Dose-Dependent Effect of L-Methionine on Caffeine Accumulation in Callus Cultures under In Vitro Conditions
DOI:
https://doi.org/10.65761/jbs.2025.16Keywords:
Caffeine; L-methionine; Callus culture; In vitro culture; Secondary metabolites.Abstract
Background: Caffeine is an important plant secondary metabolite with considerable nutritional, commercial, and pharmacological significance. Plant callus cultures provide a controlled system for studying and enhancing secondary metabolite production. L-methionine may stimulate caffeine biosynthesis through its association with S-adenosyl-L-methionine-dependent methylation reactions.
Objective: The present study evaluated the dose-dependent effect of L-methionine on caffeine accumulation in callus cultures under in vitro conditions.
Methods: Callus cultures were established on Murashige and Skoog medium supplemented with suitable plant growth regulators. The developed callus was treated with L-methionine at 0, 5, 10, 15, 20, and 25 mg/L, with five replicates per treatment. Caffeine was extracted from harvested callus and qualitatively identified using the Parry reagent method. Quantitative caffeine estimation was performed using UV–Visible spectrophotometry at 272 nm. Treatment effects were statistically evaluated using ANOVA followed by Duncan’s Multiple Range Test at p < 0.05.
Results: L-methionine significantly influenced caffeine accumulation. Caffeine content increased progressively with increasing L-methionine concentration and reached its highest level at 20 mg/L, whereas a slight decline occurred at 25 mg/L.
Conclusion: L-methionine supplementation effectively enhanced caffeine accumulation, with 20 mg/L identified as the optimum concentration under the tested conditions.
References
1. Zhang S, Jin J, Chen J, Ercisli S, Chen L. Purine alkaloids in tea plants: component, biosynthetic mechanism and genetic variation. Beverage Plant Research. 2022;2(1):1-9. doi:10.48130/BPR-2022-0013.
2. Jia X, Luo S, Ye X, Liu L, Wen W. Evolution of the biochemistry underpinning purine alkaloid metabolism in plants. Philos Trans R Soc B Biol Sci. 2024;379(1914):20230366. doi:10.1098/rstb.2023.0366.
3. Čižmárová B, Kraus V Jr, Birková A. Caffeinated beverages—unveiling their impact on human health. Beverages. 2025;11(1):18. doi:10.3390/beverages11010018.
4. Starling-Soares B, Pereira M, Renke G. Extrapolating the coffee and caffeine (1, 3, 7-trimethylxanthine) effects on exercise and metabolism—a concise review. Nutrients. 2023;15(24):5031. doi:10.3390/nu15245031.
5. Sharma VK, Sharma A, Verma KK, Gaur PK, Kaushik R, Abdali B. A comprehensive review on pharmacological potentials of caffeine. J Appl Pharm Sci Res. 2023;6(3):16-26. doi:10.31069/japsr.v6i3.04.
6. Wari D, Aboshi T, Shinya T, Galis I. Integrated view of plant metabolic defense with particular focus on chewing herbivores. J Integr Plant Biol. 2022;64(2):449-475. doi:10.1111/jipb.13204.
7. Bai Y, Liu X, Baldwin IT. Using synthetic biology to understand the function of plant specialized metabolites. Annu Rev Plant Biol. 2024;75(1):629-653. doi:10.1146/annurev-arplant-060223-013842.
8. Divekar PA, Narayana S, Divekar BA, Kumar R, Gadratagi BG, Ray A, et al. Plant secondary metabolites as defense tools against herbivores for sustainable crop protection. Int J Mol Sci. 2022;23(5):2690. doi:10.3390/ijms23052690.
9. Leibrock NV, Santegoets J, Mooijman PJ, Yusuf F, Zuijdgeest XC, Zutt EA, et al. The biological feasibility and social context of gene-edited, caffeine-free coffee: NV Leibrock et al. Food Sci Biotechnol. 2022;31(6):635-655. doi:10.1007/s10068-022-01082-3.
10. Abu-Hashem AA, Hakami O, El-Shazly M, El-Nashar HA, Yousif MN. Caffeine and purine derivatives: a comprehensive review on the chemistry, biosynthetic pathways, synthesis-related reactions, biomedical prospectives and clinical applications. Chem Biodivers. 2024;21(7):e202400050. doi:10.1002/cbdv.202400050.
11. Lin Z, Wei J, Hu Y, Pi D, Jiang M, Lang T. Caffeine synthesis and its mechanism and application by microbial degradation, a review. Foods. 2023;12(14):2721. doi:10.3390/foods12142721.
12. Jiang T, Zuo S, Liu C, Xing W, Wang P. Progress in methylxanthine biosynthesis: insights into pathways and engineering strategies. Int J Mol Sci. 2025;26(4):1510. doi:10.3390/ijms26041510.
13. Lashley A, Miller R, Provenzano S, Jarecki SA, Erba P, Salim V. Functional diversification and structural origins of plant natural product methyltransferases. Molecules. 2022;28(1):43. doi:10.3390/molecules28010043.
14. Zhou MZ, Rothenberg DO, Zeng W, Luo L, Yan CY, Zeng Z, et al. Discovery and biochemical characterization of N-methyltransferase genes involved in purine alkaloid biosynthetic pathway of Camellia gymnogyna Hung T. Chang (Theaceae) from Dayao Mountain. Phytochemistry. 2022;199:113167. doi:10.1016/j.phytochem.2022.113167.
15. Ma W, Kang X, Liu P, Zhang Y, Lin X, Li B, et al. The analysis of transcription factor CsHB1 effects on caffeine accumulation in tea callus through CRISPR/Cas9 mediated gene editing. Process Biochem. 2021;101:304-311. doi:10.1016/j.procbio.2021.01.001.
16. Ma W, Kang X, Liu P, She K, Zhang Y, Lin X, Li B, et al. The NAC-like transcription factor CsNAC7 positively regulates the caffeine biosynthesis-related gene yhNMT1 in Camellia sinensis. Hortic Res. 2022;9:uhab046. doi:10.1093/hr/uhab046.
17. Reshi ZA, Ahmad W, Lukatkin AS, Javed SB. From nature to lab: a review of secondary metabolite biosynthetic pathways, environmental influences, and in vitro approaches. Metabolites. 2023;13(8):895. doi:10.3390/metabo13080895.
18. Ozyigit II, Dogan I, Hocaoglu-Ozyigit A, Yalcin B, Erdogan A, Yalcin IE, et al. Production of secondary metabolites using tissue culture-based biotechnological applications. Front Plant Sci. 2023;14:1132555. doi:10.3389/fpls.2023.1132555.
19. Fazili MA, Bashir I, Ahmad M, Yaqoob U, Geelani SN. In vitro strategies for the enhancement of secondary metabolite production in plants: a review. Bull Natl Res Cent. 2022;46(1):35. doi:10.1186/s42269-022-00717-z.
20. Niazian M, Sabbatini P. Traditional in vitro strategies for sustainable production of bioactive compounds and manipulation of metabolomic profile in medicinal, aromatic and ornamental plants. Planta. 2021;254(6):111. doi:10.1007/s00425-021-03771-5.
21. Hazrati R, Zare N, Asghari-Zakaria R, Sheikhzadeh P, Johari-Ahar M. Factors affecting the growth, antioxidant potential, and secondary metabolites production in hazel callus cultures. AMB Express. 2022;12(1):109. doi:10.1186/s13568-022-01449-z.
22. Latunra AI, Tuwo M, Amboupe DS. Enhancement of caffeine concentration in Todolo coffee callus cultures with L-methionine and UV-Vis spectrophotometry. Pak J Biol Sci. 2024;27(12):567-576. doi:10.3923/pjbs.2024.567.576.
23. Di Bonaventura A, Marchetti S, Petrussa E, Braidot E, Colomban S, Navarini L, et al. A protocol for the development and maintenance of Coffea arabica (L.) cell suspension cultures. Plant Cell Tissue Organ Cult. 2024;158(3):48. doi:10.1007/s11240-024-02848-9.
24. Indu BK, Balasubramanya S, Anuradha M, Shilpa P. Callus and cell suspension cultures for secondary metabolite production. In: In Vitro Production of Plant Secondary Metabolites: Theory and Practice. Singapore: Springer Nature Singapore; 2025. p. 71-88. doi:10.1007/978-981-97-8808-8_5.
25. Ahmadpoor F, Zare N, Asghari R, Sheikhzadeh P. Sterilization protocols and the effect of plant growth regulators on callus induction and secondary metabolites production in in vitro cultures Melia azedarach L. AMB Express. 2022;12(1):3. doi:10.1186/s13568-022-01343-8.
26. Esteban-Campos P, Vela P, Rodríguez-Solana R, López-Sánchez JI, Salinero C, Pérez-Santín E. Influence of the culture conditions on Camellia sinensis cell cultures. Foods. 2024;13(15):2461. doi:10.3390/foods13152461.
27. Mohaddab M, El Goumi Y, Gallo M, Montesano D, Zengin G, Bouyahya A, et al. Biotechnology and in vitro culture as an alternative system for secondary metabolite production. Molecules. 2022;27(22):8093. doi:10.3390/molecules27228093.
28. Khan W, Zheng P, Sun B, Liu S. Transcriptomics analysis reveals differences in purine and phenylpropanoid biosynthesis pathways between Camellia sinensis var. shuchazao and Camellia ptilophylla. Horticulturae. 2024;11(1):8. doi:10.3390/horticulturae11010008.
29. Mi X, Yang C, Qiao D, Tang M, Guo Y, Liang S, et al. De novo full length transcriptome analysis of a naturally caffeine-free tea plant reveals specificity in secondary metabolic regulation. Sci Rep. 2023;13(1):6015. doi:10.1038/s41598-023-32435-5.
30. Yao X, Chen H, Ai A, Wang F, Lian S, Tang H, Jiang Y, Jiao Y, He Y, Li T, Lu L. The transcription factor CsS40 negatively regulates TCS1 expression and caffeine biosynthesis in connection to leaf senescence in Camellia sinensis. Hortic Res. 2023;10(9):uhad162. doi:10.1093/hr/uhad162.
Downloads
Published
Issue
Section
License
Copyright (c) 2025 Sobia Naz, Hoorish, Sabahat Jan
This work is licensed under a Creative Commons Attribution 4.0 International License.
This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
