Unique biosynthetic abilities revealed by plants determine in vitro cultures of hairy roots as a suitable source of pharmaceutically relevant bioactive compounds. The basic aim of the study was to examine the applicability of aerogel composed of methyltrimethoxysilane (MTMS) for immobilization of Rindera graeca hairy roots by identifying quantitative effects of biomass proliferation and naphthoquinones extracellular secretion in the aerogel-supported culture system. R. graeca hairy roots were simultaneously cultured for 28-days, as (i) nonimmobilized biomass (reference system), (ii) biomass immobilized on macroporous polyurethane foam (PUF), (iii) biomass with disintegrated MTMS aerogel, (iv) biomass immobilized on polypropylene (PP) fibers (as control), and (v) biomass immobilized on monolithic PP-reinforced MTMS aerogel. MTMS aerogel exhibited high level of biocompatibility toward R. graeca hairy roots which grew into the structure of monolithic aerogel-based constructs. Monolithic MTMS-based constructs significantly promoted the proliferation of hairy roots, resulting in 55% higher fresh mass than the reference system. The highest level of naphthoquinones productivity, i.e., 653 µg g_DW⁻¹, was noted for PUF-supported culture system.

Unique phytochemical profile of plants belonging to Boraginaceae family provides a prolific resource of lipophilic pigments from the group of naphthoquinone derivatives. To overcome low compound content, the major obstacle of plant-based production, immobilization of Rindera graeca roots in in vitro cultures was implemented for efficient production of rinderol, novel furanonaphthoquinone derivative with anticancer properties. Chromatographic procedures revealed rinderol presence in extracts of all investigated root lines, derived both from root biomass and post-culture medium. Unexpectedly, in the second stage of the experiment, rinderol production was ceased in control, unmodified culture systems. On the contrary, roots immobilized on PUF rafts uniformly and stably produced rinderol, and its highest amount was noted for transformed root lines after 42 days of cultivation (222.98 ± 10.47 µg/flask). PUF occurred to be the main place of compound accumulation. Moreover, investigation of rinderol biological activity revealed its fast-acting cell death induction in HeLa cervical cancer cells at relatively low concentrations. Presented results revealed successful application of R. graeca roots immobilization on PUF rafts for production and in situ product removal of rinderol, novel lipophilic furanonaphthoquinone with suggested proapoptotic activity.

The in vitro cultures of Rindera graeca, a rare endemic plant, were developed as a sustainable source of phenolic acids. Various shoot and root cultures were established and scaled up in a sprinkle bioreactor. A multiplication rate of 7.2 shoots per explant was achieved. HPLC–PDA–ESI–HRMS analysis revealed the presence of rosmarinic acid (RA) and lithospermic acid B (LAB) as the main secondary metabolites in both the shoot and root cultures. The maximum RA (30.0 ± 3.2 mg/g DW) and LAB (49.3 ± 15.5 mg/g DW) yields were determined in root-regenerated shoots. The strongest free radical scavenging activity (87.4 ± 1.1%), according to 2,2-diphenyl-1-picrylhydrazyl-hydrate assay, was noted for roots cultivated in a DCR medium. The highest reducing power (2.3 µM ± 0.4 TE/g DW), determined by the ferric-reducing antioxidant power assay, was noted for shoots cultivated on an SH medium containing 0.5 mg/L 6-benzylaminopurine. A genetic analysis performed using random amplified polymorphic DNA and start codon targeted markers revealed genetic variation of 62.8% to 96.5% among the investigated shoots and roots. This variability reflects the capacity of cultivated shoots and roots to produce phenolic compounds.

In vitro plant cell and tissue culture systems allow for controlling a wide range of culture environmental factors selectively influencing biomass growth and the yield of secondary metabolites. Among the most efficient methods, complex supplementation of the culture medium with elicitors, precursors, and other functional substances may significantly enhance valuable metabolite productivity through a stress induction mechanism. In the search for novel techniques in plant experimental biotechnology, the goal of the study was to evaluate stress-inducing properties of novel biodegradable ester-based scaffolds made of poly(glycerol sebacate) (PGS) and poly(lactic acid) (PLA) influencing on the growth and deoxyshikonin productivity of Rindera graeca hairy roots immobilized on the experimental constructs. Rindera graeca hairy roots were maintained under the dark condition for 28 days in three independent systems, i.e., (i) non-immobilized biomass (a reference system), (ii) biomass immobilized on PGS scaffolds, and (iii) biomass immobilized on PLA scaffolds. The stress-inducing properties of the applied polymerized esters selectively impacted R. graeca hairy roots. The PGS scaffolds caused the production of deoxyshikonin, which does not occur in other culture systems, and PLA promoted biomass proliferation by doubling its increase compared to the reference system.

Chitin deacetylase is the only known enzyme which is able to deacetylate N-acetyl-D-glucosamine units in chitin or chitosan chains. As chitin can hardly be dissolved in organic/inorganic solvents, new solvents are still searched. Ionic liquids are promising for that application and for homophase enzymatic deacetylation. The aim of the work was to investigate the influence of selected ionic liquids on activity of chitin deacetylase. It has been shown that [Amim] ionic liquids increase the activity of chitin deacetylase. The highest activity was observed for [Amim][Cl]. Ionic liquids with shorter (ethyl (C2)) and longer side chain (buthyl (C4)) only insignificantly influenced the activity of the enzyme. All tested ionic liquids with [Br] anion increased the activity of chitin deacetylase while the [Emim] and [Bmim] cation in combination with [Cl] anion inhibited the activity of the enzyme.

In situ extraction is a method for separating plant secondary metabolites from in vitro systems of plant biomass cultures. The study aimed to investigate the MTMS-based xerogels morphology effect on the growth kinetics and deoxyshikonin productivity in xerogel-supported in vitro culture systems of Rindera graeca hairy root. Cultures were supplemented with three types of xerogel, i.e., mesoporous gel, microporous gel, and agglomerated precipitate, in the disintegrated or monolithic form. Structure, oil sorption capacity, and SEM analyses for xerogel-based additives were performed. Application of monolithic macroporous xerogel resulted in the highest biomass proliferation, i.e., 5.11-fold fresh biomass increase after four weeks of the screening culture. The highest deoxyshikonin production (i.e., 105.03 µg) was noted when hairy roots were maintained with particles of disintegrated mesoporous xerogel. The detailed kinetics investigations (6-week culture) revealed the highest growth of hairy root biomass and secondary metabolite production, equaling 9.46-fold fresh weight biomass and 204.08 µg deoxyshikonin, respectively. MTMS-based xerogels have been recognized as selective biocompatible scaffolds for boosting the proliferation of transgenic roots or for productivity enhancement of naphthoquinones without detrimental effects on biomass growth, and their successful applicability in in situ removal of secondary plant metabolites has been experimentally confirmed.