Inorganic source of selenium in the culture of calluses of Catharanthus roseus (L.)

Catharanthus roseus, also known as vinca, is a perennial herbaceous species with a high ornamental and cultural value due to its use in traditional medicine. This species is used in different cultures worldwide to treat various ailments such as stomach infections, gastritis, or asthma. Leaf and root extracts have also been reported to have anticancer, antioxidant, anti-inflammatory, and antimicrobial activities (Das et al., 2020).

In addition, several alkaloids produced in this species, such as ajmalicine, vinblastine, and vincristine, have anticancer activities (Dhyani et al., 2022). There are pharmaceutical products from these compounds that generate more than 100 million dollars per year (Rojas-Sandoval, 2022). Thus, the study of C. roseus has gained great importance in scientific research focused on the production of drugs that involve the secondary metabolites produced by this species (Dhyani et al., 2022).

Although this plant has high yields in seed reproduction, asexual propagation is necessary to ensure less genetic variability and greater homogeneity of plant material in large-scale production. It is necessary to standardize crop conditions such as temperature and photoperiod in regions where species do not grow naturally and concentrations and types of growth regulators according to plant species (Salas-Valdivia et al., 2023).

Nonetheless, traditional propagation is not recommended for large-scale production due to low multiplication rates (Rojas-Sandoval, 2022). Over the past few decades, in vitro culture technologies have been explored in C. roseus, where callus production stands out (Rahman et al., 2019).

On the other hand, calluses have been studied in plants due to their positive effects on different plant species. In the culture of C. roseus suspension cells, a higher amount of alkaloids, such as ajmalicine and serpentine, was produced when the growth medium had some source of Se (Na₂SeO₃ or Na₂SeO₄), compared to the control (Arvy et al., 1995). Na₂SeO₃ improved the growth of Calendula officinalis (L.), increasing seedling height, leaf area, and number of leaves (Hernández-Díaz et al., 2021). Se, in the form of nanoparticles, had a biostimulant effect on the development and yield of C. roseus biomass and C. officinalis flowers (Garza-García et al., 2023).

The different sources of Se are associated with changes in plant development, the production of secondary metabolites, tolerance to different types of stress, hormonal balance and photosynthetic efficiency, among others (Behbahani et al., 2020). There are reports indicating that high concentrations of Se can cause phytotoxicity in different plant species (Garza-García et al., 2023).

Therefore, the effect of Se on plants depends on the species, the stage of development, and the source of selenium (León-Morales et al., 2019). This study aimed to induce callus formation in different explants of C. roseus through the combination of growth regulators and to evaluate the effect of two inorganic sources of Se on callus growth in order to identify the best range of concentrations.

This work was carried out at the facilities of the Plant Biotechnology Unit, Zapopan headquarters, of the Center for Research and Assistance in Technology and Design of the State of Jalisco (CIATEJ, for its acronym in Spanish). The seeds of C. roseus, Mediterranean Burgundy Halo variety (BallSeed, USA), were superficially disinfested with commercial 3% sodium hypochlorite (NaClO) for 8 and 12 min.

Subsequently, the seeds were washed three times with sterile distilled water for 3 min. On March 15, 2022, seeds were sown in 10% MS (Murashige and Skoog, 1962) medium (with 3 g L^-1 sucrose and 8 g L^-1 agar) and kept incubated at 25 °C. The germination percentage was determined 10 days after the experiment was established.

Explants of hypocotyls and leaves of seedlings obtained from in vitro culture were placed in Petri dishes with MS medium (30 g L^-1 sucrose, 8 g L^-1 agar) and with treatments with growth regulators (Table 1). Five leaves and three hypocotyls were placed on each dish, with a total of 10 replications per treatment. The dishes were kept incubated at 25 °C for 40 days.

To evaluate the different Se concentrations, subcultures of the calluses obtained were performed with the culture medium of treatment 1 and 1, 10, 50, or 100 mg L^-1 of Na₂SeO₃ or Na₂SeO₄ was added. After analyzing the data obtained from the first trial, a second experiment was carried out with a narrower range of concentrations (5, 10, 15, or 20 mg L^-1 from both sources of Se) and the control.

In both experiments, the calluses were kept incubated at 25 °C with a photoperiod of 16 h light and 8 h dark for 60 days. Callus induction was determined using photographic images, and the area of each callus was measured using ImageJ software (Version 1.53s, May 2022).

Growth was assessed with fresh callus weight on an analytical balance (BP121S, Sartorius, 120 g., Goettingen, Germany). An analysis of variance and a comparison of means were performed with Duncan’s multiple range test, in Statgraphics Centurion XV (StatPoint Inc., 2007), with a significance level of p≤ 0.05.

Ninety-three percent germination was obtained after 10 days of sowing; germination was determined by the emergence of the radicle. With the two treatments (8 and 12 min with NaClO), there was a 95% success rate in seed disinfestation. Treatment with 8 min was used for the establishment of in vitro culture.

For callus induction, no significant differences were found in the type of explant used (hypocotyls or leaves) (p= 0.3665). The growth regulator factor was statistically significant (p= 0.0001) for the callus area. Treatments 1 and 3 generated callogenesis, with an average callus area of 0.72 cm² with treatment 1 in both explants. There was no callus formation with treatment 2.

With treatment 3, 0.66 and 0.56 cm² were obtained in the calluses obtained from hypocotyls and leaves, respectively. Qualitative differences were observed in the calluses of treatments 1 and 3 (Figure 1). The mixture of growth regulators used in treatment 1 stimulated the formation of white and friable calluses (Figure 1a); these characteristics of the plant material are suitable for mass plate callus culture or for the establishment of suspension cell culture.

Figure 1

Figure 1. Callus induction in C. roseus. a) calluses obtained with treatment 1 (3 mg L^-1 of 2,4-D, 2 mg L^-1 of BA and 2 mg L^-1 of NAA); and b) calluses from treatment 3 (1.5 mg L^-1 BA and 0.5 mg L^-1 NAA).

The results of this study are consistent with those reported by Rahman et al. (2019), who obtained calluses of C. roseus with similar characteristics in leaf explants from seedlings grown ex vitro in a culture medium with 3 mg L^-1 of 2,4-D and 1 mg L^-1 of BA. The calluses of treatment 3 (Figure 1b) had a compact and green consistency, which may be due to the production of chlorophyll; after 60 days, organogenesis processes were observed. Singh et al. (2011) generated calluses with similar characteristics, with 1.5 mg L^-1 of BA and 1 mg L^-1 of NAA.

Calluses with these properties are helpful for micropropagation due to the observed differentiation process (Das et al., 2020). To determine the effect of Se on callus growth, without inducing a differentiation process, calluses obtained with treatment 1 were used. The best growth of calluses was obtained with 1 and 10 mg L^-1 of Na₂SeO₄ (0.52 and 0.53 g fresh weight, respectively) and with 1 mg L^-1 of Na₂SeO₃ (0.5 g fresh weight); these results were superior (p= 0.0001) to the control (0.36 g).

Higher Se concentrations (50 and 100 mg L^-1) had a negative impact on callus growth (0.18 and 0.1 g with Na₂SeO₃ and 0.3 and 0.07 g with Na₂SeO₄, respectively) (Figure 2).

Figure 2

Figure 2. Fresh weight of calluses grown with two inorganic sources of selenium. The letters above the bars indicate significant differences, using Duncan’s multiple range test with a significance level of p≤ 0.05.

Similar results were obtained in other studies with different plant species, where the application of Se positively affected plant development within a specific range of concentrations (Garza-García et al., 2023). Sotoodehnia-Korani et al. (2020) reported higher root and leaf growth of Capsicum annuum with 0.5 and 1 mg L^-1 of Na₂SeO₄, while plants showed symptoms of toxicity with doses greater than 10 mg L^-1.

This contradicts what was observed in C. roseus, since 10 mg L^-1 of Na₂SeO₄ or Na₂SeO₃ had the highest growth of calluses. In turn, Behbahani et al. (2020) reported larger leaf, stem, and root sizes of Momordica charantia (L.) grown in vitro when using 1 and 4 mg L^-1 of Na₂SeO₄; however, concentrations greater than 10 mg L^-1 decreased seedling size.

The results obtained in the first experiment allowed us to propose another trial with a smaller range of concentrations. In the second experiment, the calluses with the highest fresh weight were obtained with 5 and 10 mg L^-1 of both Se sources (p= 0.0001). From 15 mg L^-1 onwards, there was a decrease in the fresh weight of the calluses, although it was not statistically different from the control (p= 0.2385) (Figure 3).

Figure 3

Figure 3. Fresh weight of calluses grown with two inorganic sources of selenium. The letters above the bars indicate significant differences, using Duncan’s multiple range test with a significance level of p≤ 0.05.

In addition, differences were found between the sources of Se; the fresh weight of calluses grown with 50 mg L^-1 of Na₂SeO₃ was statistically lower than those grown at the same concentration of Na₂SeO₄. Therefore, Na₂SeO₃ was more toxic to C. roseus calluses than Na₂SeO₄. Other studies have also established a comparative effect between Na₂SeO₃ and Na₂SeO₄.

In C. annuum plants, 5 μM of Na₂SeO₃ increased the fresh and dry weight of roots, leaves, and stem, while 5 and 10 μM of Na₂SeO₄ resulted in similar effects on growth, finding that C. annuum was more tolerant to Na₂SeO₄. Nevertheless, both sources of Se had a negative impact with 20 μM of Na₂SeO₃ or Na₂SeO₄ (Hernández-Hernández et al., 2018).

Similarly, León-Morales et al. (2019) found that 2.5 μM of Na₂SeO₃ or Na₂SeO₄ improved the germination percentage of C. annuum. Nonetheless, the growth of the aerial part was higher only in seedlings treated with Na₂SeO₃. On the other hand, in Raphanus sativus (L.), there were no significant differences in germination or seedling growth with Na₂SeO₃ or Na₂SeO₄. The effect of Se on plants depends on the source, concentration, species, and cell type.