and contain a single seed with a hard seed (Newman, 2008). Tempisque is distributed from Mexico to Panama, except for Belize; its presence is fundamental at an environmental and ecological level in the ecosystems where it lives (García and Di Stefano, 2005).

The S. capiri ecosystem involves complex interactions between humans and animals. The leaves and fruits of this species are used as a food source by a wide variety of animals (Hiramatsu et al., 2009; Almazán-Núñez et al., 2021). On the other hand, in the regions where it grows, residents use its fruits in the preparation of condiments for gastronomy; furthermore, its wood is widely used not only as firewood, but also as material for the construction of homes and fences (Díaz et al., 2011; Lazos-Monterrosa et al., 2015). Another use that stands out in some regions of Central America is its medicinal application, since the bark is used to treat kidney infections (Martínez-Silvestre et al., 2022), which confirms its multifaceted function in these societies.

In recent years, interest in tempisque has grown due to its potential use as a source of secondary metabolites. Robles-García et al. (2016) reported the presence of phenols and flavonoids in methanolic extracts of the leaves. Recently, Martinez-Silvestre et al. (2022), demonstrated that the stimulation of tempisque callus with UV-B light and laser significantly increases the concentration of phenols and flavonoids in callus, which distinguishes the potential of this species as a natural source of these compounds for their application in biotechnology. These compounds are highly valued in various industries such as pharmaceutical, food and cosmetics, for their antioxidant, anti-inflammatory and anti-cancer activity (Mierziak et al., 2014; Martínez-Silvestre et al., 2022). Many of them are produced in response to stress, although their presence in vivo can vary significantly (Li et al., 2020).

However, promoting its use requires rethinking the management of natural populations and plantations, since in recent decades a worrying reduction has been observed in the population of tempisque trees in their natural habitat, to the extent that it has been classified in Mexico as a threatened species in NOM-059-SEMARNAT-2010 (Semarnat, 2010). This is attributed to various anthropogenic causes, among which excessive logging, the invasion of its ecosystem and the poor management of the species stand out, combined with the pressures exerted by climate change (Cordero et al., 2003; Antúnez, 2022). Due to the above, the search for conventional propagation strategies to promote seed germination has intensified, such as mechanical and chemical scarification and imbibition, which have not managed to significantly increase germination rates, since they are reported below 30 % (Lazos-Monterrosa et al., 2015).

The application of biotechnological techniques to propagate woody species, such as plant tissue culture, presents challenges due to the low response of tissues to in vitro manipulations and the high rate of phenolization and oxidation of explants (Mihai et al., 2023). The high oxidation rate of the explants used (leaf and seeds) in the induction process in the tempisque prevents them from generating reactions that trigger the morphogenic response in vitro, therefore the establishment of an efficient regeneration system in which obtaining complete plants has not been possible (Martínez-Silvestre et al., 2022). To date there is no record of in vitro regeneration of S. capiri, Martinez-Silvestri et al. (2022) described obtaining non-friable and compact calli without being able to regenerate shoots and/or somatic embryos due to the high rate of phenolization and necrosis that the calli presented after 30 days of culture. In this same work, the callus obtained were stimulated by UV-B radiation with the aim of obtaining secondary metabolites of interest in in vitro systems. An option to solve or reduce the problem of oxidation in in vitro systems is the use of antioxidants such as L-Cysteine added to the culture medium, which represents an alternative for the control of oxidative processes in explants thanks to the effect of this amino acid as a reducing agent (Vásquez-Hernández et al., 2021).

The objective of this research was to evaluate the effect of 6-Benzylaminopurine and Picloram on the formation, proliferation and development of embryogenic callus and/or pro-embryogenic structures, as well as the effect of L-Cysteine on the reduction of callus oxidation of tempisque previously stimulated with UV-B radiation.

Non-irradiated and UV-B light-irradiated callus from tempisque leaf explants obtained by Martínez-Silvestre et al. (2022) were used in a previous study. The irradiated calli had been exposed to UV-B light for 0, 5, 10 and 15 min week^-1, for 2 weeks. Subsequently, they were maintained in solid Murashige and Skoog (MS) medium supplemented with 4 mg L^-1 of 6-Benzylaminopurine (BAP) (Sigma Aldrich^®, Germany), 30 g L^-1 of sucrose, 2.5 g L^-1 of Phytagel (Sigma Aldrich^®, Germany) at a pH of 5.7 for 2 months, and replanting was carried out every 15 days. All the material was preserved in a culture room at 25+2 °C, with a 16 h light-8 h dark photoperiod and a light intensity of 35 µmol m² s^-1.

To assess the effect of L-Cysteine (Sigma Aldrich^®, Germany) and UV-B radiation on oxidation in callus, a multifactorial experimental design was used. The first factor was the concentration of L-Cysteine, with 5 levels, which were: 0, 25, 50, 100 and 200 mg L^-1. The second factor was the exposure time, with 4 levels that were 0, 5, 10 and 15 min week^-1. Each experimental unit contained a callus and all treatments were carried out with four replications each. Calli were cultured in 15×20 mm test tubes (Pyrex^®, 9800-15) containing 10 mL of MS culture medium described above, supplemented with the corresponding concentrations of L-Cysteine. The callus were kept for 30 days in the previously mentioned conditions.

The oxidation percentage was calculated by dividing the callus into four parts and the presence of oxidation in each quadrant was taken as a positive value. The data were analyzed using a multifactorial ANOVA and the means were compared using the Tukey test (p<0.05) using the Statgraphics Centurion XVI^® statistical software (StatPoint Technologies Inc., 2010).

To obtain methanolic extracts the methodology of Martínez-Silvestre et al. (2022) with some modifications was carried out. To do this, 1 g of fresh callus from each L-Cysteine exposure treatment was macerated with liquid nitrogen in a mortar until obtaining a fine powder. 10 mL of 99 % v/v methanol were added and maceration continued for 1 min and then allowed to rest for 10 min. The extracts were placed in 15 mL Falcon^® tubes and allowed to settle for 24 h at a temperature of 25 °C. Subsequently, they were sonicated (CO-Z^®, PS-30) for 20 min and centrifuged (Eppendorf^®, 5430 R) at 3 500 rpm for 10 min at 4 °C. The supernatant was recovered from each sample and concentrated in a rotary evaporator (BUCHI^® R-100) and then resuspended in 2 mL of methanol. The final extract was placed in Eppendorf^® tubes and stored at 4 °C until use. To avoid photooxidation, all steps were carried out in the dark or covering the materials in aluminum foil to limit light exposure.

The determination of total phenols was carried out based on the methodology described by Koufan et al. (2020) with some modifications. A standard curve of gallic acid (Fermont^®, Mexico) was performed, dilutions of 0.1 mg mL^-1 were made from a stock solution (1 mg mL^-1). For each sample, 2.1 mL of distilled water, 0.250 µL of Folin-Ciocalteu reagent (Sigma Aldrich^®, Germany) and 50 µL of extract were used, which were homogenized in a vortex (IKA^®, Vortex 2). Subsequently, 0.5 mL of 20 % (w/v) sodium carbonate was added, homogenized again and allowed to react for 2 h at 24 °C in complete darkness to avoid photooxidation. Finally, the absorbance was measured at 765 nm in a UV-VIS spectrophotometer (Beckman Coulter^®, DU 730) using methanol as a blank. The results were expressed as equivalents in mg of gallic acid per gram (mg GAE g^-1). Three repetitions were performed per sample and the data obtained were analyzed using a simple ANOVA with the Statgraphics Centurion XIV^® statistical software (StatPoint Technologies Inc., 2010).

Two treatments were tested for the induction of somatic embryos, and callus previously stimulated with 2 mg L^-1 of Picloram (Sigma Aldrich^®, Germany) were used as explants. The first treatment (IN1) consisted of MS medium supplemented with 0.5 mg L^-1 of BAP and 1.5 mg L^-1 of Picloram. The second treatment (IN2) was MS medium supplemented with 1 mg L^-1 of BAP and 2 mg L^-1 of Picloram. For both treatments, 25 mg L^-1 of L-Cysteine, 30 g L^-1 of sucrose and 2.5 g L^-1 of Phytagel were added as a gelling agent. The evaluation period was 30 days and replanting was done 15 days after starting the crop. The response variables were the number of pro-embryos g^-1 of callus. The pro-embryos were considered as friable, smooth structures, with defined and semicircular edges, white-yellow in color. The data were analyzed by a simple ANOVA and comparison of means using the Tukey test (p£0.05) using the Statgraphics Centurion XIV^® statistical software (StatPoint Technologies Inc., 2010).

The addition of L-Cysteine to the culture medium resulted in a reduction in callus oxidation and promoted callus development. The statistical analysis showed that there are significant differences between the concentrations of L-Cysteine supplied to the culture medium at 15 days (p=0.0009) and at 30 days (p=0.0001). Furthermore, a multivariate ANOVA demonstrated that both factors and their interaction have significant effects on oxidation, in particular the effect of L-Cysteine concentrations (p=0.0002) and UV-B light exposure times (p=0.0043) in contrast to the effect of the interaction between factors (p=0.076). Table 1 shows that the highest percentage of oxidation was verified in the callus without the addition of L-Cysteine to the culture medium and exposed to a time of 5 min week^-1 of radiation. In contrast, the lowest percentages of oxidation at 15 days were observed in calli exposed to 10 and 15 min week^-1 of radiation, which were developed in MS culture medium supplemented with 100 and 200 mg L^-1 of L-Cysteine (Table 1). However, after 30 days, 100 % reduction in oxidation was obtained for all exposure times to UV-B light with the addition of 200 mg L^-1 of L-Cysteine, which was the best treatment.

Table 1. Effect of L-Cysteine on the oxidation of Sideroxylon capiri (A. DC.) Pittier callus after 15 and 30 days of culture.

L-Cysteine (mg L^-1)	Radiation frequency (min week^-1)	% oxidation
L-Cysteine (mg L^-1)	Radiation frequency (min week^-1)	15 days	30 days
0	0	50±20.41^ab	50±0.00^b
	5	75±14.43^b	100±0.00^c
	10	25±14.43^ab	50±20.41^b
	15	25±14.43^ab	25±0.00^ab
25	0	25±14.43^ab	25±14.43^ab
	5	50±20.41^ab	25±17.68^ab
	10	25±0.00^ab	25±0.00^ab
	15	25±0.00^ab	0±0.00^a
50	0	50±17.68^ab	50±17.68^b
	5	25±0.00^ab	25±0.00^ab
	10	25±0.00^ab	0±0.00^a
	15	25±10.21^ab	0±0.00^a
100	0	25±10.21^ab	25±10.21^ab
	5	0±0.00^a	0±0.00^a
	10	0±0.00^a	0±0.00^a
	15	25±0.00^ab	0±0.00^a
200	0	25±0.00^ab	0±0.00^a
	5	25±0.00^ab	0±0.00^a
	10	0±0.00^a	0±0.00^a
	15	0±0.00^a	0±0.00^a

Different letters indicate a significant difference between treatments according to the Tukey test (p≤0.05). Statistical analyzes were performed separately for each evaluation time.

Variations in the color and consistency of the callus were observed throughout the cultivation process, which are related to the concentration of L-Cysteine. Specifically, it was detected that an increase in the concentration of L-Cysteine resulted in a slight loss of pigmentation in the callus, in addition to causing changes in their texture, from a compact to a friable consistency (Figure 1).

A-E = Callus 15 days after culture establishment in MS medium; F-J = Callus 30 days after culture establishment on MS medium; A and F = Control callus; B and G = 25 mg L^-1; C and H = 50 mg L^-1; D and I = 100 mg L^-1; E and J = 200 mg L^-1. The bar is equivalent to 1 cm.

Figure 1. Effect of L-Cysteine on Sideroxylon capiri (A. DC.) Pittier callus.

Regarding the content of total phenols in the tempisque callus, significant differences are recognized with respect to the control treatment (Figure 2). The treatment with 200 mg L^-1 of L-Cysteine recorded the lowest content of total phenols (1.06 mg GAE g^-1), and the control treatment the highest (8.312 mg GAE g^-1). The rest of the treatments did not show a significant difference between themselves, but a significant decrease can be observed for all treatments and an inversely proportional behavior depending on the amount of L-Cysteine to which the callus were subjected.

Different letters indicate a significant difference according to the Tukey test (p£0.05).

Figure 2. Effect of L-Cysteine on the phenol content in Sideroxylon capiri (A. DC.) Pittier callus.

Callus development in media treated with 4 mg^-1 of BAP and different concentrations of Picloram did not result in the formation of somatic organs or embryos; instead, callus growth and the formation of friable, smooth-textured, semicircular, white-yellow structures were promoted (Figure 3).

A = Control callus; B = Pro-embryogenic masses from the IN1 treatment seeded in MS medium supplemented with 0.5 mg L^-1 BAP+1.5 mg L^-1 Picloram; C = Pro-embryogenic masses from the IN2 treatment in MS medium supplemented with 1 mg L^-1 BAP+2 mg L^-1 Picloram. The bar is equivalent to 2 cm.

Figure 3. Pro-embryogenic masses formed in Sideroxylon capiri (A. DC.) Pittier calli after 21 days of induction.

In the treatments where the concentration of BAP was reduced and that of Picloram was increased, small semicircular structures formed in the callus, with defined edges that disintegrated compared to the callus of the control treatment (Figure 3C); however, these structures did not progress to more advanced stages of development, and had characteristics of globular embryos in a primary stage of somatic embryogenesis, with a white-yellow coloration (pro-embryogenic masses).

The IN2 treatment exhibited the best response in terms of the number of pro-embryogenic structures formed, with an average of 9 g^-1 callus, followed by the IN1 treatment that showed an average of 5 g^-1 callus; both treatments recorded a significantly greater formation of pro-embryogenic structures compared to the control treatment, in which an average of 1 g^-1 callus was counted (Figure 4).

INC = Regulator-free control treatment; IN1 = MS medium supplemented with 0.5 mg L^-1 BAP+1.5 mg L^-1 Picloram; IN2 = MS medium supplemented with 1 mg L^-1 BAP+2 mg L^-1 Picloram. Different letters indicate a significant difference according to the Tukey test (p£0.05).

Figure 4. Effect of different concentrations of regulators on the formation of pro-embryos of Sideroxylon capiri (A. DC.) Pittier at 21 days of induction.

A fundamental challenge for the in vitro culture of S. capiri is to achieve control of callus oxidation and de novo induction of somatic embryos or adventitious shoots. Callus oxidation is observed as a darkening/browning of the tissue. It is a common phenomenon in the in vitro culture of tropical species (Pancaningtyas, 2015) and is attributed to the accumulation of phenolic compounds, which when released into the culture medium, induce the activation of enzymes involved in their degradation, such as polyphenol oxidases and peroxidases, which generate quinones, highly reactive compounds that often cause cellular stress (Azofeifa, 2009; Lezcano et al., 2020). These can interfere with the culture from its establishment, its growth and the ability to form pro-embryogenic structures or somatic embryos.

In this work, it was observed that L-Cysteine effectively decreases oxidation in callus cultures treated with UV-B light of S. capiri and the subsequent induction of pro-embryos. The positive effect of using L-Cysteine to control phenol synthesis has been recorded in bud cultures of Pimenta dioica L. Merr. during the in vitro establishment phase with concentrations of 100 and 200 mg L^-1 L-Cysteine (Vásquez-Hernández et al., 2021), as well as in callus cultures, at a concentration of 25 mg L^-1, in which, in addition, they generated callus with a compact texture and green color (Rajput et al., 2023).

The decrease in the content of total phenols in the callus reaffirms the inhibitory effect of L-Cysteine on the synthesis of phenols. This effect is attributed to the thiol group present in L-Cysteine, which traps free radicals and quinones (Cruz-Gutiérrez et al., 2020), and thus inhibits the action of the polyphenol oxidase enzyme (Richard-Forget et al., 1992). Likewise, the amino acid L-Cysteine acts as a precursor of glutathione, a powerful reducing agent that plays a crucial role in processes of assimilation of reactive oxygen species (ROS) in plant cells (Iqbal et al., 2021; Koramutla et al., 2021; Vásquez-Hernández et al., 2021).

This inhibitory effect of L-Cysteine on the production of phenols, in addition to reducing the oxidation of tempisque callus, caused their discoloration, according to the increase in the concentration used and the exposure time (Figure 1). This decrease in coloration is attributed to the alteration of various enzymatic processes linked to secondary metabolism, synthesis of phenols and pigments, as a response to oxidative stress (Azofeifa, 2009).

It is important to note that the cultivation of tempisque callus is an option for the production of phenols and other compounds with pharmacological and biotechnological potential (Martínez-Silvestre et al., 2022); however, the application of antioxidants such as L-Cysteine directly affects the accumulation of these compounds, in favor of the development of callus and pro-embryogenic structures, so it may be counterproductive for this purpose. Therefore, the application of these antioxidant agents will depend on the objective pursued, whether micropropagation or the production of secondary metabolites in vitro.

On the other hand, the combination of growth regulators can affect the characteristics of the crop, in addition to the oxidation itself (Kamarul et al., 2020; Cabañas-García et al., 2021). For tempisque, the joint use of auxins and cytokinins is necessary for the induction and proliferation of callus (Martínez-Silvestre et al., 2022); the results of the present work show that they are also necessary for the induction of pro-embryogenic structures. In particular, combinations of BAP and Picloram are recorded for the growth of callus and for the production of masses or structures with characteristics of globular embryos, in an early phase, of white-yellow color, in Polyalthia bullata King (Kamarul et al., 2020) and in Euterpe precatoria Mart. (Barbosa et al., 2022).

Picloram, at concentrations of 2-4 mg L^-1 in the culture medium, stimulates the growth of callus masses and embryo formation in woody species (Chukwunalu et al., 2018; Kamarul et al., 2020; Barbosa et al., 2022), although effective concentrations may vary depending on the species (Manoharan et al., 2016). This regulator increases the endogenous concentrations of auxin, which allows the formation of the first embryonic stages, by effectively promoting cell elongation and division (Rodríguez et al., 2014), although subsequently it is necessary to decrease the concentration of auxin for it to be allow the transition of the different phases of histodifferentiation (Liu et al., 2021).

In addition to auxins, cytokinins also play a positive role in the acquisition of embryonic capacity by somatic cells, which is why the presence of both regulators is required (Rodríguez et al., 2014; Liu et al., 2021). Cytokinins actively participate in the regulation of plant growth and development through the signal transduction pathway, they help cell differentiation, tissue differentiation, the formation of lateral shoots and help mitigate stress generated by abiotic factors (Li et al., 2021). During the induction stage of somatic embryogenesis, the incorporation of cytokinins, together with auxins, is essential for some woody species such as Picea abies (L.) H. Karst., in which these structures are formed after of the elimination of both regulators (Larsson et al., 2008). Likewise, Sánchez et al. (2019) report that in the case of coffee (Coffea arabica L.), the formation of pro-embryogenic masses is related to the reduction of the concentration of BAP in the culture medium. In contrast, the suppression of cytokinin activity, as a consequence of the overexpression of auxin response regulator (ARR) genes, reduces the formation of somatic embryos or induces their abnormal development since they lack a root meristem (Su et al., 2015). This demonstrates that cytokinins act as an important factor for auxin-induced somatic embryo formation.

The lack of organogenic or embryogenic response during the cultivation of woody species may be a result of the limited receptivity of plant tissue as a consequence of in vitro manipulations (Lazos-Monterrosa et al., 2015). However, the inhibitory effect on development should not be ruled out, which may be linked to the production of phenolic compounds, the concentration and time of exposure to growth regulators that could affect the synthesis of proteins involved in the formation and development of organs and embryos (George et al., 2008; Quiñones et al., 2020). Therefore, the incorporation of compounds such as L-Cysteine represents an alternative to control its excessive accumulation.

In this study, it was demonstrated that for the in vitro culture of S. capiri callus, the incorporation of L-Cysteine in concentrations of 100 and 200 mg L^-1to the medium was efficient to manage oxidation since the synthesis of phenols totals is considerably reduced. On the other hand, the combination of 0.5 mg L^-1 of BAP with levels between 1.5 and 2 mg L^-1 of Picloram promotes the formation of pro-embryogenic structures in tempisque callus.

These findings offer valuable information that can be used in the formulation of future regeneration protocols, whether through embryogenic or organogenic pathways. This contribution will potentially boost conservation programs, allowing better use of this species, which is a significant source of secondary metabolites with relevance in the biotechnological field.

The authors thank the Tecnológico Nacional de México Tuxtla Gutiérrez Campus for providing its facilities to carry out this research.

Víctor Rubén López Santos, Federico Antonio Guitérrez Miceli and María del Carmen Silverio Gómez: statistical analysis and interpretation of results; Carlos Alberto Lecona Guzmán and Federico Antonio Guitérrez Miceli: conception and design of the study; Carlos Alberto Lecona Guzmán and María del Carmen Silverio Gómez: writing and correction the manuscript. All authors reviewed the results and approved the final version of this paper.

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