Colorimetric evaluation of callus with embryogenic potential and biomass accumulation in the woody medicinal plant Polyalthia bullata suspension culture

Abstract

Identifying embryogenic callus in plant tissue culture, particularly in woody species, such as Polyalthia bullata, is often hindered by its morphological similarity to non-embryogenic callus. This study introduced a rapid, cost-effective colorimetric method using the triphenyl tetrazolium chloride (TTC) assay to evaluate embryogenic callus induction in P. bullata via cell suspension culture. Suspension cultures were established using 30 µM dicamba in combination with varying concentrations of thidiazuron (TDZ: 2 to 10 µM). Among the treatments, 30 µM dicamba with 8 µM TDZ induced the highest biomass and a distinct yellowish green callus morphology, typically associated with embryogenic potential. The TTC assay confirmed high cell viability, with red staining indicating elevated metabolic activity in embryogenic cultures. These findings validate the TTC assay as an efficient proxy for embryogenic callus detection, providing a valuable alternative to labor-intensive histological or molecular methods. This approach enhances tissue culture protocols for this woody medicinal plant, supporting its large-scale propagation, biomass productivity, and conservation.

Full Article

Colorimetric Evaluation of Callus with Embryogenic Potential and Biomass Accumulation in the Woody Medicinal Plant Polyalthia bullata Suspension Culture

Nancy N. Marianyagam  , Munirah Adibah K. Zaman  , Noor A. Shaharuddin  , and Azzreena M. Azzeme  ,*

Identifying embryogenic callus in plant tissue culture, particularly in woody species, such as Polyalthia bullata, is often hindered by its morphological similarity to non-embryogenic callus. This study introduced a rapid, cost-effective colorimetric method using the triphenyl tetrazolium chloride (TTC) assay to evaluate embryogenic callus induction in P. bullata via cell suspension culture. Suspension cultures were established using 30 µM dicamba in combination with varying concentrations of thidiazuron (TDZ: 2 to 10 µM). Among the treatments, 30 µM dicamba with 8 µM TDZ induced the highest biomass and a distinct yellowish green callus morphology, typically associated with embryogenic potential. The TTC assay confirmed high cell viability, with red staining indicating elevated metabolic activity in embryogenic cultures. These findings validate the TTC assay as an efficient proxy for embryogenic callus detection, providing a valuable alternative to labor-intensive histological or molecular methods. This approach enhances tissue culture protocols for this woody medicinal plant, supporting its large-scale propagation, biomass productivity, and conservation.

DOI: 10.15376/biores.21.1.973-984

Keywords: Auxin; Biomass; Cytokinin; Embryogenic cell suspension; Polyalthia bullata

Contact information: Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; *Corresponding author: azzreena@upm.edu.my

Graphical Abstract

INTRODUCTION

Polyalthia bullata is a woody medicinal plant of high therapeutic value. It is traditionally used for its antimicrobial, antioxidant, and anticancer properties. Its bioactive compounds have shown promise in treating various ailments, generating interest in its large-scale propagation for pharmaceutical development and conservation purposes (Kamarul Zaman et al. 2020a, 2020b, 2021). However, tissue culture-based propagation of P. bullata remains challenging, particularly due to difficulties in distinguishing embryogenic callus from non-embryogenic callus, as both often exhibit similar morphological characteristics. This limitation compromises the accuracy of callus selection, thereby reducing regeneration efficiency and hindering the development of optimized protocols. Moreover, cell suspension culture was employed in lieu of callus culture, as it yields a uniform population of rapidly proliferating cells that exhibit enhanced interaction with nutrients and growth regulators, hence augmenting embryogenic potential and facilitating large-scale propagation (Kong et al. 2020).

Plant growth regulators, especially auxins and cytokinins, play a critical role in inducing embryogenic callus in many plant species. Dicamba, a synthetic auxin, has been reported to induce embryogenic callus in Triticum aestivum L. (Ren et al. 2010) and Zea mays L. (Akoyi et al. 2013). Thidiazuron (TDZ), a phenylurea compound with cytokinin-like activity, has proven effective in promoting somatic embryogenesis in Vaccinium corymbosum × V. angustifolium (Ghosh et al. 2018), Simmondsia chinensis (El-Ashry et al. 2017), and Saccharum spp. (Gallo-Meagher et al. 2000). While hormonal strategies have improved embryogenesis, the identification of callus with embryogenic potential still depends on conventional methods such as histological observation or molecular analysis.

Although precise, these traditional techniques are labor-intensive, time-consuming, and often require costly reagents and skilled personnel. Histological analysis involves complex sample preparation steps including fixation, embedding, sectioning, and staining, while molecular methods, such as polymerase chain reaction (PCR) or gene expression profiling demand advanced laboratory infrastructure (Mikuła et al. 2010; Kaushal and Sidana 2018). Such limitations reduce their practicality for large-scale or routine applications in tissue culture laboratories.

To address this bottleneck, the present study explores a TTC-based colorimetric assay as a rapid and practical alternative for identifying embryogenic callus in P. bullata suspension cultures. It is hypothesized that the TTC (2,3,5-triphenyltetrazolium chloride) is reduced by dehydrogenase enzymes in viable cells to produce red formazan, serving as a reliable visual marker of metabolic activity and cell viability, as shown in Fig. 1 (Wang et al. 2023). When paired with morphological indicators, such as yellowish-green coloration, commonly associated with embryogenic competence, TTC offers a simple, non-destructive screening tool for the identification of embryogenic callus characterized by intense red staining which indicates a higher glycolytic activity and metabolic rate in viable enzymatic tissues.

Fig. 1. Schematic representation of the TTC reduction reaction. Viable P. bullata callus reduces colorless TTC to red formazan (TTF) through dehydrogenase enzyme activity

This study is the first to develop a TTC-based colorimetric method tailored to P. bullata. Through optimizing auxin–cytokinin combinations and validating results through visual morphology, viability staining, and microscopic examination, this work presents a novel and scalable strategy for enhancing tissue culture protocols for recalcitrant woody medicinal plants.

EXPERIMENTAL

Plant Materials

The research was performed in the Plant Biochemistry and Biotechnology Laboratory at the Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor. The calli were derived from the P. bullata leaf with the identification number of PID 170820-13, provided by the Forest Research Institute Malaysia (FRIM).

Multiplication of P. bullata Callus in MS Media Supplemented with 30 µM Dicamba

The callus was multiplied using MS media (Murashige and Skoog 1962) containing 30 µM dicamba. All equipment used were autoclaved before subculturing callus on MS media supplemented with dicamba under laminar air-flow cabinet. Flasks containing callus were wrapped with aluminum foil and then sealed with plastic wrap to be incubated under dark condition for 3 weeks at 24 ± 2 °C.

Multiplication of P. bullata Cell Suspension Culture

Based on preliminary study (data not shown), a series of treatments were tested to determine the optimal combination of plant growth regulators for inducing embryogenic cell suspension culture in P. bullata. The experiment involved using two auxins, 30 µM dicamba and 30 µM NAA, in combination with three cytokinins at concentrations of 2 µM TDZ, 2 µM BAP, and 2 µM kinetin. The effects of these combinations were evaluated based on the increase in fresh and dry weight of the cultures and morphology of suspension cultures. Among the treatments, the combination of 30 µM dicamba and 2 µM TDZ exhibited the highest increase in both fresh and dry weights as well as favourable morphology, indicating high embryogenic potential. Based on these findings, the final study focused on optimizing the concentrations of TDZ while maintaining dicamba at a fixed concentration of 30 µM to further enhance the induction and development of embryogenic cell suspension culture.

One gram of 3-week-old friable calli was cultured in 100-mL conical flask containing 25 mL of ½ MS media supplemented with hormonal combinations as described in Table 1. The flasks were wrapped with aluminum foil and placed on orbital shaker at 120 rpm at 24 ± 2 °C for 18 days (Farezol et al. 2024). Based on a study by Pinto (2023), the growth of P. bullata cells in cell suspension culture had entered stationary phase after incubation for 18 days.

Determination of Cell Suspension Growth

The growth of cell suspension was determined by constructing a growth curve based on the data from the measurement of fresh weight and dry weight of suspension cells (Pinto 2023). The cell suspension was harvested by filtering the cells with filter paper (Whatman No. 1). The initial weight of a clean and dry container was initially recorded. By scraping the cells with a spatula, the filtered cells were transferred to the pre-weighed container to measure the fresh weight. For determination of dry weight, the cells were dried in an oven at 50 ºC for 2 days or until the constant weight was reached to determine the dry weight. The calculation for the dry weight of cells was done using Eq. 1:

 (1)

The growth curve was constructed based on the data recorded for both fresh and dry weight. Moreover, the data were recorded at 3-day intervals for 18 days.

Identification of Embryogenic Callus

Microscopic analysis

The embryogenic callus formations were observed using a light microscope (Zeiss Stemi DV4; Carl Zeiss Microscopy, Göttingen, Germany). The calli in the suspension cultures were removed from the flasks and then blotted dry on a sterile tissue paper. Afterwards, the embryogenic tissue portions were positioned on a glass slide. The appearance of the cells as well as the arrangement and organization of the cells were observed under a microscope. The callus with embryogenic potential is identified by the presence of isodiametric clusters of cells; these clumps are spherical globules surrounded by a layer of organised cells (Woo et al. 2021).

Determination of Cell Viability

Morphology of cell suspension culture

The morphology of cell suspension cultures of all treatments throughout the 18 days period of incubation were observed and recorded at 3-day intervals.

TTC test

To assess the viability of the cells in suspension cultures, a TTC assay was carried out (Nagananda and Satishchandra 2023). The TTC solution of 1% was prepared by dissolving 0.2 g in 20 mL sterile phosphate buffer. Then, the pH of the solution was adjusted to 7.5 using potassium hydroxide (KOH). Afterwards, TTC solution was stored in the dark at 4 °C. A total of 500 µL of cultures were transferred into microcentrifuge tubes. After addition of 1 mL of TTC to the tubes, the tubes were incubated at room temperature in dark conditions. After 1 h, the colour change was observed and recorded.

RESULTS AND DISCUSSION

Establishment of Cell Suspension Culture

The effects of varying concentrations of dicamba and TDZ on biomass accumulation in P. bullata suspension cultures were assessed over an 18-day period (Figs. 1 and 2). Cultures maintained in MS medium without hormones (MSO) showed limited proliferation, with fresh weight peaking at day 12 (2.715 ± 0.839 g) before declining, reflecting the absence of exogenous hormonal stimulation.

In treatments where auxin was the only hormone, 30 µM dicamba resulted in the highest biomass accumulation by day 18 (3.144 ± 0.603 g FW and 0.135 ± 0.025 g DW), consistent with its role in promoting cell division and callus proliferation (Dong et al. 2024). However, this likely favored non-embryogenic callus formation due to the absence of cytokinin.

When applied alone, TDZ (2 to 10 µM) resulted in variable growth. Notably, 4 µM TDZ yielded the highest fresh weight (3.009 ± 1.017 g FW) on day 15, while 10 µM TDZ produced steady increase in dry biomass, suggesting its role in morphogenesis and biomass stability (Usman et al. 2022; Zaytseva et al. 2022). However, higher TDZ concentrations also slowed initial growth, possibly due to inhibitory effects on early cell proliferation (Bernula et al. 2020).

Auxin-cytokinin combinations demonstrated synergistic effects. The combination of 30 µM dicamba with 10 µM TDZ produced the highest fresh weight (2.258 ± 0.363 g FW), whereas the 8 µM TDZ combination yielded the highest dry weight (0.087 ± 0.025 g DW) on day 18. These results support the importance of balanced auxin-cytokinin ratios in promoting embryogenic biomass accumulation (Tripathi et al. 2021; Kaňuková et al. 2024).

Fig. 2. Growth curve of fresh weight (a) and dry weight (b) in P. bullata cell suspensions cultured in ½ MS medium supplemented with 2 µM TDZ and 30 µM Dicamba over 18 days

Fig. 3. Growth curve of fresh weight (a) and dry weight (b) in P. bullata cell suspensions cultured in ½ MS medium supplemented with 4 µM TDZ and 30 µM Dicamba over 18 days

Fig. 4. Growth curve of fresh weight (a) and dry weight (b) in P. bullata cell suspensions cultured in ½ MS medium supplemented with 6 µM TDZ and 30 µM Dicamba over 18 days

Fig. 5. Growth curve of fresh weight (a) and dry weight (b) in P. bullata cell suspensions cultured in ½ MS medium supplemented with 8 µM TDZ and 30 µM Dicamba over 18 days

Fig. 6. Growth curve of fresh weight (a) and dry weight (b) in P. bullata cell suspensions cultured in ½ MS medium supplemented with 10 µM TDZ and 30 µM Dicamba over 18 days

Growth patterns were consistent with typical suspension culture dynamics, exhibiting lag (0 to 3 days), exponential (3 to 12 days), stationary (12 to 15 days), and aging (15 to 18 days).

This pattern is consistent with findings reported in Pinus thunbergii and P. europaea suspension cultures (Beigmohammadi et al. 2019; Sun et al. 2024). Notably, the extended exponential phase observed in P. bullata suggests a high regenerative capacity under optimal hormonal conditions.

Morphological Indicators of Embryogenic Potential

Changes in culture color served as visual cues of cellular differentiation and viability (Table 1). The MSO and dicamba-only cultures gradually turned yellowish-brown, consistent with stress-induced phenolic accumulation common in woody species (Gemechu and Amante 2021; Permadi et al. 2024). Similarly, TDZ-only treatments followed the same browning pattern, suggesting that cytokinin alone was insufficient to sustain embryogenic development.

In contrast, the 30 µM dicamba + 8 µM TDZ treatment retained a yellowish-green color from day 9 to 15, which is a visual trait often associated with embryogenic potential (Mishra et al. 2021; Hamza et al. 2024). This observation was further substantiated by TTC viability staining and microscopy.

Validation by TTC Assay and Microscopy

The TTC assay provided a rapid colorimetric method to evaluate metabolic activity and embryogenic viability. Cultures treated with 30 µM dicamba + 8 µM TDZ produced the most intense red staining by day 12 (Table 2), reflecting high dehydrogenase activity in viable, embryogenic cells (Wang et al. 2023). Lower intensity in 2 and 4 µM TDZ combinations indicated moderate activity, while 30 µM dicamba alone and 10 µM TDZ treatments showed diminished staining, potentially due to stress or cytotoxicity (Naseem et al. 2020).

Microscopic analysis further confirmed these findings, revealing globular structures characteristic of early-stage somatic embryo in the 30 µM dicamba + 8 µM TDZ treatment as early as day 9 (Fig. 4). Similar structures were observed in 30 µM dicamba + 4 µM and 30 µM dicamba + 2 µM TDZ combinations at later stages (days 12 and 15), indicating a dose-dependent response. The observed features, such as isodiametric cell clusters and yellowish-green pigmentation, aligned with known markers of embryogenic callus (Silveira et al. 2013; Woo et al. 2021).

These results affirm that the TTC assay, supported by morphology and microscopy, offers reliable and non-destructive methods for identifying embryogenic callus. Although the TTC colorimetric assay alone effectively reflects cell viability and metabolic activity, it cannot distinguish embryogenic from non-embryogenic tissues. Nevertheless, it serves as a useful preliminary screening tool for selecting viable cells to be further examined microscopically. Therefore, microscopic observation remains indispensable for confirming the presence of callus with embryogenic potential. The optimized hormonal combination of 30 µM dicamba and 8 µM TDZ is therefore recommended for the efficient establishment of embryogenic suspension cultures in P. bullata, a woody medicinal plant with significant biotechnological value.

Fig. 7. Formation of yellowish-green globular calli in the treatment of the combination of 30 µM dicamba and 8 µM TDZ

Table 1. Morphology of Cell Suspension Cultures for 18 days

Table 2. Colour Changes of P. bullata Suspension Cells After TTC Assay

CONCLUSIONS

1. The combination of 30 µM dicamba and 8 µM thidiazuron (TDZ) produced the highest dry weight (0.087 ± 0.025 g) and distinct yellowish green callus, indicating strong embryogenic potential.
2. Microscopic analysis detected globular embryogenic structures as early as day 9, validating the effectiveness of the hormonal treatment in inducing somatic embryogenesis.
3. A rapid and cost-effective triphenyl tetrazolium chloride (TTC) colorimetric assay was successfully established to identify embryogenic callus in P. bullata cell suspension cultures.
4. The TTC staining yielded intense red coloration, confirming high cellular viability and metabolic activity in embryogenic callus.
5. The integration of TTC-based viability testing and morphological screening provided a practical and scalable alternative to conventional histological and molecular techniques.
6. The developed method enhances tissue culture protocols for P. bullata, supporting efficient propagation and conservation of this recalcitrant woody medicinal species.

ACKNOWLEDGEMENTS

The authors would like to express their gratitude to Universiti Putra Malaysia for supporting this research through Putra Graduate Initiative (IPS) grant GP-IPS/2018/9630400.

REFERENCES CITED

Akoyi, J., Mgutu, A. J., Machuka, J., van Lijsebettens, M., Taracha, C., and Anami, S. E. (2013). “Dicamba growth regulator promotes genotype independent somatic embryogenesis from immature zygotic embryos of tropical maize inbred lines,” Journal of Life Sciences 7(7), 677-689.

Beigmohammadi, M., Movafeghi A., Sharafi, A., Jafari, S., and Danafar, H. (2019). “Cell suspension culture of Plumbago europaea L. towards production of plumbagin,” Iranian Journal of Biotechnology 17(2), 46-54. https://doi.org/10.21859/ijb.2169

Bernula, D., Benkő, P., Kaszler, N., Domonkos, I., Valkai, I., Szőllősi, R., and Gémes, K. (2020). “Timely removal of exogenous cytokinin and the prevention of auxin transport from the shoot to the root affect the regeneration potential of Arabidopsis roots,” Plant Cell Tissue Organ Culture 140, 327-339. https://doi.org/10.1007/s11240-019-01730-3

Dong, S., Zheng, W., Wang, Z., Li, J., Zhang, X., Liu, Z., Feng, H., and Zhang, Y. (2024). “Low-toxicity herbicide dicamba promotes microspore embryogenesis and plant regeneration for doubled haploid production in purple cauliflower,” Scientia Horticulturae 336, article 113405. https://doi.org/10.1016/j.scienta.2024.113405

El-Ashry, A. A. E., Gabr, A. M. M., and Bekheet, S. A. E. (2017). “Zeatin and thidiazuron induced embryogenic calli from in vitro leaf and stem of Jojoba (Simmondsia chinensis),” Pakistan Journal of Biological Sciences 20(7), 355-364. https://doi.org/10.3923/pjbs.2017.355.364

Farezol, N. F., Zaman, M. A. K., and Azzeme, A. M. (2024). “Effect of auxins on growth enhancement of cell suspension culture of Tongkat Ali Hitam (Polyalthia bullata),” Malaysian Applied Biology 53(5), 49-61. https://doi.org/10.55230/mabjournal.v53i5.3044

Gallo-Meagher, M., English, R. G., and Abouzid, A. (2000). “Thidiazuron stimulates shoot regeneration of sugarcane embryogenic callus,” In Vitro Cellular & Developmental Biology-Plant 36, 37-40. https://doi.org/10.1007/s11627-000-0009-3

Gemechu, E. C., and Amante, G. (2021). “Control of browning in plant tissue culture: A review,” Journal of Scientific Agriculture 5, 67-71. https://doi.org/10.25081/jsa.2021.v5.7266

Ghosh, A., Igamberdiev, A. U., and Debnath, S. C. (2018). “Thidiazuron-induced somatic embryogenesis and changes of antioxidant properties in tissue cultures of half-high blueberry plants,” Scientific Reports 8(1), article 16978. https://doi.org/10.1038/s41598-018-35233-6

Hamza, H. M., Diab, R. H., Khatab, I. A., Gaafar, R. M., and Elhiti, M. (2024). “Enhancing in vitro regeneration via somatic embryogenesis and Fusarium wilt resistance of Egyptian cucumber (Cucumis sativus L.) cultivars,” Journal of Genetic Engineering and Biotechnology 22(1), article ID 100360. https://doi.org/10.1016/j.jgeb.2024.100360

Kamarul Zaman, M. A., Azzeme, A. M., Ramle, I. K., Normanshah, N., Ramli, S. N., Shaharuddin, N. A., Ahmad, S., and Abdullah, S. N. (2020b). “Induction, multiplication, and evaluation of antioxidant activity of Polyalthia bullata callus, a woody medicinal plant,” Plants 9(12), article 1772. https://doi.org/10.3390/plants9121772

Kamarul Zaman, M. A., Azzeme, A. M., Ramle, I. K., Normanshah, N., Shaharuddin, N. A., Ahmad, S., and Abdullah, S. N. A. (2021). “Prolonged incubation of callus on auxin herbicide 2, 4-D displayed significant effect on alkaloid production in callus of the woody medicinal plant Polyalthia bullata,” In Vitro Cellular & Developmental Biology-Plant 57, 749-759. https://doi.org/10.1007/s11627-021-10194-0

Kamarul Zaman, M. A., Azzeme, A. M., Ramli, S. N., Shaharuddin, N. A., Ahmad, S., and Abdullah, S. N. A. (2020a). “Solvent extraction and its effect on phytochemical yield and antioxidant capacity of woody medicinal plant, Polyalthia bullata,” BioResources 15(4), 9555-9568. https://doi.org/10.15376/biores.15.4.9555-9568

Kaňuková, Š., Lenkavská, K., Gubišová, M., and Kraic, J. (2024). “Suspension culture of stem cells established of Calendula officinalis L,” Scientific Reports 14, article 441. https://doi.org/10.1038/s41598-023-50945-0

Kaushal, S., and Sidana, A. (2018). “Somatic embryogenesis and plant regeneration from cell suspension cultures of Gentiana kurroo Royle,” Annal of Plant Sciences 7(5), 2239-2246. https://doi.org/10.21746/aps.2018.7.5.6

Kong, E. Y., Biddle, J., Foale, M., and Adkins, S. W. (2020). “Cell suspension culture: A potential in vitro culture method for clonal propagation of coconut plantlets via somatic embryogenesis,” Industrial Crops and Products 147, article 112125. https://doi.org/10.1016/j.indcrop.2020.112125

Mikuła, A., Tomiczak, K., and Rybczyński, J. J. (2010). “Cryopreservation enhances embryogenic capacity of Gentiana cruciata (L.) suspension culture and maintains (epi)genetic uniformity of regenerants,” Plant Cell Report 30, 565-574. https://doi.org/10.1007/s00299-010-0970-1

Mishra, N., Tripathi, M. K., Tiwari, S., Tripathi, N., Sapre, S., Ahuja, A., and Tiwari, S. (2021). “Cell suspension culture and in vitro screening for drought tolerance in soybean using poly-ethylene glycol,” Plants 10(3), article 517. https://doi.org/10.3390/plants10030517

Murashige, T., and Skoog, F. (1962). “A revised medium for rapid growth and bio assays with tobacco tissue cultures,” Physiologia Plantarum 15, 473-497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x

Nagananda, G., and Satishchandra, N. (2023). “In vitro direct plant regeneration from somatic embryos developed from the leaf explants of Flickingeria nodosa (Dalz.) Seidenf. through synthetic seed technology,” South African Journal of Botany 153, 77-82. https://doi.org/10.1016/j.sajb.2022.12.012

Naseem, M., Othman, E. M., Fathy, M., Iqbal, J., Howari, F. M., AlRemeithi, F. A., Kodandaraman, G., Stopper, H., Bencurova, E., Vlachakis, D., et al. (2020). “Integrated structural and functional analysis of the protective effects of kinetin against oxidative stress in mammalian cellular systems,” Scientific Reports 10, article 13330. https://doi.org/10.1038/s41598-020-70253-1

Permadi, N., Akbari, S. I., Prismantoro, D., Indriyani, N. N., Nurzaman, M., Alhasnawi, A. N., Doni, F., and Julaeha, E. (2024). “Traditional and next-generation methods for browning control in plant tissue culture: Current insights and future directions,” Current Plant Biology 38, article 100339. https://doi.org/10.1016/j.cpb.2024.100339

Pinto, E. L. (2023). Establishment of Cell Suspension Culture of Polyalthia Bullata for Alkaloid Production, Bachelor’s Thesis, Universiti Putra Malaysia, Selangor, Malaysia.

Ren, J. P., Wang, X. G., and Jun, Y. I. N. (2010). “Dicamba and sugar effects on callus induction and plant regeneration from mature embryo culture of wheat,” Agricultural Sciences in China 9(1), 31-37. https://doi.org/10.1016/S1671-2927(09)60064-X

Silveira, V., De Vita, A. M., Macedo, A. F., Dias, M. F. R., Floh, E. I. S., and Santa-Catarina, C. (2013). “Morphological and polyamine content changes in embryogenic and non-embryogenic callus of sugarcane,” Plant Cell Tissue and Organ Culture 114, 351-364. https://doi.org/10.1007/s11240-013-0330-2

Sun, T., Wu, X., and Ye, J. (2024). “Establishment of embryogenic Pinus thunbergii Parl. suspension cultures: Growth parameters, dynamic analysis, and plant regenerative capacities,” BMC Plant Biology 24(1), article 1200. https://doi.org/10.1186/s12870-024-05938-0

Tripathi, M. K., Tripathi, N., Tiwari, S., Tiwari, G., Mishra, N., Bele, D., Patel, R. P., Sapre, S., and Tiwari, S. (2021). “Optimization of different factors for initiation of somatic embryogenesis in suspension cultures in Sandalwood (Santalum album L.),” Horticulturae 7(5), article 118. https://doi.org/10.3390/horticulturae7050118

Usman, H., Jan, H., Zaman, G., Khanum, M., Drouet, S., Garros, L., Tungmunnithum, D., Hano, C., and Abbasi, B. H. (2022). “Comparative analysis of various plant-growth-regulator treatments on biomass accumulation, bioactive phytochemical production, and biological activity of Solanum virginianum L. callus culture extracts,” Cosmetics 9(4), article 71. https://doi.org/10.3390/cosmetics9040071

Wang, S., Wu, M., Zhong, S., Sun, J., Mao, X., Qiu, N., and Zhou, F. (2023). “A rapid and quantitative method for determining seed viability using 2,3,5-triphenyl tetrazolium chloride (TTC): With the example of wheat seed,” Molecules 28(19), article 6828. https://doi.org/3390/molecules28196828

Woo, H., Ku, S. S., Jie, E. Y., Kim, H., Kim, H., Cho, H. S., Jeong, W., Park, S. U., Min, S. R., and Kim, S. W. (2021). “Efficient plant regeneration from embryogenic cell suspension cultures of Euonymus alatus,” Scientific Reports 11, article15120. https://doi.org/10.1038/s41598-021-94597-4

Zaytseva, Y., Petruk, A., and Novikova, T. (2022). “Thidiazuron and LED lighting enhance taxifolin and rutin production in Rhododendron mucronulatum Turcz. microshoot culture,” Journal of Plant Growth Regulation 42, 2933-2942. https://doi.org/10.1007/s00344-022-10757-4

Article submitted: July 12, 2025; Peer review completed: October 25, 2025; Revised version received: November 28, 2025; Accepted: November 30, 2025; Published: December 12, 2025.

DOI: 10.15376/biores.21.1.973-984