Suspension Cultures Of Daucus Carota Research Articles

Medium composition potentially regulates the anthocyanin production from suspension culture of Daucus carota.

In the present study, an effort has been made to optimize various culture conditions for enhanced production of anthocyanin. Nutrient content of MS medium (ammonium to potassium nitrate ratio and phosphate concentration) had a profound influence on the cell biomass and anthocyanin accumulation in cell suspension cultures of Daucus carota. Suspension cultures were carried out in shake flasks for 18days and examined for cell growth, anthocyanin synthesis, anthocyanin yield and development of pigmented cells in relation to the uptake of total sugar, extracellular phosphate, nitrate and ammonia. The addition of NH4NO3 to KNO3 ratio (20.0mM: 37.6mM) in the suspension culture media resulted in a 2.85-fold increase in anthocyanin content at day 3. Similarly, a lower concentration of KH2PO4 (0.45mM) in the MS medium resulted in 1.63-fold increase in anthocyanin content at day 9. The total sugar uptake was closely associated with a significant increase in anthocyanin accumulation. Total sugar and nitrate were consumed until 9-12days, while ammonia and phosphate were completely consumed within 3days after inoculation. After 9days, cell lysis was observed and resulted in the leakage of intracellular substances. These observations suggest that anthocyanin was synthesized only by viable pigmented cells and degraded rapidly after cell death and lysis. This study signifies the utility of D. carota suspension culture for further up-scaling studies of anthocyanin.

Enhanced accumulation of phytosterols and phenolic compounds in cyclodextrin-elicited cell suspension culture of Daucus carota

In this work, suspension-cultured cells of Daucus carota were used to evaluate the effect of β-cyclodextrins on the production of isoprenoid and phenolic compounds. The results showed that the phytosterols and phenolic compounds were accumulated in the extracellular medium (15100μgL−1 and 477.46μgL−1, respectively) in the presence of cyclodextrins. Unlike the phytosterol and phenolic compound content, β-carotene (1138.03μgL−1), lutein (25949.54μgL−1) and α-tocopherol (8063.82μgL−1) chlorophyll a (1625.13μgL−1) and b (9.958 (9958.33μgL−1) were mainly accumulated inside the cells. Therefore, cyclodextrins were able to induce the cytosolic mevalonate pathway, increasing the biosynthesis of phytosterols and phenolic compounds, and accumulate them outside the cells. However, in the absence of these cyclic oligosaccharidic elicitors, carrot cells mainly accumulated carotenoids through the methylerythritol 4-phosphate pathway.Therefore, the use of cyclodextrins would allow the extracellular accumulation of both phytosterols and phenolic compounds by diverting the carbon flux towards the cytosolic mevalonate/phenylpropanoid pathway.

N-acetyl glutamate kinase from Daucus carota suspension cultures: embryogenic expression profile, purification and characterization

In Daucus carota, N-acetylglutamate-5-phosphotransferase (NAGK; E.C. 2.7.2.8) specific activity was shown to correlate with the progression of somatic embryogenesis and was highest in the latter stages, where growth was most rapid. The enzyme was subsequently purified greater than 1200-fold using heat treatment, ammonium sulfate fractionation, gel filtration, anion exchange and dye ligand chromatography. Carrot NAGK was shown to have a subunit molecular weight of 31 kDa and form a hexamer. The Kms for NAG and ATP are 5.24 and 2.11 mM, respectively. Arginine (Arg) is a K-type allosteric inhibitor of the enzyme, and Hill coefficients in the order of 5 in the presence of Arg suggest that the enzyme is highly cooperative. D. carota NAGK does not bind to Arabidopsis thaliana PII affinity columns, nor does the A. thaliana PII increase NAGK specific activity, indicating its cellular location is probably different.

Image analysis and in vivo imaging as tools for investigation of productivity dynamics in anthocyanin‐producing cell cultures of Daucus carota

An anthocyanin-producing suspension culture of Daucus carota (L.) cv. Flakkese was used as model system to study secondary metabolite production in cell culture at the individual cell level. An approach was set up in which growth and production of anthocyanins were investigated using a combination of biochemical analysis, image (colour) analysis and in vivo imaging. This novel approach was used to segment the culture in different subpopulations and dissect the productive process in the cell culture grown under two different conditions, known to differ mainly for oxygen supply and mixing intensity (volume of 50 ml or 20 ml in 250 ml flasks). The 20 ml batch cultures gave a higher content and yield of anthocyanins, which depended on a complex balance between events that positively or negatively affected anthocyanin production. A model is proposed in which the different ability of cells to respond to environmental stimuli and stress depends on the different amount of anthocyanins accumulated within cells.

AC electrokinetic characterisation and separation of cells with high and low embryogenic potential in suspension cultures of carrot (Daucus carota)

Suspension cultures of Daucus carota L. were established, and cells with embryogenic potential were separated from those without by density gradient centrifugation in Ficoll at different stages in the growth curve. In order to obtain information about the electrical properties of individual cells, electrorotation spectra of single plant cells from different fractions were measured before and after induction of embryogenesis. The data were analysed using models based on Maxwell–Wagner's theories of interfacial polarisation. It was found that the denser cells had a higher embryogenic potential, a darker appearance and a higher internal conductivity (>1 S m−1) than the less dense cells, which had less or no embryogenic potential and a lower internal conductivity (<1 S m−1). Modelling the dielectrophoretic (DEP) response on the basis of the electrorotation data suggested that separation of cells with high embryogenic potential may be achievable in the frequency range 1–10 MHz. Actual dielectrophoretic separation of cells with high embryogenic potential from suspensions in which embryogenesis had not yet been induced was achieved using steric as well as hyperlayer dielectrophoretic Field-Flow Fractionation (DEP-FFF).

Regulation of glutamate dehydrogenase activity by manipulation of nucleotide supply in Daucus carota suspension cultures

Regulation of glutamate dehydrogenase activity by manipulation of nucleotide supply in Daucus carota suspension cultures

Mechanisms of the proliferation and differentiation of plant cells in cell culture systems.

Plant cell functions have been investigated in various cell culture systems. In this review, we summarize results obtained from investigations of gene expression during the cell cycle in synchronized cultures of Catharanthus roseus during somatic embryogenesis in suspension cultures of Daucus carota, during organogenesis in tissue cultures of Arabidopsis thaliana and during the transdifferentiation of isolated mesophyll cells to tracheary elements in single-cell cultures of Zinnia elegans.

Enhanced somatic embryo production by conditioned media in cell suspension cultures ofDaucus carota

The addition of conditioned media extracted from 8 day old embryo culture accelerated growth and production of torpedo embryos and plantlets in cell suspension cultures ofDaucus carota. The production of late-stage embryos was increased a maximum threefold (up to 1500 embryos/ml) compared with that of control culture, when spent medium was added during inoculation.

Anthocyanins from cell suspension cultures of Daucus carota

Six anthocyanins were isolated from cell suspension cultures of an Afghan cultivar of Daucus carota by PC or HPLC. The structures of these compounds were elucidated by spectroscopic methods as cyanidin 3- O-lathyroside, cyanidin 3- O-(2″- O-β- d-Xylopyranosyl-6″- O-β- d-glucopyranosyl-β- d-galactopyranoside), and the latter acylated with 4-coumaric, ferulic, 4-hydroxybenzoic or sinapic acid. Unusual 1H NMR chemical shifts and 1H NOE data indicate an intramolecular copigmentation of the aglycone with these aromatic residues.

Volatiles identified from five stages of embryo development separated from a heterogeneous suspension culture of Daucus carota

Five stages of embryo development were fractionated from a mature culture of Daucus carota (Gelbe Rheinsche), using a series of metal sieves. The composition of the population of embryos in each fraction was determined quantitatively from microscopic investigations. Volatiles from samples of tissue from six stages of development were trapped on activated charcoal cartridges. These volatiles, some of which may play a significant role in the interaction of the plant with the carrot root fly (Psila rosae), were analysed using gas chromatography/mass spectroscopy. The resulting chromatograms are arranged in order of embryo development. The progressive elaboration of the volatile profile reflects the increased biosynthetic capacity of the developing embryo.

Daucus carota cells contain a dihydrofolate reductase: thymidylate synthase bifunctional polypeptide

Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) activities from cell suspension cultures of Daucus carota were shown to copurify on (NH4)2SO4 fractionation, DEAE Sephadex and methotrexate-Sepharose affinity chromatography and to share approximately the same Mr(183 kDa and 185 kDa respectively) as judged by gel filtration on Sephacryl S-200.The copurified protein migrated as a single band on polyacrylamide gel electrophoresis under denaturing conditions.Both activities could be eluted from the same position of the native gel.Moreover, methotrexate-resistant cell lines which overproduce DHFR revealed to have a parallel higher level of TS. It is therefore proposed and discussed that in carrot, similarly to protozoa, TS and DHFR are present on a single bifunctional polypeptide of 58 kDa.

The potential of cell suspension cultures of Daucus carota L. as a source of isotope labelled gibberellins. I. Metabolism of [3H]GA5.

Gibberellin A5 (GA5), which is not known to be native to carrot (Daucus carota L.), was fed as either high specific activity [1-3H]GA5 (HSA, 3.2 Ci/mmol) or low specific activity [1,2-3H]GA5 (LSA, 74 mCi/mmol) to cell suspension cultures of carrot. Gibberellins A1, A3, A6, A8 and their glucosyl conjugates were found as metabolites of HSA [3H]GA5, as were GA22, GA29 and GA5 glucoside and glucosyl ester, with all tentative identifications based on retention times on reversed phase C18 HPLC with radiocounting. From LSA [1,2-3H]GA5 feeds, GA1, GA3, GA6, GA8 and GA29 were identified by GC/SIM. For cells or medium, only 24 to 27%, respectively, of [3H]GA5 was metabolized; major metabolites were [3H]GA5 glucosyl conjugates (HSA and LSA feeds) and free [3H]GA1/3 (LSA feeds). The system may thus be useful for production of labelled GAs and GA conjugates, and especially for conjugates of GA5 and free GA6 without dilution by endogenous GAs.

Effects of methyl mercury on arrays of microtubules and macromolecular synthesis in Daucus carota cultures

Cell suspension cultures of Daucus carota were exposed to methyl mercury at concentrations between 0 and 6 μg/ml for 1, 3, or 24 hr. Microtubule arrays exhibited no detectable disruption (as compared with controls) when treated with 1, 2, and 3 μg/ml methyl mercury. Disorganization of microtubules did occur at higher concentrations (4–6 μg/ml) in a concentration/time-dependent manner. No recovery of microtubule arrays was evident when cells were placed in methyl mercury-free medium for up to 7 days. Analyses of soluble protein and carbohydrate content, dry weight, and cell viability (reducing capacity) indicate that methyl mercury exposure has inhibitory effects on cell metabolism. The observed disruption of plant cell microtubules, induced by exposure to methyl mercury, may be secondary in response to an initial inhibition of synthetic pathways and membrane perturbations.

Facilitation of electrofusion of plant protoplasts by membrane-active agents

Protoplasts isolated from a suspension culture of Daucus carota were subjected to electrofusion by means of a combination of dielectrophoresis to align the cells and brief d.c. shocks to induce fusion. The protoplasts were treated with agents known to alter membrane structure and function in order to define the factors that limit electrofusion. Lysophosphatidylcholine, dimethylsulfoxide and calcium chloride enhanced electrofusion; low temperature reduced fusion. However, lysophosphatidylcholine, calcium chloride, and low temperature all decreased membrane fluidity as measured by electron spin resonance spectroscopy of intact carrot protoplasts labeled with 5-doxylstearic acid. These spectra were taken in the presence of Fe(CN) 6 3− and thus largely reflect the fluidity of the plasma membrane. Treating the protoplasts with pronase or proteinase K also facilitated electrofusion. The proteinase K effect was largely reversed by the specific protease inhibitor phenylmethylsulfonyl fluoride. Protoplasts labeled with fluorescein isothiocyanate-concanavalin A did not exhibit any perceptible capping or clustering of membrane proteins in response to protease treatment. Enhancement of protoplast electrofusion by proteases may be due to improvement of cell-cell contacts through the removal of membrane surface determinants. However, the possibility that proteases enhance electrofusion by causing aggregation of membrane proteins cannot be eliminated.

Marker proteins for embryogenic differentiation patterns in pea callus

Polypeptide pattern alterations during somatic embryogenesis were investigated using callus cultures of two Pisum sativum genotypes. Both genotypes show the formation of two different callus lines from the same explant after six to eight weeks in culture: a nodular yellowish callus line, which forms somatic embryoids in suspension cultures (e(+)) and a white compact callus line with no regenerative capacity (e(-)). The cytosol proteins of the two different callus lines were separated in a semi-preparative two-dimensional system and the polypeptide patterns were compared. Two protein bands were found (P1: Mr=45000 D, pI=7.0-7.1; P2: Mr=7000 D, pI=<4.5), which were characteristic of the putatively embryogenic (e(+)) callus line in all tissues investigated (two genotypes × two explant sources). These proteins found in nodular (e(+)) pea cultures are very similar to two proteins found in Daucus carota suspension cultures preceding the formation of somatic embryos.

The uptake of acylated anthocyanin into isolated vacuoles from a cell suspension culture of Daucus carota

Anthocyanin-containing vacuoles were isolated from protoplasts of a cell suspension culture of Daucus carota. The vacuoles were stable for at least 2 h as demonstrated by the fact that they showed no efflux of anthocyanin. The uptake of radioactively labelled anthocyanin was time-dependent with a pH optimum at 7.5, and could be inhibited by the protonophore carbonylcyanide m-chlorophenylhydrazone. Furthermore, the transport was specific, since vacuoles from other plant species showed no uptake of labelled anthocyanin, and strongly depended on acylation with sinapic acid, as deacylated glycosides were not taken up by isolated vacuoles. Hence, it is suggested that the acylation of anthocyanin, which is also required for the stabilization of colour in vacuoles, is important for transport, and that acylated anthocyanin is transported by a selective carrier and might be trapped by a pH-dependent conformational change of the molecule inside the acid vacuolar sap.

An endonuclease activity from suspension cultures of Daucus carota which acts upon pyrimidine dimers

A chromatin-bound ultra-violet (UV)-endonuclease has been partially purified from suspension cultures of the carrot Daucus carota. The enzyme is maximally active at pH 7.5–8.0 in the presence of 5–20 mM Mg 2+ ions and has a molecular weight of 140 000. It incisivenative DNA containing bulky lesions including pyrimidine dimers, psoralen and 2-acetylaminofluorene adducts had has no activity towards unmodified, single-stranded, alkylated or depurinated DNA or DNA containing 5,6-dihydroxydihydrothymidine residues. Its specificity is similar to that of the Escherichia coli uvr ABC endonuclease, however the carrot enzyme does not require ATP for activity. Further characterisation has proved difficult owing to the instability of the enzyme, a property it shares with other eukaryotic UV-endonucleases. The presence of this enzyme confirms the existence of a pyrimidine dimer excision-repair system in higher plants.

Chalcone synthase from cell suspension cultures of Daucus carota L

Chalcone synthase (CHS) has been partially purified about 35-fold. Withdrawal of 2-mercaptoethanol after precipitation with ammonium sulfate led to higher stability during further purification steps. In order to determine CHS activity, two procedures [according to Schröder et al. (1979) Plant Sci. Lett. 14, 281–286 ] were applied. The radioactivity extracted with ethyl acetate from the assay mixture (total products) was compared to 14C-labeled flavanone purified by TLC. The activity of CHS increased with bovine serum albumin (BSA) or 2-mercaptoethanol in the assay. Both effects were synergistic, but BSA did not promote “side products” as 2-mercaptoethanol did. BSA (10 mg ml −1) and 2-mercaptoethanol (1.4 m m) were components of the standard assay. Under these conditions, the CHS from Daucus carota had different pH optima for naringenin formation (7.9) and eriodictyol formation (6.8). The apparent K m values were 0.6 m m for 4-coumaroyl-CoA (pH 7.9), 7.7 μ m for caffeoyl-CoA (pH 6.8), and 3.0 μ m for malonyl-CoA (pH 7.9). Substrate inhibition was observed with 4-coumaroyl-CoA (>10 μ m) and malonyl-CoA (>50 μ m). The inhibitory activity of various flavonoids and related compounds (100 μ m) was investigated. Naringenin and naringenin-chalcone inhibited eriodictyol formation totally and naringenin formation by 50%. In contrast, eriodictyol and eriodictyol-chalcone inhibited only eriodictyol formation by 40%. It was shown that the inhibition with naringenin was fully uncompetitive. These in vitro data support the view that the true substrate of CHS in D. carota is 4-coumaroyl-CoA.

Organelle DNA compositions and isoenzyme expression in an interspecific somatic hybrid of Daucus

Cell suspension cultures of Daucus carota, D. capillifolius and a somatic hybrid of these lines were analyzed to determine their chloroplast and mitochondrial DNA compositions. The plastid DNAs (pDNA) from the somatic hybrid and D. carota were identical and were different from that of D. capillifolius when analyzed on agarose electrophoretic gels after digestion by the restriction endonuclease HpaII. The endonuclease restriction patterns of the mitochondrial DNAs (mtDNA) from each cell line were different. Although the restriction pattern of the mtDNA from the somatic hybrid contained fragments in common with one or both parents, unique fragments not found in the restriction pattern of either parent were also present. The amounts and feedback regulation of aspartokinase, homoserine dehydrogenase and dihydrodipicolinic acid synthase were quantified to define the effects of somatic hybridization upon the pathway leading to the biosynthesis of lysine, threonine, methionine and isoleucine. Regulation of each enzyme by end product inhibitors was not altered in the somatic hybrid, but levels of each enzyme appeared to be increased. However, isoenzyme analysis indicated two major forms of homoserine dehydrogenase were present in the hybrid, including one unique form not present in either parent.

Sensitivity of Carrot Cell Cultures and RNA Polymerase II to Amatoxins

Protoplast and cell suspension cultures of Daucus carota L. were evaluated for their sensitivity toward the three amatoxin derivatives, alpha-amanitin, 6'-deoxy-alpha-amanitin, and 6'-O-methyl-alpha-amanitin using inhibition of DNA synthesis to measure cell viability. Protoplasts appeared approximately 10-fold more refractory than suspension cells and alpha-amanitin was much less effective than the other two amatoxins, even though K(i) values for isolated RNA polymerase II were similar (4-5 nanomolar). Additional studies evaluating the recoveries of all three amatoxins from cell suspension supernates indicate one basis for these differences to be the selective degradation of alpha-amanitin. A mechanism involving the activation of the hydroxyindole moiety of the alpha-amanitin is thus invoked to explain these differences and we postulate the involvement of plant oxidases in this role.