Characterization of a new continuous cell line from the flood water mosquito,Aedes vexans

A new cell line, UM-AVE1, was established from embryos of the mosquito Aedes vexans. Banding patterns for the isozymes lactate dehydrogenase (LDH), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH), xanthine dehydrogenase (XDH), and esterases were compared with those of larval Aedes vexans tissues as well as those of four other mosquito cell lines and one moth cell line. Karyotype analyses confirmed that the dipteran cell lines were not contaminated with lepidopteran cells, because in all mosquito lines the modal number of chromosomes was 6 (=2n) or 7. Isozyme electrophoresis established a specific profile for each cell line. Two isozymes present in UM-AVE1 (LDH, IDH) were not detected in larvae; this could be a reflection of the different stages used for cell line isolation and enzyme analysis, or lability of sample preparations. It is significant that extracts from UM-AVE1 cells and Aedes vexans larvae had an identical double band for XDH, while all other cell lines examined exhibited only a single band.     

Visual selection and maintenance of the cell lines with high plant regeneration ability and low ploidy level in Dianthus acicularis by monitoring with flow cytometry analysis

Efficient plant regeneration system from cell suspension cultures was established in D. acicularis (2n=90) by monitoring ploidy level and visual selection of the cultures. The ploidy level of the cell cultures closely related to the shoot regeneration ability. The cell lines comprising original ploidy levels (2C+4C cells corresponding to DNA contents of G1 and G2 cells of diploid plant, respectively) showed high regeneration ability, whereas those containing the cells with 8C or higher DNA C-values showed low or no regeneration ability. The highly regenerable cell lines thus selected consisted of compact cell clumps with yellowish color and relatively moderate growth, suggesting that it is possible to select visually the highly regenerable cell lines with the original ploidy level. All the regenerated plantlets from the highly regenerable cell cultures exhibited normal phenotypes and no variations in ploidy level were observed by flow cytometry (FCM) analysis.     

Sodium Valproate Facilitates the Propagation of Granulocytic Ehrlichiae (Anaplasma phagocytophilum) in HL-60 Cells

As already shown, some inducers of the differentiation of promyelocytic cells along the granulocytic pathway, such as dimethylsulphoxide (DMSO) or all-trans retinoic acid, can enhance propagation of granulocytic ehrlichiae in HL-60 cell cultures. This study was conducted to prove whether sodium valproate, a salt of di-n-propylacetic acid (VPA) known to trigger cellular differentiation in several solid and hematopoietic malignancies is similarly efficient in ehrlichial cultures. Two cell lines derived from HL-60, that is, low-passage undifferentiated HL-60 (HL-60F) and high-passage HL-60 spontaneously differentiated towards monocytic phenotype (HL-60J) were grown in RPMI 1640 medium supplemented with 10% FBS. The respective HL-60F and HL-60J IC₅₀-values for NaVPA were estimated to be 0.8 and 2.2 mM under these culture conditions; to stimulate the differentiation, the respective doses of 0.3 and 1.2 mM were then applied. When the NaVPA-treated cells of both lines were challenged with an ehrlichial laboratory strain (HGE), maintained in splenectomized NMRI mice, the respective 1–2 and ≤0.1% primary infection rates in HL-60F and HL-60J cultures were observed 3 days post-inoculation. In comparison, only rare (≤0.1%) infected HL-60F and no infected HL-60J cells were recorded under the same experimental conditions in untreated control cultures. HGE continuously propagated in NaVPA-supplemented HL-60F cultures remained infectious to mice at least up to the 95th passage (12 months). NaVPA can thus facilitated continuous propagation of granulocytic ehrlichiae in cell cultures without a substantial loss of infectiveness. This revised version was published online in July 2006 with corrections to the Cover Date.     

Somatic hybridization in the gramineae: Pennisetum americanum (L.) K. Schum. (Pearl millet) +Panicum maximum Jacq. (Guinea grass)

Summary Protoplasts from Pennisetum americanum resistant to S-2-amino-ethyl-l-cysteine (AEC) were fused with protoplasts of Panicum maximum utilizing polyethylene glycol-dimethylsulfoxide after inactivation of the Pennisetum protoplasts with 1 mM iodoacetic acid. The iodoacetate treatment prevented division of Pennisetum protoplasts; therefore, only Panicum protoplasts and heterokaryons potentially could give rise to colonies. A second level of selection was imposed by plating 3–4-week-old colonies on AEC medium. Putative somatic hybrid calli were analyzed for alcohol dehydrogenase, 6-phosphogluconate dehydrogenase, aminopeptidase, and shikimate dehydrogenase isozymes. Three somatic hybrid cell lines (lines 2, 3, and 67) were identified which showed two bands of alcohol dehydrogenase activity representing homodimers of P. maximum and P. americanum as well as a novel intermediate band of activity where Panicum-Pennisetum heterodimers would be expected. Aminopeptidase and shikimate dehydrogenase were useful for identifying presumptive hybrid calli but the isozyme patterns were additive-evidence which would not preclude the selection of chimeric callus. A more complex isozyme pattern which varied among the somatic hybrids was observed for 6-phosphogluconate dehydrogenase. In the hybrid calli, the presence of DNA sequences homologous to both P. maximum and P. americanum sequences was confirmed by hybridization of a maize ribosomal DNA probe to XbaI and EcoRI restriction fragments. Growth of hybrid lines on various concentrations of AEC was either similar to the AEC-resistant parent (hybrid line 2) or intermediate between the resistant and sensitive parents (hybrid lines 3, 67).     

Development and Characterization of a Nonmorphogenetic Cell Line of Wheat (Triticum aestivum)

This study was conducted to compare characteristics of a wheat (Triticum aestivum L.) cell line to those of the maize (Zea mays L.) black Mexican sweet (BMS) cell line and to compare protoplasts isolated from suspension cells of these cell lines. The wheat cell line was established from immature-embryo derived callus of the experimental line ‘ND7532’ and was conditioned for growth in suspension culture. For both cell lines, measurements of packed cell volume (PCV), fresh weight (FW), and dry weight (DW) were taken at 3 day intervals from suspension cultures. Measurements of FW of calluses cultured from suspension cells of both cell lines were taken at 6 day intervals. The morphogenetic potential of the wheat ND7532 cell line was tested in both callus and suspension cultures using media promoting regeneration and/or organogenesis. Growth rates of ND7532 cells in suspension culture were comparable to those of BMS cells. However, relative growth rates of calluses recovered from ND7532 suspension cells were slower than those of calluses recovered from BMS suspension cells. The ND7532 cell line has very limited morphogenetic potential and has been maintained as rapidly growing callus tissue for 11 years. Yields of protoplasts from suspension cells of the two cell lines were comparable, though ND7532 protoplasts were typically smaller. The wheat cell line has is now designated ND7532-NM (nonmorphogenetic) and is available for cellular and molecular biology research.     

Capsaicin formation in p-fluorophenylalanine resistant and normal cell cultures ofCapsicum frutescens and activity of phenylalanine ammonia lyase

Callus cultures ofCapsicum frutescens capable of producing a maximum of 53 μg capsaicin/g FW were exposed to various levels of p-fluorophenyialanine (PFP) at 100, 400, 1000 and 2000 μM to develop a resistant cell line that over produces capsaicin. After 15 days of culturing on media lacking PFP, cell lines resistant to 100, 400 and 1000 μM registered 18%, 34.5% and 45% increase in capsaicin content over normal cell line (cells not exposed to PFP). Capsaicin accumulation was inhibited in 2000 μM PFP resistant cell line. The profile of phenylalanine ammonia lyase (PAL), the key enzyme in pheny1propanoid pathway in resistant cell cultures was studied and compared with normal cell cultures to understand its role in capsaicin formation. Importantly increased production of capsaicin was obtained using PFP resistant cell lines. The activity profile of PAL had no correlation with capsaicin content in both control and PFP resistant cells.     

Chromosomal variation in dividing protoplasts derived from cell suspensions of barley (Hordeum vulgare L.)

Summary Numerical and structural chromosome variation was analysed in dividing protoplasts isolated from suspension cells of barley. Five cell lines exhibited distribution patterns in chromosome number with different peaks and ranges. Embryogenic/morphogenic cell lines showed a peak at 2n = 14 (ca. 50%) after 6–7 months in culture, while older non-embryogenic cell lines had peaks at aneuploid or polyploid chromosome numbers. Culture duration had a clear effect on numerical and structural chromosome variation in embryogenic cell lines. With ageing of the cultures chromosome variation accumulated and the proportion of 2n = 14 cells decreased. The effect of protoplast isolation and culture on chromosome variation was examined; more cells with normal chromosome sets (12%) were maintained in protoplast-derived colonies than in source suspension cells (4%) of the same culture age.Abbreviations DC Dicentric - F fragment - T telocentric     

Factors affecting transformation of cell cultures from three dicotyledonous pigment-producing species using microprojectile bombardment

Gene transfer methods were established for cell suspension cultures of sweet potato (Ipomoea batatas), ohelo (Vaccinium pahalae) and carrot (Daucus carota, two lines) using micro-projectile bombardment. Several parameters were studied (particle size/type, helium pressure, stage height, DNA concentration, pre-culture period) to determine which significantly affected transformation efficiency. All the physical parameters influenced transient gene expression, with particle size and type having the greatest effect. Cell culture age also affected transformation efficiency in all cell lines. Nuclear DNA conformation (relaxation) as measured by flow cytometry showed no change associated with culture age or transformation efficiency. Agrobacterium-based methods were also tested and in most experiments produced no GUS-expressing loci. Microprojectile bombardment will now be used to study anthocyanin biosynthesis in cell cultures as an alternative source of natural food colourants.     

Regulation of lysine and threonine synthesis in carrot cell suspension cultures and whole carrot roots

Aspartokinase (EC 2.7.2.4), homoserine-dehydrogenase (EC 1.1.1.3) and dihydrodipicolinic-acid-synthase (EC 4.2.1.52) activities were examined in extracts from 1-year-old and 11-year-old cell suspension cultures and whole roots of garden carrot (Daucus carota L.). Aspartokinase activity from suspension cultures was inhibited 85% by 10 mM L-lysine and 15% by 10mM L-threonine. In contrast, aspartokinase activity from whole roots was inhibited 45% by 10 mM lysine and 55% by 10 mM threonine. This difference may be based upon alterations in the ratios of the two forms (lysine-and threonine-sensitive) of aspartokinase, since the activity is consistently inhibited 100% by lysine+threonine. Only one form each of homoserine dehydrogenase and of dihydrodipicolinic acid synthase was found in extracts from cell suspension cultures and whole roots. The regulatory properties of either enzyme were identical from the two sources. In both the direction of homoserine formation and aspartic--semialdehyde formation, homoserine dehydrogenase activities were inhibited by 10mM threonine and 10 mM L-cysteine in the presence of NADH or NADPH. KCl increased homoserine dehydrogenase activity to 185% of control values and increased the inhibitory effect of threonine. Dihydrodipicolinic acid synthase activities from both sources were inhibited over 80% by 0.5 mM lysine. Aspartokinase was less sensitive to inhibition by low concentrations of lysine and threonine than were dihydrodipicolinic acid synthase and homoserine dehydrogenase to inhibition by the respective inhibitors.     

Characterization of established,cell lines derived from mutagen-sensitiveDrosophila strains

Summary Six established cell lines have been generated from embryos ofDrosophila melanogaster homozygous for different X-linked mutations. Four of these mutants, confer hypersensitivity to chemical mutagens in larvae. The cell lines derived from the two mutageninsensitive stocks, serve as controls in the analyses of DNA metabolism. One cell line (UCD-Dm-mei-9-2) is uniquely identified by a strong hypersensitivity to ultraviolet radiation. Another (UCD-Dm-mus104-1) expresses an enzyme variant not found in the other lines. The population doubling time for these cultures varies between 24 and 47 h. Labeling indices of 24.4 to 37.5% were found. The duration of the S phase in one of the control cell lines is estimated to be about 9 h. Karyotype stability was monitored for five lines over a period of about 1 y. In general these cultures each, became hypotetraploid with a preferential loss of the Y and fourth chromosomes. DNA synthesis in two of the lines fails to exhibit the pattern of sensitivity to mutagens or caffeine that is observed in the corresponding primary cultures. In primary cultures three classes of cells can be identified by autoradiography. About 50% of the cells label at a moderate rate, 20% do not label within the first 1.5 d of culture, and the remaining cells exhibit a burst of labeling shortly after the cultures are initiated. This research was supported by NIH Grants GM16298 and GM22221 and by DOE Contract AT(04-3)-34 PA 210.     

Initiation of morphogenic cell-suspension and protoplast cultures of barley (Hordeum vulgare L.)

Cell-suspension cultures were initiated from embryogenic calli of various barley cultivars. Seven fast-growing suspension lines were obtained from four different cultivars (cvs. Dissa, Emir, Golden Promise and Igri). Two of these cell suspensions showed morphogenic capacity. From a cell suspension of cv. Dissa, albino plantlets were regenerated when aggregates were cultured on solid medium. Aggregates of cv. Igri usually stopped differentiation at the globular stage, but occasionally formed scutellum-like structures. Five suspension lines were used for protoplast isolation and culture. Dividing protoplasts were obtained from all lines, but with cv. Igri a few divisions only and no further development were observed. Protoplasts from the various lines differed in the time of first division (2–14 d), division frequency (0.09–70.9%) and efficiency of colony formation (0.09–7.3%). Protoplasts isolated from the morphogenic cell suspension of cv. Dissa developed compact calli which sporadically regenerated albino plantlets.Abbreviations CC, MS, N6, SH, Kao8p culture media; see Material and methods - cv chltivar - dicamba 3,6-dichloro-o-anisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Differential accumulation of monolignol-derived compounds in elicited flax (Linum usitatissimum) cell suspension cultures

Lignin and lignans share monolignols as common precursors and are both potentially involved in plant defence against pathogens. In this study, we investigated the effects of fungal elicitors on lignin and lignan metabolism in flax (Linum usitatissimum) cell suspensions. Cell suspension cultures of flax were treated with elicitor preparations made from mycelium extracts of Botrytis cinerea, Phoma exigua and Fusarium oxysporum F ssp lini. Elicitors induced a rapid stimulation of the monolignol pathway, as confirmed by the increase in PAL (phenylalanine ammonia-lyase, EC 4.1.3.5), CCR (cinnamoyl-CoA reductase EC 1.2.1.44) and CAD (cinnamyl alcohol dehydrogenase EC 1.1.1.195) gene expression and PAL activity. At the same time, CCR activity only increased significantly in F. oxysporum-treated cells 24 h post elicitation. On the other hand, CAD activity measured for coniferyl alcohol formation was transiently decreased but a substrate-specific activation of CAD activity was observed in F. oxysporum-treated cells when using sinapyl alcohol as substrate. The accumulation of monolignol-derived products varied according to the elicitor used. B. cinerea or P. exigua-elicited cell cultures were characterised by a reinforcement of the cell wall by a deposit of 8-O-4′-linked non-condensed lignin structures and phenolic monomers, while at the same time no stimulation of 8-8′-linked lignan or 8-5′-linked phenylcoumaran lignan accumulation was observed. Additionally, elicitation of cell cultures with F. oxysporum extracts even triggered a strong incorporation of monolignols in the non condensed labile ether-linked lignin fraction concomitantly with a decrease in lignan and phenylcoumaran lignan accumulation. Several hypotheses are proposed to explain the putative role of these compounds in the defence response of flax cells against pathogens. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. C. Hano and M. Addi contributed equally to this work.     

Selection of cell lines for the production of rosmarinic acid from cell suspension cultures of Orthosihon stamineus benth

Summary Cell suspension cultures of Orthosiphon stamineus were established from friable calluses produced from leaf pieces of in vitro plantlets that were derived from nodal segments of the mother plants collected from three different geographical locations. Eight lines were eventually selected after seven subculture cycles based on the growth characteristic (plant height) of the plantlets from the three locations: two fast-growing lines (>5.1 cm tall), three intermediate-growing lines (3.1–5.0 cm tall), and three slow-growing lines (<3.0 cm tall). All eight lines grew well in liquid Murashige and Skoog medium supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.4 μM 1-naphthaleneacetic acid (NAA). All cell lines exhibited the same growth pattern but produced different maximum cell biomass when cultured in this medium. The time of harvesting the plant cells from the culture medium and the geographical source of the original plant material were both found to affect the production of rosmarinic acid (RA) in cell cultures. Two cell lines were successfully selected and identified to produce high amounts of RA. These cell lines were a fast-growing cell line from Air Itam, Penang and an intermediate-growing cell line from Relau Agriculture Research Centre, Penang which could produce 5% [(w/w) dry weight] and 4.5% [(w/w) dry weight] of RA, respectively.     

Rapid recovery of photosynthetic cell suspension cultures following heterotrophic bleaching

Summary Seven suspension-cultured lines of five different species (Amaranthus powellii Datura innoxia, Glycine max, Gossypium hirsutum, andNicotiana tabacum × Nicotiana glutinosa fusion hybrid), which had been grown under photomixotrophic conditions, were placed under heterotrophic conditions (darkness and media with 3% sucrose or starch) where the chlorophyll levels declined to near zero. After three transfers over a 70-d period, the cells were placed back into photomixotrophic or photoautotrophic conditions where regreening occurred rapidly and continued growth was observed. This rapid adaptation to photosynthetic conditions contrasts with the original initiation process for these cultures, which required many months and an apparent selection since many of the original cells died. Thus, these seven photosynthetic cell suspension cultures appear to be different from the original cultures due possible to genetic or adaptive changes.     

Stable transformation and long-term maintenance of transgenic <Emphasis Type="Italic">Taxus</Emphasis> cell suspension cultures

A cell line of Taxus cuspidata has been transformed with wild-type Agrobacterium rhizogenes ATCC strain 15834 containing binary vector pCAMBIA1301 and, separately, with A. tumefaciens strain EHA105 containing binary vector pCAMBIA1305.2. Additionally, a cell line of T. chinensis has been transformed with wild-type A. rhizogenes ATCC strain 25818 containing binary vector pCAMBIA1301. The two transgenic T. cuspidata cell lines have been maintained in culture for more than 20 months, and the transgenic T. chinensis cell line for more than 9 months, with no loss of reporter gene expression or antibiotic resistance. The introduced genes had no discernable effect on growth or Taxol production in the transgenic cell lines when compared to the parent control. The methods for transforming non-embryogenic Taxus suspension cultures are described.     

Bioreactor studies on the effect of dissolved oxygen concentrations on growth and differentiation of carrot (Daucus carota L.) cell cultures

A bioreactor control system was used to investigate the effects of two dissolved oxygen concentrations (10% and 100%) on the growth and differentiation of Daucus carota L. cell cultures. The strategy used allowed the dissolved oxygen concentration to be controlled without the need for changing either the agitator speed or the total gas flow rate. During the proliferation phase, reducing oxygen resulted in a lower growth rate and in a delay in sugar uptake kinetics. Nonetheless, varying levels of oxygen were observed to have no effect on the final dry biomass. The higher alcohol dehydrogenase activity obtained under reduced oxygen conditions suggests that proliferating cultures adapted to the hypoxic environment by inducing alcoholic fermentation. Cell differentiation was highly sensitive to reduced oxygen since under this condition, the somatic embryo production was inhibited by about 75%. Sugar uptake and embryo formation were also delayed.Abbreviations ADH alcohol dehydrogenase - 2,4-D 2,4-dichlorophenoxyacetic acid - DO2 dissolved oxygen - SE somatic embryos - Tris tris(hydroxymethyl)-aminoethane     

Yew (Taxus x media Rehd.) cell suspension cultures as a source of taxanes

Natural resources of paclitaxel, an effective anticancer compound, were threatened with extinction soon after the discovery of this valuable substance. Cell suspension cultures derived from different Taxus species have rapidly become an alternative source of paclitaxel and other taxanes. In this paper we provide some insight into cell growth characteristics in cell suspension culture of Taxus x media cv. Hicksii, with emphasis on the effects of jasmonic acid (JA) on taxane production in cell lines with different initial taxane content. Additionally cell growth characteristics of two cell lines was followed during cultivation of cell suspension culture of Taxus x media cv. Hicksii. Packed cell volume (PCV) was shown to be a reliable and efficient alternative for measuring cell growth instead of fresh and dry weight. The initial total taxane content was screened in a number of cell lines, followed by observing the effect of JA on cell mass and total taxane production of selected lines. We showed a great variability in initial taxane content in different cell lines, which decreased during cell suspension maintenance. JA was shown to inhibit cell growth and increase total taxane production (14 to 106 fold).     

Efficient plant regeneration from cell cultures of ornamental statice, Limonium sinuatum mill.

Summary A plant regeneration system from cell suspension cultures was established in an important ornamental crop, Limonium sinuatum Mill. cv. ‘Early Rose’. Friable callus was initially induced from leaf segments of in vitro-cultured seedlings on 0.25% gellan gum-solidified half-strength Murashige and Skoog [1/2MS] medium containing 1.0 mg l⁻¹ (4.14 μM) picloram. These calluses were maintained as cell suspension cultures, which showed high proliferation ability with about 80 times increase in fresh weight during the 2-wk interval of subculture. Shoot regeneration from these cell cultures was achieved by cytokinins, especially zeatin, which was the most effective in producing normal shoots with reduced hyperhydration when used in combination with 0.5% gellan gum. Shoot regeneration ability was different among the cell lines originated from each different seedling. Shoot formation was observed at different frequencies on four of five cell lines whereas one cell line showed no shoot differentiation. Regenerated shoots detached from callus readily rooted 1 mo. after the transfer onto 0.5% gellan gum-solidified 1/2MS medium lacking plant growth regulators. The plantets were successfully transferred to the greenhouse after acclimatization. No ploidy changes were observed in the callus induced or in the regenerated plantlets. The regenerated plantlets that were transferred to the greenhouse after acclimatization grew normally and did not any morphological signs of somaclonal variation.     

Culture of Isolated Single Cells from <Emphasis Type="Italic">Taxus</Emphasis> Suspensions for the Propagation of Superior Cell Populations

Single cells isolated from aggregated Taxus cuspidata cultures via enzymatic digestion were grown in suspension culture. High seeding density (4×10⁵ cells/ml) and the addition of cell-free conditioned medium were essential for growth. Doubling the concentration of the nutrients [ascorbic acid (150 g/l), glutamine (6.25 mm), and citric acid (150 g/l)] had no effect on single cell growth or viability. A specific growth rate of 0.11 days⁻¹ was achieved, which is similar to the observed growth rate of aggregated Taxus suspensions. The biocide, Plant Preservative Mixture, added at 0.2% (v/v) to all single cell cultures to prevent microbial contamination, had no significant effect on growth or viability. Following cell sorting, single cell cultures can be used to establish new cell lines for biotechnology applications or provide cells for further study.