Screening the Key Region of Sunlight Regulating the Flavonoid Profiles of Young Shoots in Tea Plants (Camellia sinensis L.) Based on a Field Experiment.

Both UV and blue light have been reported to regulate the biosynthesis of flavonoids in tea plants; however, the respective contributions of the corresponding regions of sunlight are unclear. Additionally, different tea cultivars may respond differently to altered light conditions. We investigated the responses of different cultivars ('Longjing 43', 'Zhongming 192', 'Wanghai 1', 'Jingning 1' and 'Zhonghuang 2') to the shade treatments (black and colored nets) regarding the biosynthesis of flavonoids. For all cultivars, flavonol glycosides showed higher sensitivity to light conditions compared with catechins. The levels of total flavonol glycosides in the young shoots of different tea cultivars decreased with the shade percentages of polyethylene nets increasing from 70% to 95%. Myricetin glycosides and quercetin glycosides were more sensitive to light conditions than kaempferol glycosides. The principal component analysis (PCA) result indicated that shade treatment greatly impacted the profiles of flavonoids in different tea samples based on the cultivar characteristics. UV is the crucial region of sunlight enhancing flavonol glycoside biosynthesis in tea shoots, which is also slight impacted by light quality according to the results of the weighted correlation network analysis (WGCNA). This study clarified the contributions of different wavelength regions of sunlight in a field experiment, providing a potential direction for slightly bitter and astringent tea cultivar breeding and instructive guidance for practical field production of premium teas based on light regimes.

Dissection of the general two-step di-C-glycosylation pathway for the biosynthesis of (iso)schaftosides in higher plants.

Schaftoside and isoschaftoside are bioactive natural products widely distributed in higher plants including cereal crops and medicinal herbs. Their biosynthesis may be related with plant defense. However, little is known on the glycosylation biosynthetic pathway of these flavonoid di-C-glycosides with different sugar residues. Herein, we report that the biosynthesis of (iso)schaftosides is sequentially catalyzed by two C-glycosyltransferases (CGTs), i.e., CGTa for C-glucosylation of the 2-hydroxyflavanone aglycone and CGTb for C-arabinosylation of the mono-C-glucoside. The two enzymes of the same plant exhibit high homology but remarkably different sugar acceptor and donor selectivities. A total of 14 CGTa and CGTb enzymes were cloned and characterized from seven dicot and monocot plants, including Scutellaria baicalensis, Glycyrrhiza uralensis, Oryza sativa ssp. japonica, and Zea mays, and the in vivo functions for three enzymes were verified by RNA interference and overexpression. Through transcriptome analysis, we found homologous genes in 119 other plants, indicating this pathway is general for the biosynthesis of (iso)schaftosides. Furthermore, we resolved the crystal structures of five CGTs and realized the functional switch of SbCGTb to SbCGTa by structural analysis and mutagenesis of key amino acids. The CGT enzymes discovered in this paper allow efficient synthesis of (iso)schaftosides, and the general glycosylation pathway presents a platform to study the chemical defense mechanisms of higher plants.

A spatially-resolved approach to visualize the distribution and biosynthesis of flavones in Scutellaria baicalensis Georgi.

Imaging the spatial distributions and dynamics of flavones in heterogeneous plant tissues is significant for our understanding of plant metabolism. Here, we proposed a spatially-resolved approach to map the locations and biosynthesis of flavones in S. baicalensis. A total of 11 flavones, 5 flavone glycosides, 6 carbohydrates, and a variety of flavone synthesis-related metabolites were imaged. Most of these flavone-related metabolites presented stronger ion intensities in root phloem. The biosynthetic network of flavones and their glycosides in S. baicalensis were visualized for the first time. Moreover, we characterized the region-specific activities of four crucial enzymes in flavone synthesis pathway, including l-phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, 4-coumarate coenzyme A ligase, and flavone synthase. In line with the spatial characteristic of flavones, all these four enzymes exhibit higher activity in the root phloem of S. baicalensis. The combination of spatially-resolved metabolites and enzymes information greatly broadens our understanding of flavone biosynthetic network.

[Isolation and identification of endophytic fungi producing harpagoside and harpagide from Scrophularia ningpoensis].

The endophytic fungi from root,main stem,branch and leaf of Scrophularia ningpoensis were isolated from Zhejiang,whether these strains could yield harpagide or harpagoside were tested by HPLC and LC-MS. According to the morphological characteristic and the similarity of the nucleotide sequence of internal transcribed spacer( ITS) between r DNAs,the strains producing harpagide or harpagoside were identified. The results showed that 210 strains were isolated from the samples,which were classified into 9 orders,13 families and 17 genera by morphological study. Harpagide was detected in endogenous fungi ZJ17 and harpagoside was detected in endogenous fungi ZJ25 by HPLC coupled with LC-MS. ZJ17 was identified as Alternaria alternate and ZJ25 was identified as A.gaisen by its morphology and authenticated by ITS( ITS4 and ITS5 regions and the intervening 5. 8 S rDNA region).

Effects of nitrogen supply on flavonol glycoside biosynthesis and accumulation in tea leaves (Camellia sinensis).

Widely distributed in tea plants, the flavonoid flavonol and its glycosylated derivatives have important roles in determining tea quality. However, the biosynthesis and accumulation of these compounds has not been fully studied, especially in response to nitrogen (N) supply. In the present study, 'Longjing 43' potted tea seedlings were subjected to N deficiency (0g/pot), normal N (4g/pot) or excess N (16g/pot). Quantitative analyses using Ultra Performance Liquid Chromatography-Triple Quadrupole Mass Spectrometry (UPLC-QqQ-MS/MS) revealed that most flavonol glycosides (e.g., Quercetin-3-glucoside, Kaempferol-3-rgalactoside and Kaempferol-3-glucosyl-rhamnsoyl-glucoside) accumulated to the highest levels when treated with normal N. Results from metabolomics using Gas Chromatography-Mass Spectrometer (GC-MS) suggested that the levels of carbohydrate substrates of flavonol glycosides (e.g., sucrose, sucrose-6-phosphate, D-fructose 1,6-bisphosphate and glucose-1-phosphate) were positively correlated with flavonol glycoside content in response to N availability. Furthermore, Quantitative Real-time PCR analysis of 28 genes confirmed that genes related to flavonoid (e.g., flavonol synthase 1, flavonol 3-O-galactosyltransferase) and carbohydrate (e.g., sucrose phosphate synthase, sucrose synthase and glucokinase) metabolism have important roles in regulating the biosynthesis and accumulation of flavonol glycosides. Collectively, our results suggest that normal N levels promote the biosynthesis of flavonol glycosides through gene regulation and the accumulation of substrate carbohydrates, while abnormal N availability has inhibitory effects, especially excess N.

Modification of isoprene synthesis to enable production of curcurbitadienol synthesis in Saccharomyces cerevisiae.

Cucurbitane-type triterpenoids such as mogrosides and cucurbitacins that are present in the plants of Cucurbitaceae are widely used in Asian traditional medicine. Cucurbitadienol is the skeleton of cucurbitane-type triterpenoids. As an alternative production strategy, we developed baker's yeast Saccharomyces cerevisiae as a microbial host for the eventual transformation of cucurbitadienol. The synthetic pathway of cucurbitadienol was constructed in Saccharomyces cerevisiae by introducing the cucurbitadienol synthase gene from different plants, resulting in 7.80 mg cucurbitadienol from 1 L of fermentation broth. Improving supplies of isoprenoid precursors was then investigated for increasing cucurbitadienol production. Cucurbitadienol production increased to 21.47 mg/L through the overexpression of a global regulatory factor (UPC2) gene of triterpenoid synthase. In addition, knockout of the ERG7 gene increased cucurbitadienol production from 21.47 to 61.80 mg/L. Finally, fed-batch fermentation was performed, and 63.00 mg/L cucurbitadienol was produced. This work is an important step towards the total biosynthesis of valuable cucurbitane-type triterpenoids and demonstrates the potential for developing a sustainable and secure yeast biomanufacturing platform for triterpenoids.

Short-chain dehydrogenase/reductase governs steroidal specialized metabolites structural diversity and toxicity in the genus Solanum.

Thousands of specialized, steroidal metabolites are found in a wide spectrum of plants. These include the steroidal glycoalkaloids (SGAs), produced primarily by most species of the genus Solanum, and metabolites belonging to the steroidal saponins class that are widespread throughout the plant kingdom. SGAs play a protective role in plants and have potent activity in mammals, including antinutritional effects in humans. The presence or absence of the double bond at the C-5,6 position (unsaturated and saturated, respectively) creates vast structural diversity within this metabolite class and determines the degree of SGA toxicity. For many years, the elimination of the double bond from unsaturated SGAs was presumed to occur through a single hydrogenation step. In contrast to this prior assumption, here, we show that the tomato GLYCOALKALOID METABOLISM25 (GAME25), a short-chain dehydrogenase/reductase, catalyzes the first of three prospective reactions required to reduce the C-5,6 double bond in dehydrotomatidine to form tomatidine. The recombinant GAME25 enzyme displayed 3ß-hydroxysteroid dehydrogenase/Δ5,4 isomerase activity not only on diverse steroidal alkaloid aglycone substrates but also on steroidal saponin aglycones. Notably, GAME25 down-regulation rerouted the entire tomato SGA repertoire toward the dehydro-SGAs branch rather than forming the typically abundant saturated α-tomatine derivatives. Overexpressing the tomato GAME25 in the tomato plant resulted in significant accumulation of α-tomatine in ripe fruit, while heterologous expression in cultivated eggplant generated saturated SGAs and atypical saturated steroidal saponin glycosides. This study demonstrates how a single scaffold modification of steroidal metabolites in plants results in extensive structural diversity and modulation of product toxicity.

Synthesis of rebaudioside D, using glycosyltransferase UGTSL2 and in situ UDP-glucose regeneration.

Steviol glycosides from Stevia rebaudiana leaves are used in stevia-based sweeteners for their intense sweetness and low calories. Rebaudioside D is present in leaves in minute quantities (∼0.4-0.5% w/w total dry weight), but it is ∼350 times sweeter than sucrose, and sweeter than the more abundant rebaudioside A and stevioside. In the present study, pathways for rebaudioside D synthesis and UDP-glucose recycling were developed by coupling recombinant UDP-glucosyltransferase UGTSL2 from Solanum lycopersicum and sucrose synthase StSUS1 from Solanum tuberosum. Reaction parameters, including substrate ratio, sucrose concentration, temperature, crude extract concentration, and reaction time, were evaluated, and 17.4 g/l of rebaudioside D (yield = 74.6%) was obtained from 20 g/l of rebaudioside A after 20 h, using UDP or UDP-glucose in recombinant cell crude extracts. Extending the reaction time generated rebaudioside M2 from further glycosylation of rebaudioside D. Km values for UGTSL2 indicated a higher affinity for rebaudioside D than for rebaudioside A.

Wound Stress, an Unheeded Factor for Echinacoside Accumulation in Cistanche deserticola Y. C. Ma.

Cistanche deserticola Y. C. Ma, a precious parasitic medicinal herb distributed in desert areas in the Northwest of China, also known as "desert ginseng", has been used in China for thousands of years for its nourishing effects. The phenylethanoid glycosides (PeGs) have been proven as the main effective compounds due to their neuroprotective effects and were used for quality control. In this study, echinacoside content, a representative PeG, total phenolic content, DPPH scavenging activity, and PAL activity were determined in different tissues of C. deserticola. Our results showed that most indices had a similar pattern of scale > cambium ring > pith and bottom part > middle part > upper part. Besides, stereomicroscopic observation showed that the scale surface was densely covered with physical wounds formed during vertical and broadwise growth in sand. Thus, wound area was quantified and a linear regression analysis was conducted between wound area and PAL activity, total phenolics, and echinacoside content. Our results suggested that physical wounding caused by sand might play an important role in echinacoside biosynthesis which has never been noticed in C. deserticola development. Furthermore, the coexistence of the highest PAL activity and highest echinacoside accumulation in scale tissue might indicate that the biosynthetic site of echinacoside in C. deseticola Y. C. Ma is mainly in the scale tissue.

[Diversity of Secondary Metabolites from Some Medicinal Plants and Cultivated Lichen Mycobionts].

Studies on the structural determination, biosynthesis, and biological activities of secondary metabolites from natural sources are significant in the field of natural products chemistry. This review focuses on diverse secondary metabolites isolated from medicinal plants and cultivated mycobionts of lichens in our laboratory. Monoterpene-tetrahydroisoquinoline glycosides and alkaloids isolated from Cephaelis acuminata and Alangium lamarckii gave important information on the biosynthesis of ipecac alkaloids. A variety of glycosides linked with a secologanin unit and indole alkaloids were obtained from medicinal plants belonging to the families of Rubiaceae, Apocynaceae, and Loganiaceae. Plant species of the four genera Fraxinus, Syringa, Jasminum, and Ligustrum of the family Oleaceae were chemically investigated to provide several types of secoiridoid and iridoid glucosides. The biosynthetic pathway leading from protopine to benzophenanthridine alkaloids in suspension cell cultures of Eschscholtzia californica was elucidated. The structures and biological activities of the bisbenzylisoquinoline alkaloids of Stephania cepharantha and Nelumbo nucifera were also investigated. In addition, the mycobionts of lichens were cultivated to afford various types of metabolites that differ from the lichen substances of intact lichens but are structurally similar to fungal metabolites. The biosynthetic origins of some metabolites were also studied. These findings suggest that cultures of lichen mycobionts could be sources of new bioactive compounds and good systems for investigating secondary metabolism in lichens.

Synthesis and pharmacological evaluation of glycosides of resveratrol, pterostilbene, and piceatannol.

To enhance their water solubility and pharmacological activities, the stilbenes resveratrol, pterostilbene, and piceatannol were glycosylated to their monoglucosides (ß-glucosides) and diglycosides (ß-maltosides) by cultured cells and cyclodextrin glucanotransferase (CGTase). Cultured cells of Phytolacca americana and glucosyltransferase (PaGT) were capable of glucosylation of resveratrol to its 3- and 4'-ß-glucosides. Pterostilbene was slightly transformed into its 4'-ß-glucoside by P. americana cells. Piceatannol was readily converted into piceatannol 4'-ß-glucoside, with the highest yield among the three substrates. The 3- and 4'-ß-glucosides of resveratrol were subjected to further glycosylation by CGTase to give 3- and 4'-ß-maltoside derivatives. The inhibitory action of resveratrol and pterostilbene toward histamine release induced with compound 48/80 from rat peritoneal mast cells was improved by ß-glucosylation and/or ß-maltosylation (i.e., the inhibitory activity for histamine release of the 3- and 4'-ß-glucosides of resveratrol, the 3- and 4'-ß-maltosides of resveratrol, and the 4'-ß-glucoside of pterostilbene was higher than that of the corresponding aglycones, resveratrol and pterostilbene, respectively). In addition, the phosphodiesterase (PDE) inhibitory activity of resveratrol and pterostilbene was enhanced by ß-glucosylation and/or ß-maltosylation (i.e., the PDE inhibitory activities of the 3- and 4'-ß-glucosides of resveratrol, the 4'-ß-maltoside of resveratrol, and the 4'-ß-glucoside of pterostilbene were higher than those of the corresponding aglycones, resveratrol and pterostilbene, respectively).

Volatile Glycosylation in Tea Plants: Sequential Glycosylations for the Biosynthesis of Aroma ß-Primeverosides Are Catalyzed by Two Camellia sinensis Glycosyltransferases.

Tea plants (Camellia sinensis) store volatile organic compounds (VOCs; monoterpene, aromatic, and aliphatic alcohols) in the leaves in the form of water-soluble diglycosides, primarily as ß-primeverosides (6-O-ß-D-xylopyranosyl-ß-D-glucopyranosides). These VOCs play a critical role in plant defenses and tea aroma quality, yet little is known about their biosynthesis and physiological roles in planta. Here, we identified two UDP-glycosyltransferases (UGTs) from C. sinensis, UGT85K11 (CsGT1) and UGT94P1 (CsGT2), converting VOCs into ß-primeverosides by sequential glucosylation and xylosylation, respectively. CsGT1 exhibits a broad substrate specificity toward monoterpene, aromatic, and aliphatic alcohols to produce the respective glucosides. On the other hand, CsGT2 specifically catalyzes the xylosylation of the 6'-hydroxy group of the sugar moiety of geranyl ß-D-glucopyranoside, producing geranyl ß-primeveroside. Homology modeling, followed by site-directed mutagenesis of CsGT2, identified a unique isoleucine-141 residue playing a crucial role in sugar donor specificity toward UDP-xylose. The transcripts of both CsGTs were mainly expressed in young leaves, along with ß-primeverosidase encoding a diglycoside-specific glycosidase. In conclusion, our findings reveal the mechanism of aroma ß-primeveroside biosynthesis in C. sinensis. This information can be used to preserve tea aroma better during the manufacturing process and to investigate the mechanism of plant chemical defenses.

Production of oleanolic acid glycosides by hairy root established cultures of Calendula officinalis L.

In order to initiate hairy root culture initiation cotyledons and hypocotyls of Calendula officinalis L. were infected with Agrobacterium rhizogenes strain ATCC 15834 or the same strain containing pCAMBIA 1381Z vector with ß-glucuronidase reporter gene under control of promoter of NIK (Nematode Induced Kinase) gene. The efficiency of induction of hairy roots reached 33.8% for cotyledons and 66.6% for hypocotyls together for both transformation experiments. Finally, eight control and nine modified lines were established as a long-term culture. The hairy root cultures showed the ability to synthesize oleanolic acid mainly (97%) as glycosides; control lines contained it at the average 8.42 mg · g(-1) dry weight in tissue and 0.23 mg · dm(-3) in medium; modified lines: 4.59 mg · g(-1) for the tissue, and 0.48 mg · dm(-3) for the medium. Additionally lines showed high positive correlation between dry/fresh weight and oleanolic acid concentration in tissue. Using the Killiani mixture in acidic hydrolysis of oleanolic acid glycosides released free aglycones that were partially acetylated in such conditions.

Synthesis of resveratrol glycosides by cultured plant cells.

Incubation of cultured cells of Glycine max with trans-resveratrol gave its 3-O-beta-D- and 4'-O-beta-D-glucosides. Cultured Gossypium hirsutum cells glycosylated trans-resveratrol to its 3-O-beta-D-, 4'-O-beta-D-, and 3,4'-O-beta-D-diglucosides. On the other hand, trans-resveratrol was converted into cis-resveratrol 4'-O-beta-D-glucoside, together with trans-resveratrol 3-O-beta-D-glucoside and trans-resveratrol 4'-O-beta-D-glucoside, by Eucalyptus perriniana.

Use of the cryptogein gene to stimulate the accumulation of Bacopa saponins in transgenic Bacopa monnieri plants.

Genetic transformation of the Indian medicinal plant, Bacopa monnieri, using a gene encoding cryptogein, a proteinaceous elicitor, via Ri and Ti plasmids, were established and induced bioproduction of bacopa saponins in crypt-transgenic plants were obtained. Transformed roots obtained with A. rhizogenes strain LBA 9402 crypt on selection medium containing kanamycin (100 mg l(-1)) dedifferentiated forming callus and redifferentiated to roots which, spontaneously showed shoot bud induction. Ri crypt-transformed plants thus obtained showed integration and expression of rol genes as well as crypt gene. Ti crypt-transformed B. monnieri plants were established following transformation with disarmed A. tumefaciens strain harboring crypt. Transgenic plants showed significant enhancement in growth and bacopa saponin content. Bacopasaponin D (1.4-1.69 %) was maximally enhanced in transgenic plants containing crypt. In comparison to Ri-transformed plants, Ri crypt-transformed plants showed significantly (p ≤ 0.05) enhanced accumulation of bacoside A(3), bacopasaponin D, bacopaside II, bacopaside III and bacopaside V. Produced transgenic lines can be used for further research on elicitation in crypt-transgenic plants as well as for large scale production of saponins. Key message The cryptogein gene, which encodes a proteinaceous elicitor is associated with increase in secondary metabolite accumulation-either alone or in addition to the increases associated with transformation by A. rhizogenes.

Cloning and functional characterization of a chalcone isomerase from Trigonella foenum-graecum L.

Flavonoids belong to a group of plant natural products with variable phenolic structures and play important roles in protection against biotic and abiotic stress. Fenugreek (Trigonella foenum-graecum L.) seeds and stems contain flavonol glycosides and isoflavone derivatives. Up to now, the molecular features of fenugreek flavonoid biosynthesis have not been characterized. Here we present cloning of a cDNA encoding a chalcone isomerase (namely TFGCHI-1) from the leaves of T. foenum-graecum which convert chalcones to flavanones in vitro. Transformation of Arabidopsis loss-of-function TT5 (CHI) mutant with a TFGCHI-1 cDNA complemented TT5 and produced higher levels of flavonol glycosides than wild-type Col-0.

Production of cinnamyl glycosides in compact callus aggregate cultures of Rhodiola rosea through biotransformation of cinnamyl alcohol.

Rhodiola rosea is a multipurpose medicinal plant with adaptogenic properties: it increases the body's nonspecific resistance and normalizes body functions. The commercial interest for roseroot-based products has quickly increased worldwide. Nearly all raw-materials originate from natural populations. As a result of the intensive collection the species has become endangered. Production of the pharmaceutical compounds from the medicinal plants in cell cultures is an alternative to field cultivation. The present protocol describes the establishment of compact callus aggregate culture of Rhodiola rosea and the production of cinnamyl alcohol glycosides through biotransformation.

Environmentally benign synthesis of natural glycosides using apple seed meal as green and robust biocatalyst.

Salidroside is a natural glycoside with pharmacological activities of resisting anoxia, microwave radiation and fatigue, improving oxygen lack, and postponing ageing. In this work, salidroside and other natural glucosides such as cinnamyl O-beta-d-glucopyranoside and 4-methoxybenzyl O-beta-d-glucopyranoside were efficiently synthesized via an environmentally benign and energy economic process. In the synthetic process, apple seed, easily available from discards of fruit processing factories, was employed as a natural and green catalyst. Moreover, all of the catalyst, solvent and excessive substrate was reused or recycled. The biocatalytic reaction was carried out in a clean and less toxic medium of aqueous tert-butanol and the glucoside produced was selectively removed from reaction mixture by alumina column adsorption, making excessive substrate (aglycon) recyclable for a repeated use in the next batch of reaction. For improvement of the biocatalyst stability, apple seed meal was further cross-linked by glutaraldehyde, yielding a net-like porous structure within which the dissociating proteins were immobilized, resulting in improved permeability of the biocatalyst. After the simple cross-linking treatment, the half-life of apple seed catalyst was significantly improved from 29 days to 51 days. The productivity of the bioreactor in the case of salidroside can reach ca. 1.9 gl(-1)d(-1), affording the product in up to 99.3% purity after refinement.

Enhancement of phenylethanoid glycosides biosynthesis in cell cultures of Cistanche deserticola by osmotic stress.

The effect of osmotic stress on cell growth and phenylethanoid glycosides (PeGs) biosynthesis was investigated in cell suspension cultures of Cistanche deserticola Y. C. Ma, a desert medicinal plant grown in west region of China. Various initial sucrose concentrations significantly affected cell growth and PeGs biosynthesis in the suspension cultures, and the highest dry weight and PeGs accumulation reached 15.9 g l(-1)-DW and 20.7 mg g(-1)-DW respectively at the initial osmotic stress of 300 mOsm kg(-1) where the sucrose concentration was 175.3 mM. Stoichiometric analysis with different combinations of sucrose and non-metabolic sugar (mannitol) or non-sugar osmotic agents (PEG and NaCl) revealed that osmotic stress itself was an important factor for enhancing PeGs biosynthesis in cell suspension cultures of C. deserticola. The maximum PeGs contents of 26.9 and 23.8 mg g(-1)-DW were obtained after 21 days at the combinations of 87.6 mM sucrose with 164.7 mM mannitol (303 mOsm kg(-1)) or 20 mM PEG respectively, which was higher than that of C. deserticola cell cultures grown under an initial sucrose concentration of 175.3 mM after 30 days. The stimulated PeGs accumulation in the cell suspension cultures was correlated to the increase of phenylalanine ammonium lyase (PAL) activity induced by osmotic stress.

Improvement of pseudojujubogenin glycosides production from regenerated Bacopa monnieri (L.) Wettst. and enhanced yield by elicitors.

Bacopa monnieri (L.) Wettst. was studied for shoot induction and regeneration on Murashige and Skoog (MS) medium supplemented with different plant growth regulators. Stem explants cultured on medium containing 0.1 mg/l thidiazuron (TDZ) resulted in the highest number of shoots (117 shoots/explant). Regenerated plants from medium with 0.5 mg/l TDZ contained the highest level of pseudojujubogenin glycosides [(30.62 +/- 1.29) mg/g dry wt] which was 2-fold higher than that of in vitro grown plants of the same age [(16.96 +/- 1.49) mg/g dry wt]. Plantlets regenerated from 0.1 mg/l TDZ also showed a high level of pseudojujubogenin glycosides [(27.94 +/- 1.19) mg/g dry wt]. The effect of elicitor on pseudojujubogenin glycosides accumulation in B. monnieri whole plant cultures was investigated. Chitosan at 150 mg/l and yeast extract at 2 mg/ml increased the pseudojujubogenin glycosides production [(40.83 +/- 2.24) mg/g dry wt and (40.05 +/- 2.37) mg/g dry wt, respectively] after 7 days, which was 6-fold higher than in the control cultures.