Media and amino acids supplementation alter morphological, development, and bioactive metabolite profiling in Alocasia cuprea callus cultures

The study aimed to evaluate how Murashige and Skoog (MS) medium with combinations of plant growth regulators (PGRs) [TB9: MS with the addition of 9.80 μM thidiazuron (TDZ) and 4.40 μM 6-benzyl amino purine (BAP), and IA9: MS supplemented with 9.80 μM TDZ and 4.52 μM 2,4-D)] and five different of amino acids (L-arginine, L-asparagine, L-cysteine, L-glutamine, and L-proline) affect callus proliferation, differentiation, and bioactive metabolite production in Alocasia cuprea. The morphological development and metabolic changes in callus cultures induced by MS medium with different combinations of PGRs and amino acid supplementation are discussed. The medium composition strongly affected callus proliferation and differentiation: compact organogenic callus was induced in TB9, whereas IA9 induced embryogenic callus with a distinct morphology. Among amino acids, L-arginine, in combination with TB9, gave maximum productivity and shoot initiation, while L-cysteine supported embryogenesis on IA9 but inhibited shoot development. Metabolic profiling through GC–MS revealed significant differences in the production of bioactive compounds. Treatment IA9 increased variation among callus in metabolites produced in comparison to TB9. The key compound was squalene, which was highly produced in TB9 with L-asparagine, whereas octadecadienoic acid was exclusively found in IA9. PCA and heatmap clustering highlighted the activation of biosynthetic pathways by specific culture conditions, supporting the production of targeted bioactive compounds. Among the bioactive metabolites, the identification of squalene is particularly notable for its commercial applications. Our study reveals promising media with different combinations of growth regulators and amino acid combinations for high production of A. cuprea embryogenic and organogenic callus and also produced high-value compounds such as conjugated linoleic acid/CLA and squalene.

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