Carrot Cell Suspension Cultures Research Articles

Effect of diflufenican on total carotenoid and phytoene production in carrot suspension-cultured cells.

Diflufenican increased 493-fold the level of phytoene. Diflufenican-induced inhibition of phytoene desaturase gene expression in carrot cells resulted in an increased production of phytoene. This work analyzes the effect of diflufenican, an inhibitor of phytoene desaturase, on the gene expression profiles of the biosynthetic pathway of carotenoids related with the production of these compounds in carrot cell cultures. The results showed that the presence of 10µM diflufenican in the culture medium increased phytoene levels, which was 493-fold higher than in control cells after 7days of treatment but did not alter cell growth in carrot cell cultures. The maximal production of phytoene was reached with 10µM diflufenican after 7days of incubation in the presence of light and with 30g/L sucrose in the culture medium. Moreover, diflufenican decreased the expression of phytoene synthase and phytoene desaturase genes at all the times studied. This diflufenican-induced inhibition of phytoene desaturase gene expression in carrot cell cultures resulted in an increased production of phytoene. Our results provide new insights into the action of diflufenican in carrot cell cultures, which could represent an alternative more sustainable and environmentally friendly system to produce phytoene than those currently used.

Effect of terbinafine on the biosynthetic pathway of isoprenoid compounds in carrot suspension cultured cells.

Terbinafine induced a significant increase of squalene production. Terbinafine increased the expression levels of squalene synthase. Cyclodextrins did not work as elicitors due to the gene expression levels obtained. Plant sterols are essential components of membrane lipids, which contributing to their fluidity and permeability. Besides their cholesterol-lowering properties, they also have anti-inflammatory, antidiabetic and anticancer activities. Squalene, which is phytosterol precursor, is widely used in medicine, foods and cosmetics due to its anti-tumor, antioxidant and anti-aging activities. Nowadays, vegetable oils constitute the main sources of phytosterols and squalene, but their isolation and purification involve complex extraction protocols and high costs. In this work, Daucus carota cell cultures were used to evaluate the effect of cyclodextrins and terbinafine on the production and accumulation of squalene and phytosterols as well as the expression levels of squalene synthase and cycloartenol synthase genes. D. carota cell cultures were able to produce high levels of extracellular being phytosterols in the presence of cyclodextrins (12mg/L), these compounds able to increase both the secretion and accumulation of phytosterols in the culture medium. Moreover, terbinafine induced a significant increase in intracellular squalene production, as seen after 168h of treatment (497.0 ± 23.5µgg dry weight-1) while its extracellular production only increased in the presence of cyclodextrins.The analysis of sqs and cas gene expression revealed that cyclodextrins did not induce genes encoding enzymes involved in the phytosterol biosynthetic pathway since the expression levels of sqs and cas genes in cyclodextrin-treated cells were lower than in control cells. The results, therefore, suggest that cyclodextrins were only able to release phytosterols from the cells to the extracellular medium, thus contributing to their acumulation. To sum up, D. carota cell cultures treated with cyclodextrins or terbinafine were able to produce high levels of phytosterols and squalene, respectively, and, therefore, these suspension-cultured cells of carrot constitute an alternative biotechnological system, which is at the same time more sustainable, economic and ecological for the production of these bioactive compounds.

Production of Uniformly Labeled 13C‐Lutein and 13C‐α‐Tocopherol in vitro Using Carrot Cell Suspension Culture

The xanthophyll lutein (Lut) is present in high concentrations in primate retinae, protecting against photooxidation and enhancing visual performance. Interestingly, in the elderly, diets high in Lut slow cognitive decline and circulating levels of both Lut and the lipid‐soluble antioxidant α‐tocopherol (αT) are inversely associated with cognitive impairment. However, Lut deposition, metabolism, and function in the brain are poorly understood, and cost‐effective isotopically labeled Lut tracers are not commercially available. The goal of this work, therefore, is to establish a system for efficient bioproduction of uniformly labeled 13C‐Lut and 13C‐αT. Seven carrot (Daucus carota) cultivars were germinated, separated into tissue subtypes, dedifferentiated into solid callus, and initiated in liquid suspension culture. Conditions for production of Lut and αT in suspension culture were optimized and uniformly labeled 13C‐glucose was provided as the sole media carbon source for four serial growth cycles. Lut and αT yields were 2.6 and 3.3 μg/g cells, respectively, as measured by reverse‐phase HPLC. Cell extracts were fractionated by preparatory reverse‐phase HPLC and analyzed for 13C enrichment by mass spectrometry. 58% of Lut and 50% of αT in carrot extract fractions were uniformly labeled with 13C. These tracers can be utilized in studies of Lut and αT in the brain, enhancing our understanding of their function in cognition.

Efficient Regeneration of Carrot (Daucus carota L.) plants from cell suspension derived-callus

This study succeeded in establishment carrot cell suspension cultures from stem callus in liquidMS medium containing 1.0 mg L-1of each NAA and BA. Its density approach 3.4 ×105cell ml-1atthe third day of culture. These cells ,continued, when embedded in agar drop using MultipleDrop Array (MDA) technique, in division and forming cellular colonies which producingnumerous callus primordia that developed to callus cultures. When transferred to the soliddifferentiation medium (MS+ 1.0 mg L-1 NAA+ 1.5 mg L-1 BA), two hundreds and seventy-threeshoots produced. They readily rooted in agar-solidified MSO medium after three weeks andadapted in conditions of culture room. They were not transferred to field due to the unfavorableenvironmental conditions at that time. Cell suspension-derived callus tissues contained 82.62 μganthocyanin gm-1 of callus fresh weight, and 0.555 mg beta-carotene 100 gm-1of callus freshweight in the third period of extraction compared with their quantity calculated in stems calluswhich recorded 52.4 μg gm-1and 1.988 mg gm-1respectively.

Use of β-cyclodextrins to enhance phytosterol production in cell suspension cultures of carrot (Daucus carota L.)

Phytosterols are isoprenoid-derived compounds that play essential roles in plant growth and development as they are integral components of the plant cell membranes, and responsible for their permeability and fluidity. In this study, the effect of different types of beta-cyclodextrins (β-CD) on phytosterol production was evaluated using Daucus carota cell suspension cultures. A detailed analysis provides the optimal type and concentration of β-CD, elicitation time, and the optimal cell age and density. The highest levels of phytosterols produced by cells and secreted to the culture medium were obtained when 10 day-old D. carota cell suspension cultures, with an initial cell density of 200 g of fresh weight (FW) l−1, were incubated in the presence of 50 mM of methylated-β-CD (M-β-CD) for 144 h. In addition, M-β-CD significantly promoted the accumulation of phytosterol in the extracellular medium of D. carota cell suspension cultures, which was not observed in control cell suspension cultures. Moreover, these high phytosterol levels did not improve when methyl jasmonate (MJ) was added to the cell suspension cultures alone or combined with 50 mM M-β-CD, although MJ stimulated the formation of defense-related compounds. Therefore, the use of carrot cell suspension cultures seems a promising biotechnological alternative since the addition of β-CD to the culture medium not only induced the biosynthesis of phytosterols but also promoted their secretion into the culture medium, where they were accumulated and could be isolated easily.

Isolation of Daucus carota ethylene insensitive3-like (DcEIL) involved in stress-inducible DcMYB1 expression in suspension-cultured carrot cells

In carrot suspension-cultured cells, the expression of phenylalanine ammonia-lyase 1 (DcPAL1) gene was crucially regulated by the transcription regulatory factor Daucus carota MYB1 (DcMYB1). To elucidate the regulatory mechanism of DcMYB1 expression, we isolated and identified the transcription regulatory factor D. carota ethylene-insensitive3 (EIN3)-like protein (DcEIL) from a cDNA library prepared from suspension-cultured carrot cells treated with the “dilution effect” using a yeast one-hybrid system. DcEIL bound to a region of the DcMYB1 promoter containing a putative cis-element, which suppressed DcMYB1 promoter expression. The amino acid sequence of DcEIL contained the predicted signal sequence for nuclear localization, and transportation to the nucleus was confirmed using a green fluorescent protein-DcEIL fusion protein expressed in suspension-cultured Arabidopsis thaliana cells. The DcEIL gene was constitutively expressed irrespective of the elicitor treatment or the dilution effect. These results suggest that the binding of DcEIL to a cis-element of the DcMYB1 promoter might be regulated at the posttranslational level as a consequence of the regulation of DcPAL1 gene expression via DcMYB1 expression.

Characterization of MYB proteins acting as transcriptional regulatory factors for carrot phenylalanine ammonia-lyase gene (DcPAL3)

In suspension-cultured carrot cells, a phenylalanine ammonia-lyase gene, DcPAL3, plays an important role in regulation of anthocyanin synthesis. In the DcPAL3 promoter region, a putative cis-element, box-L, which is committed to the upregulation of promoter activity, has been identified. Here, we isolated DcMYB5 cDNA using yeast one-hybrid screening with box-L as a bait from a cDNA library prepared from cells of a variant cultured cell line constitutively synthesizing anthocyanin. Although expression of DcMYB2, 3, and 4 was observed, all of which were previously isolated by plaque hybridization from a subtracted cDNA library of anthocyanin-synthesizing cells of a normal cultured cell line cultured in medium lacking 2,4-dichlorophenoxyacetic acid, DcMYB5 expression was not observed in cells of the normal cultured cell line; high expression of DcMYB5 was observed in cells of the variant cultured cell line. Although not only DcMYB5 but also DcMYB2, 3, and 4 could bind to the box-L sequence in yeast, DcMYB3 and 5 showed strong transcriptional activation activity for DcPAL3 promoter in carrot protoplasts. These results suggest that DcMYB3 and 5 might play an important role in the upregulation of DcPAL3 promoter activity in the different regulatory paths between the normal and variant cultured cell lines.

ニンジン培養細胞におけるストレス誘導性 DcMYB1 遺伝子の発現制御に関与する転写調節因子 DcEIL の機能解析

ニンジン培養細胞におけるストレス誘導性 DcMYB1 遺伝子の発現制御に関与する転写調節因子 DcEIL の機能解析

Upregulation of the promoter activity of the carrot (Daucus carota) phenylalanine ammonia-lyase gene (DcPAL3) is caused by new members of the transcriptional regulatory proteins, DcERF1 and DcERF2, which bind to the GCC-box homolog and act as an activator to the DcPAL3 promoter

The phenylalanine ammonia-lyase (PAL) gene, DcPAL3, was expressed during the synthesis of anthocyanin in suspension-cultured cells of carrot (Daucus carota). There were two putative cis-elements in the DcPAL3 promoter region: the box-L and GCC-box homologs. Both of these are committed to the upregulation of promoter activity. Although box-L is known as the conserved cis-element present in the promoter region of most PAL genes of many plant species targeted by the R2R3-MYB protein, among PAL genes, the GCC-box homolog is unique to the promoter region of the DcPAL3 gene. We have isolated two proteins belonging to the ethylene-responsive element-binding factor (ERF) family, DcERF1 and DcERF2, from two different cDNA libraries prepared from anthocyanin-synthesizing cells of different cultured cell lines of carrot. The methodology employed was yeast one-hybrid screening with the GCC-box homolog as a bait. Both DcERF1 and DcERF2 bound to the GCC-box homolog sequence in vitro. Transient expression analysis showed that, in carrot protoplasts, DcERF1 was able bind to the GCC-box homolog and act as an activator of the DcPAL3 promoter. In contrast, DcERF2 itself had no ability to activate DcPAL3 promoter activity, possibly because transiently expressed DcERF2 may not be exported into the nucleus. These results suggest that DcERF1 and DcERF2 may function in different ways in committing to the upregulation of the DcPAL3 promoter activity in anthocyanin-synthesizing cells of carrot.

Response surface methodology in media optimization for production of β-carotene from Daucus carota

Plants are a valuable source of a vast array of chemical compounds including fragrances, flavours, food additives, colours, natural sweeteners, industrial feedstocks, anti-microbials and pharmaceuticals. The present study reports on application of Response Surface Methodology (RSM) in media optimization for suspension culture for the production of β-carotene. Growth kinetics of carrot cells in suspension culture has been carried out to understand the relationship between growth and β-carotene formation. The maximum production of β-carotene obtained using the optimized medium was 13.614 μg/g dry weight cell mass. The μ (specific growth rate) and t d (doubling time) were found to be higher for 20 g DW/l inoculum size.

Role of miniature inverted repeat transposable elements inserted into the promoter region of a carrot phenylalanine ammonia-lyase gene and its gene expression

The expression of the phenylalanine ammonia-lyase gene (PAL) was induced in anthocynanin-producing suspension-cultured carrot cells. In our previous study, we isolated two PAL genes, DcPAL3 and DcPAL4, from a carrot genomic library, the nucleotide sequences of which are highly similar. Here, the complete nucleotide sequence of DcPAL4 was determined and compared with that of DcPAL3, revealing two miniature inverted repeat transposable elements (MITEs), here designated MITE1 and MITE2, inserted into the proximal promoter region of DcPAL3, but not into that of DcPAL4. The expression of DcPAL4 was not detectable by RT–PCR in cultured carrot cells or the tissues of carrot plants, whereas DcPAL3 expression was detected. Transient expression experiment in carrot protoplasts showed that the DcPAL4 promoter actively produces transcripts, as does the DcPAL3 promoter. To characterize the roles of MITE1 and MITE2 (MITE1/2) in the transgenes of stable transformants, they were inserted into a plant transformation vector containing the 35S–β-glucuronidase (GUS) reporter gene and selectable antibiotic-resistance marker genes, and then introduced into tobacco BY-2 cells using an Agrobacterium method. More regenerated calli carrying the MITE1/2 construct survived on medium containing kanamycin than calli lacking MITE1/2. Regenerated calli containing MITE1/2 were more numerous having stronger GUS activity than those without MITE1/2. These results suggest that MITE1/2 enhances the expression of adjacent transgenes introduced into stable transformants and possibly into the endogenous DcPAL3 gene.

Production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of Gaucher's disease using a plant cell system

Gaucher's disease, a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy using recombinant GCD (Cerezyme) expressed in Chinese hamster ovary (CHO) cells. As complex glycans in mammalian cells do not terminate in mannose residues, which are essential for the biological uptake of GCD via macrophage mannose receptors in human patients with Gaucher's disease, an in vitro glycan modification is required in order to expose the mannose residues on the glycans of Cerezyme. In this report, the production of a recombinant human GCD in a carrot cell suspension culture is described. The recombinant plant-derived GCD (prGCD) is targeted to the storage vacuoles, using a plant-specific C-terminal sorting signal. Notably, the recombinant human GCD expressed in the carrot cells naturally contains terminal mannose residues on its complex glycans, apparently as a result of the activity of a special vacuolar enzyme that modifies complex glycans. Hence, the plant-produced recombinant human GCD does not require exposure of mannose residues in vitro, which is a requirement for the production of Cerezyme. prGCD also displays a level of biological activity similar to that of Cerezyme produced in CHO cells, as well as a highly homologous high-resolution three-dimensional structure, determined by X-ray crystallography. A single-dose toxicity study with prGCD in mice demonstrated the absence of treatment-related adverse reactions or clinical findings, indicating the potential safety of prGCD. prGCD is currently undergoing clinical studies, and may offer a new and alternative therapeutic option for Gaucher's disease.

Assignment of UVB-responsive cis-element and Protoplastization- (Dilution-) and Elicitor-responsive Ones in the Promoter Region of a Carrot Phenylalanine Ammonia-lyase Gene (gDcPAL1)¶

Expression of a carrot phenylalanine ammonia-lyase (PAL) gene (gDcPAL1) in suspension-cultured carrot cells is induced by dilution of the culture or by application of a fungal elicitor, as well as by ultraviolet B (UVB) irradiation. We demonstrated that among its upstream cis-elements (Takeda et al.[1997] Photochem. Photobiol. 66, 464–470), L4 is UVB responsive, and L1 is protoplastization- (dilution-) and elicitor responsive, from studies with transiently transformed mutated or truncated gDcPAL1 promoter–luc constructs. This conclusion is consistent with our observation that PAL activities induced by UVB and by protoplastization (dilution) or elicitor are additive.

DcMYB1 Acts as a Transcriptional Activator of the Carrot Phenylalanine Ammonia-lyase Gene (DcPAL1) in Response to Elicitor Treatment, UV-B Irradiation and the Dilution Effect

Expression of a carrot phenylalanine ammonia-lyase (PAL) gene (DcPAL1) in suspension-cultured carrot cells is induced by treatment with a fungal elicitor, ultraviolet B (UV-B) irradiation, and by transferring and diluting cells with fresh medium (the dilution effect). Box-L-like sequences are known as important cis-elements of genes for enzymes involved in the phenylpropanoid biosynthetic pathway. Six sequences, box-L0 to box-L5, exist in the DcPAL1 gene promoter region. In this study, we isolated cDNA encoding the R2R3 type of MYB transcription factor, DcMYB1, using yeast one-hybrid screening with box-L1 or box-L5 as target elements. DcMYB1 bound to boxes-L0, L1, L3/4, and L5 sequences (ACC(A/T)(A/T)CC) in vitro, and in yeast cells and carrot protoplasts. Transient expression of DcMYB1 could up-regulate DcPAL1 promoter activity in carrot protoplasts. Results of the transient expression experiment for the deletion-mutated promoters of boxes-L0, L1, L3, and L5 suggest that these box-L-like sequences were required for the complete activation of the DcPAL1 promoter by DcMYB1. Expression of DcMYB1 transcripts was induced 0.5 h after elicitor treatment or UV-B irradiation, and 2 h after the dilution effect. Induction of DcPAL1 expression occurred 1 h after DcMYB1 expression in all stress treatments, and repression of DcMYB1 expression by RNA interference caused cessation of the up-regulation of DcPAL1 expression in the elicitor treatment or with UV-B irradiation. These results suggest that DcMYB1 is the main regulatory factor acting on box-L sequences in the DcPAL1 gene that respond to environmental cues.

Identification and characterization of plant glycerophosphodiester phosphodiesterase

GPX-PDE (glycerophosphodiester phosphodiesterase; EC 3.1.4.46) is a relatively poorly characterized enzyme that catalyses the hydrolysis of various glycerophosphodiesters (glycerophosphocholine, glycerophosphoethanolamine, glycerophosphoglycerol, glycerophosphoserine and bis-glycerophosphoglycerol), releasing sn-glycerol 3-phosphate and the corresponding alcohol. In a previous study, we demonstrated the existence of a novel GPX-PDE in the cell walls and vacuoles of plant cells. Since no GPX-PDE had been identified in any plant organism, the purification of GPX-PDE from carrot cell walls was attempted. After extraction of cell wall proteins from carrot cell suspension cultures with CaCl2, GPX-PDE was purified up to 2700-fold using, successively, ammonium sulphate precipitation, gel filtration and concanavalin A-Sepharose. Internal sequence analysis of a 55 kDa protein identified in the extract following 2700-fold purification revealed strong similarity to the primary sequence of GLPQ, a bacterial GPX-PDE. To confirm the identity of plant GPX-PDE, an Arabidopsis thaliana cDNA similar to that encoding the bacterial GPX-PDE was cloned and overexpressed in a bacterial expression system, and was used to raise antibodies against the putative Arabidopsis thaliana GPX-PDE. Immunochemical assays performed on carrot cell wall proteins extracted by CaCl2 treatment showed a strong correlation between GPX-PDE activity and detection of the 55 kDa protein, validating the identity of the plant GPX-PDE. Finally, various properties of the purified enzyme were investigated. GPX-PDE is a multimeric enzyme, specific for glycerophosphodiesters, exhibiting a K(m) of 36 microM for glycerophosphocholine and active within a wide pH range (from 4 to 10). Since these properties are similar to those of GLPQ, the bacterial GPX-PDE, the similarities between plant and bacterial enzymes are also discussed.

Long‐term conservation of embryogenic competence by induction and disorganization of somatic embryos in carrot

AbstractA simple procedure was established to maintain the rate of somatic embryogenesis in carrot cell strains. Secondary cell strains were induced by the disorganization of heart‐shaped somatic embryos by transferring them into a medium containing 2,4‐dichlorophenoxyacetic acid and the rate of somatic embryogenesis was restored to the original level. It was possible to keep the original rate of embryogenesis by repeated induction and disorganization of somatic embryos, as the restored rate of embryogenesis decreased gradually during subcultures. In the carrot cell suspension culture used in this work, it was possible to keep high rates of somatic embryogenesis for at least 3 years by using this method at every 50th subculture. The method presented here should be useful to maintain regeneration competence in certain important cell strains.

High-affinity binding proteins for N-acetylchitooligosaccharide elicitor in the plasma membranes from wheat, barley and carrot cells: conserved presence and correlation with the responsiveness to the elicitor.

Binding experiments as well as affinity labeling with an (125)I-labeled 2-(4-aminophenyl)ethylamino derivative of N-acetylchitooctaose revealed the presence of high-affinity binding sites/proteins for N-acetylchitooligosaccharide elicitor in the plasma membrane preparation from suspension-cultured carrot cells, barley cells and wheat leaves. Their binding specificity corresponded with the elicitor activity of N-acetylchitooligosaccharides and related sugars in these plant cells/tissues, and was similar to that reported for the binding site/protein previously reported for suspension-cultured rice cells. The molecular size of the binding proteins identified in carrot, barley and wheat was slightly smaller than that of rice. These plant cells were shown to respond to N-acetylchitooligosaccharides and generate reactive oxygen species, induced medium alkalinization, or previously shown to initiate lignification (wheat leaves, Barber et al. (1989) Physiol. Mol. Plant Pathol. 34: 3). No elicitor-binding protein nor the elicitor-induced cellular responses was detected for a cell line of tobacco BY-2 (BY-2T). On the other hand, another cell line of tobacco BY-2 (BY-2N) showed the presence of elicitor-binding protein and also elicitor-induced medium alkalinization. Thus, there was a good correlation between the existence of high-affinity binding proteins for the elicitor and elicitor-induced cellular responses among tested plant cells. These results indicated the wide distribution of N-acetylchitooligosaccharide elicitor-binding protein among various plants and added further support for the function of these plasma membrane proteins in the perception of the elicitor signal.

Assignment of UVB-responsive cis-element and protoplastization-(dilution-) and elicitor-responsive ones in the promoter region of a carrot phenylalanine ammonia-lyase gene (gDcPAL1).

Expression of a carrot phenylalanine ammonia-lyase (PAL) gene (gDcPAL1) in suspension-cultured carrot cells is induced by dilution of the culture or by application of a fungal elicitor, as well as by ultraviolet B (UVB) irradiation. We demonstrated that among its upstream cis-elements (Takeda et al. [1997] Photochem. Photobiol. 66, 464-470), L4 is UVB responsive, and L1 is protoplastization- (dilution-) and elicitor responsive, from studies with transiently transformed mutated or truncated g-DcPAL1 promoter-luc constructs. This conclusion is consistent with our observation that PAL activities induced by UVB and by protoplastization (dilution) or elicitor are additive.

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.

The integration of a major hepatitis B virus gene into cell- cycle synchronized carrot cell suspension cultures and its expression in regenerated carrot plants

The percentage of cells successfully transformed by Agrobacteria is usually very low (not more than 10% and usually much less; Thomas et al., 1989). In this report we show that in carrot (Daucus carota L., ssp. sativus) cell suspensions transformation efficiency was strongly improved by using cell cycle synchronized cells. Fluorodesoxyuridine (FDU) was added for 24 h to inhibit thymidine synthesis. This blocked the cell cycle at the transition from G1- to S-phase. Then the block was released by applying thymidine. A high rate of transformation was obtained when Agrobacterium tumefaciens was added concurrently with thymidine. As examples of efficient and long term foreign gene expression in transgenic cells, the reporter enzyme β-glucuronidase (GUS) as a model as well as the major hepatitis B virus surface protein were used. Both genes were linked to the MAS promoter. In carrot cell suspensions containing the viral gene, the corresponding viral protein was produced. In roots of mature transgenic carrot plants generated through somatic embryogenesis and raised in soil as well as in callus cultures derived thereof, the viral protein was also produced.