Fuente: PubMed "medicinal and aromatic plants" Sci Rep. 2026 Jul 17. doi: 10.1038/s41598-026-61913-9. Online ahead of print.ABSTRACTSaffron (Crocus sativus L.) is one of the most valuable medicinal and aromatic plants because of its apocarotenoids such as crocin, picrocrocin, crocetin, and safranal. However, the limited availability of stigmas and the low multiplication rate of this sterile species restrict the large-scale production of these bioactive compounds. This study aimed to optimize in vitro callus induction and regeneration from different saffron explants, identify suitable culture conditions for establishing cell suspension cultures and differentiated tissues, and evaluate the effects of silver nanoparticles (AgNPs) as nano-elicitors on carotenoid/apocarotenoid biosynthetic gene expression and metabolite accumulation in different tissue differentiation states. Corm, cormlet, ovary, and leaf explants were cultured on Murashige and Skoog (MS) medium containing sixteen auxin-cytokinin combinations under continuous darkness or a 16 h light/8 h dark photoperiod for callus induction. Regeneration was performed using six corm regeneration media and three ovary regeneration media to produce cormlets and direct and indirect stigma-like structures (DSLS and INDSLS). After the establishment of suspension cultures, AgNPs (0, 55, and 110 mg L⁻¹) were applied for 24 and 72 h. The relative expression of CsPDS, CsLYC, CsBCH, CsZCD, and CsUGT2 was quantified by qRT-PCR, while crocin, picrocrocin, crocetin, and safranal were quantified by HPLC-DAD. Among the responsive explants, cormlet-derived tissues cultured under continuous darkness on T2B1 medium (2 mg L⁻¹ 2,4-D + 1 mg L⁻¹ BAP) demonstrated the highest callus induction (69.28%), fresh weight (590.89 mg), surface area (457.44 mm²), and callus quality, producing large, friable calli suitable for suspension culture. For regeneration, COEX4 medium (0.1 mg L⁻¹ NAA + 2.0 mg L⁻¹ BAP) produced the highest regeneration frequency (75.20%), cormlet fresh weight (1.38 g), and cormlet diameter (9.72 mm). Ovary explants regenerated both DSLS and INDSLS, with ROVR2 (5 mg L⁻¹ NAA + 5 mg L⁻¹ BAP) producing the highest number of INDSLS (23.5 per explant) and ROVR3 (10 mg L⁻¹ NAA + 10 mg L⁻¹ BAP) producing the highest number of DSLS (6.6 per explant). No detectable expression of the target genes was observed in undifferentiated suspension-cultured cells, demonstrating that AgNPs elicitation alone was insufficient to activate carotenoid and apocarotenoid biosynthetic pathways in the absence of tissue differentiation. In contrast, differentiated tissues indicated significant dose- and time-dependent transcriptional responses, with the greatest induction occurring after treatment with 110 mg L⁻¹ AgNPs for 72 h. DSLS showed the strongest induction of CsPDS, CsLYC, and CsBCH, whereas greenhouse-grown stigmas indicated the highest expression of CsZCD and CsUGT2. HPLC-DAD analysis confirmed corresponding increases in crocin, picrocrocin, crocetin, and safranal following AgNPs elicitation, with crocin identified as the predominant metabolite. The highest metabolite accumulation was consistently observed under the 110 mg L⁻¹ AgNPs treatment after 72 h, with greenhouse-grown stigmas containing the greatest concentrations, followed by DSLS and INDSLS. This study revealed that successful optimization of tissue culture and regeneration is a prerequisite for effective nano-elicitation in saffron. AgNPs enhanced carotenoid/apocarotenoid biosynthetic gene expression and metabolite accumulation only in differentiated tissues, indicating that tissue differentiation is essential for metabolic activation. The optimized regeneration protocols combined with AgNPs nano-elicitation provide a promising platform for improving the in vitro production of high-value saffron apocarotenoids and support future applications in saffron biotechnology and metabolic engineering.PMID:42469371 | DOI:10.1038/s41598-026-61913-9