The effects of thidiazuron (TDZ) pretreatment of shoot tips on Harpagophytum procumbens shoot proliferation and successive stages of micropropagation, i.e. rooting of regenerated shoots and acclimatization of plantlets to ex vitro conditions, were described in the present study. The best response in terms of shoot proliferation (about seven shoots/explant) and shoot length (3.2 ± 0.4 cm) was obtained when explants pretreated with 25 µmol L⁻¹ TDZ for 6 h were cultured on Schenk and Hildebrandt medium containing indole-3-acetic acid (IAA) (0.57 µmol L⁻¹) and 6-benzylaminopurine (BAP) (8 µmol L⁻¹). Under these conditions, a 330 % increase in shoot multiplication over TDZ non-pretreatment culture was achieved and TDZ pretreatment shoots were longer compared to those in control culture (2.6 ± 0.3 cm). The TDZ pretreatment did not affect the percentage of rooted shoots, length of roots and number of roots formed per shoot. The rooted plantlets were transplanted from in vitro to pots with soil and grown during 1 year in the greenhouse. The hardening process was difficult and time-consuming. We found that the plants developed from the TDZ pretreated culture were superior to plants from non-pretreated culture in terms of survival rate and morphological features, such as shoot length, leaf size, flowering and earlier root tuberisation. Random amplified polymorphic DNA and inter-simple sequence repeat analyses of pretreatment with TDZ plants showed genetic similarity to non-pretreatment plants. We conclude that applying the strategy of initial explant pretreatment with TDZ may be valuable for the improvement in H. procumbens in vitro propagation.

Methyl jasmonate (MJA), a signaling molecule in stress pathways, can be used to induce secondary metabolite synthesis in plants. The present study examines its effects on the growth of Salvia viridis hairy roots, and the accumulation of bioactive compounds, and correlates it with the expression of genes involved in the phenylpropanoid pathway. To our knowledge, this study represents the first exploration of elicitation in S. viridis culture and the first comprehensive analysis of MJA’s influence on such a wide array of genes within the polyphenol metabolic pathway in the Salvia genus. Plants were treated with 50 and 100 µM MJA, and samples were collected at intervals of one, three, five, and seven days post-elicitation. HPLC analysis revealed that MJA stimulated the accumulation of all tested compounds, with a 30% increase (38.65 mg/g dry weight) in total polyphenol content (TPC) on day five. Quantitative real-time polymerase chain reaction (RT-PCR) analysis demonstrated a significant increase in the expression of the phenylpropanoid pathway genes—TAT (tyrosine aminotransferase), HPPR (4-hydroxyphenylpyruvate reductase), PAL (phenylalanine ammonia-lyase), C4H (cinnamic acid 4-hydroxylase), 4CL (4-coumarate-CoA ligase), and RAS (rosmarinic acid synthase)—following MJA treatment. For the majority of the genes, this increase was observed after the first day of treatment. Importantly, our present results confirm strong correlations of the analyzed gene expression with polyphenol biosynthesis. These findings support the notion that hairy roots provide a promising biotechnological framework for augmenting polyphenol production. Additionally, the combination of elicitor treatment and transgenic technology emerges as a viable strategy to enhance the biosynthesis of these valuable metabolites.

Salvia miltiorrhiza is a medicinal plant that synthesises biologically-active tanshinones with numerous therapeutic properties. An important rate-limiting enzyme in the biosynthesis of their precursors is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). This study presents the organ-specific expression profile of the S. miltiorrhiza HMGR4 gene and its sensitivity to potential regulators, viz. gibberellic acid (GA₃), indole-3-acetic acid (IAA) and salicylic acid (SA). In addition, it demonstrates the importance of the HMGR4 gene, the hormone used, the plant organ, and the culture environment for the biosynthesis of tanshinones. HMGR4 overexpression was found to significantly boost the accumulation of dihydrotanshinone I (DHTI), cryptotanshinone (CT), tanshinone I (TI) and tanshinone IIA (TIIA) in roots by 0.44 to 5.39 mg/g dry weight (DW), as well as TIIA in stems and leaves. S. miltiorrhiza roots cultivated in soil demonstrated higher concentrations of the examined metabolites than those grown in vitro. GA₃ caused a considerable increase in the quantity of CT (by 794.2 µg/g DW) and TIIA (by 88.1 µg/g DW) in roots. In turn, IAA significantly inhibited the biosynthesis of the studied tanshinones in root material.

This study reports the first successful establishment of Perovskia atriplicifolia hairy root cultures using Rhizobium rhizogenes and evaluates their potential for bioactive phenolic acid production, particularly rosmarinic acid (RA). Hairy roots were induced using two R. rhizogenes strains, A4 and ATCC 15834; transformation was confirmed by PCR analysis targeting the rol and aux genes. The A4 strain exhibited higher transformation efficiency (41.3%) than ATCC 15834 (30.2%). Eight transgenic root clones (C1–C8) were established and confirmed as transformed. The clones exhibited significant variation in biomass accumulation and phenolic acid production. RA production was most strongly correlated with PAL, RAS, and CYP98A14 expression. Hierarchical clustering clustered the clones into three groups based on growth, metabolite content, and gene expression. Lines C1 and C2 exhibiting the highest RA, total polyphenol content, and the highest productivity were selected for further experiments. McCown Woody Plant (WP) and Schenk and Hildebrandt (SH) media demonstrated the greatest biomass accumulation, with growth indexes exceeding 13. Conversely, Gamborg (B5) medium enhanced RA content, achieving 38.3 and 40.8 mg/g dry weight (DW) for clones C1 and C2, respectively, representing a fourfold increase compared to the least favorable Murashige and Skoog (MS) medium. These findings establish P. atriplicifolia hairy roots as efficient systems for RA biosynthesis and can provide a basis for metabolic engineering and scale-up production of phenolic acids in medicinal plants.