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Plants have a memory function for the environmental stress they have suffered. When they are subjected to repeated environmental stress, they can quickly and better activate the response and adaptation mechanism to environmental stress, thus realizing long-term stable reproduction. However, most of the relevant studies are applied to crops and Arabidopsis thaliana rather than medicinal plants about the improvement of plant growth status and the effect on phytoalexin biosynthesis. In this study, yeast extract(YE) was used as an elicitor to simulate biotic stress, and the changes in biomass and the content of some secondary metabolites were measured by giving repeated stresses to Sorbus aucuparia suspension cell(SASC). The results showed that the accumulation levels of biomass and some secondary metabolites in SASC subjected to repeated stress are significantly increased at some time points compared with single stress. A phenomenon that SASC can memorize biotic stress is confirmed in this study and influences phytoalexin accumulation in SASC. Furthermore, the work laid the groundwork for research into the transgenerational stress memory mechanism of medicinal plant.
Subject(s)
Cells, Cultured , Secondary Metabolism , Sorbus , Stress, PhysiologicalABSTRACT
Objective: To explore the functions of pathogenesis related protein 1-like (PR1-like) in suspension cell of Sorbus aucuparia (SaPR1-like) under biotic stress. Method: The full sequence of SaPR1-like gene was cloned and analyzed by multiple bioinformatic tools. The expression of SaPR1-like in the suspension cell of S. aucuparia in response to harpin protein stress was analyzed by real-time fluorescence quantitative polymerase chain reaction (PCR). Result: A SaPR1-like gene was identified and cloned, which contained a complete open reading frame and encoded 161 amino acids. The coding protein was hydrophilic and stable, had transmembrane structure and signal peptide, and belonged to secretory protein. Amino acid sequence alignment data indicated that the C-terminal of SaPR1-like contained a highly conserved domain[cysteine-rich secretory protein, antigen 5, and pathogenesis-related 1 peptides (CAPE)], which could induce defense genes to produce immune responses against biotic stresses. SaPR1-like gene expression was significantly increased after harpin protein induced suspension cell of S. aucuparia for 24 h. Conclusion: A SaPR1-like gene derived from the PR1 family is found to induce significant responses to biotic elicitors in S. aucuparia. This study highlights a role for PR1 in immune signaling and suggests the potential application of PR1 in efforts to defeat biotic stress in plants.
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Objective To study the effects of abiotic elicitors methyl jasmonate (MeJA) and salicylic acid (SA) on the alkaloids accumulation and related enzymes metabolism inPinellia ternata suspension cell cultures. Methods Using the leaf petioles-derived suspension cell cultures as the study object, the culture duration, concentrations of MeJA and SA were determined to get the optimal alkaloids accumulation, and the activities of metabolic enzymes IMP dehydrogenase and sAMP synthase were also measured.Results A 9-fold of dried biomass and a 3-fold of alkaloids accumulation were observed inP. ternata suspension cell cultures after culture for 21 d. Both MeJA and SA could significantly promote the accumulation of alkaloids inP. ternata suspension cells. 150 μmol/L MeJA enhanced alkaloids content (4.7 mg/gDW) by 3.6 folds in comparison with control group, whereas 50 μmol/L SA showed a 2.5-fold increase. Meanwhile, 100 μmol/L MeJA and 50 μmol/L SA promoted the increase in IMP dehydrogenase activity by 3.0 and 3.7 fold respectively, and 150 μmol/L MeJA and 100 μmol/L SA showed the increase by 2.6 and 4.4 fold respectively.Conclusion Proper adding exogenous MeJA and SA can promote the accumulation of alkaloids inPinellia ternata suspension cell cultures.
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Objective: To study the establishment of cell suspension culture system and build a platform for exploring the functional genes in the biosynthesis pathway for Carthamus tinctorius. Methods: Using the cotyledon of C. tinctorius as explants for callus induction, callus with good quality was selected for suspension cultivation. The factors influencing the establishment of C. tinctorius cell suspension culture system were investigated, such as hormone, inoculum amount, pH, and cell activity. The effect of MeJA at different concentration on suspension cell growth rate was studied. Chemical compounds in suspension cell system were preliminary identified by UPLC-Q-TOF/MS. Results: The optimal medium for the cell suspension culture was MS + 1.0 mg/LTDZ + 0.1 mg/L NAA, the pH was 5.5-6.0, and inoculum amount was 0.02 g/mL. MeJA at different concentration had effects on suspension cell growth rate: 50 μmol/L MeJA increased cell growth rate, 500 μmol/L MeJA depressed cell growth rate greatly. Thirteen potential chemical compounds in suspension cell system were preliminary identified. Conclusion: The cell suspension culture system of C. tinctorius is established, and MeJA with optimal concentration has effects on suspension cell growth rate.
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Las líneas celulares de neem (Azadirachta indica A. Juss.) cultivadas en suspensión líquida han demostrado producir metabolitos secundarios bioactivos, particularmente triterpenoides. En consecuencia, se han realizado estudios para el control de microorganismos de importancia médica, como los hongos dermatofitos. El objetivo principal de este trabajo fue evaluar a través de un método de referencia in vitro la actividad antifúngica de diferentes extractos de cultivos celulares de neem sobre varios aislamientos de Trichophyton mentagrophytes, Trichophyton rubrum y Epidermophyton floccosum. Se realizó un escalado de cultivos de suspensiones celulares de neem, a partir de los cuales se obtuvo un extracto crudo metanólico. Éste extracto fue fraccionado posteriormente por cromatografía en columna de silica gel. Con los extractos obtenidos se determinó la Concentración Mínima Inhibitoria (CMI), siguiendo el método de microdilución en caldo M38-2, con cinco aislamientos de T. mentagrophytes, cinco de T. rubrum y tres de E. floccosum. Se usó como control positivo el antimicótico Terbinafina. Los resultados mostraron que el extracto crudo de biomasa celular de neem inhibe el crecimiento hasta en 100 % de T. mentagrophytes, T. rubrum y E. floccosum. Al evaluar las fracciones por separado, se observó que las de menor polaridad exhibieron en general mayor actividad antifúngica (CMI=109 μg/mL) que el extracto crudo per se (CMI=2500 μg/ mL) y las fracciones más polares (CMI=7000 μg/mL). Lo anterior indica que las células de neem cultivadas en suspensión producen compuestos con actividad antifúngica, siendo más bioactivos los presentes en las fracciones de menor polaridad.
Cell lines of neem (Azadirachta indica A. Juss.) grown in liquid suspension have shown to produce bioactive secondary metabolites, particularly triterpenoids. In consequence, its use as a control of medical microorganisms (like dermatophytes) is proposed. The main goal of this study was to assess the antifungal activity of methanolic extracts from neem cultured cell suspensions on several isolates of Trichophyton mentagrophytes (five isolates), Trichophyton rubrum (five isolates) and Epidermophyton floccosum (three isolates). Neem cell suspension cultures were scaled up, from which a raw methanolic extract was obtained. This extract was fractionated by silica gel column chromatography. The raw methanolic extract and its fractions were used in order to determine the Minimal Inhibitory Concentration (MIC) on the dermatophytes isolates by following M38-A2 broth microdilution method. Antimycotic Terbinafine was used as positive control. The results shown that neem raw cellular biomass extract inhibits the growth of T. mentagrophytes, T. rubrum and E. floccosum in at least 100%. In the evaluations of the separated fractions, it was observed that the low polarity fractions had higher antifungal activity (MIC=109 μg/mL) than the raw extract per se (MIC=2500 μg/mL) and the most polar ones (MIC=7000 μg/mL). The latter suggest that neem cells cultured in liquid suspension produces compounds with antifungal activity, being more active those present in the low polarity fractions.
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Objective: To analyze the effect of exogenous polyamine on the growth of Betula platyphylla suspension cells and triterpenoid accumulation. Methods: At the end of the growth stage, 0.1 mmol/L and 1.0 mmol/L putrescine (Put), spermine (Spm), and spermidine (Spd) were added to the suspension cells of B. platyphylla, the triterpenoid content and gene expression of lupeol synthase (LUS) related to the synthesis of triterpenoid were analyzed by chemical colorimetry and RT-PCR, respectively. Results: Except for the treatment with 1.0 mmol/L Spm, the cell viability, dry weight, and triterpenoid production in B. platyphylla suspension cells induced by Put and Spd were increased, and the dry weight and triterpenoid production of B. platyphylla cells were increasing with the extension of polyamine treatment time. Among them, dry weight and triterpenoid production were the highest after 2 d Put induction, which were 38.89% and 116.35% higher than those in the control, respectively. Triterpenoid production in B. platyphylla cells induced by polyamine was further confirmed by RT-PCR of LUS gene. Conclusion: Put could effectively enhance the growth of B. platyphylla suspension cells and triterpenoid accumulation.
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PURPOSE: Most chemical transfection reagents are ineffective for the transfection of cells in suspension, such as leukemic cell and stem cell lineages. We developed two different types of viroplexes, cationic Sendai F/HN viroplexes (CSVs) and protamine sulfate-condensed cationic Sendai F/HN viroplexes (PCSVs) for the efficient transfection of T-leukemic cells. MATERIALS AND METHODS: The viroplex systems were prepared by reconstitution of fusogenic Sendai F/HN proteins in DMKE (O,O'-dimyristyl-N-lysyl glutamate) cationic liposomes. The viroplexes were further optimized for plasmid DNA and siRNA delivery to suspension cells. The particle size and surface charge of the viroplexes were analyzed with a zeta-sizer. Transfection of plasmid DNA (pDNA) and small interfering RNA (siRNA) by CSVs or PCSV was evaluated by measurement of transgene expression, confocal microscopy, FACS, and RT-PCR. RESULTS: The optimized CSVs and PCSVs exhibited enhanced gene and siRNA delivery in the tested suspension cell lines (Jurkat cells and CEM cells), compared with conventional cationic liposomes. In the case of pDNA transfection, the CSVs and PCSVs show at least 10-fold and 100-fold higher transgene expression compared with DMKE lipoplexes (or lipofectamine 2000), respectively. The CSVs showed more effective siRNA delivery to the suspension cells than cationic liposomes, as assessed by confocal microscopy, FACS, and RT-PCR. The effective transfection by the CSVs and PCSVs is presumably due to fusogenic activity of F/HN proteins resulting in facilitated internalization of pDNA and siRNA. CONCLUSION: This study suggests that Sendai F/HN viroplexes can be widely applicable for the transfection of pDNA and siRNA to suspension cell lines.
Subject(s)
Humans , Cell Line, Tumor , HN Protein/genetics , Jurkat Cells , RNA, Small Interfering , Sendai virus/genetics , Transfection/methods , Viral Fusion Proteins/genetics , VirosomesABSTRACT
Object To select the cell line of Ginkgo biloba L that may produce high yield of ginkgolide B (GB) in suspension culture and to study the stability of GB in subculture Methods Calculus was induced with stem, root and leaf of selected elite species and high yield suspension cell lines chosen by hypoxia stress Results Seven suspension cell lines with improved yield of GB were obtained Among which cell line MH 3 gave a 3 71 fold increase of cellular bioproduct with an increased content up to 302 ?g/g DW after culturing for 18 d, a leading record country wide In shaking bottle culture, it showed a consistent yield of GB with a content of 291 ?g/g DW in 6 successive subcultures, coefficient of variation=0 131 Conclusion The suspension cell line selected by hypoxia stress gave a higher yield and stability in successive transfer culture
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Objective To establish the suspension cell line of Atractylodes lancea and to study the effect of two endophytic fungal elicitors on its essential oil production.Methods The essential oil was extracted by using ultrasonic wave after suspension cell was treated with endophytic fungal elicitors.Then,the determination of four compounds(atractylone,hinesol,?-eudesmol,and atractylodin) was carried out by gas chromatography.Results By testing in various conditions,the suspension cell line with a rapid growth rate was established.Its highest biomass(6.95 g/L) was obtained on day 21.?-Eudesmol was the only detection in the control suspension cell,and its highest content(17.469 ?g/g) was also reached on day 21.The effect of crude elicitors of two endophytic fungi(belong to Cunninghamella sp.and Gilmaniella sp.respectively,named AL4 and AL12) on the cell growth and the production of essentia1 oil were investigated.Overall AL4 elicitor got better effect.When suspension cell of 14-day-old cultures was exposed to AL4 elicitor(carbohydrate 20 mg/L medium) for 7 d,the biomass increased 3.31% over the control,and the four compounds(atractylone: 14.715 ?g/g,hinesol: 28.395 ?g/g,?-eudesmol: 38.794 ?g/g,and atractylodin: 8.310 ?g/g) were all detected.Among them,the content of ?-eudesmol reached 2.22 times as much as the control.Conclusion The cell growth and the accumulation of essential oil of A.lancea could also be promoted by adding crude elicitors of the endophytic fungi AL4 and AL12.