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...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.展开更多
A high taxol yield cell line of Taxus yunnanensis Cheng et L. K. Fu keeps a high taxol_producing level after successive subcultures for more than eight years. In this study, eight taxanes were isolated from the su...A high taxol yield cell line of Taxus yunnanensis Cheng et L. K. Fu keeps a high taxol_producing level after successive subcultures for more than eight years. In this study, eight taxanes were isolated from the suspension cell cultures of this cell line. Based on NMR and MS analyses, and comparison with literature data and standards, their structures were determined to be 2α,5α,10β_triacetoxy_14β_propionyloxy_4(20),11_taxadiene (1), 2α,5α,10β_triacetoxy_14β_(2′_methyl)_butyryloxy_4(20),11_taxadiene (2), 2α,5α,10β_14β_tetra_acetoxy_4 (20),11_taxadiene (3, taxuyunnanine C), 2α,5α,10β_triacetoxy_14β_(2′_methyl_3′_hydroxy)_butyryloxy_4(20),11_taxadiene (4, yunnanxane) and its 3′_epimer (5), baccatin Ⅳ (6), baccatin Ⅲ (7) and taxol (8), respectively. Among those compounds, 3, 5, 6 and 7 were reported to be isolated from the suspension cell cultures of T. yunnanensis for the first time. TLC and HPLC analyses indicated that the chemical constituents of the culture solution were similar to those of cultured cells. Moreover, the highest taxol content of this cell line reached 0.3% and the cell line could be applied for a large_scale culture.展开更多
[Objective] The aim of this article is to establish a cell suspension culture system for eggplant. [Method] Using orthogonal design, appropriate media were screened for callus induction, subculture and cell suspension...[Objective] The aim of this article is to establish a cell suspension culture system for eggplant. [Method] Using orthogonal design, appropriate media were screened for callus induction, subculture and cell suspension culture. [Result] The appropriate medium for callus induction was MS supplemented with 0.2 mg/L 6-BA and 0.2 mg/L NAA; the appropriate subculture medium was MS supplemented with0.2 mg/L 6-BA, 0.2 mg/L NAA, and 0.1 mg/L KT; the suitable medium for cell suspension culture was liquid MS medium supplemented with 0.4 mg/L NAA and 0.2mg/L. Cell growth curve was similar to ＂S＂ shape in the above suspension medium.The cell growth included four phases, the initial phase（1-3 d）, the logarithmic phase（3-7 d）, the steady phase（7-8 d）, and the decline phase（8-11 d）. With the increasing culture time, the number of suspension cells increased, and it reached the maximum value at the 7thd, about 3.8 ×105cells/ml. Then the number of cells began to decline rapidly. The cell vigor was the highest at the initial phase. Suspension cells grew best in the liquid MS medium supplemented with NAA（0.4 mg/L）and 6-BA（0.2 mg/L）. The mitotic index reached the maximum, about 4.1% in the above medium, which suggested that this medium was suitable for cell suspension culture of eggplant. [Conclusion] Cell suspension culture system of eggplant provides a significant method for eggplant biotechnology. Genetic transformation and mutants screening can be carried out with this system.展开更多
[Objective] This study aimed to investigate the browning of T. cuspidata cells in suspension culture and provide the guidance for the cell suspension culture of T. cuspidata. [Method] T. cuspidata callus was used as e...[Objective] This study aimed to investigate the browning of T. cuspidata cells in suspension culture and provide the guidance for the cell suspension culture of T. cuspidata. [Method] T. cuspidata callus was used as experimental materials, to explore the effect of different medium, N/P ratio, pH, shaking speed, illumination time and light intensity and other factors on browning of T. cuspidata cells in suspension culture. [Result] Non-browning callus was transferred to 2MB5 medium （pH 7.0） for illumination culture at 22℃ under light intensity of 1 500 lx with shaking speed of 90 r/min for 24 h. Results showed that the cell browning was significantly inhibited. [Conclusion] This study laid the foundation for cell suspension culture of T. cuspidata and had important significance to the large-scale industrial production of paclitaxel.展开更多
This paper puts forward a physical and mathematical model for the rheological properties of a plant cell suspension culture system.The model can explain why the system is pseudoplastic satisfactorily,thus the rheologi...This paper puts forward a physical and mathematical model for the rheological properties of a plant cell suspension culture system.The model can explain why the system is pseudoplastic satisfactorily,thus the rheological properties of the system as the effect of the flow behavior index on plant cell concentration are interpreted correctly and the mechanism of the rheological properties of the system is further understood.Therefore the model can be applied in the technological design and optimum conditions of the system and the reformation,evaluation and scale up of reactors.展开更多
Aim To modify the structure of resibufogenin by using Ginkgo bilobasuspension. Methods Young leaves of Ginkgo biloba were differentiated into callus in MS medium withonly 2,4-D as plant growth regulator. The callus wa...Aim To modify the structure of resibufogenin by using Ginkgo bilobasuspension. Methods Young leaves of Ginkgo biloba were differentiated into callus in MS medium withonly 2,4-D as plant growth regulator. The callus was then transferred aseptically to liquid MSmedium exoge-nously supplemented with appropriate concentration of 6-BA, NAA and 2,4-D to establishsuspension cell culture system. Resibufogenin was administered into the well-grown cell cultures andincubated for 4 d. The products dissolved in the liquid phase of the cultures were extracted andpurified by silica gel column chromatography gradiently eluted with petroleum ether and acetonesystem. Results One transformed product was obtained in 40% yield after 4 d incubation, which wasidentified as 3-epi-resibufogenin on the basis of FAB MS, ~1H NMR and ^(13)C NMR spectroscopicanalysis and corresponding data reported in literature. Conclusion G. biloba suspension cultures canbe used as an enzyme system to biotransform resibufogenin, an animal-originated bufadienolide, into3-epi-resibufogenin.展开更多
Genetic transformation of embryogenic suspension cultures of sweetpotato cv. Lizixiang was conducted by using Agrobacterium tumefaciens strain A208SE harboring the binary vectors pROA93 with β-glucronidase(GUS)and ne...Genetic transformation of embryogenic suspension cultures of sweetpotato cv. Lizixiang was conducted by using Agrobacterium tumefaciens strain A208SE harboring the binary vectors pROA93 with β-glucronidase(GUS)and neomycin phosphotransferase(NPTⅡ)genes. The results indicated that embryogenic suspension cultures precultured for 1 - 3 d were suitable for the transformation. The optimal cocultivation time was 4 - 5 d. The optimal concentration of kanamycin was 50-75 mg L-1 for suspension culture and 100 mg L-1 for embryogenic callus proliferation and plant regeneration. The optimal concentration of carbencillin was 100 mg L-1. Transgenic plants identified with GUS assays and PCR analyses were obtained.展开更多
Objective: To evaluate the impact of plant growth regulators including kinetin(KN),benzyl adenine and naphthalene acetic acid, yeast extract and casein hydrolyzate on biomass accumulation of Vietnamese ginseng Panax v...Objective: To evaluate the impact of plant growth regulators including kinetin(KN),benzyl adenine and naphthalene acetic acid, yeast extract and casein hydrolyzate on biomass accumulation of Vietnamese ginseng Panax vietnamensis(P. vietnamensis) in cell suspension culture.Methods: Cell suspension cultures were established from friable calluses derived from leaves and petioles of 3-year-old in-vitro P. vietnamensis plants. The cell suspension cultures were grown in Murashige and Skoog basal media supplemented with various concentrations of KN, benzyl adenine, naphthalene acetic acid, and yeast extract and casein hydrolyzate.Results: All tested factors generated an increase in the cell biomass of P. vietnamensis in suspension culture, but the impact of each varies depended on the factor type, concentration, and incubation period. Addition of 2.0 mg/L KN resulted in the largest biomass increase after 24 d,(57.0 ± 0.9) and(3.1 ± 0.1) mg/m L fresh and dry weight, respectively,whereas addition of benzyl adenine or naphthalene acetic acid produced optimum levels of Panax cell biomass at 1.0 and 1.5 mg/L, respectively. Addition of the elicitor yeast extract led to a 1.4–2.4 fold increase in biomass of P. vietnamensis, while addition of casein hydrolyzate enhanced biomass accumulation 1.8–2.6 fold.Conclusions: The addition of each factor causes significant changes in biomass accumulation of P. vietnamensis. The largest biomass accumulation is from cultures grown in MS media containing 2.0 mg/L KN for 24 d. The outcome of the present study provides new insights into the optimal suspension culture conditions for studies on the in vitro cell biomass production of P. vietnamensis.展开更多
Plant natural products including alkaloids,polyphenols,terpenoids and flavonoids have been reported to exert anticancer activity by targeting various metabolic pathways.The biological pathways regulated by plant produ...Plant natural products including alkaloids,polyphenols,terpenoids and flavonoids have been reported to exert anticancer activity by targeting various metabolic pathways.The biological pathways regulated by plant products can serve as novel drug targets.Plant natural compounds or their derivatives used for cancer treatment and some novel plant-based compounds which are used in clinical trials were discussed.Callus suspension culture with secondary metabolites can provide a continuous source of plant pharmaceuticals without time and space limitations.Previous research has shown that rice callus suspension culture can kill>95%cancer cells with no significant effect on the growth of normal cells.The role of candidate genes and metabolites which are likely to be involved in the process and their potential to serve as anticancer and anti-inflammatory agents were discussed.Large scale production of plant callus suspension culture and its constituents can be achieved using elicitors which enhance specific secondary metabolites combined with bioprocess technology.展开更多
The hallmark of apoptosis, in suspension cultures of Taxus spp. cells induced by fungal extractive or by abiotic means, was studied by total DNA agarose gel electrophoresis and in situ end-labeling. The cleavage of nu...The hallmark of apoptosis, in suspension cultures of Taxus spp. cells induced by fungal extractive or by abiotic means, was studied by total DNA agarose gel electrophoresis and in situ end-labeling. The cleavage of nuclear DNA (nDNA) into oligonucleosomal fragments(DNA laddering) was a characteristic of apoptosis, which involved cell shrinkage, condensation of cytoplasm and tracheary elements differentiation. Terminal deoxynucleotidy transferase-mediated dUTP nick end in situ labeling (TUNEL) assay of Taxus spp. cells showed that fungal extractive or abiotic elicltors (Ce4+, Taxol, H2O2) induced TUNEL positive. Also, the increase of the apoptotic cell ratio was accompanied by the increase of secondary metabolites (especially Taxol). These results suggest that apoptosis may have some coincidence with biosynthesis of Taxol. The implication of apoptosis for the production of secondary metabolites in plant cell cultures is discussed.展开更多
Nitraria sibirica Pall. is a shrub that grows in saline-alkali soil and has traditional medicinal value and potential commercial value. The objectives of this study include induction and multiplication of callus, esta...Nitraria sibirica Pall. is a shrub that grows in saline-alkali soil and has traditional medicinal value and potential commercial value. The objectives of this study include induction and multiplication of callus, establishment of a suspension cell line, and isolation of protoplasts from cell suspensions. Murashige and Skoog (MS) medium was used for callus induction from mature seeds of N. sibirica. Seed-derived calluses were further multiplied on MS medium augmented with 0.5 mg L-1 6-benzylaminopurine (6-BA) and 1.0 mg L-1 2,4-dichlorophenoxy (2,4-D) acetic acid. Suspension cultures of N. sibirica were initiated by transferring friable calli to the same liquid multiplication medium. Characterization of the suspension culture was assessed based on fresh mass, dry mass, cell viability and pH value of the culture. A typical growth curve was observed after inoculating 1.5 g of callus in 40 mL liquid medium, including a lag phase, an exponential growth phase, a stationary phase, and a negative acceleration phase. The effect of factors such as pre-plasmolysis, enzyme combination, enzymolysis time and mannitol concentration, on the isolation of cell-derived protoplasts were evaluated to determine the usefulness of suspension cultures. The maximum yield (9.79 x 10(6) cells/g) and highest viability (79.97%) of protoplast were reached when approximately 1 g of cell suspension (cultured for 6 days) was inoculated for 12 h in cell and protoplast washing solution made of 0.8 mol L-1 mannitol mixture solution, cellulose onozuka R-10 2% (w/v), hemicellulose 0.2%, macerozyme R-10 1%, and pectolyase Y-23 0.5%. Protoplast yield was significantly influenced by pre-plasmolysis and cellulose onozuka R-10 (P < 0.05).展开更多
Vitis vinifera cv.Chardonnay suspension cultures were established from proembryogenic mass and employed for optimizing Agrobacterium-mediated transformation system.One-factor-at-a-time experiment revealed that OD600 o...Vitis vinifera cv.Chardonnay suspension cultures were established from proembryogenic mass and employed for optimizing Agrobacterium-mediated transformation system.One-factor-at-a-time experiment revealed that OD600 of Agrobacterium,time of inoculation,co-cultivation,and cell-drying before inoculation significantly affected the transformation efficiency which reached maximum 21.5% at the following conditions：0.8 of OD600,25 min of inoculation,2 d of co-cultivation,and 10 min of cell drying.Response surface methodology experiments based on a five-level,four-factor central-composite rotatable design were then used to optimize these selected factors.The optimized conditions for Chardonnay grape transformation were：0.8711 of OD600,28.9 min of inoculation,2.25 d of co-cultivation and 11.76 min of cell drying.After optimization,transformation efficiency was 26.2% and there were no interactions among different factors.展开更多
[ Objective] This study aimed to establish an optimized suspension culture for hairy roots of Aconitum coreanum （Levi.） Raipaics. [ Method ] Hairy root of Aconitum coreanum （ Levl. ） Raipaics A0489 was selected as...[ Objective] This study aimed to establish an optimized suspension culture for hairy roots of Aconitum coreanum （Levi.） Raipaics. [ Method ] Hairy root of Aconitum coreanum （ Levl. ） Raipaics A0489 was selected as experimental material, to screen the optimal medium for suspension culture. [ Result] Aconitum coreanum （Levl.） Raipaics hairy roots showed different growth conditions in various liquid medium; salt concentration, nitrogen content, sugar content, rotation speed and room temperature had great influences on the growth of Aconitum coreanum （Levi.） Raipaics hairy roots. [ Conclusion] The optimal suspension culture system for Aconitum coreanum （ Levi. ） Raipaics hairy roots was established ： B5 was used as basic medium, culture temperature was 20℃ under dark conditions, rotation speed was 140 r/m in the first 15 d and 110 r/m in the latter 15 d.展开更多
The standard detection hallmarks of apoptosis of Taxus cuspidata cells in suspension culture with Ce 4+ were studied. The condensation and margination of chromatin were observed under the electron microscopy....The standard detection hallmarks of apoptosis of Taxus cuspidata cells in suspension culture with Ce 4+ were studied. The condensation and margination of chromatin were observed under the electron microscopy. DNA fragmentation ranged 'DNA ladder' on agarose gel electrophoresis. TdT mediated dUTP nick end labeling (TUNEL) analysis of the cells reveals that the nuclear DNA strand breaks can be identified by labeling free 3′ OH termini. These results suggest that Ce 4+ can induce apoptosis of Taxus cuspidata cells and also indicate that there is a certain relationship between apoptosis and secondary metabolite product Taxol.展开更多
Friable callus was induced when immature seeds of floribunda rose were inoculated on MS medium supplemented with 2,4-D 3.0 mg.L^-1. When transfered onto subculture media, friable callus developed into embryogenic call...Friable callus was induced when immature seeds of floribunda rose were inoculated on MS medium supplemented with 2,4-D 3.0 mg.L^-1. When transfered onto subculture media, friable callus developed into embryogenic callus, which was used to establish cell suspension lines. Cell suspensions had to be subcultured at a interval of 4-5 days at the first several culture cycles. The best subculturing cycle for the stable cell suspensions was 8-10 days. The best inoculum quantity was 1 mL PCV（Packed Cell Volume） per 40 mL culture fluid.展开更多
In order to find suspension culture conditions suitable for Premna microphylla Turea, with the leaves of experimented P. microphylla as a material, em- bryogenic callus induction, subculture, dispersion of embryogenic...In order to find suspension culture conditions suitable for Premna microphylla Turea, with the leaves of experimented P. microphylla as a material, em- bryogenic callus induction, subculture, dispersion of embryogenic callus and suspension culture were performed. The results showed that the calli induced from leaves were different. The medium most suitable for induction of loose embryogenic callus for suspension culture was MS ＋ 0.4 mg/L 2,4-D ＋ 0.4 mg/L NAA ＋ 3% sucrose ＋0.8% agar. A better cell line could be obtained with cellulase and pectinase for dispersion of P. microphyUa callus. The medium most suitable for sus- pension culture was MS ＋0.4 mg/L 2,4-D ＋0.8 mg/L KT ＋3% sucrose. In this medium, the fresh weight of the suspensian-cultured cells increased by 6.46 times per 7 d. Dark culture was more suitable for cell proliferation. The average contents of pectin and protein were, respectively, 6.57% and 0.12% in the sus- pended ceils of P. microphylla, which had been cultured for 3 weeks under the optimum culture conditions.展开更多
The major factors affecting plant regeneration in suspension culture were investigated.The resultsshow that,in order for cell clusters to differentiate directly in liquid medium,it is essential toestablish an embryoge...The major factors affecting plant regeneration in suspension culture were investigated.The resultsshow that,in order for cell clusters to differentiate directly in liquid medium,it is essential toestablish an embryogenic callus line with high potential for plant regeneration.Embryogenic calliwere suspended in AA basic medium supplemented with 2,4-D 1 mg/L,6-benzylaminopurine 0.5mg/L,CH 300 mg/L,sucrose 3％ and mannitol 3％ and subcultured 7 days for each passage.Aftermore than 6 months of culturing,a fine suspension culture of small cell clusters(SCC)wasestablished.The SCC,80-270μin diameter,were then transferred to a liquid medium(MSsupplemented with NAA 0.01 mg/L and 4-pyridylurae(4-PU)0.5 mg/L)and allowed to grow instationary culture.Finally direct differentiation from SCC was observed.Several factors in suspension-subculture exerted strong after-effects on differentiation ofSCC.Basicmedia,kinds and concentration combinations of auxin and cytokinin in the subculture media,duration of shaking at each subculture passage,and amount of packed cell volume transferred to newmedium at each subculture passage and so on,all these affected the frequency of differentiation ofSCC.Higher concentrations of NAA and kinetin in the differentiation medium inhibited the directdifferentiation of SCC.Low concentrations(0.01-0.1 mg/L)of NAA with 4-PU in thedifferentiation medium were helpful for the direct differentiation of SCC.The differentiated clusterspossessed typical embryogenic structure from which normal plantlets could develop after transferringto agar medium.展开更多
Suspension cell cultures of Maytenus hookeri Loos. (Celastraceae) in SH media were established from the calli induced from the leaves and young steins of M. hookeri on MS media with the supplement of 2 mg/L 2,4-D and ...Suspension cell cultures of Maytenus hookeri Loos. (Celastraceae) in SH media were established from the calli induced from the leaves and young steins of M. hookeri on MS media with the supplement of 2 mg/L 2,4-D and 0.1 mg/L KIN (kinetin). Ethyl acetate extract of the cultures showed inhibitory activities against Penicillium avellaneum UC-4376 which was sensitive to maytansinoids. Exhaustive isolation of natural products from a large scale of suspension cell cultures did not yield maytansine instead of affording nine compounds including one novel triterpenoid, named 2, 3-diacetoxyl maytenusone (1), and eight known ones including squalene (2), beta-sitosterol (3), 2', 3', 4-triacetyl-sitoindoside I (4), salaspermic acid (5), maytenonic acid (6), 2alpha-hydroxy-maytenonic acid (7), 6, 11,12-trihydroxy-8, 11, 13-abietrien-7-one (8) and 11, 12-dihydroxy-8, 11, 13-abietatrien-7-one (9) elucidated on the basis of 1D and 2D NMR data. The H-1-NMR and C-13-NMR assignments were made for 1, 5, 6 and 7, while the C-13-NMR assignments for 5 and 6 were revised. The chemical results suggested that the suspension cell cultures of M. hookeri did not produce maytansinoids under the reported experiment conditions.展开更多
AIM: To investigate the effects of feeding phenylalanine(Phe) and tyrosine(Tyr) on the accumulation of total phenolic compounds and four phenylethanoid glycosides(PeGs) to a cell suspension culture of the parasitic pl...AIM: To investigate the effects of feeding phenylalanine(Phe) and tyrosine(Tyr) on the accumulation of total phenolic compounds and four phenylethanoid glycosides(PeGs) to a cell suspension culture of the parasitic plant Cistanche deserticola. METHOD: A cell suspension culture of C. deserticola was established and precursors of different concentrations were fed. In each group, the cell was sampled at the 24th day after inoculation. The content of total phenolic compounds and four PeGs compounds were determined using the Folin-Ciocalteu method and an HPLC method, respectively. RESULTS: In the Phe fed cells, the maximum PeGs yield was achieved when Phe was fed at 1.5 mmol·L-1 and the yield reached 1.13 times the control cell concentration. In the Tyr fed cells, the maximum yield of PeGs was 1.60 times of control when 0.75 mmol·L-1 Tyr was fed to the cells. Furthermore, it was found that the salidroside yield was 4.01 times of control group when 5 mmol·L-1 Tyr was fed. CONCLUSION: Tyr is a better precursor for PeGs accumulation compared with Phe, and the rate limiting enzymes might be involved in the Tyr branch.展开更多
基金partly funded by the Department of Science and Technology Fund for Improvement of S&T Infrastructure (Grant No. SR/FST/LS-I/2018/125)。
文摘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.
文摘A high taxol yield cell line of Taxus yunnanensis Cheng et L. K. Fu keeps a high taxol_producing level after successive subcultures for more than eight years. In this study, eight taxanes were isolated from the suspension cell cultures of this cell line. Based on NMR and MS analyses, and comparison with literature data and standards, their structures were determined to be 2α,5α,10β_triacetoxy_14β_propionyloxy_4(20),11_taxadiene (1), 2α,5α,10β_triacetoxy_14β_(2′_methyl)_butyryloxy_4(20),11_taxadiene (2), 2α,5α,10β_14β_tetra_acetoxy_4 (20),11_taxadiene (3, taxuyunnanine C), 2α,5α,10β_triacetoxy_14β_(2′_methyl_3′_hydroxy)_butyryloxy_4(20),11_taxadiene (4, yunnanxane) and its 3′_epimer (5), baccatin Ⅳ (6), baccatin Ⅲ (7) and taxol (8), respectively. Among those compounds, 3, 5, 6 and 7 were reported to be isolated from the suspension cell cultures of T. yunnanensis for the first time. TLC and HPLC analyses indicated that the chemical constituents of the culture solution were similar to those of cultured cells. Moreover, the highest taxol content of this cell line reached 0.3% and the cell line could be applied for a large_scale culture.
基金Supported by the National Key Technology R&D Program of China(2011BAD12B03)the National Natural Science Foundation of China(30700002,31101536)~~
文摘[Objective] The aim of this article is to establish a cell suspension culture system for eggplant. [Method] Using orthogonal design, appropriate media were screened for callus induction, subculture and cell suspension culture. [Result] The appropriate medium for callus induction was MS supplemented with 0.2 mg/L 6-BA and 0.2 mg/L NAA; the appropriate subculture medium was MS supplemented with0.2 mg/L 6-BA, 0.2 mg/L NAA, and 0.1 mg/L KT; the suitable medium for cell suspension culture was liquid MS medium supplemented with 0.4 mg/L NAA and 0.2mg/L. Cell growth curve was similar to ＂S＂ shape in the above suspension medium.The cell growth included four phases, the initial phase（1-3 d）, the logarithmic phase（3-7 d）, the steady phase（7-8 d）, and the decline phase（8-11 d）. With the increasing culture time, the number of suspension cells increased, and it reached the maximum value at the 7thd, about 3.8 ×105cells/ml. Then the number of cells began to decline rapidly. The cell vigor was the highest at the initial phase. Suspension cells grew best in the liquid MS medium supplemented with NAA（0.4 mg/L）and 6-BA（0.2 mg/L）. The mitotic index reached the maximum, about 4.1% in the above medium, which suggested that this medium was suitable for cell suspension culture of eggplant. [Conclusion] Cell suspension culture system of eggplant provides a significant method for eggplant biotechnology. Genetic transformation and mutants screening can be carried out with this system.
基金Supported by National Natural Science Foundation of China (31070164)Young Scientists Fund of Dalian (2006J23JH031)~~
文摘[Objective] This study aimed to investigate the browning of T. cuspidata cells in suspension culture and provide the guidance for the cell suspension culture of T. cuspidata. [Method] T. cuspidata callus was used as experimental materials, to explore the effect of different medium, N/P ratio, pH, shaking speed, illumination time and light intensity and other factors on browning of T. cuspidata cells in suspension culture. [Result] Non-browning callus was transferred to 2MB5 medium （pH 7.0） for illumination culture at 22℃ under light intensity of 1 500 lx with shaking speed of 90 r/min for 24 h. Results showed that the cell browning was significantly inhibited. [Conclusion] This study laid the foundation for cell suspension culture of T. cuspidata and had important significance to the large-scale industrial production of paclitaxel.
文摘This paper puts forward a physical and mathematical model for the rheological properties of a plant cell suspension culture system.The model can explain why the system is pseudoplastic satisfactorily,thus the rheological properties of the system as the effect of the flow behavior index on plant cell concentration are interpreted correctly and the mechanism of the rheological properties of the system is further understood.Therefore the model can be applied in the technological design and optimum conditions of the system and the reformation,evaluation and scale up of reactors.
文摘Aim To modify the structure of resibufogenin by using Ginkgo bilobasuspension. Methods Young leaves of Ginkgo biloba were differentiated into callus in MS medium withonly 2,4-D as plant growth regulator. The callus was then transferred aseptically to liquid MSmedium exoge-nously supplemented with appropriate concentration of 6-BA, NAA and 2,4-D to establishsuspension cell culture system. Resibufogenin was administered into the well-grown cell cultures andincubated for 4 d. The products dissolved in the liquid phase of the cultures were extracted andpurified by silica gel column chromatography gradiently eluted with petroleum ether and acetonesystem. Results One transformed product was obtained in 40% yield after 4 d incubation, which wasidentified as 3-epi-resibufogenin on the basis of FAB MS, ~1H NMR and ^(13)C NMR spectroscopicanalysis and corresponding data reported in literature. Conclusion G. biloba suspension cultures canbe used as an enzyme system to biotransform resibufogenin, an animal-originated bufadienolide, into3-epi-resibufogenin.
基金supported by the National Natural Science Foundation of China(30170587)the National High Tech R&D Program,China(863 Program,2002AA241031,2001AA241181)the Teaching and Research Award Program for Outstanding Young Teachers in Higher Education Institutions of Ministry of Education,China.
文摘Genetic transformation of embryogenic suspension cultures of sweetpotato cv. Lizixiang was conducted by using Agrobacterium tumefaciens strain A208SE harboring the binary vectors pROA93 with β-glucronidase(GUS)and neomycin phosphotransferase(NPTⅡ)genes. The results indicated that embryogenic suspension cultures precultured for 1 - 3 d were suitable for the transformation. The optimal cocultivation time was 4 - 5 d. The optimal concentration of kanamycin was 50-75 mg L-1 for suspension culture and 100 mg L-1 for embryogenic callus proliferation and plant regeneration. The optimal concentration of carbencillin was 100 mg L-1. Transgenic plants identified with GUS assays and PCR analyses were obtained.
基金the Ministry of Science and Technology,Vietnam for financial support
文摘Objective: To evaluate the impact of plant growth regulators including kinetin(KN),benzyl adenine and naphthalene acetic acid, yeast extract and casein hydrolyzate on biomass accumulation of Vietnamese ginseng Panax vietnamensis(P. vietnamensis) in cell suspension culture.Methods: Cell suspension cultures were established from friable calluses derived from leaves and petioles of 3-year-old in-vitro P. vietnamensis plants. The cell suspension cultures were grown in Murashige and Skoog basal media supplemented with various concentrations of KN, benzyl adenine, naphthalene acetic acid, and yeast extract and casein hydrolyzate.Results: All tested factors generated an increase in the cell biomass of P. vietnamensis in suspension culture, but the impact of each varies depended on the factor type, concentration, and incubation period. Addition of 2.0 mg/L KN resulted in the largest biomass increase after 24 d,(57.0 ± 0.9) and(3.1 ± 0.1) mg/m L fresh and dry weight, respectively,whereas addition of benzyl adenine or naphthalene acetic acid produced optimum levels of Panax cell biomass at 1.0 and 1.5 mg/L, respectively. Addition of the elicitor yeast extract led to a 1.4–2.4 fold increase in biomass of P. vietnamensis, while addition of casein hydrolyzate enhanced biomass accumulation 1.8–2.6 fold.Conclusions: The addition of each factor causes significant changes in biomass accumulation of P. vietnamensis. The largest biomass accumulation is from cultures grown in MS media containing 2.0 mg/L KN for 24 d. The outcome of the present study provides new insights into the optimal suspension culture conditions for studies on the in vitro cell biomass production of P. vietnamensis.
文摘Plant natural products including alkaloids,polyphenols,terpenoids and flavonoids have been reported to exert anticancer activity by targeting various metabolic pathways.The biological pathways regulated by plant products can serve as novel drug targets.Plant natural compounds or their derivatives used for cancer treatment and some novel plant-based compounds which are used in clinical trials were discussed.Callus suspension culture with secondary metabolites can provide a continuous source of plant pharmaceuticals without time and space limitations.Previous research has shown that rice callus suspension culture can kill>95%cancer cells with no significant effect on the growth of normal cells.The role of candidate genes and metabolites which are likely to be involved in the process and their potential to serve as anticancer and anti-inflammatory agents were discussed.Large scale production of plant callus suspension culture and its constituents can be achieved using elicitors which enhance specific secondary metabolites combined with bioprocess technology.
文摘The hallmark of apoptosis, in suspension cultures of Taxus spp. cells induced by fungal extractive or by abiotic means, was studied by total DNA agarose gel electrophoresis and in situ end-labeling. The cleavage of nuclear DNA (nDNA) into oligonucleosomal fragments(DNA laddering) was a characteristic of apoptosis, which involved cell shrinkage, condensation of cytoplasm and tracheary elements differentiation. Terminal deoxynucleotidy transferase-mediated dUTP nick end in situ labeling (TUNEL) assay of Taxus spp. cells showed that fungal extractive or abiotic elicltors (Ce4+, Taxol, H2O2) induced TUNEL positive. Also, the increase of the apoptotic cell ratio was accompanied by the increase of secondary metabolites (especially Taxol). These results suggest that apoptosis may have some coincidence with biosynthesis of Taxol. The implication of apoptosis for the production of secondary metabolites in plant cell cultures is discussed.
基金supported by the National Science&Technology Pillar Program during the 12th Five-year Plan Period(2011AA10020102)
文摘Nitraria sibirica Pall. is a shrub that grows in saline-alkali soil and has traditional medicinal value and potential commercial value. The objectives of this study include induction and multiplication of callus, establishment of a suspension cell line, and isolation of protoplasts from cell suspensions. Murashige and Skoog (MS) medium was used for callus induction from mature seeds of N. sibirica. Seed-derived calluses were further multiplied on MS medium augmented with 0.5 mg L-1 6-benzylaminopurine (6-BA) and 1.0 mg L-1 2,4-dichlorophenoxy (2,4-D) acetic acid. Suspension cultures of N. sibirica were initiated by transferring friable calli to the same liquid multiplication medium. Characterization of the suspension culture was assessed based on fresh mass, dry mass, cell viability and pH value of the culture. A typical growth curve was observed after inoculating 1.5 g of callus in 40 mL liquid medium, including a lag phase, an exponential growth phase, a stationary phase, and a negative acceleration phase. The effect of factors such as pre-plasmolysis, enzyme combination, enzymolysis time and mannitol concentration, on the isolation of cell-derived protoplasts were evaluated to determine the usefulness of suspension cultures. The maximum yield (9.79 x 10(6) cells/g) and highest viability (79.97%) of protoplast were reached when approximately 1 g of cell suspension (cultured for 6 days) was inoculated for 12 h in cell and protoplast washing solution made of 0.8 mol L-1 mannitol mixture solution, cellulose onozuka R-10 2% (w/v), hemicellulose 0.2%, macerozyme R-10 1%, and pectolyase Y-23 0.5%. Protoplast yield was significantly influenced by pre-plasmolysis and cellulose onozuka R-10 (P < 0.05).
基金supported by the 948 Program,Ministry of Agriculture,China(2006-G26)the National Grape Industry Technology System,China(nycytx-30-zy-05)
文摘Vitis vinifera cv.Chardonnay suspension cultures were established from proembryogenic mass and employed for optimizing Agrobacterium-mediated transformation system.One-factor-at-a-time experiment revealed that OD600 of Agrobacterium,time of inoculation,co-cultivation,and cell-drying before inoculation significantly affected the transformation efficiency which reached maximum 21.5% at the following conditions：0.8 of OD600,25 min of inoculation,2 d of co-cultivation,and 10 min of cell drying.Response surface methodology experiments based on a five-level,four-factor central-composite rotatable design were then used to optimize these selected factors.The optimized conditions for Chardonnay grape transformation were：0.8711 of OD600,28.9 min of inoculation,2.25 d of co-cultivation and 11.76 min of cell drying.After optimization,transformation efficiency was 26.2% and there were no interactions among different factors.
基金Supported by Project of Applied Basic Research from the Institute of Special Wild Economic Animal and Plant Science,Chinese Academy of Agricultural Sciences(tcs2004-08)Project of Jilin Science and Technology Bureau(201222306)
文摘[ Objective] This study aimed to establish an optimized suspension culture for hairy roots of Aconitum coreanum （Levi.） Raipaics. [ Method ] Hairy root of Aconitum coreanum （ Levl. ） Raipaics A0489 was selected as experimental material, to screen the optimal medium for suspension culture. [ Result] Aconitum coreanum （Levl.） Raipaics hairy roots showed different growth conditions in various liquid medium; salt concentration, nitrogen content, sugar content, rotation speed and room temperature had great influences on the growth of Aconitum coreanum （Levi.） Raipaics hairy roots. [ Conclusion] The optimal suspension culture system for Aconitum coreanum （ Levi. ） Raipaics hairy roots was established ： B5 was used as basic medium, culture temperature was 20℃ under dark conditions, rotation speed was 140 r/m in the first 15 d and 110 r/m in the latter 15 d.
文摘The standard detection hallmarks of apoptosis of Taxus cuspidata cells in suspension culture with Ce 4+ were studied. The condensation and margination of chromatin were observed under the electron microscopy. DNA fragmentation ranged 'DNA ladder' on agarose gel electrophoresis. TdT mediated dUTP nick end labeling (TUNEL) analysis of the cells reveals that the nuclear DNA strand breaks can be identified by labeling free 3′ OH termini. These results suggest that Ce 4+ can induce apoptosis of Taxus cuspidata cells and also indicate that there is a certain relationship between apoptosis and secondary metabolite product Taxol.
文摘Friable callus was induced when immature seeds of floribunda rose were inoculated on MS medium supplemented with 2,4-D 3.0 mg.L^-1. When transfered onto subculture media, friable callus developed into embryogenic callus, which was used to establish cell suspension lines. Cell suspensions had to be subcultured at a interval of 4-5 days at the first several culture cycles. The best subculturing cycle for the stable cell suspensions was 8-10 days. The best inoculum quantity was 1 mL PCV（Packed Cell Volume） per 40 mL culture fluid.
基金Supported by Nature Science Foundation of Hubei Province(2008CDZ085)
文摘In order to find suspension culture conditions suitable for Premna microphylla Turea, with the leaves of experimented P. microphylla as a material, em- bryogenic callus induction, subculture, dispersion of embryogenic callus and suspension culture were performed. The results showed that the calli induced from leaves were different. The medium most suitable for induction of loose embryogenic callus for suspension culture was MS ＋ 0.4 mg/L 2,4-D ＋ 0.4 mg/L NAA ＋ 3% sucrose ＋0.8% agar. A better cell line could be obtained with cellulase and pectinase for dispersion of P. microphyUa callus. The medium most suitable for sus- pension culture was MS ＋0.4 mg/L 2,4-D ＋0.8 mg/L KT ＋3% sucrose. In this medium, the fresh weight of the suspensian-cultured cells increased by 6.46 times per 7 d. Dark culture was more suitable for cell proliferation. The average contents of pectin and protein were, respectively, 6.57% and 0.12% in the sus- pended ceils of P. microphylla, which had been cultured for 3 weeks under the optimum culture conditions.
基金This work was completed in Tohoku University,Japan
文摘The major factors affecting plant regeneration in suspension culture were investigated.The resultsshow that,in order for cell clusters to differentiate directly in liquid medium,it is essential toestablish an embryogenic callus line with high potential for plant regeneration.Embryogenic calliwere suspended in AA basic medium supplemented with 2,4-D 1 mg/L,6-benzylaminopurine 0.5mg/L,CH 300 mg/L,sucrose 3％ and mannitol 3％ and subcultured 7 days for each passage.Aftermore than 6 months of culturing,a fine suspension culture of small cell clusters(SCC)wasestablished.The SCC,80-270μin diameter,were then transferred to a liquid medium(MSsupplemented with NAA 0.01 mg/L and 4-pyridylurae(4-PU)0.5 mg/L)and allowed to grow instationary culture.Finally direct differentiation from SCC was observed.Several factors in suspension-subculture exerted strong after-effects on differentiation ofSCC.Basicmedia,kinds and concentration combinations of auxin and cytokinin in the subculture media,duration of shaking at each subculture passage,and amount of packed cell volume transferred to newmedium at each subculture passage and so on,all these affected the frequency of differentiation ofSCC.Higher concentrations of NAA and kinetin in the differentiation medium inhibited the directdifferentiation of SCC.Low concentrations(0.01-0.1 mg/L)of NAA with 4-PU in thedifferentiation medium were helpful for the direct differentiation of SCC.The differentiated clusterspossessed typical embryogenic structure from which normal plantlets could develop after transferringto agar medium.
文摘Suspension cell cultures of Maytenus hookeri Loos. (Celastraceae) in SH media were established from the calli induced from the leaves and young steins of M. hookeri on MS media with the supplement of 2 mg/L 2,4-D and 0.1 mg/L KIN (kinetin). Ethyl acetate extract of the cultures showed inhibitory activities against Penicillium avellaneum UC-4376 which was sensitive to maytansinoids. Exhaustive isolation of natural products from a large scale of suspension cell cultures did not yield maytansine instead of affording nine compounds including one novel triterpenoid, named 2, 3-diacetoxyl maytenusone (1), and eight known ones including squalene (2), beta-sitosterol (3), 2', 3', 4-triacetyl-sitoindoside I (4), salaspermic acid (5), maytenonic acid (6), 2alpha-hydroxy-maytenonic acid (7), 6, 11,12-trihydroxy-8, 11, 13-abietrien-7-one (8) and 11, 12-dihydroxy-8, 11, 13-abietatrien-7-one (9) elucidated on the basis of 1D and 2D NMR data. The H-1-NMR and C-13-NMR assignments were made for 1, 5, 6 and 7, while the C-13-NMR assignments for 5 and 6 were revised. The chemical results suggested that the suspension cell cultures of M. hookeri did not produce maytansinoids under the reported experiment conditions.
文摘AIM: To investigate the effects of feeding phenylalanine(Phe) and tyrosine(Tyr) on the accumulation of total phenolic compounds and four phenylethanoid glycosides(PeGs) to a cell suspension culture of the parasitic plant Cistanche deserticola. METHOD: A cell suspension culture of C. deserticola was established and precursors of different concentrations were fed. In each group, the cell was sampled at the 24th day after inoculation. The content of total phenolic compounds and four PeGs compounds were determined using the Folin-Ciocalteu method and an HPLC method, respectively. RESULTS: In the Phe fed cells, the maximum PeGs yield was achieved when Phe was fed at 1.5 mmol·L-1 and the yield reached 1.13 times the control cell concentration. In the Tyr fed cells, the maximum yield of PeGs was 1.60 times of control when 0.75 mmol·L-1 Tyr was fed to the cells. Furthermore, it was found that the salidroside yield was 4.01 times of control group when 5 mmol·L-1 Tyr was fed. CONCLUSION: Tyr is a better precursor for PeGs accumulation compared with Phe, and the rate limiting enzymes might be involved in the Tyr branch.
