Hairy roots of Hypericum perforatum L.: a promising system for xanthone production

Hypericum perforatum L. is a common perennial plant with a reputed medicinal value. Investigations have been made to develop an efficient protocol for the identification and quantification of secondary metabolites in hairy roots (HR) of Hypericum perforatum L. HR were induced from root segments of in vitro grown seedlings from H. perforatum, after co-cultivation with Agrobacterium rhizogenes A4. Transgenic status of HR was confirmed by PCR analysis using rolB specific primers. HR had an altered phenolic profile with respect to phenolic acids, flavonol glycosides, flavan-3-ols, flavonoid aglycones and xanthones comparing to control roots. Phenolics in control and HR cultures were observed to be qualitatively and quantitatively distinct. Quinic acid was the only detectable phenolic acid in HR. Transgenic roots are capable of producing flavonol glycosides such as quercetin 6-C-glucoside, quercetin 3-O-rutinoside (rutin) and isorhamnetin O-hexoside. The HPLC analysis of flavonoid aglycones in HR resulted in the identification of kaempferol. Transformed roots yielded higher levels of catechin and epicatechin than untransformed roots. Among the twenty-eight detected xanthones, four of them were identified as 1,3,5,6-tetrahydroxyxanthone, 1,3,6,7-tetrahydroxyxanthone, γ-mangostin and garcinone C were de novo synthesized in HR. Altogether, these results indicated that H. perforatum HR represent a promising experimental system for enhanced production of xanthones.     

Root cultures of <Emphasis Type="Italic">Hypericum perforatum</Emphasis> subsp. <Emphasis Type="Italic">angustifolium</Emphasis> elicited with chitosan and production of xanthone-rich extracts with antifungal activity

Hypericum perforatum is a well-known medicinal plant which contains a wide variety of metabolites, including xanthones, which have a wide range of biological properties, including antifungal activity. In the present study, we evaluated the capability of roots regenerated from calli of H. perforatum subsp. angustifolium to produce xanthones. Root biomass was positively correlated with the indole-3-butyric acid concentration, whereas a concentration of 1 mg l⁻¹ was the most suitable for the development of roots. High auxin concentrations also inhibited xanthone accumulation. Xanthones were produced in large amounts, with a very stable trend throughout the culture period. When the roots were treated with chitosan, the xanthone content dramatically increased, peaking after 7 days. Chitosan also induced a release of these metabolites into the culture. The maximum accumulation (14.26 ± 0.62 mg g⁻¹ dry weight [DW]) and release (2.64 ± 0.13 mg g⁻¹ DW) of xanthones were recorded 7 days after treatment. The most represented xanthones were isolated, purified, and spectroscopically characterized. Antifungal activity of the total root extracts was tested against a broad panel of human fungal pathogen strains (30 Candida species, 12 Cryptococcus neoformans, and 16 dermatophytes); this activity significantly increased when using chitosan. Extracts obtained after 7 days of chitosan treatment showed high antifungal activity (mean minimum inhibitory concentration of 83.4, 39.1, and 114 μg ml⁻¹ against Candida spp., C. neoformans, and dermatophytes, respectively). Our results suggest that root cultures can be considered as a potential tool for large-scale production of extracts with stable quantities of xanthones.     

Identification and quantification of phenolic compounds in Hypericum perforatum L. transgenic shoots

Regeneration of transgenic shoots was achieved from Hypericum perforatum L. hairy roots on hormone-free MS/B₅ medium for a period of 4 weeks under a photoperiod of 16-h light. A control experiment was set up with root segments obtained from in vitro grown seedlings. Investigations have been made to study the production of phenolic compounds in non transgenic and transgenic shoot cultures. Six groups of phenolic compounds such as phenolic acids, flavonols, flavan-3-ols, naphtodianthrones, phloroglucinols, and xanthones were recorded in the transgenic shoots. Chlorogenic acid was found as the most representative phenolic acid in shoot extracts. With regard to the class of quercetin derivatives in transformed shoots, quercetin 6-C-glucoside usually dominated among the glycosides followed by quercitrin and hyperoside. The analysis of flavan-3-ols in transgenic shoots resulted in the identification of epicatechin and proanthocyanidin dimers. One of the main achievements in this study was considerably enhanced hypericin and pseudohypericin production in transgenic shoots. The concentration of identified naphtodianthrones was about 12-fold higher in transformed shoots compared to control. Chromatographic analysis of phloroglucinols in transgenic shoots resulted in the identification of hyperforin, while its homolog adhyperforin was detected in traces. A twofold higher content of hyperforin was observed in transgenic shoots compared to control. Although mangiferin was found as the main representative xanthone in shoot extracts, several other xanthones identified as γ-mangostin isomers, trihydroxy-1-methoxy-C-prenyl xanthone, garcinone E, and banaxathone E were de novo synthesized in transformed shoots. Therefore, H. perforatum transgenic shoots could be considered as a source for rapid and increased production of naphtodianthrones and other specific phenolic compounds.     

<i>In Vitro</i> and <i>in Silico</i> Insights on the Biological Activities,Phenolic Compounds Composition of <i>Hypericum perforatum</i> L. Hairy Root Cultures

Three Hypericum perforatum hairy root lines (HR B, HR F and HR H) along with non-transformed roots were analyzed for phenolic compounds composition and in vitro enzyme inhibitory properties. In silico molecular modeling was performed to predict the interactions of the most representative phenolic compounds in HR clones with enzymes related to depression, neurodegeneration and diabetes. Chromatographic analyses revealed that HR clones represent an efficient source of quinic acid and hydroxybenzoic acids, epicatechin and procyanidin derivatives, quercetin and kaempferol glycosides, as well numerous xanthones. In vitro antidepressant activity of HR extracts through monoamine oxidase A (MAO-A) inhibition was attributed to the production of oxygenated and prenylated xanthones. The neuroprotective potential of HR extracts was related to the accumulation of quercetin 6-C-glucoside, epicatechin, procyanidins and γ-mangostin isomers as potential inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Vanillic acid and prenylated xanthones in HR clones as promising inhibitors of tyrosinase additionally contributed to the neuroprotective activity. Five preeminent xanthones in HR (γ-mangostin, mangiferin, garcinone C, garcinone E and 1,3,7-trihydroxy-6-metoxy-8-prenyl xanthone) along with the flavonol quercetin 6-C-glucoside effectively inhibited α-amylase and α-glucosidase indicating the antidiabetic properties of HR extracts. Transgenic roots of H. perforatum can be exploited for the preparation of novel phytoproducts with multi-biological activities.     

Anthocyanins and xanthones in the calli and regenerated shoots of Hypericum perforatum var. angustifolium (sin. Fröhlich) Borkh

The present paper reports on the production of anthocyanins and xanthones in different in vitro systems of Hypericum perforatum var. angustifolium (sin. Fröhlich) Borkh. Undifferentiated calli and regenerated shoots at different developmental stages were analyzed by applying an extractive and an analytical procedure capable of detecting and quantifying anthocyanins. The findings revealed, for the first time, the co-presence of hypericins and anthocyanins in shoots at initial and more developed stages of H. perforatum var. angustifolium L. Moreover, a high production of xanthones was found in the undifferentiated calli.     

Metabolite profiling and fingerprinting of Hypericum species: a comparison of MS and NMR metabolomics

Hypericum perforatum, commonly known as St. John’s wort, is a popular herbal supplement used for the treatment of mild to moderate depression. The major secondary metabolites of St. John’s wort extracts include phenylpropanoids, flavonoids, xanthones, phloroglucinols, and naphthodianthrones. There are over 400 species in the genus Hypericum world-wide, most of which are little or not characterized in terms of phytochemical or pharmacological properties. Metabolomics techniques were used to investigate the natural product diversity within the genus Hypericum (Hypericaceae) and its correlation to bioactivity, exemplified by cytotoxic properties. Utilizing nuclear magnetic resonance (NMR) fingerprinting and mass spectrometry (MS) metabolic profiling techniques, MS and NMR spectra of extracts from H. perforatum, H. polyphyllum, H. tetrapterum, H. androsaemum, H. inodorum, H. undulatum and H. kouytchense were evaluated and submitted to statistical multivariate analyses. Although comparable score plots in principal component analysis were derived from both MS and NMR datasets, loading plots reveal, that different set of metabolites contribute for species segregation in each dataset. Major peaks in ¹H NMR and MS spectra contributing to species discrimination were assigned as those of hyperforins, lipids, chlorogenic and shikimic acid. Shikimic acid and its downstream phenylpropanoids were more enriched in H. perforatum, H. androsaemum, H. kouytchense and H. inodorum extracts; whereas a novel hyperforin was found exclusively in H. polyphyllum. Next to H. perforatum, H. polyphyllum and H. tetrapterum show the highest levels of hypericins, and H. perforatum and H. polyphyllum are highest in phloroglucinols, suggesting that the latter species might be used as an alternative to St. John’s wort. However, the major hyperforin-type compound in H. polyphyllum possesses a novel constitution of yet unknown bioactivity. Anti-cancer in vitro assays to evaluate the ability of extracts from Hypericum species in inhibiting prostate and colon cancer growth suggest that such bioactivity might be predicted by gross metabolic profiling.     

Shoot and root culture of Hypericum perforatum L. transformed with Agrobacterium rhizogenes A4M70GUS

Hairy root cultures of Hypericum perforatum were obtained following inoculation of aseptically germinated seedlings with A. rhizogenes strain A4M70GUS. Effect of sucrose on the growth and biomass production of hairy root cultures was investigated. Hairy root cultures spontaneously regenerated shoots buds from which a number of shoot culture clones was established. Transformed shoot cultures exhibited good shoot multiplication, elongation and rooting on a hormone-free woody plant medium. Plants regenerated from hairy roots were similar in appearance to the normal, nontransformed plants.     

Production of adventitious root biomass and secondary metabolites of Hypericum perforatum L. in a balloon type airlift reactor

The effects of inoculum density, aeration volume and culture period on accumulation of biomass and secondary metabolites in adventitious roots of Hypericum perforatum in balloon type airlift bioreactors (3 l capacity) were investigated. The greatest increment of biomass as well as metabolite content occurred at an inoculum density of 3 g l⁻¹ and an aeration volume of 0.1 vvm. After 6 weeks of culture, an approximately 50-fold increase in biomass was recorded containing 60.11 mg g⁻¹ dry weight (DW) of phenolics, 42.7 mg g⁻¹ DW of flavonoids and 0.80 mg g⁻¹ DW of chlorogenic acid. Liquid chromatography–mass spectroscopy/mass spectroscopy demonstrated that the presence of quercetin and hyperoside in adventitious roots at a level of 1.33 and 14.01 μg g⁻¹ DW, respectively after 6 weeks of culture. The results suggest scale-up of adventitious root culture of H. perforatum for the production of chlorogenic acid, quercetin and hyperoside is feasible.     

Acetic acid acts as an elicitor exerting a chitosan-like effect on xanthone biosynthesis in <Emphasis Type="Italic">Hypericum perforatum</Emphasis> L. root cultures

Key message

Acetic acid acts as a signal molecule, strongly enhancing xanthone biosynthesis in Hypericum perforatum root cultures. This activity is specific, as demonstrated by the comparison with other short-chain monocarboxylic acids.

Abstract

We have recently demonstrated that Hypericum perforatum root cultures constitutively produce xanthones at higher levels than the root of the plant and that they respond to chitosan (CHIT) elicitation with a noteworthy increase in xanthone production. In the present study, CHIT was administered to H. perforatum root cultures using three different elicitation protocols, and the increase in xanthone production was evaluated. The best results (550 % xanthone increase) were obtained by subjecting the roots to a single elicitation with 200 mg l^?1 CHIT and maintaining the elicitor in the culture medium for 7 days. To discriminate the effect of CHIT from that of the solvent, control experiments were performed by administering AcOH alone at the same concentration used for CHIT solubilization. Unexpectedly, AcOH caused an increase in xanthone production comparable to that observed in response to CHIT. Feeding experiments with ¹³C-labeled AcOH demonstrated that this compound was not incorporated into the xanthone skeleton. Other short-chain monocarboxylic acids (i.e., propionic and butyric acid) have little or no effect on the production of xanthones. These results indicate that AcOH acts as a specific signal molecule, able to greatly enhance xanthone biosynthesis in H. perforatum root cultures.

     

Production of phenolic compounds,antioxidant and antimicrobial activities in hairy root and shoot cultures of <Emphasis Type="Italic">Hypericum perforatum</Emphasis> L.

Three hairy root clones of Hypericum perforatum (HR 2, HR 15 and HR 27) transformed with Agrobacterium rhizogenes A4M70GUS and their corresponding regenerated shoot culture clones (HRRS) were compared for differences in growth, production of phenolic compounds, antioxidant and antimicrobial activities. Transgenic clones were selected on the basis of morphological evaluation, genetic and molecular analyses. The clone HR 2 had the highest biomass accumulation, while HR 27 showed the highest shoot regeneration potential. The total phenolics and flavan-3-ols were enhanced in all tested transgenic cultures, while total flavonoids and hypericins were augmented in HRRS clones compared to non-transformed shoots. The HRRS clones produced substantial amounts of chlorogenic acid and 3-p-coumaroylquinic acid. Regarding the flavonoids, they produced significant contents of luteolin hexoside (HRRS 2), quercitrin and quercetin (HRRS 15) and isoquercetin (HRRS 27), while HR 2 and 15 accumulated 4-O-methylkaempferol-O-hexoside and quercetin 6-C-glucoside, respectively. The HR 15 was promising for the production of catechin and procyanidin derivatives and together with its HRRS clone exhibited a high potential for hyperforin and adhyperforin production. All identified naphtodianthrones were confirmed in HRRS 2 and 15 clones. Among xanthones, mangiferin was found as the major compound in HRRS, while trihydroxy-1-metoxy-C-prenyl xanthone was dominant in HR clones. Antimicrobial activity of transgenic cultures revealed that HRRS 15 strongly inhibited the growth of Bacillus cereus, Micrococcus flavus, Pseudomonas aeruginosa and Escherichia coli. Altogether, H. perforatum HR and HRRS cultures could be proposed as promising experimental systems for enhanced production of phenolic compounds with antioxidant and antibacterial properties.     

Metabolic profiling analysis of the degradation of phenol and 4-chlorophenol by Pseudomonas sp. cbp1-3

To investigate the enhancement of phenol on the biodegradation of 4-chlorophenol (4-cp), metabolic profiling approach was performed for the first time to analyze metabolite changes of Pseudomonas sp. cbp1-3 using single substrate (succinate, phenol, and 4-cp) and dual substrate (mixtures of phenol and 4-cp). Phosphoric acid, γ-aminobutyric acid, 4-cp, 4-chlorocatechol, and catechol were shown to change significantly. Results indicated that phenols, especially 4-cp, depressed cell growth by inhibiting its primary metabolic pathway. In addition, the addition of phenol into the 4-cp-containing medium had a global influence on cells including the accumulation of amino acids, amines, saturated fatty acids, and monoacylglycerols as well as the concentration changes of metabolite participating in phenols biodegradation, thus enhancing the degradation of 4-cp. This study provided novel insights into the biodegradation of mixed phenolic compounds and the method could be used to investigate the biodegradation of complicated multi-pollutants.     

Relation métabolique entre l'arginine et l'acide γ-aminobutyrique dans le fruit de Castanea sativa: étude préliminaire de l'arginase

Analysis of the free amino acid pool in the chestnut fruit (Castanea sativa) shows that it is high in γ-aminobutyric acid. A metabolic connection between arginine and γ-aminobutyric acid is evident. Three enzymatic activities are involved: L-arginine ureohydrolase, L-ornithine 2-oxo-acid aminotransferase and L-glutamate carboxylyase.     

Optimal reaction conditions for the production of γ-aminobutyric acid by the marine yeast isolate Pichia anomala MR-1 strain

The optimal conditions for the production of γ-aminobutyric acid (GABA) by the marine yeast isolate Pichia anomala MR-1 strain appeared to be completely different from those required for growth and biomass production of the yeast strain. By proper reaction temperatures and pH levels, the efficiency of GABA production was improved dramatically. Supplying enough energy for the reaction was another important technical element to improve the production efficiency of GABA. The MR-1 isolate was found to use various saccharides, including glucose and fructose, as well as intermediate metabolites in the tricarboxylic acid (TCA) cycle to promote GABA production. On the other hand, ethanol, various free amino acids, and organic acids were detected along with GABA in the reaction solution. These results suggest that production of GABA by the MR-1 strain is not due to a single enzymatic reaction. Rather it is a fermentation reaction, possibly involving a combination of intracellular metabolic pathways.     

Xanthone biosynthesis in Hypericum perforatum cells provides antioxidant and antimicrobial protection upon biotic stress

Xanthone production in Hypericum perforatum (HP) suspension cultures in response to elicitation by Agrobacterium tumefaciens co-cultivation has been studied. RNA blot analyses of HP cells co-cultivated with A. tumefaciens have shown a rapid up-regulation of genes encoding important enzymes of the general phenylpropanoid pathway (PAL, phenylalanine ammonia lyase and 4CL, 4-coumarate:CoA ligase) and xanthone biosynthesis (BPS, benzophenone synthase). Analyses of HPLC chromatograms of methanolic extracts of control and elicited cells (HP cells that were co-cultivated for 24 h with A. tumefaciens) have revealed a 12-fold increase in total xanthone concentration and also the emergence of many xanthones after elicitation. Methanolic extract of elicited cells exhibited significantly higher antioxidant and antimicrobial competence than the equivalent extract of control HP cells indicating that these properties have been significantly increased in HP cells after elicitation. Four major de novo synthesized xanthones have been identified as 1,3,6,7-tetrahydroxy-8-prenyl xanthone, 1,3,6,7-tetrahydroxy-2-prenyl xanthone, 1,3,7-trihydroxy-6-methoxy-8-prenyl xanthone and paxanthone. Antioxidant and antimicrobial characterization of these de novo xanthones have revealed that xanthones play dual function in plant cells during biotic stress: (1) as antioxidants to protect the cells from oxidative damage and (2) as phytoalexins to impair the pathogen growth.     

Effect of plant growth regulators on growth and biosynthesis of phenolic compounds in genetically transformed hairy roots of<Emphasis Type="Italic">Panax ginseng</Emphasis> C. A. Meyer

In this study, morphological alterations, biomass growth, and secondary metabolite production of genetically transformed hairy roots ofPanax ginseng C. A. Meyer, were evaluated after administration of plant growth regulators. The addition of benzylamino purine and kinetin to the culture media increased biomass formation and phenolic compound biosynthesis in the hairy roots. α-Naphthaleneacetic acid and indole-3-butyric acid inhibited hairy root growth, however, low concentrations of indole-3-acetic acid slightly increased hairy root growth. Low concentrations of 2,4-Dichlorophenoxyacetic acid profoundly inhibited growth of hairy roots. The addition of plant growth regulators, such as auxin, did not increase total phenolic compounds in hairy roots that did not contain gibberellic acid and cytokinins. Callus formation was induced in cultures suspended in liquid medium amended with benzylamino purine and kinetin. Hairy roots regenerated from these calluses exhibited an active growth pattern with extensive lateral branching in non-amended medium, similar to the growth pattern of the original hairy roots.     

Secoiridoids and xanthones in the shoots and roots of <Emphasis Type="Italic">Centaurium pulchellum</Emphasis> cultured <Emphasis Type="Italic">in vitro</Emphasis>

Summary The qualitative and quantitative composition of secondary metabolites was studied in the shoots and roots of Centaurium pulchellum cultured in vitro. Secoiridoids (gentiopierin, swertiamarin, and sweroside) and xanthones (methylbellidifolin, demethyleustomin, and deccussatin) were isolated. Sweroside was found to be the major secoiridoid compound in the aerial parts of plants growing in nature. while swertiamarin dominated in plants cultured in vitro. In roots of all plants, genciopicrin was the major compound. Xanthone demethyleustomin was the major compound both in the shoots and roots of plants growing in nature and cultured in vitro. Different sugars (glucose, fructose, and sucrose) added in different concentrations in the medium affected the production of secondary compounds.     

The variability of the hypericin content in the regenerants of Hypericum perforatum

The hypericin content of in vitro regenerated plants of Hypericum perforatum L. was determined by spectrophotometry. A significant variability of some of the morphological characters, of the biomass production and the hypericin formation was found within the somaclones of the same genetic origin and among the regerants of different genotypes. The concentration of BAP which promoted the shoot differentiation did not affect the hypericin content and the gland density. New information on the ultrastructure of hypericin-containing multicellular glands is presented here.     

Influence of carbohydrate source on xanthone content in root cultures of Gentiana dinarica Beck

The effects of different types and concentrations of sugars on root growth and xanthone production in root culture of Gentiana dinarica were investigated. The results showed that sucrose, glucose and fructose all supported root growth, and sucrose was superior in terms of growth index, dry mass and fresh/dry mass ratio then fructose or glucose at the same concentrations. However, considering equimolar concentration of sugars, their contribution to the root growth was similar. The HPLC analysis of roots indicated the presence of xanthone compounds, and the contents of norswertianin-1-O-primeveroside (1), norswertianin-1-O-glucoside (2), gentioside (3) and norswertianin (4) were evaluated. In all samples, norswertianin-1-O-primeveroside (1) was present in highest concentration, followed by norswertianin-1-O-glucoside (2), whereas gentioside (3) and norswertianin (4) were present in lower amounts. The production of xanthones was affected by both type and concentration of sugar. In general, roots growing in media supplemented with sucrose contained higher levels of xanthones. The amounts of xanthone primeveroses (1) and (3) increased with the increase of concentrations of all types of sugars, whereas higher sugar concentrations resulted in reduction of the contents of norswertianin-1-O-glucoside (2) and aglycone norswertianin (4). The roots were also evaluated regarding the content of total phenolics and higher accumulation of total phenolic compounds was observed in roots grown in fructose-containing medium. Antioxidant activity was determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, and high correlation between total phenolic content and antiradical activity was observed (r = ?0.83).     

Foliar spraying of elicitors in pear trees induced resistance to Cacopsylla bidens

The pear psylla, Cacopsylla bidens (?ulc, 1907) (Psylloidea, Psyllidae, Psyllinae) is a serious pest of pear worldwide. In this research, the effects of salicylic acid (SA), chitosan, γ-aminobutyric acid (GABA), and Serenade Aso (Bacillus subtilis QST713) were studied on the induced resistance of pear trees to C. bidens. The treatments showed a significant difference in terms of population growth of C. bidens after 7 and 14 days during two years. In 2019, the mean population growth of total life stages of C. bidens in the studied treatments was significantly lower than control after 7 and 14 days of spraying. In 2020 (7 and 14 days after spraying), the percent decline in the mean population of pear psylla (total life stages) was highest on chitosan treatment (75 and 85 %, respectively) and the lowest on control (30 and 48 %, respectively). GC–MS analysis revealed the range of chemicals in the pear leaves under different treatments. Based on the results, 23 compounds were identified, including 9 alkaloids, 6 flavonoids, 4 polyphenols, 3 terpenoids, and one glycoside. In all treatments except Serenade, the amounts of alkaloids increased compared to the control. Furthermore, the activity of catalase and glutathione S- transferase enzymes in C. bidens under chitosan treatment was significantly higher than others; but the activity of peroxidase significantly decreased on chitosan compared to SA and GABA. The lower levels of peroxidase in C. bidens reared on trees treated with chitosan indicate this insect has not been able to detoxify plant secondary metabolites effectually. In addition, our findings suggested that chitosan on pear trees could act effectively in reducing the C. bidens population and can be considered in integrated management programs of this pest.     

Metabolic changes and improved growth in micropropagated red raspberry “Indian summer” are tied to improved mineral nutrition

Mineral nutrition is directly involved in plant metabolism and greatly affects growth and development. An initial study modeling Murashige and Skoog (MS) medium mineral components revealed that the quality of red raspberry shoot cultures was significantly affected by CaCl₂, MgSO₄, and KH₂PO₄ (mesos components). This study investigated the effects of increased mesos components on shoot growth and metabolism. Rubus idaeus L. “Indian summer” shoots grown on standard MS medium (1.0× MS mesos components) were compared to shoots grown with 1.5× and 2.5× MS mesos components. After 9 wk, shoots were evaluated for shoot quality, multiplication, elongation, and metabolic changes. Metabolic changes were determined by liquid chromatography (LC) coupled with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). Shoots grown on increased mesos components had improved quality, shoot length, and leaf color compared to shoots grown on MS medium. Metabolomic analysis indicated that shoots grown on high mesos component medium had reduced amounts of some free amino acids (glutamine, arginine, histidine, and proline) and some secondary metabolites (epicatechin, quercetin, and ellagic acid) compared to shoots on MS medium, which indicated reduced stress. Shoots grown on high mesos component also had increases in fructose 1-phosphate and glutathione associated with biosynthetic pathways, plant defense mechanisms, and redox homeostasis. Another factor involved in improved growth responses may be that increased glutamine was also found in high mesos component treatments, possibly influenced by ammonium accumulated from photorespiration. These metabolic changes provide initial insights into medium optimization and in vitro mineral nutrition, and the impact of nutrients on plant growth and development in micropropagated red raspberry shoots.