Establishment of <i>Rhodiola quadrifida</i> Hairy Roots and Callus Culture to Produce Bioactive Compounds

Rhodiola quadrifida is a rare mountain medicinal plant whose root extracts are used in traditional Chinese medicine as a hemostatic, antitussive, and tonic in the treatment of gynecological diseases. The aim of the study was to obtain R. quadrifida cultures at different degrees of differentiation in vitro and compare their growth characteristics and the content of salidroside and rosavin. Hairy roots were obtained by incubating cotyledons and hypocotyls in a suspension of Agrobacterium rhizogenes strain A4. The presence of the rolB and rolC genes was proven by polymerase chain reaction. The obtained roots were cultivated in Murashige-Skoog medium (MS). Calluses were obtained from the hairy roots in MS medium with the addition of hormones: 3 mg/L 2,4 D and 0.5 mg/L BAP. The presence of the main secondary metabolites of R. quadrifida, salidroside and rosavin, in calluses and salidroside in hairy roots by HPLC/MS was confirmed. The content of salidroside in callus culture was significantly higher than in hairy roots, 0.158 and 0.047%, respectively. The content of rosavin in callus culture was 0.07%. The content of rosavin and salidroside in callus culture was close to the level of these substances in the rhizomes of R. quadrifida plants growing in vivo, making this culture promising for its possible biotechnological use.     

Biotransformation of cinnamyl alcohol to rosavins by non-transformed wild type and hairy root cultures of Rhodiola kirilowii

Non-transformed wild type (NTWT) and hairy root cultures of Rhodiola kirilowii were grown in medium supplemented with 2.5 mM cinnamyl alcohol as a precursor and/or sucrose (1 %) on the day of inoculation or on the 14th day of culture. Rosarin, rosavin, and rosin were produced by NTWT root culture but only rosarin and rosavin by hairy roots. Approximately 80 and 95 % of the glycosides were released into the medium for NTWT and hairy root cultures, respectively. The highest rosavin yield, 505 ± 106 mg/l, was in hairy root culture when cinnamyl alcohol was applied on the day of inoculation with the addition of sucrose on the 14th day of culture. For rosin production, supplementation with cinnamyl alcohol alone on day 14 was more favourable with the highest amount 74 ± 10 mg/l in NTWT root culture. Only traces of rosarin were detected.     

Effects of yeast extract on the production of phenylpropanoid metabolites in callus culture of purple basil (Ocimum Basilicum L. var purpurascens) and their in-vitro evaluation for antioxidant potential

Ocimum basilicum L. var. purpurascens is an enriched reservoir of pharmaceutically important compounds with plenty of health and therapeutic attributes such as phenolic acids and anthocyanins. However, the inefficient production of aforementioned metabolites in wild has restricted its commercial utilization. Herein, commercially viable phytochemicals have been enhanced through elicitation of in-vitro cultures of O. basilicum using yeast extract.The impact of various concentrations (YE 1 mg/L,YE 10 mg/L, YE 25 mg/L, YE 50 mg/L, YE 100 mg/L, YE 200 mg/L and YE 400 mg/L) of yeast extract on biomass accumulation, phytochemical production, and antioxidant activities were assessed in callus cultures. Moderate concentration of yeast extract (100 mg/L) enhanced biomass accumulation i.e. fresh weight (FW 216.28 g/L) and dry weight (DW 15.49 g/L) up to 1.5 folds as compared to control (FW 167.14 g/L and DW 10.25 g/L). Similarly, yeast extract (100 mg/L) increased total phenolic and flavonoid contents as well as enhanced antioxidant activities such as ABTS (2,2 azinobis 3-ethylbenzthiazoline-6-sulphonic acid), FRAP (ferric reducing antioxidant power) and DPPH (2,2-diphenyl-1-picryhydrazyl). High performance liquid chromatography (HPLC) analysis was elucidated for further phytochemical investigation. HPLC analysis showed an increase of almost 1.9 folds as compared to control in rosmarinic acid (15.19 mg/g DW), chicoric acid (2.13 mg/g DW), peonidin (2.70 mg/g DW) and cyanidin (1.57 mg/g DW). Likewise, 1.8 fold and 2.4 folds increase was observed in eugenol essential oils (0.25 mg/g DW) and chavicol (0.037 mg/g DW), respectively. For cellular antioxidant activity, reactive oxygen specie or reactive nitogen specie (ROS/RNS) was induced in yeast cells and the effect of O. basilicum callus culture was further investigated in stressed yeast cells. A positive correlation exists between the antioxidant activities, TPC and TFC analysis. In short, these results showed that yeast extract could act as an efficient elicitor to enhance pharmacologically important metabolites in callus cultures of Ocimum basilicum.

Graphical abstract

     

Cell line selection through gamma irradiation combined with multi-walled carbon nanotubes elicitation enhanced phenolic compounds accumulation in Salvia nemorosa cell culture

The current study focused on improving the production of phenolic acids in the Woodland Sage cell suspension culture (CSC) through attaining high-yielding cell lines and carboxyl functionalized multi-walled carbon nanotubes (MWCNT-COOH) elicitation. The leaf-derived callus was irradiated at different doses of gamma irradiation 10 to 100 Gy. The maximum content of rosmarinic acid (RA), salvianolic acid B (SAB), ferulic acid (FA), and cinnamic acid (CA) was recorded in callus cultures irradiated with 70 Gy, which was 18.53, 5.21, 1.9, and 7.59 mg/g DW, respectively. The CSC that established from 70 Gy γ-irradiated calli accumulated 1.7-fold RA more higher irradiated callus culture. The CSC elicited with various concentrations of MWCNT-COOH in range 25 to 100 mg/l significantly increased fresh weight (FW), dry weight (DW), and phenolic acid contents of cells. The highest FW with 268.47 g/l and DW with 22.17 g/l was obtained in 100 mg/l MWCNT-COOH treatment. The RA, SAB, CA and FA content of CSC treated with 100 mg/l MWCNT-COOH were 13-fold, 14.2-fold, 20-fold, and 3- fold higher than wild S. nemorosa plant at flowering stage, respectively. The antioxidant activity of cultures significantly enhanced with both gamma and MWCNT-COOH based on DPPH and FRAP assay. Our results showed that the combination of cell line selection and MWCNT-COOH elicitation significantly improved the production of secondary metabolites in Woodland Sage, which is useful for large-scale production of phenolic compounds.

     

Effect of mycelial morphology on ergothioneine production during liquid fermentation of Lentinula edodes

The morphological type of a microorganism generally influences its metabolite production. In the present study, we investigated the effects of the mycelial morphology of shiitake (Lentinula edodes) on the production of 2-mercaptohistidine trimethylbetaine (ergothioneine, ESH) during liquid fermentation. Analyses of the distribution of ESH in mycelial cells of different morphological types revealed that the ESH content of pellets obtained from the liquid fermentation media was much greater than the content in the free mycelia and clumps. The concentration of ESH in pellets on day 15 of liquid fermentation reached 0.79 mg/g dry weight (DW), which is approximately three times the concentration found in mycelia clumps (0.28 mg/g DW) and free mycelia (0.31 mg/g DW). Macroscopic image analysis of the development and morphological changes of the pellets during a liquid fermentation period of up to 25 days indicated that pellet growth showed a highly positive correlation with the increase in ESH concentration (r ² = 0.9851). A reduced agitation rate of 50 rpm for the culture medium was suitable for pellet formation and size enlargement. The results obtained in this work would be helpful in guiding the intentional manipulation of the distribution and enrichment of ESH in L. edodes through changes in liquid fermentation conditions.     

Valepotriates accumulation in callus, suspended cells and untransformed root cultures of Valeriana glechomifolia

Summary Valeriana glechomifolia is an endemic species of southern Brazil, capable of accumulating, in all of its organs, the terpene derivatives known as valepotriates, the presumed sedative components of the roots of pharmaceutically used species of Valeriana. In vitro cultures of the plant were established and the accumulation of acevaltrate, didrovaltrate, and valtrate in callus, cell suspension, and untransformed root cultures was studied. Leaves of in natura plants and roots of micropropagated plantlets were used as the explants for callus induction and root culture establishment, respectively, on Gamborg B5 basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or with kinetin (KIN). Culture growth and secondary metabolite yields were enhanced with 2,4-D (4.52μM) and KIN (0.93μM). Maximum valepotriate contents, quantified by HPLC, of acevaltrate (ACE) 2.6mg g⁻¹ DW, valtrate (VAL) 10.2mgg⁻¹ DW, and didrovaltrate (DID) 2.9mg g⁻¹ DW were observed in root cultures after 7–8wk of culture.     

利用高山红景天培养细胞生物转化外源酪醇生产红景天甙的研究

高山红景天（ＲｈｏｄｉｏｌａｓａｃｈａｌｉｎｅｎｓｉｓＡ．Ｂｏｒ．）培养细胞中,甙元酪醇在细胞生长静止期大量积累,而此时糖基化反应的效率很低,因而红景天甙（ｓａｌｉｄｒｏｓｉｄｅ）产量较低。考虑到培养细胞中酪醇葡萄糖基转移酶的活性在指数生长期达到最高,考察了在指数生长期添加外源酪醇生物转化生产红景天甙的可能性,并探讨了酪醇添加浓度、添加方法及细胞密度对酪醇转化率及红景天甙产量的影响。结果表明,细胞在酪醇浓度为１ｍｍｏｌ／Ｌ的培养基中培养２４ｈ后可使酪醇转化率达到９５％;过高的酪醇浓度（＞３ｍｍｏｌ／Ｌ）对细胞生长及酪醇转化率都有明显抑制作用;通过较低浓度酪醇的３次重复添加,可使细胞密度为６ｇＤＷ／Ｌ、１２ｇＤＷ／Ｌ及１８ｇＤＷ／Ｌ的培养物中的红景天甙产量分别达到１３２０ｍｇ／Ｌ、１７４０ｍｇ／Ｌ和１９８０ｍｇ／Ｌ。     

Production of Salidroside Through Biotransformation of Exogenous Tyrosol in Rhodiola sachalinensis Cell Suspension Cultures

In Rhodiola sachalinensis A. Bot. cell cultures, low yields of salidroside was supposed to be associated with the low efficiency of glucosylation reaction at the stationary phase of cell growth, when large amounts of the substrate, aglycon tyrosol, were accumulated. Considering the activity of tyrosol glucosyhransferase being the highest at the exponential growth phase, the author added exogenous tyrosol into the cultures at this time so as to produce salidroside through biotransformation. The effects of tyrosol concentration, the way of tyrosol addition as well as the cell density on the transformation rate and salidroside yield were investigated. It was found that the transformation rate attained 95 % after cells were incubated in the medium containing 1 mmol/L tyrosol for 24 h. Excess high concentrations of tyrosol in medium ( > 3 mmol/L) caused inhibition of transformation rate and cell growth. By 3 repeated additions of tyrosol in low concentrations, the salidroside yields of 1 320 mg/L, 1 740 mg/L and 1 980 mg/L to the cell densities of 6 g DW/L, 12 g DW/L and 18 g DW/L were obtained respectively.     

Improved production of dibenzocyclooctadiene lignans in the elicited microshoot cultures of Schisandra chinensis (Chinese magnolia vine)

Dibenzocyclooctadiene lignans are a specific group of secondary metabolites that occur solely in Schisandra chinensis. The aim of the presented work was to boost the accumulation of lignans in the agitated microshoot cultures of S. chinensis, using different elicitation schemes. The experiments included testing of various concentrations and supplementation times of cadmium chloride (CdCl₂), chitosan (Ch), yeast extract (YeE), methyl jasmonate (MeJa), and permeabilizing agent—dimethylsulfoxide (DMSO). After 30 days, the microshoots were harvested and evaluated for growth parameters and lignan content by LC-DAD method. The analyses showed enhanced production of lignans in the elicited S. chinensis microshoots, whereas the respective media samples contained only trace amounts of the examined compounds (< 5 mg/l). Elicitation with CdCl₂ caused up to 2-fold increase in the total lignan content (max. ca. 730 mg/100 g DW after the addition of 1000 μM CdCl₂ on the tenth day). Experiments with chitosan resulted in up to 1.35-fold increase in lignan concentration (max. ca. 500 mg/100 g DW) after the supplementation with 50 mg/l on the first day and 200 mg/l on the tenth day. High improvement of lignan production was also recorded after YeE elicitation. After the elicitation with 5000 mg/l of YeE on the first day of the growth period, and with 1000 and 3000 mg/l on the 20th day, the lignan production increased to the same degree—about 1.8-fold. The supplementation with 1000 mg/l YeE on the 20th day of the growth cycle was chosen as the optimal elicitation scheme, for the microshoot cultures maintained in Plantform temporary immersion system—the total content of the estimated lignans was equal to 831.6 mg/100 g DW.

     

The effect of light, salinity, and nitrogen availability on lipid production by Nannochloropsis sp

We examined responses of batch cultures of the marine microalga Nannochloropsis sp. to combined alterations in salinity (13, 27, and 40 g/l NaCl) and light intensity (170 and 700 μmol photons/m²·s). Major growth parameters and lipid productivity (based on total fatty acid determination) were determined in nitrogen-replete and nitrogen-depleted cultures of an initial biomass of 0.8 and 1.4 g/l, respectively. On the nitrogen-replete medium, increases in light intensity and salinity increased the cellular content of dry weight and lipids due to enhanced formation of triacylglycerols (TAG). Maximum average productivity of ca. 410 mg TFA/l/d were obtained at 700 μmol photons/m²·s and 40 g/l NaCl within 7 days. Under stressful conditions, content of the major LC-PUFA, eicosapentaenoic acid (EPA), was significantly reduced while TAG reached 25% of biomass. In contrast, lower salinity tended to improve major growth parameters, consistent with less variation in EPA contents. Combined higher salinity and light intensity was detrimental to lipid productivity under nitrogen starvation; biomass TFA content, and lipid productivity amounted for only 33% of DW and ca. 200 mg TFA/l/day, respectively. The highest biomass TFA content (ca. 47% DW) and average lipid productivity of ca. 360 mg TFA/l/day were achieved at 13 g/l NaCl and 700 μmol photons/m²·s. Our data further support selecting Nannochloropsis as promising microalgae for biodiesel production. Moreover, appropriate cultivation regimes may render Nannochloropsis microalgae to produce simultaneously major valuable components, EPA, and TAG, while sustaining relatively high biomass growth rates.     

Salicylic acid‐enhanced biosynthesis of pharmacologically important lignans and neo lignans in cell suspension culture of Linum ussitatsimum L

Linum usitatsimum L. (flax) is a perennial herb with magnitude of medicinal and commercial applications. In the present study, we investigated the effects of salicylic acid (SA) on biosynthesis of lignans (secoisolariciresinol diglucoside (SDG) and lariciresinol diglucoside (LDG)) and neolignans (dehydrodiconiferyl alcohol glucoside (DCG) and guaiacylglycerol‐β‐coniferyl alcohol ether glucoside (GGCG)) in cell cultures of flax. Moderate concentration of SA (50 μM) enhanced biomass accumulation (10.98 g/L dry weight (DW)), total phenolic content (37.81 mg/g DW), and antioxidant potential (87.23%) to two‐fold than their respective controls after 72 h of exposure. However, higher levels of total flavonoid content (5.32 mg/g DW) were noted after 48 h of exposure to 50 μM of SA. HPLC analyses revealed that 50 μM SA, significantly enhanced biosynthesis of SDG (7.95 mg/g DW), LDG (7.52 mg/g DW), DCG (54.90 mg/g DW), and GGCG (16.78 mg/g DW), which was almost 2.7, 1.8, 3.88, and 3.98 fold higher than their respective controls after 72 h of exposure time, respectively. These results indicated that moderate concentrations of SA had significant effects on biosynthesis and productivity of lignans and neolignans in cell culture of L. usitatissimum.     

Production of high-density Chlorella culture grown in fermenters

The freshwater microalga Chlorella vulgaris was grown heterotrophically in fed-batch 50–600-L fermenters at 36°C, on aerated and mixed nutrient solution with urea as a nitrogen and glucose as a carbon and energy source. Cell density increased from the initial value 6.25 to 117.18 g DW L⁻¹ in 32 h in the fermenter 50 L at a mean growth rate 3.52 g DW L⁻¹ h⁻¹. The DW increase in the fermenter 200 L was from 7.25 to 94.82 g DW L⁻¹ in 26.5 h at a mean growth rate 3.37 g DW L⁻¹ h⁻¹. Mean specific growth rate μ was about 0.1 h⁻¹ in the both fermenters, if nutrients and oxygen were adequately supplied. The DW increase in the fermenter 600 L was from 0.8 to 81.6 g DW L⁻¹ in 66.5 h at a mean growth rate 1.22 g DW L⁻¹ h⁻¹ and μ = 0.07 h⁻¹. A limitation of the cell growth rate in 600 L fermenter caused by a low dissolved oxygen concentration above cell densities higher than 10 g DW L⁻¹) occurred. Specific growth rate decreased approximately linearly with increasing glucose concentration (25–80 g glucose L⁻¹) at the beginning of cultivation and decreased with the time of cultivation. The cell yield was 0.55–0.69 g DW (g glucose)⁻¹. The content of proteins, β-carotene, and chlorophylls in the cells steadily increased and starch content decreased, by keeping aerated and mixed culture another 12 h in fermenter after the cell growth was stopped due to glucose deficiency.     

Improved growth and quality of <Emphasis Type="Italic">Siraitia grosvenorii</Emphasis> plantlets using a temporary immersion system

The effects of temporary immersion system (TIS) culture on the growth and quality of Siraitia grosvenorii plantlets were investigated. The TIS promoted the growth and quality of S. grosvenorii plantlets. Proliferation rate, shoot length, fresh weight (FW) and dry weight (DW) of shoots, and total biomass production were significantly (P ≤ 0.05) higher in the TIS than in gelled and liquid medium, respectively. The TIS also decreased callus formation at the base of shoots. Callus diameter was significantly (P ≤ 0.05) lower in the TIS (3.30 mm) than in gelled medium (6.31 mm) and liquid medium (6.77 mm), respectively. FW (50.83 mg) and DW (7.08 mg) of callus in the TIS were also significantly (P ≤ 0.05) lower than those in gelled medium (80.00 and 10.56 mg, respectively) and liquid medium (218.75 and 23.75 mg, respectively). During rhizogenesis, minimal callus was evident at the base of shoots in the TIS, with a well-developed root system. However, the plantlets in gelled medium just produced thick, brown and easily broken roots with obvious callus and fewer secondary roots. The natural-like plantlets of S. grosvenorii obtained in the TIS would probably have positive effects on ex vitro rooting and transplanting in large-scale commercial production.     

Influence of endophytic fungal elicitation on production of inophyllum in suspension cultures of <Emphasis Type="Italic">Calophyllum inophyllum</Emphasis> L.

The influence of dried cell powder and culture filtrates of endophytic fungi on production of inophyllum in cell suspension cultures of leaf- and stem-derived callus of Calophyllum inophyllum was investigated. Two fungi, Nigrospora sphaerica and Phoma spp., endophytic to C. inophyllum, were isolated from leaf tissues, and were identified by both 18S rRNA gene amplification and sequencing. Elicitation of suspension cultures of both callus types of C. inophyllum with dried cell powder and culture filtrates of both fungi consistently elicited production of inophyllum A, B, C, and P. In comparison to stem-derived callus, suspension cultures of leaf-derived callus enhanced production of most inophyllum. Of the four inophyllum studied, the highest production of inophyllum A, C, and P was achieved in elicited suspension cultures of leaf-derived callus. Suspension cultures of stem-derived callus enhanced production only of inophyllum B. When suspension cultures of leaf-derived callus were elicited with 40 mg dried cell powder of Phoma spp., a level of 751-fold (6.84 mg/100 g elicited biomass) of inophyllum A was produced, compared to control. Whereas, a level of 414-fold (6.22 mg/100 g elicited biomass) of inophyllum B was produced when suspension cultures of stem-derived callus were elicited with 20 mg dried cell powder of N. sphaerica. When compared to control, a 10% culture filtrate of N. sphaerica in suspension cultures of leaf-derived callus elicited inophyllum C and P production by 928-fold (7.43 mg/100 g elicited biomass) and 750-fold (1.5 mg/100 g elicited biomass), respectively.     

Analysis of capsaicinoid biosynthesis pathway genes expression in callus cultures of Capsicum chinense Jacq. cv. ‘Umorok’

The placental tissue of the highly pungent chilli cultivar, Capsicum chinense Jacq. cv. ‘Umorok’, is used as explants for callus induction. Callus cultures were subcultured after every 32 days and growth curves for a period of six consecutive growth cycles were studied till a stable capsaicinoids producing callus cultures were obtained. The capsaicinoids content in placental tissue explants decreased gradually during the first 2 months of culture as the explants dedifferentiated to form friable callus while the biomass and capsaicinoid content did not show much change in the subsequent growth cycles. The maximum callus biomass of 7.8 g freshweight (FW) or 0.56 g dry weight (DW) per culture were obtained on the 24th day of every growth cycle and the maximum average capsaicinoids content (1.6 mg g^?1 FW capsaicin and 0.78 mg g^?1 FW dihydrocapsaicin) were obtained on the 20th day of every growth cycle. To investigate the underlying dynamics for capsaicinoid biosynthesis during callus formation, comparative gene expression analysis of the genes involved in capsaicinoid biosynthesis pathway were also studied by qRT-PCR analysis. When compared with placental tissue, all the studied genes showed reduced expression during callus formation, especially putative aminotransferase (pAMT) and pungent gene 1 (Pun1), which were extensively down regulated from the 3rd month onwards in the callus cultures. Therefore, the present study revealed that the down-regulated expression of mainly two putative genes in capsaicinoid biosynthetic pathway (pAMT and Pun1) resulted in lower accumulation of capsaicinoids in callus cultures compared to placental tissues of fruits.

     

Stimulation of anthocyanin synthesis in grape (<Emphasis Type="Italic">Vitis vinifera</Emphasis>) cell cultures by pulsed electric fields and ethephon

Anthocyanin from grape cell cultures can be used as a natural alternative to synthetic dyes; particularly due to their reported health-promoting properties. In this study, production of anthocyanin in cell suspension culture of Vitis vinifera was evaluated following treatment with either ethephon and/or pulsed electric fields (PEF). Overall, total production of anthocyanin increased in treated cells compared to untreated cells. Treatment of cell suspension with PEF at day 14 of culture resulted in 1.7-fold increase (1.42 mg/g DW) in anthocyanin content when compared to control cells; while, treatment with ethephon resulted in 2.3-fold increase (1.99 mg/g DW) in anthocyanin content. When cells were treated with both ethephon and PEF, 2.5-fold increase in anthocyanin content (2.2 mg/g DW) was observed. These findings demonstrate that PEF induces a defense response in plant cells, and it may also alter the dielectric properties of cells and/or cell membranes, and would serve as a viable elicitor of secondary metabolites in plant cell cultures.     

Production of saponions and polysaccharide in the presence of lactoalbumin hydrolysate in <Emphasis Type="Italic">Panax quinquefolium</Emphasis> L. cells cultures

In this article, ginsenosides and polysaccharide contents in suspension cells and native roots of Panax quinquefolium L. were studied. In order to enhance the contents of ginsenosides and polysaccharide in P. quinquefolium suspension cells, we tested the effects of lactoalbumin hydrolysate on the growth of P. quinquefolium suspension cell, synthesis of ginsenosides and polysaccharide in flask and bioreactor. In flask culture, cells growth ratio was significantly enhanced by the addition of lower concentration of lactoalbumin hydrolysate. Addition of 100 mg L⁻¹ lactoalbumin hydrolysate significantly enhanced the contents of total saponins (5.44 mg g⁻¹ DW) and the contents were 3.89-fold over the control group. Addition of lactoalbumin hydrolysate significantly promoted the accumulation of polysaccharide, except 200 mg L⁻¹ lactoalbumin hydrolysate. The highest total saponins yield (36.72 mg L⁻¹ DW) and polysaccharide yield (0.83 g L⁻¹ DW) were obtained at 100 mg L⁻¹ lactoalbumin hydrolysate. In a 5-L stirred tank bioreactor, the highest contents of total saponins and TRb group ginsenosides were achieved on day 26, while the effect of lactoalbumin hydrolysate on the contents of TRg group ginsenosides were insignificant. This result suggests that lactoalbumin hydrolysate might have triggered the enzyme activities for the synthesis of TRb group ginsenosides. Overall, the highest total saponins yield (31.37 mg L⁻¹ DW) and polysaccharide yield (1.618 g L⁻¹ DW) were obtained on day 26 and day 24 respectively and the polysaccharide yield was 1.95-fold higher than the shake flask culture (0.83 g L⁻¹ DW). These results provided theoretical reference for two-stage culture in suspension cells of P. quinquefolium in bioreactor.     

Phytochemical,biochemical, and growth changes in response to salinity in callus cultures of Nigella sativa L.

Black cumin (Nigella sativa L.) is considered as a noteworthy herbal medicine. However, no study has been conducted on the physiological adaptive mechanism of it to salinity stress, especially under in vitro condition. To this aim, the callus cultures of ten different genotypes of N. sativa were applied to evaluate the changes occurring in biochemical traits under salinity stress. The calluses were exposed to the in vitro salt stress using different sodium chloride concentrations (0, 84, and 250 mM). A reduction occurred in the content of K⁺ and callus growth by enhancing the NaCl concentration. However, most of the content of Na⁺ (4 mgg^− 1 DW), malondialdehyde (1.38 μmolg^− 1 FW), total phenolic content (1.18 mg GAEg⁻¹ FW), thymol (25.26 mgg^− 1 DW), total flavonoids content (0.06 mg QEg^− 1 FW), total flavonols (TFL) content (0.023 mg QEg^− 1 FW), total anthocyanins (Ant) (0.05 μmol g^− 1 FW) and DPPH activity (58.17%) was observed at 250 mM of NaCl. In fact, two secondary metabolites including TFL and Ant can be considered as the major contributors to the potential antioxidant activity of N. sativa at the callus level. The elicitation through NaCl opens new avenues for the selection of best dosages of NaCl for the enhancement of commercially important secondary metabolites, in superior genotypes (Nig₁ and Nig₂) of N. sativa at cellular level.

     

Methyl jasmonate- and salicylic acid-induced <Emphasis Type="SmallCaps">d</Emphasis>-chiro-inositol production in suspension cultures of buckwheat (<Emphasis Type="Italic">Fagopyrum esculentum</Emphasis>)

The aim of this work is to investigate the effects of methyl jasmonate (MeJ) and salicylic acid (SA) on d-chiro-inositol (DCI) production in buckwheat (Fagopyrum esculentum) suspension cultures. In this study, adding optimal concentrations of MeJ and SA at an appropriate time markedly increased DCI production (yield 6.141 and 5.521 mg/g DW, respectively). In addition, treatment of buckwheat cultures with a combination of 0.2 mM MeJ and 0.6 mM SA on days 0 and 9 increased the DCI yield to 7.579 mg/g DW, which was 3.726 times higher than that in the control; furthermore, the former yield was higher than that achieved by the addition of either elicitor alone. Moreover, unlike MeJ, SA did not exert a negative effect on cell growth.     

Variation in polyphenolic profile and in vitro antioxidant activity of eastern teaberry (Gaultheria procumbens L.) leaves following foliar development

Seasonal dynamics in the polyphenolic composition, antioxidant activity, and their relationships during plant development were evaluated for eastern teaberry (Gaultheria procumbens L.) leaves, a traditional herbal medicine of North American natives. With the complementary UHPLC-PDA-ESI-MS³, HPLC-PDA-fingerprint, Folin-Ciocalteau, and n-butanol/HCl assays of methanol-water (75:25, v/v) extracts, the dried leaf samples harvested monthly across the growing season under Polish climate conditions were found rich in structurally diverse polyphenols (149.2–210.7 mg/g DW) including the dominating salicylates (64.6–107.5 mg/g DW), proanthocyanidins (53.0–66.8 mg/g DW), and flavonoids (17.3–25.3 mg/g DW), and the accompanying chlorogenic acid isomers (2.4–4.4 mg/g DW) and simple phenolic acids (0.9–1.1 mg/g DW). Among 28 detected analytes, gaultherin (64.6–107.5 mg/g DW), miquelianin (14.6–21.1 mg/g DW), procyanidin A-type trimer (5.5–9.5 mg/g DW), and (–)-epicatechin (5.8–7.8 mg/g DW) were the most abundant. The phenolic levels and antioxidant activity parameters in the DPPH (EC₅₀, 15.0–18.2 μg DW/mL; 0.95–1.16 mmol Trolox equivalents/g DW) and FRAP (2.3–3.4 mmol Fe ²⁺/g DW; 0.86–1.26 mmol Trolox equivalents/g DW) assays showed parallel seasonal trends with maxima in September and October. As the subsequent correlation studies confirmed the determinative impact of polyphenols on the leaf antioxidant activity and its seasonal fluctuations, the Fall season could be recommended as optimal for harvesting the plant material for medicinal purposes and cost-effective production of natural health products.