Leaf Cell Suspension Research Articles

Endophyte-based fungal elicitors for enhanced production of valepotriates and sesquiterpenoids in leaf cell suspension cultures of Valeriana jatamansi Jones.

The immense therapeutic value of Valeriana jatamansi is attributed to the presence of bioactive secondary metabolites (valepotriates and sesquiterpenoids). Its over-exploitation in wild habitats resulted in extensive depletion, necessitating alternative approaches to produce its therapeutic metabolites. This study sought to assess the ability of endophytes of V. jatamansi to boost the biosynthesis of secondary metabolites in the leaf-cell suspension (LCS) culture of V. jatamansi. A total of 11 fungal endophytes were isolated from the rhizomes of V. jatamansi. Isolated endophytes were found to belong to phylum Ascomycota, Basidiomycota, and Mucoromycota. Supplementation of extracts of endophyte Phaeosphaeriaceae sp. VRzFB, Mucor griseocyanus VRzFD, Penicillium raistrickii VRzFK, and Penicillium sajarovii VRzFL in the LCS culture of V. jatamansi increased the fresh cell biomass by 19.6%-39.1% and dry cell biomass by 23.4%-37.8%. Most of the endophytes' extract could increase the content of valepotriates (26.5%-76.5% valtrate and 40.5%-77.9% acevaltrate) and sesquiterpenoids (19.9%-61.1% hydroxyl valerenic acid) in LCS culture. However, only two endophytes, Irpex lacteus VRzFI and Fusarium oxysporum VRzFF, could increase the sesquiterpenoids acetoxy valerenic acid (36.9%-55.3%). In contrast, some endophytes' extracts caused negative or no significant effect on the cell biomass and targeted metabolites. Increased secondary metabolites were corroborated with increased expression of iridoid biosynthesis genes in LCS culture. Production of H2O2 and lipid peroxidation was also varied with different endophytes indicating the modulation of cellular oxidative stress due to endophyte elicitors. The results suggest the distinct effect of different fungal endophytes-extract on LCS culture, and endophytes can serve as biotic elicitors for increasing the secondary metabolite production in plant in vitro systems.

Targeting apoptotic anticancer response with natural glucosinolates from cell suspension culture of Lepidium sativum

BackgroundFinding natural products with anticancer activity is an effective strategy to fight this disease. In this respect, Lepidium sativum or garden cress (family Brassicaceae) has been widely used worldwide for its wide therapeutic application, including anticancer and chemoprotective agents. Plant tissue culture techniques hold great promise for natural product enhancement without any climatic boundaries. In this study, glucosinolates and petroleum ether fractions were isolated from in vitro cell cultures and used against different carcinoma cell lines to investigate their anticancer potential. MethodsIn this study, callus cultures from leaf and root explants were initiated, cell suspension cultures were established, and cell growth and viability profiles were characterized. Different amino acids were added as precursors to the cell suspension cultures to enhance glucosinolates accumulation. Gas chromatography–mass spectrometric analysis (GC–MS) of glucosinolates and petroleum ether fractions was performed, and all fractions were tested against different carcinoma cell lines. ResultsThe findings clarified that the maximum callus initiation percentage was obtained in the medium containing 1.0 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D) + 1.0 mg/l kinetin (Kin) (C1). The viable cell number of cell suspension cultures from leaves and roots increased until it reached the maximum values on day 15. Adding tyrosine and methionine to the cell suspension cultures was the most influential and recorded high glucosinolate percentages. 1H-Cyclopenta (b) pyridine-3-carbonitrile-4,5,6,7-tetrahydro-2-methylthio-4-spirocyclohexane was the main glucosinolate compound found in tyrosine-treated leaf suspension (GLT). Fifteen compounds were detected in the petroleum ether fraction in both cell suspensions initiated from the leaf and root (OL and OR). The major compounds were benzene-1,3,5-trimethyl (12.99%) in root cell suspension (OR), and benzene-2-ethyl-1,4-dimethyl (10.66%) in leaf cell suspension (OL). All glucosinolate extracts demonstrated significant anticancer activity against the prostate (PC3), lung (A-549), colorectal (caco2), and liver (HepG2) cell lines. Glucosinolates extracted from leaf cell suspension (GL) were the most active on the hepatocellular carcinoma cell line (HepG2) among all remaining glucosinolate extracts. Treated hepatocellular carcinoma with an IC50 of GL extract (47.5 ug/ml) upregulates pro-apoptotic BAX and downregulates anti-apoptotic BCL2, which disrupts the BAX/BCL2 ratio, leading to activation of caspase 3 inside treated HepG2 cells. ConclusionsThe anticancer action of the GL extract was validated by the cell cycle study of its glucosinolates, which successfully promoted apoptosis and reduced hepatocellular growth by causing S-phase arrest.

The effect of plant growth regulators on the antioxidant enzyme activity and secondary metabolite production in the cell suspension cultures of Melia azedarach L.

ABSTRACT In the present study, we investigated the effect of plant growth regulators and explant type on callus induction, cell suspension culture growth, antioxidant enzyme activity, and accumulation of rutin, quercetin, and kaempferol in M. azedarach L. cell cultures. Our studies showed that the inflorescence and petiole explants had a high percentage of callus induction compared to the leaf explants, whereas the highest callus growth was obtained from the leaf explants cultured on the MS medium containing 5 mg l−1 NAA and 5 mg l−1 BAP and 5 mg l−1 NAA and 3 mg l−1 kinetin Kin. Furthermore, the highest cell growth was obtained from the leaf and inflorescence calli transferred to liquid MS medium supplemented with 1 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D)+1 mg l−1 Kin/BAP. The highest activity of antioxidant enzymes and the lowest protein content was related to the leaf cell suspension cultures in liquid MS medium containing 3 mg l−1 NAA and 3 mg l−1 BAP. The inflorescence cell suspension cultures in the MS medium containing 3 mg l−1 NAA+1 mg l−1 BAP exhibited the highest rutin accumulation (47.536 mg g−1 FW). However, the highest amount of quercetin (8.570 mg g−1 FW) and kaempferol (5.420 mg g−1 FW) accumulation were obtained from the petiole cell suspension cultures in the MS medium with 1 mg l−1 2,4-D +1 mg l−1 Kin.

Bioprocess development for enhancing cell biomass, differential picrosides accumulation, and gene expression profiling at shake flask levels in suspension cultures of Picrorhiza kurroa

Plant cell suspension culture is an alternative and perpetual source for large-scale metabolites production in significantly less time. However, this approach has not yet been executed in Picrorhiza kurroa for their specialized metabolites production. Therefore, the present study aimed to develop the suspension culture process from the rhizome and leaf callus of P. kurroa by optimizing the physical cultivation parameters. Furthermore, the established cultures were subjected to sequential scale-up at 250, 500, 1000, 2000, and 5000 mL shake flask levels to evaluate growth, metabolites accumulation, and gene expression profiling of picrosides biosynthesis. Concurrently, the growth, metabolites content, and gene expression patterns were also assessed at the 4th, 8th, and 12th months of the incubation period. The investigation results revealed that the cell biomass in suspension cultures significantly improved solely by varying the culture parameters. The initial inoculum density (3 g/75 mL), subculture by pouring method, shaking speed (80 rpm), and pH (5.8) were suitable for enhanced production of cell biomass in rhizome and leaf suspension cultures in a 250 mL shake flask. During sequential scale-up, the cell biomass was reached in rhizome suspension (335 g FW; 13.80 g DW) and leaf suspension (263.33 g FW; 10.87 g DW) under optimized parameters at 5000 mL shake flask level. However, a maximum increase in the cell growth index and cell biomass (DW) in g/L of the medium was observed in a 250 mL flask compared to other shake flask volumes. In rhizome and leaf suspension, total picrosides (PI, PII, and PIII) (rhizome: 2.36–2.48 mg/g DW; leaf: 2.50–2.67 mg/g DW) and total precursors (vanillic acid, caffeic acid, cinnamic acid, catalpol, and aucubin) (rhizome: 1.35–1.37 mg/g DW; leaf: 1.20–1.45 mg/g DW) content range was observed during sequential scale-up at 250–5000 mL shake flask levels. Hence, no significant level of variation was observed in picrosides content (PI, PII, and PII in mg/g DW) with respect to different shake flask volumes (250–5000 mL) in rhizome and leaf suspension culture, respectively. Nevertheless, the productivity of metabolites content in mg/L of medium with respect to cell growth in g/L of medium showed a significant level of variation. The 250 mL shake flask volume culture of rhizome and leaf cell suspension accumulated the maximum total picrosides (31.07 and 31.20 mg/L of medium) and total precursors (17.04 and 16.94 mg/L of medium) content with cell biomass (12.53 and 11.69 g/L of medium), respectively. The results also revealed that the shikimate/phenylpropanoid pathway (EPSPS), mevalonate pathway (PMK and GDPS), non-mevalonate pathway (ISPD, DXPS, and ISPE), and iridoid pathway (GS, G10H, UGT, F3D, SQM, and ACT) genes showed upregulated expression in the rhizome and leaf suspension cultures. Analyzing the 1st month cultures with 4th, 8th, and 12th month cultures, the study depicted that growth index, cell viability, metabolites content, productivity in mg/L of the medium, and gene expression pattern were decreased with the age of cultures under one year of incubation. The present investigation delivered a simple yet feasible suspension culture process for metabolites enriched cell biomass production in P. kurroa.

Enhanced Production of Phenylethanoids Mediated Through Synergistic Approach of Precursor Feeding and Light Regime in Cell Suspension Culture of Rhodiola imbricata (Edgew.).

Precursor feeding is a potential strategy for increasing specialized metabolite production in plant cell culture systems. In the present study, cell suspension cultures were developed and subsequently evaluated for precursor feeding investigations. Cell suspension cultures were established in Murashige and Skoog (MS) medium containing 0.5mg/L thidiazuron (TDZ) + 1mg/L α-naphthalene acetic acid (NAA). The growth biomass and metabolite pattern were analyzed to identify specific culture days required for prolific biomass production. The maximum cell dry weight (DW) was observed in leaf cell suspension (1.22g/100mL) and root cell suspension culture (1.12g/100mL) on day 21. Afterward, the effect of precursor concentrations (tyrosol; 0.5, 1, 2, and 3mM) along with two light regimes, photoperiod (16L/8D h, 70µmol/m2/s) and dark (24h), was evaluated for cell growth and metabolite accumulation. The results revealed that leaf cell suspension treated with 3mM tyrosol concentration detected maximum salidroside content (26.05mg/g DW) on day 15, incubated under photoperiod (16L/8D h) condition. Similarly, under photoperiod (16L/8D h), root cell suspension treated with 3mM tyrosol produced maximum salidroside content (26.62mg/g DW) on day 12. Moreover, the total phenolics content increased significantly (44.21mg/g DW) on day 12 in 3mM tyrosol treatment under photoperiod (16L/8D h). However, precursor concentrations did not influence the total flavonoids content. The present investigation suggests that the immediate pathway precursor, tyrosol, has a strong effect on enhanced production of salidroside, irrespective of explant type and light regimes.

Enhanced production of cichoric acid in cell suspension culture of Echinacea purpurea by silver nanoparticle elicitation

This study was conducted to optimize callus induction, cell suspension culture, and enhance cichoric acid production in Echinacea purpurea. For this purpose, leaf and root explants were grown in ½ MS medium supplemented with different concentrations of plant growth regulators. The highest percentage of callus induction (100%) was obtained with different concentrations of Kin in combination with 2,4-D and or NAA, in both explants. Then interaction slicing method was applied to analyze the effects of different concentrations of Kin in combination with 2,4-D and or NAA on total phenol contents. The maximum amount of total phenol was observed in 0.5 mg/L kin and 1 mg/L 2,4-D, in both leaf and root-derived callus. Data from HPLC–UV analysis showed that cichoric, chlorogenic, and caffeic acids are present in the callus. While cichoric acid content was 2.11 and 4.73 mg/g dry weight (DW) in leaf explant and its derived callus, it was 3.47 and 5.67 mg/g DW in root explant and its derived callus, respectively. Cell suspension culture was established and treated by AgNO3 and AgNPs (0, 2 and 4 mg/L). Overall, AgNPs’ elicitation was more effective in cichoric acid production compared to AgNO3. The highest amount of cichoric acid (9.54 mg/g DW) was found in root cell suspension culture after 48 h of exposure to 2 mg/L AgNPs. But leaf cell suspension culture reached the maximum cichoric acid level (8 mg/g DW) 72 h after treatment with 2 mg/L AgNPs. The current protocol can be used for the production of cichoric acid from callus and cell suspension cultures. Optimization of callus induction, cell suspension culture and cichoric acid production by AgNPs elicitation in Echinacea purpurea.

Creating Leaf Cell Suspensions for Characterization of Mesophyll and Bundle Sheath Cellular Features.

Imaging of mesophyll cell suspensions prepared from Arabidopsis has been pivotal for forming our current understanding of the molecular control of chloroplast division over the past 25 years. In this chapter, we provide a method for the preparation of leaf cell suspensions that improves upon a previous method by optimizing cellular preservation and cell separation. This technique is accessible to all researchers and amenable for use with all plant species. The leaf suspensions can be used for imaging chloroplast features within a cell that are important for photosynthesis such as size, number, and distribution. However, we also provide examples to illustrate how the cells in the suspensions can be easily stained to image other features, for example pit fields where plasmodesmata are located and organelles such as mitochondria, to improve our understanding of traits that are important for photosynthetic physiology.

Application of Separated Leaf Cell Suspension to Xenobiotic Metabolism in Plant

Metabolic profiles of (14)C-labeled primary metabolites from several pesticides, 4-cyanophenol (1), 3-phenoxybenzoic acid (2), 3-phenoxybenzyl alcohol (3), 3,5-dichloroaniline (4), and (1RS)-trans-2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropanecarboxylic acid (5), were examined by using enzymatically separated leaf cell suspension from seedlings of cabbage ( Brassica oleracea ) and tomato ( Lycopersicon esculentum ). After 1 day of incubation, the metabolites were extensively transformed in cabbage, whereas they were scarcely metabolized in tomato. The major metabolic pathways were the phase II reactions leading to a number of conjugates such as glucoside/malonylglucoside of 1-5, malate of 2, and glutamate of 4. The oxidation of 1 and 2 was observed as a minor reaction to produce 4-hydroxybezoic acid and 3-(4-hydroxyphenoxy)benzoic acid. The chemical identities of the secondary metabolites were determined by various spectrometric analyses (LC-MS, LC-MS/MS, and NMR) and/or HPLC cochromatography with the synthetic reference standards. As a result, this separated leaf cell suspension system was found to well reproduce the in vivo plant metabolism.

Studies on the superoxide anion production and peroxidase activity in potato leaf cell suspension exposed to salicylic acid

It is now widely accepted that salicylic acid (SA) signaling is mediated by reactive oxygen species (ROS) production. We have studied the effect of SA on peroxidase activity and superoxide anion production in potato leaf cell suspension. The results show that potato cells are insensitive to low concentrations of exogenous SA (< 1 mM) and the effect is observed at 1–5 mM SA. The cells exposed to SA exhibit higher peroxidase activity and show different peroxidase pattern when analyzed on native gels compared to the control. Superoxide anion production is enhanced after two hours of treatment and 2.5 mM SA gives the highest value. The results suggest peroxidase-mediated detoxification of ROS elicited by SA.

Regulation of the Activity of N-1-Naphthylphthalamic Acid Binding Protein by ATP and Phosphatase

N-1-naphthylphthalamic acid (NPA), an auxin transport inhibitor, was found to bind specifically to a crude membrane preparation from sugar beet seedling leaf cell suspension cultures. The dissociation constant (Kd) and binding protein concentration were found to be 1.71 &mu;mol dm-3 and 220 pmol g-1(membrane protein), respectively. The amount of specific 3H-NPA binding was significantly increased by adding Mg2+ATP to the binding assay solution. Treatment of membrane preparations with acid phosphatase, prior to the NPA binding assay, resulted in lower specific binding. ATP activation and phosphatase inactivation were culture stage dependent. Although a considerable effect could be detected when using cells from day 8 (representing the linear phase), the same treatment did not alter the binding if cells from day 1 (representing lag phase) or day 14 (representing the stationary phase) were used. These observations have strongly highlighted the possible involvement of a phosphorylation and dephosphorylation mechanism in vivo in the regulation of the activity of the NPA binding protein. High phosphatase activity was found in the supernatant, but not in the membrane pellet) after 50 000 g centrifugation. Our present study has indicated that receptor activity could be regulated by a phosphorylation and dephosphorylation mechanism in plants.

Activated charcoal affects morphogenesis and enhances sporophyte regeneration during leaf cell suspension culture of Platycerium bifurcatum.

In a leaf cell suspension culture of Platycerium bifurcatum, the incorporation of activated charcoal (AC) greatly increased the number of regenerated sporophytes even in growth regulator-free medium. The degree of improvement was dependent on cell aggregate sizes and medium composition. The maximal increase was observed in medium with 5.37 µM NAA and 4.44 µM BA, from 9 to 1520 sporophytes. The qualitative improvement by AC included: (1) regeneration of single sporophytes, which was relatively less frequent in non-AC media, (2) prevention of the formation of gametophyte clusters prior to sporophyte regeneration from 30- to 60-µm cells cultured in MS basal medium, (3) prevention of the formation of bud clusters, sporophytes with multiple bud primordia, `nodule'-like bud clusters in growth regulator containing media, and (4) prevention of the occurrence of hyperhydricity of regenerated sporophytes.

Role of oxalic acid in the Sclerotinia wilt of sunflower

Leaves of greenhouse-grown sunflower plants infected with Sclerotinia sclerotiorum frequently exhibit wilting and inter-veinal necrosis 3 to S days after inoculation. A wilt-inducing substance was found in water extracts of hypocotyl, lesions. Chemical tests, thin-layer chromatography and gas-liquid chromatography demonstrated that wilt-active aliquots of purified hypocotyl extracts contained oxalic acid. Wilted leaves from infected plants contained over 15 times more oxalic acid than leaves of healthy plants. Healthy plants fed oxalic acid through the hypocotyl showed foliar symptoms identical to those associated with fungal infections, and the concentrations of oxalic acid in leaves of treated plants were equivalent to those in infected plants. The pH of xylem sap from infected plants taken 2 cm above lesions was one unit lower than sap from healthy plants. Oxalic acid was detectable in sap from infected plants. Oxalic acid moves systemically to the leaves where it apparently accumulates to a “critical” level and elicits the wilt syndrome. A varietal screening technique using oxalic acid and whole leaf cell suspensions was developed which indicated that sunflower cultivars more resistant to Sclerotinia wilt were more tolerant to oxalic acid during their flowering period.