In vitro elicitation effectively stimulates the biosynthesis of plant specialized metabolites by using elicitors that enhance plant defense under stress. In this study, a chitosan (CTS) based elicitation protocol was established and validated in flaxseed, a rich source of lignans known to undergo de novo synthesis under stress. Two flax (Linum usitatissimum L.) genotypes, Agram and CDC Bethune, with distinct lignan profiles, were selected to assess whether elicitation enhances lignan production and antioxidant potential. The study also investigated the role of miRNAs in lignan biosynthesis under in vitro elicitation. CTS derived from shrimp shells at 10 mg L⁻¹ and 15 mg L⁻¹ was used to elicit the lignan-rich flax genotype Agram in vitro. While secoisolariciresinol content had been previously reported for these genotypes, we additionally quantified secoisolariciresinol diglucoside, pinoresinol, pinoresinol diglucoside, matairesinol, and lariciresinol using High-Performance Liquid Chromatography–Mass Spectrometry, enabling a more comprehensive evaluation of lignan composition and its genotype-dependent modulation under CTS treatment. In parallel, antioxidant activity, polyphenol levels, and phenolic acid content were assessed, with CDC Bethune exhibiting higher values than Agram under CTS treatment. Furthermore, analysis of lignan-related microRNAs revealed that lus-miR168a expression was higher in CDC Bethune, whereas lus-miR396 expression was higher in Agram in the presence of CTS. Chitosan elicitation enhances lignan biosynthesis and antioxidant capacity in flaxseed genotypes through a process regulated by specific microRNAs.
Eleutherine bulbosa (Mill.) Urb. is a medicinal plant rich in bioactive flavonoids, but the effects of salicylic acid (SA) and methyl jasmonate (MJ) elicitation on flavonoid accumulation in callus cultures remain poorly understood. This study evaluated SA and MJ separately at 50 and 100 ppm using molecular dynamics simulation, quantitative PCR (qPCR), untargeted liquid chromatography-high-resolution mass spectrometry (LC-HRMS), and total flavonoid content (TFC) analysis. Exploratory molecular dynamics simulations using Arabidopsis-derived receptor models indicated sustained SA retention in the Non-expressor of Pathogenesis-Related genes 1 (NPR1) model, whereas MJ was not retained in isolated Coronatine Insensitive 1 (COI1), a simplified system excluding the jasmonate perception complex. qPCR showed higher relative expression of chalcone synthase (CHS), flavonoid 3’5’-hydroxylase (F3’5’H), flavonol synthase (FLS), and anthocyanidin synthase (ANS) under elicitor treatments than in the control. SA 50 ppm showed the highest relative FLS expression (223.38-fold) and F3’5’H expression (25.75-fold), while MJ 50 ppm showed the highest relative ANS expression (7.93-fold). LC-HRMS showed higher relative total flavonoid peak areas for all treatments, with SA 100 ppm showing the highest fold change (3.40-fold). SA 50 ppm showed the strongest flavonol response (47.44-fold) and highest flavonol diversity, whereas MJ 50 ppm generated the highest compound diversity. TFC increased only under SA treatments, with the highest value under SA 50 ppm. These findings indicate that SA and MJ modulate flavonoid responses in a pathway- and concentration-dependent manner. SA 50 ppm showed the strongest flavonol-oriented metabolic response, while MJ 50 ppm supported flavonoid profile diversification. SA 50 ppm showed the strongest flavonol-oriented response, with the highest FLS expression, while MJ 50 ppm diversified the flavonoid profile without increasing total flavonoid content in E. bulbosa callus.
Efficient in vitro regeneration and genetic transformation are crucial prerequisites for biotechnological improvement of ryegrass (Lolium spp.). However, these processes are often genotype-dependent and constrained by explant responsiveness and endophytic fungal contamination. In this study, callus induction and regeneration potential were systematically evaluated in different ryegrass varieties using different explant sources, such as leaves, anthers and seeds. Among these, caryopsis-derived explants exhibited the highest callus induction and regeneration efficiency, identifying these as the most suitable explant source for ryegrass in vitro culture. A surface sterilization protocol was established to eliminate contamination without the use of fungicides or other additional treatments commonly required for Lolium explant production. Regeneration efficiency was quantified as the percentage of explants producing regenerated shoots, revealing significant variation among seven perennial, three Italian and one hybrid ryegrass cultivar. Genetic transformation was then performed on callus, leaves and anthers. While transient transformation was observed in leaves and anther tissues (indicated by red betalain pigmentation due to RUBY and fluorescent protein expression), these tissues failed to regenerate callus or whole plants. In contrast, callus-based transformation enabled stable RUBY expression. Incorporation of developmental regulators (DRs) significantly increased transformation efficiency by 40
Phenolic browning and microbial contamination are major constraints to the in vitro establishment of woody perennials including pomegranate (Punica granatum L.) explants during micropropagation. The present study aimed to develop an efficient protocol for culture establishment by optimizing pretreatment, anti-browning strategies, and culture conditions. In the first experiment to standardize nodal explant pre-treatments, different antimicrobial pretreatments, with or without ascorbic acid and citric acid supplementation, were evaluated in cv. ‘Solapur Lal’ to establish an effective aseptic culture initiation protocol. In the second experiment, the standardized explant pretreatment protocol was followed and eleven anti-browning treatment strategies were evaluated for successful culture initiation and selection of contamination-free explants, in the cultivars ‘Solapur Lal’ and ‘Bhagwa’ using Murashige and Skoog (1962) (MS) or Woody Plant Medium (WPM) as basal media under dark or light incubation conditions. These strategies included antioxidant pretreatment, paraffin sealing, polyvinylpyrrolidone (PVP), activated charcoal, explant repositioning after three days, and their combinations. The bacteriostatic chemical treatment 2-Bromo-2-nitro-1,3-propanediol (Bronopol) combined with antioxidants was the most effective pretreatment by reducing contamination while improving aseptic culture establishment to 75.69
Patchouli (Pogostemon cablin Benth.) is an industrially valued aromatic medicinal plant of the Lamiaceae family, native to tropical Asia and widely cultivated for its essential oil. The plant produces a complex array of bioactive compounds, including patchouli alcohol, pogostone, α-/β-patchoulene, α-bulnesene, seychellene, and pachypodol, that underpin its significance in the perfume, cosmetic, pharmaceutical, and aromatherapy industries. Indonesia supplies more than 80
