Mechanical purification of torpedo stage somatic embryos of Daucus carota L.

A filtering unit was designed and constructed that concentrates and purifies torpedo-stage embryos, from carrot (Daucus carota L.) embryo suspensions. The filtering unit was composed of three sections, each containing a screen disk. Purification efficiency of a series of three screen size combinations was evaluated using 10 to 15 ml of 23- to 30-day-old embryo suspensions. Under optimal conditions where the first screen was a 1000 micron in size, >84% of the total number of torpedo embryos were concentrated in the purified fraction. Purified fractions contained more than double the percentage of torpedo embryos when compared to unfiltered suspensions. This filtering unit provided a simple method for concentrating and purifying torpedo embryos from a suspension.     

Morphogenetic and Histological Events during Somatic Embryogenesis in Intact Carrot Plantlets (Daucus carota L.) in Various Nutrient Media

It is shown that the induction of somatic embryogenesis neither requires the isolation of explants from intact carrot plants nor mechanical or chemical “wounding”.     

Production and analysis of plants that are somatic hybrids of barley (Hordeum vulgare L.) and carrot (Daucus carota L.)

In order to obtain plants that were somatic hybrids of barley (Hordeum vulgare L.) and carrot (Daucus carota L.), we fused protoplasts that had been isolated from 6-month-old suspension cultures of carrot cells with protoplasts isolated from barley mesophyll by electrofusion. After culture for 1 month at 25°C , the cells were cultured for 5 weeks at 4°C , and were then returned to 25°C for culture on a shoot-inducing medium. Three plants (nos. 1, 2 and 3) were regenerated from the cells. The morphology of the regenerated plants closely resembled that of the parental carrot plants. A cytological analysis of callus cultures induced from these plants indicated that most of the cells had about 24 chromosomes, fewer than the sum of the numbers of parent chromosomes which was 32. Southern hybridization analysis with fragments of the rgp1 gene used as probe showed that the regenerated plants contained both barley and carrot genomic DNA. Chloroplast (ct) and mitochondrial (mt) DNAs were also analyzed with several probes. The ctDNA of the regenerated plants yielded hybridization bands specific for both barley and carrot when one fragment of rice ctDNA was used as probe. Furthermore, the regenerated plants yielded a barley specific band and a novel band with another fragment of rice ct DNA as a probe. One of the regenerated plants (no. 1) yielded a novel pattern of hybridized bands of mt DNA (with an atp6 probe) that was not detected with either of the parents. These results indicated that the regenerated plants were somatic hybrids of barley and carrot and that recombination of both the chloroplast genomes and the mitochondrial genomes might have occurred. Received: 28 May 1996 / Accepted: 2 August 1996     

Embryogenesis of photoautotrophic cell cultures of Daucus carota L.

In this paper photoautotrophic carrot (Daucus carota L.) suspension cultures are described which are able to produce somatic embryos. The development of somatic embryos, however, requires a sucrose supplement. Although an elevation of the CO₂ concentration up to 2.3% results in the same level of dry weight production as with sucrose in the medium, somatic embryos could not be observed.Results on the influence of sucrose on some aspects of the photosynthetic apparatus of cultured cells are discussed.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DW dry weight - ELISA enzyme-linked-immunosorbent-assay - FW fresh weight - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - PEPCase phosphoenol-pyruvate carboxylase - Rubisco Ribulose- 1,5-bisphosphate carboxylase/oxygenase - se somatic embryogenesis     

Stage-specific nitrogen metabolism in developing carrot somatic embryos

The physiology of individual somatic embryo developmental stages otDaucus carota L. was examined by in vivo nuclear magnetic resonance (NMR) spectroscopy, amino acid analysis and ¹⁴C-labeling. ¹⁵N NMR spectroscopy was used to examine the uptake and incorporation of ¹⁵N isotopically labeled inorganic nitrogen sources. NMR spectra of proembryogenic masses (PEMs) contained resonances for histidine, amino sugars, glutamine, arginine, urea, alanine. α-amino nitrogen, serine, aliphatic amines and several unknowns. Similar resonances were found in various embryo developmental stages. However, resonances for arginine and aliphatic amines peaked during globular and torpedo stages and substantially decreased in germinating stage embryos. The dominant resonances observed in non-embryogenic cells and germinating embryos were glutamine and α-amino nitrogen. Amino acid analysis of the various embryo stages showed that glutamate, glutamine and arginine were the major contributors to the soluble amino acid profiles. During development, glutamate and glutamine continued to increase in concentration whereas arginine and its related metabolites (i.e. ornithine and y-aminobutyric acid [GABA]) were biphasic; increasing in globular and torpedo stage embryos and decreasing in germinating embryos. Carbon-14 labeling indicated that labeled glutamine pools in non-embryogenic and germinating embryos were greatest compared to other embryo stages, whereas labeled GABA pools were greatest in globular and torpedo stage embryos. Taken together, these data indicate that the physiology of each embryo developmental stage is distinct. They also suggest that during somatic embryo development, a switch takes place in metabolism whereby the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway is predominant in non-embryogenic cells and germinating stage embryos. Furthermore, during early to mid-embryo development (PEMs, globular and torpedo stage embryos), metabolism utilizing the omithine cycle is enhanced and predominant.     

Transient reduction in secreted 32 kD chitinase prevents somatic embryogenesis in the carrot (Daucus carota L.) variant ts11

At the nonpermissive temperature, somatic embryos of the temperature-sensitive (ts) carrot (Daucus carota L.) cell variant ts11 only proceed beyond the globular embryo stage in the presence of medium conditioned by wild-type cells. The causative component in the conditioned medium has been identified as an acidic 32 kD endochitinase. An antiserum raised against the 32 kD chitinase detected this protein in culture medium from ts11 embryo cultures grown at the permissive temperature as well as at the nonpermissive temperature. No difference in biochemical characteristics or in effect on ts11 embryo development could be detected between the 32 kD chitinase purified from wild-type cultures and the chitinase from ts11 cultures grown at the permissive or at the nonpermissive temperature. Compared to the amount present in a ts11 embryo culture at the permissive temperature, a reduction in the amount of 32 kD chitinase was observed during the temperature-sensitive period at the nonpermissive temperature. These results imply that the arrested embryo phenotype of ts11 is not the result of a structural difference in its 32 kD chitinase, but is the result of a transient decrease in the amount of 32 kD chitinase present. Morphological observations indicate that the ts11 phenotype is pleiotropic and also affects the cell wall of nonembryogenic cells. © 1995 Wiley-Liss, Inc.     

Ionic basis of currents in somatic embryos of Daucus carota

A vibrating probe was used to measure extracellular electrical currents around developing somatic embryos in two lines (RCC27, RCC48) of cultured cells of Daucus carota L. at the heart and torpedo stages. At pH 5.5, an inward current of 1.2±0.1 A·cm^-2 (n=23) was detected at the cotyledon, and an outward current of 1.0±0.1 A·cm^-2 (n=22) was found at the radicle in torpedostage embryos from the RCC27 line. At a pH of 5.75 the currents increased by 0.2–0.3 A·cm^-2 (n=60–62). In a few cases an additional small inward current was detected at the tip of the radicle in toepedo-stage embryos from RCC27 line. Such an inward current at the radicle seemed to appear earlier, some time after the heart stage, in embryos from the RCC48 line.Both extracellular pH measurements (using microelectrodes filled with ion-sensitive resin) and ion-substitution studies were carried out in order to ascertain the ionic composition of the currents in torpedo-stage embryos from the RCC27 line. Regions adjacent to the cotyledon and radicle, at the points of current entry and exit, were found to be more acidic by 0.02±0.01 (n=14) and 0.07±0.01 (n=12) pH units, respectively, than the bulk medium. Removal of K⁺ from the medium reversibly reduced the currents to about 25% of their original value at both cotyledon and radicle. Deletion of Cl^- decreased the currents slightly. Removal of Ca²⁺ resulted in a rapid doubling of currents. Addition of either N,N-dicyclohexylcarbodiimide or tetraethyl ammonium chloride substantially reduced overall currents, and their removal resulted in partial recovery of the currents. It is suggested that the inward current at the cotyledon is comprised largely of K⁺ influx and the outward current at the radicle is mainly the result of active H⁺ efflux.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog     

Extracts of Marine cyanobacteria stimulated somatic embryogenesis of Daucus carota L.

Twenty five strains of marine cyanobacteria were screened for their ability to promote carrot somatic embryogenesis. Hot water extracts prepared from 21 of these strains promoted plantlet formation. Extracts from four strains increased plantlet numbers to an average of over 3.7-fold. Dialysates and nondialysates of each of these extracts also increased plantlet formation. For extracts from filamentous cyanobacteria, Nostoc sp. and Anabaena sp., dialysate was more effective (4.2-fold increase) than nondialysate (3.0-fold increase), whereas for unicellular strains Synechococcus sp. and Xenococcus sp., nondialysate was more effective (5.2-fold increase) than the dialysate (3.2-fold increase). These cyanobacterial extracts also promoted embryolike structure formation from two-year old carrot cell cultures which were unable to produce plantlets using the usual methods. Here, we demonstrate the existence in marine cyanobacterial extracts of low and high molecular weight factors which strongly promote somatic embryogenesis in carrot cell cultures.Abbreviations MS Murashige and Skoog medium - 2, 4-D 2, 4-dichlorophenoxyacetic acid - PCV packed cell volume     

Production of somatic hybrids between Daucus carota L. and Nicotiana tabacum

Protoplasts of a kanamycin-resistant (KR, nuclear genome), streptomycin-resistant (SR, chloroplast genome) and chlorophyll-deficient (A1, nuclear genome) Nicotiana tabacum (KR-SA) cell suspension cultures or X-ray-irradiated mesophyll protoplasts of kanamycin- and streptomycin-resistant green plants (KR-SR) were fused with protoplasts of a cytoplasmic male-sterile (CMS) Daucus carota L. cell suspension cultures by electrofusion. Somatic hybrid plants were selected for kanamycin resistance and the ability to produce chlorophyll. Most of the regenerated plants had a normal D. carota morphology. Callus induced from these plants possessed 23–32 chromosomes, a number lower than the combined chromosome number (66) of the parents, and were resistant to kanamycin, but they segregated for streptomycin resistance, which indicated that N. tabacum chloroplasts had been eliminated. Genomic DNA from several regenerated plants was analyzed by Southern hybridization for the presence of the neomycin phosphotransferase gene (NPTII); all of the plants analyzed were found to contain this gene. Mitochondrial (mt) DNA was analyzed by Southern hybridization of restriction endonuclease digests of mtDNA with two DNA probes, PKT5 and coxII. The results showed that the two plants analyzed possessed the mitochondria of D. carota. These results demonstrate that the regenerated plants are interfamilial somatic hybrids.     

Bioreactor studies of the effect of medium pH on carrot (Daucus carota L.) somatic embryogenesis

Daucus carota cell differentiation was examined under different medium pH conditions in a controlled bioreactor. Somatic embryogenesis was affected by pH changes. Embryo production was greatest when the pH of the hormone-free medium was maintained at 4.3. However, the same level was not favourable to development since most embryos did not progress to the torpedo and plantlet stages. In contrast, although there was about a threefold decrease in embryo yield in cultures on the same free 2,4-dichlorophenoxyacetic acid medium maintained at pH 5.8, cells differentiated into fully developed plantlets. Changes in embryo development appeared to be associated with alterations in ammonium loss from the medium and sugar uptake.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid     

胡萝卜与川西獐牙菜不对称体细胞杂种的核/质基因组特征

胡萝卜(Daucus carota var.sativa)原生质体与经260μW/cm2紫外线照射的川西獐牙菜(Swertia mussotii)原生质体用PEG法诱导融合.对融合再生的克隆的5SrDNA间隔序列和RAPD分析结果得知,各再生克隆均存在双亲的核DNA及重组DNA.杂种的核基因组组成以胡萝卜(受体)为主,供体川西獐牙菜DNA谱带较少;UV照射剂量对体细胞杂种核基因组组成没有明显的影响.进一步对再生克隆叶绿体DNA的SSR分析表明,杂种细胞中双亲叶绿体基因组随机分离并发生重组.     

Patterns of expression of the JIM4 arabinogalactan-protein epitope in cell cultures and during somatic embryogenesis in Daucus carota L.

Spatiotemporal patterns of expression of the cell-surface arabinogalactan-protein epitope defined by monoclonal antibody JIM4 (J.P. Knox et al., 1989, Development 106, 47–56) have been characterized by indirect immunofluorescence during the process of somatic embryogenesis in Daucus carota L. The JIM 4 epitope (J4e) occurred on cells established in culture from hypocotyl explants which appeared to derive, at least in part, from the epidermal cells of the hypocotyl. Cultures maintained in the presence of 2,4-dichlorophenoxyacetic acid developed proembryogenic masses of which only infrequent cells at the surface expressed J4e. Sub-culture at a low cell density and withdrawl of the synthetic auxin resulted in an increase in J4e expression in most surface cells and most abundantly in surface layers of cells at the future shoot end of developing embryos. The transition to heart-shaped embryos occurred concurrently with the expression of J4e by groups of cells beneath the developing cotyledons, at the junction of the future root and shoot. At this stage, J4e was also expressed by a single well-defined layer of cells at the surface of the embryos. Advancement to the mature torpedo stage was accompanied by the expression of the epitope on cells forming two regions of the future stele and of cells associated with the cotyledonary provascular tissue characteristic of the carrot seedling. At this stage there was substantially less expression of the marker antigen by epidermal cells, although infrequent expression by isolated cells of the epidermis was maintained. The correlation of J4e expression with the development and distinction of plant tissue patterns during somatic embryogenesis indicates a role for plasma-membrane arabinogalactan proteins in these processes.Abbreviations AGP arabinogalactan protein - 2,4-D 2,4-di-chlorophenoxyacetic acid - J4e JIM 4 epitope - PEM proembryogenic mass We thank Andrew Davis for photographic assistance and Roger Pennell for useful discussions.     

Morphogenetic effects of Brefeldin A on embryogenic cell cultures of Daucus carota L.

Brefeldin A, an inhibitor of protein secretion, caused typical alterations to the endomembrane system with limited effects on viability when given to unorganized carrot cells growing in suspension. When given to the same cells during particular stages of embryogenesis, it caused similar endomembrane lesions and an almost complete arrest of the embryogenic process. Addition of conditioned medium containing extracellular secreted proteins to the embryos during treatment with Brefeldin A allowed acquisition of polarity and the continuation of a quasi-normal embryogenic process. Received: 4 December 1996 / Accepted: 4 March 1997     

Application of image analysis to fed-batch cultures of somatic embryos

Summary Nutrient limitation and inhibitor accumulation have been shown to impede the development of somatic embryos of carrots in batch cultures. To improve the development of embryos, semicontinuous cultures with daily medium replenishment were performed. An image analysis system capable of identifying normal and abnormal embryos was used to facilitate the kinetic study. At a high daily medium replenishment rate of 20%, the biomass production was also higher, but not the embryo concentration. Total embryo and torpedo embryo concentrations were both higher at a 10% medium replenishment rate than at 5% and 20%. The profiles of embryo concentrations under three medium replenishment rates appeared similar until a late stage of cultivation; however, statistical analysis of the morphological features distribution revealed that significant differences were discernible earlier. For the formation of mature embryos, the optimal daily medium replenishment is judged to be in the range of 10–20%. In this study, more than 20000 embryos were classified and counted. The information on population kinetics and statistical comparison were made possible by the availability of our image analysis system.     

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.     

Evidence for maternal inheritance of the chloroplast genome in cultivated carrot (Daucus carota L. ssp. sativus)

 The incidence and inheritance of a chloroplast DNA (cpDNA) mutation/marker, BP10U, was studied in crosses among cultivated carrots (Daucus carota ssp. sativus). BP10U is about 400 bp larger than the more common BP10L allele. The occurrence of BP10U among carrot inbreds was widespread. Individual plants exhibited only one form of BP10, and cpDNA inheritance was strictly maternal. BP10U only occurred in male-fertile plants. Some male-fertile inbreds and all cytoplasmically male-sterile (petaloid) carrots had the BP10L allele. Alloplasmic cpDNA variation has been reported previously in Daucus, but this is the first report of variation and inheritance of cpDNA within cultivated carrot. Received: 11 August 1998 / Accepted: 8 September 1998