Callus Suspension Cultures Research Articles

Effect of salt stress on growth and phenolic compounds production in callus suspension culture of the dioecious species thyrse sorrel (Rumex thyrsiflorus Fingerh.)

The sex-dependent differences in the response to salt stress of the dioecious Rumex thyrsiflorus and the influence of different concentrations of sodium chloride (NaCl) on the biosynthesis of phenolic compounds in callus suspension cultures were evaluated. The cultures originated from callus obtained on hypocotyls isolated from male and female seedlings were cultured on Murashige and Skoog medium supplemented with 0.4 mg/L 6-benzylaminopurine and 1 mg/L 2,4-dichlorophenoxyacetic acid. The results showed that the response to salt stress depends on both the sex of the plant and the NaCl concentration in the culture medium. The analysis of the main morphometric parameters showed that callus tissue derived from the hypocotyls of female seedlings was less sensitive to salt stress than that from male seedlings, which was correlated with higher concentrations of phenolic compounds. The optimal NaCl concentration to increase the production of phenolic compounds was 129 mM for females and 43 mM for males. In the methanolic tissue extracts 22 compounds were determined using the HPLC–DAD method. In general, higher amounts of compounds were detected in the extracts from the female tissue. The following dominated: catechin (max. 213.31), cryptochlorogenic acid (max. 76.35) and epicatechin (max. 54.84) (mg/100 g DW). This comprehensive phytochemical analysis of the sex-related aspects of the response to salt stress was performed for the first time in this dioecious model species. The results revealed potential application of NaCl as the ecological friendly and inexpensive elicitor to increase the production of pharmaceutically valuable compounds and highlight the importance of dioecy in phytochemistry.

Anticancer Activity of Rice Callus Suspension Cultures from Aromatic Varieties and Metabolites Regulated in Treated Cancer Cell Lines

Tissue culture techniques were used to produce large amounts of bioactive compounds with medicinal potential, overcoming space and time constraints for cancer prevention. Rice callus suspension cultures (RCSC) and seed extracts prepared from aromatic rice varieties were used to evaluate the cytotoxic impact on human colon and lung cancer cell lines, as well as a normal control cell line, using Taxol as a positive control. RCSC and seed extracts from two Indian aromatic rice varieties were applied at different concentrations to treat the cancer cell lines and normal lung fibroblasts over varying time intervals. Apoptosis was assessed in 1:5 dilutions of the A549 and HT-29 cell lines treated with RCSC for 72 h, using propidium iodide staining and flow cytometry. RCSC showed a more potent cytotoxic effect than seed extracts with minimal effect on the normal cell line, in contrast to Taxol. Confocal microscopy and flow cytometry further confirmed the apoptotic effect of RCSC. Gas chromatography-mass spectrometry-based metabolic profiling identified metabolites involved in cytotoxicity and highlighted altered pathways. RCSC is proposed as an alternative source for the development of novel anticancer drugs with reduced side effects.

In vitro production of steroidal saponin, total phenols and antioxidant activity in callus suspension culture of Paris polyphylla Smith: an important Himalayan medicinal plant.

Paris polyphylla Smith (Melanthiaceae) family, which is native to the Himalayan region, has received a lot of attention recently due to its extensive history of usage in traditional medicine. The production of steroidal saponin from callus suspension cultures of P. polyphylla was observed in the current study. The current study attempted to develop a P. polyphylla plant callus suspension culture through optimization of cultivation technique for callus suspension, quantification of total phenolic components and estimation of the extract's antioxidant activity. A light-yellow callus was formed within six weeks of cultivating rhizomes on Murashige and Skoog (MS) media supplemented with Thidiazuron (TDZ). Furthermore, the effect of TDZ, Methyl Jasmonate (MeJA), and Yeast Extract (YE) on callus growth, steroidal saponin (dioscin and diosgenin), total phenolic content, total flavonoids, total tannin, and total antioxidant activity was also measured. The medium containing 0.5 μM TDZ depicted the maximum callus biomass (2.98 g fresh weight). Significantly high phenolic and tannin content was observed in the MS medium containing 50 μM MeJA, whereas, no significant increase was observed in total tannin production in any treatment. Three in vitro assays, DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2'-azino-bis (3-ethylbenzothiazoline- 6-sulfonic acid)) and FRAP (ferric ion reducing antioxidant potential) and FC (Folin-Ciocalteu), were used to assess antioxidant potential of callus. Maximum antioxidant analysis reported in 1.0 μM TDZ (6.89 mM AAE/100 g) containing medium followed by 50 μM MeJA (6.44 mM AAE/100g). The HPLC analysis showed a high presence of dioscin and diosgenin (5.43% and 21.09%, respectively) compared to the wild sample (2.56% and 15.05%, respectively). According to the results, callus produced on media supplemented with 50 μM MeJA have significant phenolic contents and elevated antioxidant activity; nevertheless, callus growth was greater in the presence of 0.5 μM TDZ. The findings of the current study have commercial implications since greater biomass production will result in active phytochemicals that the pharmaceutical and nutraceutical sectors are in need desperately.

Integrated transcriptomic and metabolomic analyses revealed the molecular mechanism of terpenoid formation for salicylic acid resistance in Pulsatilla chinensis callus.

As a kind of traditional Chinese medicine, Pulsatilla chinensis (Bunge) Regel is well known for its anti-inflammation and anti-cancer activities, which are attributed to its active components including total saponins and monomers. To clarify the synthesis and metabolism mechanisms of class components in callus terpenes of P. chinensis, a certain concentration of salicylic acid (SA) hormone elicitor was added to the callus before being analysed by transcriptomic and metabolomic techniques. Results showed that the content of Pulsatilla saponin B4 in the callus suspension culture was significantly increased up to 1.99% with the addition of SA. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the differentially expressed genes were mainly enriched in 122 metabolic pathways, such as terpenoid metabolism-related pathways: terpenoid skeleton synthesis pathway, monoterpenoid biosynthesis pathways, diterpenoid biosynthesis pathways, and ubiquinone and other terpenoid-quinone biosynthesis pathways. A total of 31 differentially accumulated metabolites were obtained from four differential groups. Amongst 21 kinds of known chemical components in P. chinensis, deoxyloganic acid was the only monoterpenoid; the others are triterpenoids. In summary, this study found that SA elicitors can affect the metabolic changes of terpenoids in P. chinensis callus, which provided a basis for analysing the genetic regulation of terpenoid components of leucons.

In vitro propagation, shoot regeneration, callus induction, and suspension from lamina explants of Sorbus caloneura.

Sorbus caloneura has high ornamental and medicinal value but is endangered, and significant effort is required to preserve this natural resource. In this study, the stem and sterilized leaves of S. caloneura were used to explore the effects of different plant hormones basic medium type, initial callus quality, initial liquid volume, and ratio of old liquid culture medium to new on stem proliferation, regeneration and callus suspension culture. Naphthylacetic acid (NAA) and 6-benzyladenine (6-BA) significantly affected the proliferation of stem tissue. Murashige and Skoog (MS) basal medium supplemented with 1.75 mg/L 6-BA, 0.25 mg/L NAA, and 0.25 mg/L indole butyric acid (IBA) yielded a proliferation rate of 100%, with an average number of adventitious shoots per stem of 4.9. The best callus induction was observed with MS basal medium containing 0.5 mg/L 6-BA, 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.2 mg/L thidiazuron (TDZ). The adventitious shoots were directly induced by MS basal medium containing 5.0 mg/L 6-BA, 0.5 mg/L NAA, and 1.5 mg/L kinetin (KT) and indirectly induced by medium containing 3.0 mg/L 6-BA, 0.3 mg/L NAA, and 0.1 mg/L TDZ. NAA significantly affected rooting rate, with ideal conditions found to be medium supplemented with 0.2 mg/L NAA and 1.5 mg/L IBA. MS basal medium with 4.5 g of calli and 120 mL of liquid culture medium without retention of the original culture medium yielded the best suspension effect for callus proliferation. Taken together, the results of this study lay a foundation for the breeding, preservation of germplasm resources, and genetic transformation of S. caloneura.

In vitro elicitation supports the enrichment of 2H4MB production in callus suspension cultures of D. hamiltonii Wight & Arn

Influence of elicitation on in vitro production of 2-Hydroxy-4-Methoxy Benzaldehyde (2H4MB), a structural isomer of vanillin from callus suspension cultures of Decalepis hamiltonii was investigated. In vitro culture conditions were optimized to induce callus, suspension culture and biomass followed by metabolite production. Suspension cultures were established using leaf generated friable callus. Maximum content of 2H4MB production 0.079 ± 0.01 mg 100g-1 DW and biomass 197.5 ± 1.5 g1_,-1 were observed by 4th week of culturing. Elicitation was induced to suspension cultures by using m¬topolin (mT), sodium nitroprusside (SNP), and pectin individually at different concentrations. m¬topolin (15 itiM), pectin (15 itiM) and SNP (10 itiM) supported 0.31 mg100g-1DW, 0.27 mg100g-1DW and 0.21 mg100g-lof 2H4MB production respectively by 4th week. This data infers that the elicitation improves 2H4MB content in callus suspension cultures of D. hamiltonii.

Mesembrine alkaloid production in in vitro culture morphotypes of Sceletium tortuosum (L.) N.E. Br

Different routes of access to mesembrine alkaloids uniquely produced by the medicinal succulent Sceletium tortuosum (Aizoaceae) are thus highly sought after in the natural products sector. In order to develop a biotechnological method, the establishment of in vitro microshoot and callus suspension cultures was investigated using in vitro seedlings of S. tortuosum. The production of mesembrine and other alkaloid derivatives in vitro was monitored using high-throughput ultra-high performance liquid chromatography-mass spectrometry in regenerated microplant and callus cultures. Microshoots were transferred to a shoot multiplication medium after initiation on media with factorial combinations of 1-naphthylacetic acid and N6-benzyladenine at 0–1 mg/L and 1–5 mg/L, respectively. Persistent carryover effects associated with the initiation procedures led to four dominant morphotypes. A drier Micropore™ seal environment reduced the number of propagules but the cultures exhibited greater proportions of healthy plantlets (88%) even though the incidence of a red coloured pigment was higher, recorded at 100% in some of the lines. Using a semi-in vitro technique, 96% of in vitro derived plants survived ex vitro. Microshoot cultures, pre-treated with a dehydration step, accumulated four key biomarkers with mesembrine (3270.9 mg/kg), mesembranol (4738.8 mg/kg), mesembrenol (333.1 1 mg/kg), and Δ4- mesembrenone (29.9 mg/kg) (dry weight) at levels comparable to wild types. Δ7-Mesembrenone, while detected in the microshoots, was not present in callus cultures. The reported mass propagation strategies are opportune for offering an alternative source to the mesembrine alkaloids currently used in clinical herbal formulations.

Highly Efficient Bioconversion of trans-Resveratrol to δ-Viniferin Using Conditioned Medium of Grapevine Callus Suspension Cultures.

δ-Viniferin is a resveratrol dimer that possesses potent antioxidant properties and has attracted attention as an ingredient for cosmetic and nutraceutical products. Enzymatic bioconversion and plant callus and cell suspension cultures can be used to produce stilbenes such as resveratrol and viniferin. Here, δ-viniferin was produced by bioconversion from trans-resveratrol using conditioned medium (CM) of grapevine (Vitis labruscana) callus suspension cultures. The CM converted trans-resveratrol to δ-viniferin immediately after addition of hydrogen peroxide (H2O2). Peroxidase activity and bioconversion efficiency in CM increased with increasing culture time. Optimized δ-viniferin production conditions were determined regarding H2O2 concentration, incubation time, temperature, and pH. Maximum bioconversion efficiency reached 64% under the optimized conditions (pH 6.0, 60 °C, 30 min incubation time, 6.8 mM H2O2). In addition, in vitro bioconversion of trans-resveratrol was investigated using CM of different callus suspension cultures, showing that addition of trans-resveratrol and H2O2 to the CM led to production of δ-viniferin via extracellular peroxidase-mediated oxidative coupling of two molecules of trans-resveratrol. We thus propose a simple and low-cost method of δ-viniferin production from trans-resveratrol using CM of plant callus suspension cultures, which may constitute an alternative approach for in vitro bioconversion of valuable molecules.

Cytobacts: Abundant and Diverse Vertically Seed-Transmitted Cultivation-Recalcitrant Intracellular Bacteria Ubiquitous to Vascular Plants

We have recently described ‘Cytobacts’ as abundant intracellular endophytic bacteria inhabiting live plant cells based on the observations with callus and cell suspension cultures of grapevine and other plant species with the origin ascribable to field explants. In this study, we investigated the prevalence of such cytoplasmic bacterial associations in field plants across different taxa, their cultivability, and the extent of taxonomic diversity and explored the possibility of their embryo-mediated vertical transmission. Over 100 genera of field plants were surveyed for ‘Cytobacts’ through bright-field live-cell imaging as per our previous experience using fresh tissue sections from surface-sterilized shoot-tissues with parallel cultivation-based assessments. This revealed widespread cellular bacterial associations visualized as copious motile micro-particles in the cytoplasm with no or sparse colony forming units (CFU) from the tissue-homogenates indicating their general non-cultivability. Based on the ease of detection and the abundance of ‘Cytobacts’ in fresh tissue sections, the surveyed plants were empirically classified into three groups: (i) motile bacteria detected instantly in most cells; (ii) motility not so widely observed, but seen in some cells; and (iii) only occasional motile units observed, but abundant non-motile bacterial cells present. Microscopy versus 16S-rRNA V3–V4 amplicon profiling on shoot-tip tissues of four representative plants—tomato, watermelon, periwinkle, and maize—showed high bacterial abundance and taxonomic diversity (11–15 phyla) with the dominance of Proteobacteria followed by Firmicutes/Actinobacteria, and several other phyla in minor shares. The low CFU/absence of bacterial CFU from the tissue homogenates on standard bacteriological media endorsed their cultivation-recalcitrance. Intracellular bacterial colonization implied that the associated organisms are able to transmit vertically to the next generation through the seed-embryos. Microscopy and 16S-rRNA V3–V4 amplicon/metagenome profiling of mature embryos excised from fresh watermelon seeds revealed heavy embryo colonization by diverse bacteria with sparse or no CFU. Observations with grapevine fresh fruit-derived seeds and seed-embryos endorsed the vertical transmission by diverse cultivation-recalcitrant endophytic bacteria (CREB). By and large, Proteobacteria formed the major phylum in fresh seed-embryos with varying shares of diverse phyla. Thus, we document ‘Cytobacts’ comprising diverse and vertically transmissible CREBs as a ubiquitous phenomenon in vascular plants.

In vitro elicitation supports the enrichment of 2H4MB production in callus suspension cultures of D. hamiltonii Wight & Arn

Influence of elicitation on in vitro production of 2-Hydroxy-4-Methoxy Benzaldehyde (2H4MB), a structural isomer of vanillin from callus suspension cultures of Decalepis hamiltonii was investigated. In vitro culture conditions were optimized to induce callus, suspension culture and biomass followed by metabolite production. Suspension cultures were established using leaf generated friable callus. Maximum content of 2H4MB production 0.079 ± 0.01 mg 100g-1 DW and biomass 197.5 ± 1.5 gL-1 were observed by 4th week of culturing. Elicitation was induced to suspension cultures by using m-topolin (mT), sodium nitroprusside (SNP), and pectin individually at different concentrations. m-topolin (15 μM), pectin (15 μM) and SNP (10 μM) supported 0.31 mg100g-1 DW, 0.27 mg100g-1 DW and 0.21 mg100g-1 of 2H4MB production respectively by 4th week. This data infers that the elicitation improves 2H4MB content in callus suspension cultures of D. hamiltonii.

SAPONINS, GLYCOSIDES AND FLAVONOIDS IN CELLS AND TISSUES OF BALANITES AEGYPTIACA CULTURED ON SOLID AND LIQUID CULTURE MEDIA

Objectives: The main objective of the present study is to obtain callus and cell suspension culture from Balanitesa egyptiaca sterile plantlets grown in vitro and to compare growth and the biosynthetic potential of saponins, flavonoids and glycosides by callus and cell suspension culture Balanitisa egyptiaca.
 Methods: Callus was induced from the mother plants on MS culture media supplemented with 2.0 mg/l BA + 2.0 mg/l 2,4-D with and without agar gelling. Total saponins, glycosides and flavonoids were estimated in both types of cultures over a period extending from 1 to 5 weeks to compare the productivity of such secondary metabolites in callus and cell suspension cultures.
 Results: The results obtained indicated that both calli and cell suspension cultures were able to synthesize the target active ingredients and that cell suspension culture was superior to the callus culture in the biosynthesis and accumulation processes. By the end of the incubation period, the amount of total saponins in cell suspension culture reached up 51.97±0.26 dry biomass compared to 35.02 ±0.06 mg/g in callus culture. The amount of total flavonoids in cell suspension culture reached up 10.88±0.24 dry biomass compared to 6.40±0.02 mg/g in callus culture and of total glycosides reached up 6.11±0.25 dry biomass compared to 5.06 ±0.05 mg/g in callus culture.
 Conclusions: The results obtained in this study may indicate the promising role that plant cell culture will play in the future in phytopharmaceutical industry.
 Peer Review History: 
 Received 16 July 2020; Revised 12 August; Accepted 26 August, Available online 15 September 2020
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 Reviewer(s) detail:
 Name: Dr. Gehan Fawzy Abdel Raoof Kandeel
 Affiliation: Researcher at pharmacognosy Department, National Research Centre, Egypt.
 E-mail: gehankandeel9@yahoo.com
 
 Name: Ahmad Najib
 Affiliation: Department of Pharmacognosy-Phytochemystry Universitas Muslim Indonesia-Indonesia
 E-mail: ahmad.najib@umi.ac.id
 
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Large-scale production of recombinant miraculin protein in transgenic carrot callus suspension cultures using air-lift bioreactors

Miraculin, derived from the miracle fruit (Synsepalum dulcificum), is a taste-regulating protein that interacts with human sweet-taste receptors and transforms sourness into sweet taste. Since miracle fruit is cultivated in West Africa, mass production of miraculin is limited by regional and seasonal constraints. Here, we investigated mass production of recombinant miraculin in carrot (Daucus carota L.) callus cultures using an air-lift bioreactor. To increase miraculin expression, the oxidative stress-inducible SWPA2 promoter was used to drive the expression of miraculin gene under various stress treatments. An 8 h treatment of hydrogen peroxide (H2O2) and salt (NaCl) increased the expression of miraculin gene by fivefold compared with the untreated control. On the other hand, abscisic acid, salicylic acid, and methyl jasmonate treatments showed no significant impact on miraculin gene expression compared with the control. This shows that since H2O2 and NaCl treatments induce oxidative stress, they activate the SWPA2 promoter and consequently up-regulate miraculin gene expression. Thus, the results of this study provide a foundation for industrial-scale production of recombinant miraculin protein using transgenic carrot cells as a heterologous host.

Callus growth kinetics and accumulation of secondary metabolites of Bletilla striataRchb.f. using a callus suspension culture.

Bletilla striata is an endangered traditional Chinese medicinal plant with multiple uses and a slow regeneration rate of its germplasm resources. To evaluate the callus growth kinetics and accumulation of secondary metabolites (SMs), a callus suspension culture was proven to be a valuable approach for acquiring high yields of medicinal compounds. An effective callus suspension culture for obtaining B. striata callus growth and its SMs was achieved with the in vitro induction of calluses from B. striata seeds. The callus growth kinetics and accumulation of SMs were analyzed using a mathematical model. The resulting callus growth kinetic model revealed that the growth curves of B. striata suspension-cultured calluses were sigmoidal, indicating changes in the growth of the suspension-cultured calluses. Improved Murashige and Skoog callus growth medium was the most favorable medium for B. striata callus formation, with the highest callus growth occurring during the stationary phase of the cultivation period. Callus growth acceleration started after 7 days and thereafter gradually decreased until day 24 of the cultivation period and reached its highest at day 36 period in both the dry weight and fresh weight analyses. The coelonin concentration peaked during the exponential growth stage and decreased afterward during the stationary stage of the callus suspension culture. The maximum content of coelonin (approximately 0.3323 mg/g callus dry weight) was observed on the 18th day of the cultivation cycle, while dactylorhin A and militarine reached the highest concentrations at day 24, and p-hydroxybenzyl alcohol at day 39. This investigation also laid a foundation for a multimathematical model to better describe the accumulation variation of SMs. The production of SMs showed great specificity during callus growth and development. This research provided a well-organized way to increase the accumulation and production of SMs during the scaled-up biosynthesis of calluses in B. striata callus suspension cultures.

Effect of biotic and abiotic elicitors on isoflavone biosynthesis during seed development and in suspension cultures of soybean (Glycine max L.).

The present investigation aimed to look at the effects of biotic and abiotic elicitors during Soybean seed development and cell suspension culture in isoflavones accumulation. The expression levels of four major genes viz., CHS7, CHS8, IFS2, and IFS1 involved on isoflavones biosynthesis during seed developmental stages from R5L-R7 was seen in both MAUS-2 and JS-335 Soybean varieties. The R7 stage showed 1.24-fold upregulation of IFS1transcript level and considered as the control for Soybean seed development. Both varieties during R6-R8 stages responded differently to the foliar application of 10µM SA, 10µMMJ and 0.1% Aspergillus niger. The IFS2 transcripts were upregulated by SA at the R7 stage with 5.21- and 4.68-fold in JS-335 and MAUS-2, respectively. IFS1 expression was significantly increased by A. niger treatment at R7 stage with 3.98- and 3.21-fold in MAUS-2 and JS-335, respectively. The expression of CHS7 and CHS8 by 10μM SA at R7 level revealed maximum up-regulation of 0.51- and 1.01-fold in MAUS-2; 0.37- and 0.82-fold in JS-335, respectively. In the soybean callus suspension culture, biosynthetic genes were used to validate the effects of elicitor on isoflavones. Both biotic and abiotic treatments contribute to the upregulation of IFS1 and IFS2 expression, that in turn, leads to the accumulation of isoflavone in seed development as well as in suspension cultures. These data further suggested that the IFS2 is the key gene responsible for the isoflavone accumulation during elicitor treatment.

Methyl jasmonate induced polyphenols and antioxidant production in callus suspension culture of Nardostachys jatamansi

In vitro establishment of callus suspension culture and incorporation of Methyl jasmonate (MeJA) was studied on the accumulation of polyphenols and other antioxidant activity in Nardostachys jatamansi. The maximum explants establishment (95%) was recorded in Murashige and Skoog (MS) medium supplemented with naphthaleneacetic acid (NAA 21.1 µM) and kinetin (Kn 3.16 µM). Leaf explants from in vitro derived shoots when cultured on MS medium in combination with NAA (23.1 µM), 6-Benzyladenine (BA 4.0 µM) and Kn (1.16 µM), 32% of the explants formed tiny adventitious roots with callus and 52% of the explants formed green callus without adventitious roots within 60 days of inoculation. Callus cultured with a combination of NAA, BA and Kn in ½ MS liquid medium exhibited maximum fresh weight (7.9 ± 0.10 g). Incorporation of MeJA in ½ MS liquid medium containing NAA, BA and Kn enhanced polyphenolic content and antioxidant activity, however it varied with the concentration used. MeJA-treated callus showed higher accumulation of all the measured polyphenols and antioxidant activity. This protocol is suitable to harness the industrial potential of the species without destroying plants growing in the natural habitat and this, in turn, would reduce collection from the wild.

Anti-inflammatory Effects of Northern Highbush Blueberry Extract on an In Vitro Inflammatory Bowel Disease Model

Blueberry anthocyanins have the ability to efficiently reach the GI tract and exhibit a broad range of biochemical effects. In the context of inflammatory bowel disease (IBD), they remain a promising complement to current IBD treatments. Here, we investigated the anti-inflammatory and antioxidant capabilities of Highbush blueberries in-vitro on two normal colon epithelial cell lines, NCM 356 and CCD 841 CoN using fluorescent microscopy and flow cytometry following stimulation with a pro-inflammatory cytokine cocktail. Treatment with blueberry extract revealed a significant decrease in nuclear and cytoplasmic generated reactive oxygen species (ROS) compared to controls. Additionally, the blueberry extract increased cell viability following treatment with the pro-inflammatory cytokine cocktail. A comparison with previous report on rice callus suspension culture (RCSC) revealed opposing trend with reference to the levels of nuclear and cytoplasmic ROS. It is likely that blueberry extract and RCSC employ different players and pathways to mitigate inflammation.

Analysis of phenolics in Calligonum polygonoides in vitro cultured roots

Background: Calligonum polygonoides L. subsp. Comosum (L’Her.) Sosk. is an endangered plant species belonging to family Polygonaceae. Although the plant is rich in phytoconstituents and has multipurpose medicinal applications, but in vitro root culture studies and phytochemical investigations of these cultures are rare. Objectives: To establish in vitro root, callus and cell suspension cultures from in vitro germinated fruits of C. polygonoides to investigate the production of phenolics through root, callus and cell suspension cultures and attempt to enhance cell capacity to accumulate phenolics. Materials and Methods: Modified Murashige and Skoog medium supplemented with 1 mg l-1 indole-3-butyric acid was used to establish the root culture. Elicitation of cell suspension culture was performed using salicylic acid and yeast extract. The phenolic compounds in root, callus and cell suspension cultures were evaluated using reversed phase high performance liquid chromatography technique. Results: The unorganized cell suspension culture contained fewer amounts of phenolic compounds than the differentiated roots tissue. Elicitation produced quantitative reprogramming of phenolic content. Conclusion: The present study provides a chance to improve secondary metabolite yield from this valuable natural plant.

Anti-inflammatory and immune-modulating effects of rice callus suspension culture (RCSC) and bioactive fractions in an in vitro inflammatory bowel disease model

BackgroundRice callus suspension culture (RCSC) has been shown to exhibit potent antiproliferative activity in multiple cancer cell lines. RCSC and its bioactive compounds can fill the need for drugs with no side effects. Hypothesis/PurposeThe anti-inflammatory potential of RCSC and its bioactive fractions on normal colon epithelial cell lines, was investigated. Study designThree cell lines, InEpC, NCM356 and CCD841-CoN were treated with proinflammatory cytokines followed by RCSC. Cytoplasmic and nuclear ROS were assayed with fluorescent microscopy and flow cytometer. Expression analysis of immune-related genes was performed in RCSC-treated cell lines. RCSC was fractionated using column chromatography and HPLC. Pooled fractions 10–18 was used to test for antiproliferative activity using colon adenocarcinoma cell line, SW620 and anti-inflammatory activity using CCD841-CoN. Mass spectrometric analysis was performed to identify candidate compounds in four fractions. ResultsRCSC treatment showed differential effects with higher cytoplasmic ROS levels in NCM356 and CCD841-CoN and lower ROS levels in InEpC. Nuclear generated ROS levels increased in all three treated cell lines. Flow cytometry analysis of propidium iodide stained cells indicated mitigation of cell death caused by inflammation in RCSC treated groups in both NCM356 and CCD841-CoN. Genes encoding transcription factors and cytokines were differentially regulated in NCM356 and CCD841-CoN cell lines treated with RCSC which provided insights into possible pathways. Analysis of pooled fractions 10–18 by HPLC identified 8 peaks. Cell viability assay with fractions 10–18 using SW620 showed that the number of viable cells were greatly reduced which was similar to 6X and 33X RCSC with very little effect on normal cells which similar to 1X RCSC. RCSC fractions increased nuclear and cytoplasmic ROS vs. both untreated and inflammatory control. Analysis of four fractions by mass spectrometry identified 4-deoxyphloridzin, 5′-methoxycurcumin, piceid and lupeol as candidate compounds which are likely to be responsible for the antiproliferative, anti-inflammatory and immune-regulating properties of RCSC. ConclusionRCSC and its fractions showed anti-inflammatory activity on inflamed colon epithelial cells. Downstream target candidate genes which are likely to mediate RCSC effects were identified. Candidate compounds responsible for the antiproliferative and anti-inflammatory activity of RCSC and its fractions provide possible drug targets.

Phytochemical characteristics of callus suspension culture of Helicteres angustifolia L. and its in vitro antioxidant, antidiabetic and immunomodulatory activities

Helicteres angustifolia L. (H.angustifolia) is an important medicinal plant. This study investigated the phytochemical characteristics of callus suspension cultures of H.angustifolia and its bioactive functions, such as antioxidant, antidiabetic and immunomodulatory activities. Results demonstrated that the cultures were rich in phenolic compounds, flavonoids, terpenoids, saponins and triterpenoids. Compared to the equivalent wild roots, the cultures yielded larger amounts of total phenolics (17.75 mg GAE g−1), flavonoids (56.15 mg RE g−1), saponins (29.21 mg OAE g−1) and polysaccharides (13.69 mg g−1), about 1.62-, 1.90-, 1.14- and 3.41-fold higher than those from the equivalent wild roots, respectively. Furthermore, as the valuable target product from in vitro culture, phenolic compounds were subjected to HPLC analysis, reflecting that some representative phenolic compounds fluctuated significantly under the cultivation conditions. Additionally, bioactive assays showed that the extract of callus suspension cultures (CE) possessed strong antioxidant activities, and rat maltase (IC50 = 13.29 mg mL−1) and sucrase (IC50 = 13.76 mg mL−1) inhibitory activities. As for the immunomodulatory activity, CE could stimulate the proliferation of macrophages (121.93 ± 1.76%) and the production of nitric oxide (200% of the control) and phagocytic activity (178% of the control) at 6.25 μg mL−1. Moreover, CE could mitigate DOX-induced macrophages damage to some extent. In conclusion, the callus suspension culture of H.angustifolia is a potential source for bioactive phytochemical compounds and functional foods.

Different glyceollin synthesis-related metabolic content and gene expressions in soybean callus suspension cultures and cotyledon tissues induced by alginate oligosaccharides

Abstract Glyceollins are a group of inducible soybean phytoalexins. The aim of the present study was to investigate the mechanism of glyceollin production in callus suspension cultures (CS) and cotyledon tissues elicited with alginate oligosaccharides (AOS) at 1%, 2%, and 3% concentrations that were applied for three days. HPLC analysis of the extracts from 2% AOS-elicited callus suspension cultures (ECS) revealed that glyceollins content was 85.3-fold and 8.2-fold higher than that in unelicited controls and elicited cotyledon tissues (ECT), respectively. UPLC-Q-Exactive Orbitrap MS analysis showed that the 2% AOS induction resulted in elevated levels of metabolites, the intermediary products of glyceollin biosynthesis, such as daidzein, glycitein, genistein, naringenin, glycinol, glyceollidin I/II, and glyceollin I, II, and III in ECS and ECT. Glyceollin I was prominent among the three glyceollin isomers in ECS, whereas isomer II and III were dominant in the synthetic route for glyceollins in ECT. qRT-PCR analysis of related genes showed that UGT, CYP93A1, G2DT, and G4DT displayed a direct effect on glyceollin synthesis. Our results suggested that 2% AOS-induced CS produced glyceollins in higher content compared to ECT by altering metabolite content and gene expression. Elicitation of CS is thus a promising technology for glyceollin synthesis extensively.