Callus formation index, shoot, and embryo number obtained with different explant of Bonellia macrocarpa
The growth regulators used in different concentrations induced different morphogenic responses: callogenesis, organogenesis, and somatic embryogenesis depending on the explant used (root, stem, and leaf). 2,4-D at concentrations of 0.5 and 1.0 mg-l L induced the highest callogenic response when root was used as explant, while at a concentration of 2.0 mgL-l it promoted callogenesis when leaf was used as explant. Regarding the use of ANA and BAP at a concentration of 2.0 mgL-l, both regulators promoted callus formation with a higher percentage when the stem was used as an explant (Table 1). At a concentration of 1.0 mgL-l of BAP, an organogenic response was obtained with the highest number of shoots per explant at 60 days. Treatments with NAA and 2,4-D did not generate shoot formation. However, 2,4-D favored the formation of embryos at 60 days in the globular state in root explants (Table 1).
The response caused by BAP is a result that this regulator is a cytokinin and this type of hormone promotes cell division and differentiation, which is, it initiates directly process of bud formation and multiplication (Van Staden et al. 2008). The molecular mechanism of the cytokinin-induced (BAP)-induced shoot generation process in B. macrocarpa is still unknown, but previous studies have reported results that could explain the genetic events involved in the formation of shoots in plant cultures in vitro. It has been reported that a key event for this generation of cytokinin-induced budding (BAP) is the activation of the WUS gene. However, cytokinin signaling activation alone is not sufficient to induce WUS (Zhang et al. 2017). Therefore, the participation of HD Zip III transcription factors phabulosa (PHB), phavoluta (PHV), and revoluta (REV) act as necessary regulators for the induction of WUS independent of cytokinins for the subsequent formation of buds (Zhang et al. 2017). In addition, HD ZIP III interacts with type B ARRs (Zhang et al. 2017), as well as other regulatory factors STM (Shi et al. 2016) and AP2/ERF RAP2.6L (Che et al. 2007; Yang et al. 2018) that are of importance for shoot formation.
The results obtained in the formation of callogenesis in the different types of explants in Bonellia macrocarpa could be explained by the fact that there are different concentrations of endogenous regulators in the different parts of the plant and the complementary effect with the exogenous regulators induces callogenesis. According to (Gaspar et al. 1996), they mention that endogenous regulators interact with exogenous regulators, so the biological activity of exogenous regulators can be equivalent or superior to endogenous regulators. Interaction with endogenous regulators is specific, and cell and tissue responses are highly-dependent on plant species and explant sources. In B. macrocarpa no endogenous quantification studies of regulators have been carried out, which warrants further studies. I could be a consequence of the use of NAA and 2,4-D auxins regulators that play the role of cell elongation, cell division, and callus formation in culture media. The molecular mechanism in callus induction in B. macrocarpa is still unknown, but previous studies carried out in Arabidopsis plants explain how auxins induce callogenesis. Auxin signaling is transduced by transcription factors (ARF 7 and 19) (Fukaki et al. 2005), to activate (LBD 16, 18, and 29) to promote callus formation (Okushima et al. 2007; Fan et al. 2012). LBDs activate transcription factors such as (E2FA) (Berckmans et al. 2011); (EXP14) (Lee et al. 2013); (FAD-BD) and (PME2) (Xu et al. 2018). These LBDs regulate cellular processes associated with root initiation or callus formation. This molecular mechanism could be occurring in the formation of callus in B. macrocarpa.
When root was used as an explant, cream-colored compact calli were obtained (Fig. 1A). While, in explants of stem and leaf, the callus was compact and yellow in color (Fig. 1B, C). Callogenesis formation was observed 30 days after the explants were placed in the culture medium. Oxidation was observed in the callus from the beginning of the induction. In Fig. 1, photographs with calluses of compact texture are observed. In stem explants BAP induced the formation of shoots while 2,4-D induced the formation of somatic embryo in the globular stage.
In Bonellia macrocarpa we observed that the degree of compaction of the calluses can vary depending on the type of plant used, the composition of the medium, growth regulators, and the environmental conditions of the crop. In addition, the compact texture of calluses is due to the ability of plant tissue to absorb nutrients and growth regulators in the culture medium. It was observed that the root calluses were white, while the leaf and stem explants had a yellow color. The coloration of the calluses can be due to various factors, such as pigmentation, light intensity, and the explant source of the different parts of the plants.
The BAP regulator induced green organogenic callus after 30 days, this event occurs because the callus shows chlorophyll formation. According to Sugiyarto et al. (2014) and Sari et al. (2019) state that this color is the result of the influence of cytokinins on the formation of chlorophyll. In addition, the generation of shoots is observed 60 days after induction, using stems as a source of explant (Fig. 1D). With the 2,4-D regulator, using concentrations of 0.5 and 1.0 mg/L, the greatest formation of somatic embryogenesis was obtained, using root as an explant (Fig. 1E). While NAA and BAP did not favor responses of somatic embryogenesis (Table 1). The result obtained in the formation of embryogenesis is because the 2,4-D regulator is one of the exogenous auxins used in the induction of somatic embryogenesis (Jiménez 2001). Arabidopsis thaliana has been used as a model to understand the molecular mechanisms and genes involved in the process of somatic embryogenesis. According to Tsuwamoto et al. (2010), among the responsible genes related to the competition for embryogenesis, are the LEAFY COTYLEDON genes, (LEC1) (Lotan et al. 1998) and LEC2 (Stone et al. 2008), WUSCHEL (WUS) (Zuo et al. 2002), BABY BOOM (BBM) (Boutilier et al. 2002), AGAMOUS-LIKE-15 (AGL15) (Harding et al. 2003) and AINTEGUMENTA-LIKE5/PLETHORA 5/EMBRIOMAKER (AIL5/PTL5/ EMK). These are the genes that encode transcription factors, and when overexpressed they promote somatic embryogenesis. These genes could be participating in the somatic embryo formation response in root explants of B. macrocarpa; however, more specific studies are needed to confirm this. In addition, there are factors such as regulators (endogenous to the explant) that influence metabolic processes and genetic regulation that are involved in this morphogenic response.
The use of 2,4-D favored the formation of embryos in the globular stage when the root was used as an explant. The generation of the embryos occurred 60 days after the explants were placed in the culture medium (Fig. 1E).
Quantification of bonediol in callus from different explants (root, stem, and leaf)
Callus from explants (root, stem, and leaf) were analyzed for bonediol quantification in HPLC. The concentration of bonediol present in the callus, coming from the stem as an explant source, was 49.57 mg bonediol/g callus. However, in the leaf and root calluses, the presence of bonediol could not be confirmed (Table 2 and Fig. 2).
Similar studies regarding the quantification of bonediol have been carried out in Bonellia root in wild plants (Caamal-Fuentes et al. 2011), and recently in transformed roots (Ruíz-Ramírez et al. 2018). The presence of bonediol was confirmed only in callus from stem explants. It is important to perform further studies on the biosynthesis in each part of the B. macrocarpa plant and if there is the translocation of this secondary metabolite because there are no reports on the biosynthetic route of bonediol. One of the strategies for understanding the biosynthesis pathway is using proteomic techniques. These techniques allow investigating the genes and enzymes are involved in the biosynthesis of active compounds in plants (Yang et al. 2021). On the other hand, the importance of this work was to verify the presence of bonediol in calluses. The concentration of bonediol in callus from stems was higher than in transformed root, obtaining a content which was 49.57 mg bonediol/g dry callus, a concentration 17.8 times higher, compared to the content of 2.78 mg bonediol/g dry weight of transformed roots.
Quantification of bonediol in suspension cell cultures
In cells cultured suspension (Fig. 1F), it was possible to detect the metabolite of interest with 24.44 mg bonediol/g dry cells in HPLC, being the first identification of bonediol in cells in suspension. These cell suspensions are made up of meristematic cells, which is, undifferentiated cells, which have the characteristic of not separating after division and form aggregates of different sizes and shapes (Meyer et al. 2002). The tendency of these cells to aggregate is regulated by the cohesiveness of the cell wall and allows cell-cell communication, which can favor the transport of intermediates, necessary for the biosynthesis of secondary metabolites. The content of bonediol in cells is important for the upscaling of this secondary metabolite that various strategies can be used for the increase of bonediol employing several approaches to develop the production of the desired natural products, such as the selection of high-producing cell lines, optimization of the conditions of the culture medium, addition of elicitors or precursors and metabolic engineering (Yue et al. 2014). Suspension cell culture remains one of the most widely used techniques for the production of secondary metabolites. Therefore, it is important to clarify the biosynthetic route of bonediol to regulate the biosynthetic process, as well as to combine strategies such as the addition of inducers, precursors to improve the content of secondary metabolites (bonediol), in addition to seeking an adequate selection in the use of bioreactors for the scale of this secondary metabolite (Yue et al. 2014).