Mature Carrot Research Articles

Development of inoculation methods for Pythium violae to evaluate resistance of carrot cultivars and efficacy of crop protection products for cavity spot control

AbstractCarrot is a major root vegetable crop grown in many parts of the world. In Europe, cavity spot disease continues to have a major impact on marketable yield. The disease is caused by several Pythium spp., with P. violae the main pathogen in the UK and results in small black lesions on mature carrot roots. The lack of reliable inoculation methods for these Pythium pathogens has, for many years, hampered the identification of new effective crop protection products or carrot varieties that are resistant to the disease. In this research, inoculation methods were developed for P. violae using mycelium or oospores, each of which successfully induced typical cavity spot symptoms in both pot‐ and field‐grown carrots as well as consistent root stunting in the former. These methods were also used to successfully identify carrot cultivars with resistance to cavity spot and confirmed the efficacy of the fungicide metalaxyl‐M against the disease. Results therefore demonstrated that the inoculation methods should be reliable for identifying the efficacy of crop protection products, assessing cavity spot resistance and for further studies investigating the biology and epidemiology of the pathogen.

Testing the cleaning effectiveness of new ecological aqueous dispersions applied on old icons

Adherent deposits are very aggressive towards ancient heritage paintings since they affect the varnish and the painting's layers, sometimes reaching the preparative layers. The biggest problem to the restorer is their removal without affecting the patina, the transparent varnish (well preserved) and fine colour glazes made during painting. Therefore, their removal requires preliminary cleaning tests that allow the optimization of the cleaning system composition that is going to be used. The study was focused on organic natural systems, as colourless supernatants, some of them used during ages, but insufficiently studied. The paper presents an evaluation of the effectiveness of cleaning varnished icons of the nineteenth century, with complex conservation cases using supernatants derived from aqueous dispersions extracted from vegetables and dry indigenous herbal infusions. Best results, after six consecutive cleaning steps, on tempera old icon was obtained for a mixture made of mature white onion juice+extract of Soapwort flowers+corn silk tea+acacia tea. As a best result after just one cleaning step was obtained for a quaternary mixture composed from mature white onion juice+mature carrot juice+corn silk tea+aqueous extract of Soapwort flowers.

First Report of Fusarium oxysporum and F. solani Causing Fusarium Dry Rot of Carrot in China.

Carrot (Daucus carota L.) is an economically important vegetable crop in China. In August 2008, a disease was observed on carrot in Inner Mongolia. The symptoms appeared as dry rot lesions on root surface, expressing light brown cankers with defined rounded or irregular shapes (1,3). The average disease incidence was up to 80% in Tuo Ke Tuo County. The disease has been a serious problem in these two counties since then, especially where consecutive carrot cropping was practiced. Carrot roots with typical dry rot symptoms were washed with tap water. Root tissues near the margin of necrotic lesions were excised, surface sterilized with 1% NaOCl for 3 min, and rinsed with sterile distilled water three times. The disinfected tissue was placed on potato dextrose agar (PDA) in a petri dish. Plates were incubated at 25 ± 1°C in the dark for 4 days. Fusarium single spore isolates were obtained from characteristic colonies (1). Three isolates (CF1, CF2, and CF3) were used for further study. The isolates were identified as Fusarium spp. on the basis of microscopic morphology on PDA. CF1 produced pink pigment, abundant falciform macroconidia of 14.7 to 38.2 × 4.5 to 5.7 μm with 2 to 3 septates, and elliptic microconidia of 7.5 to 15.1 × 3.3 to 5.4 μm with none or one septate. CF2 and CF3 produced light blue pigment, abundant falciform macroconidia of 16.4 to 34.4 × 4.0 to 6.1 μm with 2 to 3 septates, and elliptic microconidia of 6.7 to 10.7 × 3.0 to 4.9 μm with none or one septate. They were further identified and confirmed by PCR. The PCR involved amplifying the internal transcribed spacer (ITS) region of ribosomal DNA using genomic DNA as the template with universal primers ITS1 and ITS4 (2). The PCR products were sequenced. BLAST analysis of these sequences against the GenBank database determined the taxonomy of the isolates. The sequence of CF1 was 99% identical to F. oxysporum (Accession No. KC594035); sequences of CF2 and CF3 were 99% identical to F. solani (KC215123). To confirm the pathogenicity of the isolates, mature carrot roots (cv. Hong Ying 2) were inoculated with mycelial plugs (5 mm in diameter) cut from the margin of actively growing colonies on PDA plates. One mycelial plug was placed on each carrot root, with the mycelial side facing the root. PDA plugs were used for controls. Each treatment had five replicates. The inoculated roots were incubated in a humid chamber (90% RH) at 25°C. Four days after incubation, mycelia of the isolates developed and covered most of the surface of carrot roots, and brown rot lesions were observed on all inoculated roots, while the controls remained symptomless. This experiment was repeated. In another trial, carrot seeds (cv. Hong Ying 2) were sown in sterilized soil in pots (30 × 25 cm opening) with 15 seeds per pot. The soil was infested with either CF1, CF2, or CF3 by adding spore suspension to make the final concentration of 1 × 104 CFU/g soil. Plants grown in non-infested soil served as controls. There were three replicates per treatment. All the treated pots were placed in a field. After 13 weeks, the same symptoms of dry rot were observed as previously described. No symptoms were observed on the control plants. The trial was repeated. Symptomatic tissues from the inoculated roots were sampled and the pathogen was re-isolated, and identified using PCR. To our knowledge, this is the first report of F. oxysporum and F. solani causing dry rot of carrot in China.

First Report of Pythium sulcatum Causing Cavity Spot in Processing Carrot Crops in the Columbia Basin of Washington State.

In October 2012, symptoms of cavity spot (1) were observed on roots of two 50 ha, Red Core Chantenay processing carrot (Daucus carota L. subsp. sativus (Hoffm.)) crops in the Columbia Basin of central Washington. Symptoms consisted of sunken, elliptical lesions (3 to 15 mm long) on the root surface. Approximately 6% of the roots in each crop were affected, which was sufficient to present sorting problems for the processor. Symptomatic roots were washed thoroughly in tap water, and then small sections of tissue from the lesion margins were removed aseptically and plated onto water agar (WA) without surface-sterilization. Isolates with morphological characteristics typical of Pythium sulcatum Pratt & Mitchell (2) were obtained consistently from the symptomatic tissue. The genus and species identity of seven isolates was confirmed by sequence analysis of the internal transcribed spacer (ITS) 1-5.8S-ITS2 region of ribosomal DNA (rDNA) using universal eukaryotic primers UN-UP18S42 and UN-LO28S576B with the PCR protocol described by Schroeder et al. (3). The ITS consensus sequences of the seven isolates (Accession Nos. KF509939 to KF509945) were 98 to 99% homologous to ITS sequences of P. sulcatum in GenBank. Pathogenicity of all seven isolates was confirmed by inoculating mature carrot roots of cv. Bolero. Each root was washed with tap water, sprayed to runoff with 70% isopropanol, and dried in a laminar flow hood on sterilized paper toweling. The roots were then placed in plastic bins lined with paper toweling moistened with sterilized, deionized water. Each root was inoculated by placing two 5 mm-diameter agar plugs, taken from the edge of an actively growing WA culture of the appropriate isolate, on the root surface approximately 3 cm apart. Non-colonized agar plugs were used for a non-inoculated control treatment. Four replicate roots were inoculated for each isolate and the control treatment. After inoculation, the roots were misted with sterilized, deionized water, a lid was placed on each bin, and the roots were incubated in the dark at 22°C. Roots were misted daily to maintain high relative humidity. Dark, sunken lesions were first observed 3 days post-inoculation on roots inoculated with the P. sulcatum isolates, and all inoculated roots displayed cavity spot lesions by 7 days. No symptoms were observed on the non-inoculated control roots. Colonies with morphology typical of P. sulcatum were re-isolated from the symptomatic tissue of roots inoculated with the P. sulcatum isolates, and the species identity of the re-isolates was confirmed by ITS rDNA sequence analysis, as described above. Although P. sulcatum is one of several Pythium species that can cause cavity spot of carrot (1), to our knowledge, this is the first report of P. sulcatum causing cavity spot in Washington State, which has the largest acreage of processing carrot crops in the United States (4).

First Report of Pythium recalcitrans Causing Cavity Spot of Carrot in Michigan.

During a survey of carrot (Daucus carota L.) cavity spot in Michigan in September 2010, carrot roots with typical cavity spot symptoms were collected from production fields in Fremont Co. The lesions were excised from infected roots, surface-disinfested with 0.62% NaClO for 3 min, rinsed in sterilized, distilled water three times, cut into 0.5 cm long pieces, and then plated on water agar (WA) amended with carbendazim (10 μg/ml), ampicillin (50 μg/ml), rifampicin (50 μg/ml), and pentachloronitrobenzene (10 μg/ml) (cumulatively referred to as CARP). Plates were incubated at 22 ± 1°C in the dark for 3 days. Pure cultures of the isolates were obtained by transferring a single hyphal tip of each colony to potato dextrose agar (PDA) amended with CARP. Among the 33 isolates obtained, M2-05 was identified as a Pythium sp. that differed from the known cavity spot pathogens of carrot. The isolate has spherical hyphal swellings but no other distinguishing morphological characteristics. M2-05 was further classified by analyzing the partial sequences of four genes: the internal transcribed spacer (ITS) region of ribosomal DNA, beta-tubulin (β-tub), cytochrome c oxidase subunit 2 (cox 2), and NADH dehydrogenase subunit 1 (nadh 1) (1,3). A BLAST search of these sequences for M2-05 was conducted using the nucleotide database of GenBank, resulting in 100% similarity to all four sequenced genes of P. recalcitrans (2). The DNA sequences of M2-05 were deposited in GenBank (JQ734349, JQ734229, JQ734289, and JQ734409 for ITS, β-tub, cox 2, and nadh 1, respectively). Koch's postulates were conducted by inoculating mature carrot roots (cv. Nantindo) with mycelial plugs (4 mm in diameter) cut from the margin of actively growing colonies of M2-05 on PDA plates. Two mycelial plugs were placed on each carrot root at 3-cm intervals, with the mycelial side facing the root; and two non-colonized agar plugs were placed similarly for the non-inoculated control treatment. In comparison, carrot roots also were inoculated with an isolate of each of P. sulcatum and P. violae using the same method. There were four replicate carrot roots inoculated for each isolate and each of the control treatments. The inoculated roots were placed on a plastic grid (7 mm in height) in a closed plastic container, with moist paper towels underneath the grids. The container was incubated in the dark at 22 ± 1°C, and the roots were sprayed gently daily with sterilized, distilled water to maintain high humidity. Brown lesions were observed on all inoculated carrot roots 5 days after inoculation. The lesions measured 0.68 ± 0.48, 1.20 ± 0.71, and 0.56 ± 0.31 mm2 averaged over all eight lesions for the isolates of P. recalcitrans, P. sulcatum, and P. violae, respectively. Symptomatic tissues from the inoculated roots were excised and incubated on WA-CARP plates, and the culture from each lesion confirmed as the isolates inoculated using the same molecular methods described above. The carrot tissue under the control agar plugs remained symptomless, and no Pythium was recovered from the control roots. P. recalcitrans was described in 2008 as infecting roots of Beta vulgaris and Vitis vinifera (2). To our knowledge, this is the first published report of P. recalcitrans naturally infecting carrot roots, not only in Michigan, but anywhere in the world.

Molecular Tagging and Selection for Sugar Type in Carrot Roots Using Co-dominant, PCR-based Markers

Carrot storage roots accumulate free sugars. The type of sugar accumulated is conditioned by the Rs locus so that typical carrot roots (Rs/-) accumulate predominantly glucose and fructose while rs/rs plants accumulate predominantly sucrose. We recently have found rs/rs plants in one inbred line that harbor a naturally occurring insertion sequence of 2.5 kb integrated into the first intron region of acid soluble invertase isozyme II. Using these facts, three primers were designed to differentiate Rs/Rs, Rs/rs and rs/rs carrot plants with simple PCR amplification. Co-dominant, PCR-based markers for acid soluble invertase isozyme II allowed genotyping of the Rs locus in 1-week-old carrot seedlings whereas mature carrot roots were needed to make this evaluation previously, and homozygous dominant plants could not be differentiated from heterozygotes without lengthy progeny testing. Marker-assisted evaluation and selection of carrot root sugar type were exercised in segregating families of diverse background and complete accuracy in predicting sugar type was realized in subsequent generations to further confirm that acid soluble invertase isozyme II conditions the Rs locus. These PCR-based markers will be useful in carrot breeding programs screening for this trait in segregating populations, for studying the distribution and origins of this trait in domestic and wild carrots, and for identifying seed mixtures as low as 10% Rs/- or 1% rs/rs.

Optimising production of carrot hairy roots

This research describes the production of carrot hairy roots for monoxenic culture of New Zealand arbuscular mycorrhizal fungi (AMF) Induction of hairy roots on mature carrot root sections was carried out using Agrobacterium rhizogenes isolate A4T that had been grown in either LauriaBertani (/ acetosyringone) or Yeast Mannitol medium (24 or 48 h incubation) Results showed both methods could initiate hairy root production The source of the carrots was one of the most important factors with mature freshly harvested carrots showing better hairy root production compared to coolstored carrots Dissection of the cortex to expose the cambium had a positive effect on the least optimal treatment

Effects of climatic factors, fluctuating water availability and partial defoliation on the diurnal radial growth pattern of carrot (Daucus carota) taproots

Carrot taproots are prone to radial longitudinal tissue fractures (splitting). The longitudinal alignment of the fractures is consistent with the tangential stress induced by radial growth in the annular cambium. In this study linear variable differential transformers were used to monitor the diurnal radial growth pattern of mature carrot taproots. The diurnal growth pattern was characterised by rapid expansion during the night (dark) period and small shrinkage during the light period. The magnitude of the daily shrinkage was positively correlated with both vapour pressure deficit and global radiation. A positive correlation between the rate of taproot expansion during the night period and global radiation level the preceding day was also recorded. Water stress reduced the taproot night time expansion rate, but the taproot of water stressed plants expanded rapidly when watered to field capacity. Partial defoliation of plants at midday resulted in immediate expansion of the taproot, however, the daily growth rates of the partially defoliated plants was less than untreated control plants over the following 3 days. It was concluded that the short-term changes in taproot diameter could be used to infer changes in tangential stresses and therefore taproot splitting propensity.

First Report of Rhizoctonia solani AG-2-4 on Carrot in Georgia.

Anastomosis group-2-4 (AG-2-4) of Rhizoctonia solani Kühn was formally described in 2002 (1), but it was first collected in 1983 in Georgia from corn (Zea mays L.) exhibiting symptoms of crown and brace root rot. Although occasionally present on diseased corn roots, the role that isolates of AG-2-4 play in crown and brace root rot of corn is not specifically known (2). More recently, as part of a broad multi-year (1996 to 2000) survey of root diseases in field grown carrot (Daucus carota L.), isolates of R. solani AG-2-4 were recovered from diseased carrot plants in various stages of growth from fields of sandy loam soil at many locations in southern Georgia, including commercial fields in Coffee and Tift counties. During the 1996 to 2000 growing seasons, 123 isolates of Rhizoctonia sp. (including multinucleate and binucleate types) were collected from lesions on developing and mature carrot roots. Of these, 34% were AG-2-4, 10% were AG-2-2IV, 6% were AG-4, and 32% were binucleate Rhizoctonia sp. The remaining 18% were lost prior to AG typing. An additional 40 isolates were collected from carrot seedlings or soil and of these, 55% were AG-4, 18% were AG-2-2IV but none were AG-2-4. Virulence on carrot seedlings by two isolates of AG-2-4 (777R1P5-SL2 and 758C) was compared with virulence of isolates of AG-4, AG-2-2IV, AG-2-1, and binucleate Rhizoctonia sp. Carrot seeds soaked for 5 min in 0.5% NaOCl were planted in petri dishes containing moist autoclaved sandy loam soil. Each dish was inoculated in the center with a 10-mm-diameter disk cut from a 9-day-old potato dextrose agar (PDA) culture of the appropriate isolate. Petri dishes were placed in a 26°C incubator for 9 days, and then the seedlings were rated for disease. Virulence on mature carrot root tissue was also assessed on the same set of isolates. Cross sections of carrot roots (5 to 10 mm thick) were surface disinfested in 0.5% NaClO for 5 min. Three cross sections were placed on moist filter paper in sterile petri dishes and each was inoculated with a 5-mm-diameter disk of inoculum cut from 8- to 10-day-old cultures growing on PDA. All treatments were rated for damage following incubation on a lab bench at 21 to 24°C for 7 days. Isolate 777R1P5-SL2 caused moderate damage to seedlings but minimal rotting of mature carrot root tissue. Isolate 758C did no damage to either seedlings or root tissue. The AG-4 and AG-2-2IV isolates killed all seedlings and caused extensive rot on mature root tissue. The AG-2-1 isolate caused moderate damage to seedlings and mature root tissue, whereas isolates of binucleate Rhizoctonia sp. damaged neither seedling nor mature root tissue. These data suggest that some isolates of R. solani AG-2-4 may be capable of causing minor damage to carrot seedlings in the field in Georgia, but isolates of R. solani AG-4 and AG-2-2IV pose greater threat to seedlings and mature roots of carrot. Published data shows that isolates of AG-2-4 can kill seedlings of lettuce, cauliflower, and broccoli in the laboratory (1). R. solani AG-2-4 also may be capable of killing these crops in the field, all of which are grown commercially in Georgia. To our knowledge, this is the first report of R. solani AG-2-4 on carrot in Georgia.

Endo-β-mannanase activity is associated with the completion of embryogenesis in imbibed carrot (Daucus carota L.) seeds

AbstractDevelopment of the rudimentary embryo in mature carrot (Daucus carota) seed during imbibition was characterized. The small embryo in the carrot seed, located in the micropylar region, elongated into the lateral part during imbibition and attained about two-thirds the length of the seed before radicle protrusion. Developing embryos excised from imbibed seeds were only capable of germinating in both water and Murashige–Skoog (MS) medium when they reached maximum size. The corrosion cavity into which the embryo grew enlarged concomitantly with endosperm degradation. The expression of endo-β-mannanase (EC 3.2.1.78), which is assumed to be involved in endosperm degradation, was characterized. A cDNA encoding an endo-β-mannanase was obtained by reverse transcription polymerase chain reaction (RT-PCR) using total RNA extracted from 24-h-imbibed carrot seeds. The full-length cDNA (DcMAN1) exhibited 64% deduced amino acid sequence identity with tomato (Lycopersicon esculentum) seed germination-associated mannanase (LeMAN2). DcMAN1 mRNA and endo-β-mannanase activity were first detected in the micropylar-half seed and then in the lateral-half seed. The timing of the appearance of DcMAN1 mRNA and endo-β-mannanase activity in the lateral-half seed corresponded with that of embryo development into this region. These results suggest that the expression of DcMAN1 and endo-β-mannanase activity in imbibed carrot seeds is associated with the enlargement of the corrosion cavity, which accompanies embryogenesis.

A 2.5-kb insert eliminates acid soluble invertase isozyme II transcript in carrot (Daucus carota L.) roots, causing high sucrose accumulation.

The predominant storage carbohydrates of mature carrot (Daucus carota L.) storage roots typically are the free sugars glucose and fructose. This trait is conditioned by the Rs allele. A naturally occurring recessive mutation, rs/rs, conditions a shift from these reducing sugars to sucrose. RT-PCR and sequencing revealed a unique 2.5 kb insert in the first and largest intron near the 5' end of the acid soluble invertase isozyme II gene of rs/rs carrots. This insert was not totally spliced out during mRNA processing. While the wild-type acid-soluble invertase isozyme II transcript (ca. 2 kb) was detected in Rs/Rs roots and leaves, none was observed in rs/rs roots throughout development. RT-PCR of rs/rs leaves revealed two novel transcripts (2.7 kb and 3.2 kb). A comparison of enzyme activity between the near-isogenic Rs/Rs and rs/rs carrot lines revealed very low acid-soluble invertase activity in rs/rs roots whereas neutral invertase, sucrose synthase and sucrose phosphate synthase levels were comparable. Those results and linkage analysis indicate that Rs is a candidate locus for carrot vacuolar acid-soluble invertase isozyme II. Although the 2.5 kb insert does not occur in the Rs wild-type acid-soluble invertase isozyme II allele, it does occur elsewhere in the genome of Rs/Rs plants.

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.

Abstracts, The Canadian Phytopathological Society, Annual Meeting, Toronto, Ontario, 25-28 June 1995

Abstracts, The Canadian Phytopathological Society, Annual Meeting, Toronto, Ontario, 25-28 June 1995

Chitinase profiles in mature carrot (Daucus carota) roots and purification and characterization of a novel isoform

The profile of chaitinases (EC 3.2.1.14) in mature carrot (Daucus carota L. cv. Eagle) roots was studied. Multiple chitinase bands (8–10) were observed in native and sodium dodecylsulfate‐denaturing polyacrylamide gels. The molecular masses of these chitinases were estimated to be from 20 000 to 40 000. One major chitinase was purified and found to be an acidic protein with pI at 4.3 and a molecular mass of 39 500. The optimum pH for enzymatic activity was around 5 and the optimum temperature was 25°C. The enzyme was stable at pHs below 8 and temperatures below 60°C. The protein did not have a chitin‐binding domain, but showed some similarity to the amino acid composition of tobacco class I chitinase. The N‐terminal amino acid sequence did not resemble any of the described classes of chitimases. This chitinase did not possess lysozyme activity and showed antifungal activity when tested against Trichoderma sp.

Chitinase profiles in mature carrot (Daucus carota) roots and purification and characterization of a novel isoform

Chitinase profiles in mature carrot (Daucus carota) roots and purification and characterization of a novel isoform

Simultaneous purification of chloroplast and mitochondrial DNA without isolation of intact organelles from green carrot tissue and suspension cultures

We report here the simultaneous purification of chloroplast (cpDNA) and mitochondrial DNA (mtDNA) from green tissue and suspension cultures of carrot without organelle isolation. This method is based on isolating total nucleic acids from frozen tissue and separating the nuclear, chloroplast and mitochondrial fractions using sequential isopycnic sedimentation in two gradients of cesium chloride containing bisbenzamide. From 10 g of mature carrot leaves, 10 to 30μg of organelle DNA was consistently recovered from mature carrot leaves, while 30 to 50 μg was recovered from suspension cells. The method can be used to isolate chloroplast and mitochondrial DNAs from single plants without sacrificing the individual.

Effect of Broomrape (Orobanche spp.) Infection on Sugar Content of Carrot Roots

The soluble sugar content of mature carrot roots (Daucus carota L. cv. Ti-To, Nantes type) grown in broomrape-infested or noninfested soil was measured. Orobanche aegyptiaca Pers. (Egyptian broomrape) and O. crenata Forssk. (crenate broom-rape) decreased the total sugar content similarly. Sucrose was the primary sugar in noninfected roots and its level was greatly reduced in infected roots. Concentrations of sucrose were higher in the cortex than in the core; broomrape infection reduced sucrose concentration in both portions of the root. Even broomrape-infected roots with a healthy appearance (acceptable size and color) had a greatly reduced sucrose content. These results show that broomrape infection can reduce carrot root quality even when the visual appearance of the root is not affected.

Interaction of nuclear factors with upstream sequences of a lipid body membrane protein gene from carrot.

To study the regulation of gene expression during embryo development, we isolated a gene, DC 59, expressed in embryos but not in mature carrot plants. Sequence and S1 analysis showed that the gene was composed of one exon encoding a polypeptide of 19 kilodaltons and was highly homologous to the lipid body membrane protein gene L3 from maize. The plant hormone abscisic acid regulated the accumulation of DC 59 mRNA. To understand the mechanism of embryo-specific and hormonal regulation of DC 59, 5' DNA fragments were incubated with nuclear proteins. Two adjacent regions (from -706 to -235) interacted with nuclear extracts from embryos, resulting in the formation of four complexes (C1, C2, C3, and C4). Factors involved in the formation of the C3 and C4 complexes could be competed with sequences upstream of DC 8, a gene that is coordinately expressed with DC 59 during embryo development. DNase I footprinting analysis revealed that nuclear extracts from embryos bound to four AT-rich sequences, and the protected motifs within fragment V were located in the highly homologous upstream regions of DC 59 and DC 8 genes.

Gene-expression programs in embryogenic and non-embryogenic carrot cultures

Somatic embryogenesis can be synchronized by enriching carrot (Daucus carota L.) suspension cultures for small, dense clusters of cells termed proembryogenic masses (PEMs). Gene-expression programs of PEMs were compared with those of embryonic and mature tissues by in-vitro translation of representative mRNA populations and by nucleic-acid hybridization. Analysis of invitro-translated polypeptides by two-dimensional polyacrylamide gel electrophoresis revealed striking similarities between the mRNA populations of PEM and torpedo-stage embryos; substantial differences, however, were observed when in-vitro translation products of PEMs and torpedo embryos were compared with those of hypocotyls and leaves. Northern blots of RNA isolated from PEMs, staged embryos, and mature carrot tissues were hybridized with cDNA probes for Dc3, Dc5 and Dc13; these cDNA recombinants represent mRNAs that are regulated during carrot somatic embryogenesis. The pattern of expression of these embryo-regulated transcripts was similar in PEMs and somatic embryos but differed in other carrot tissues. These results indicate that many of the molecular processes of embryogenesis are already established in PEMs in the presence of auxin. Additional experiments indicate the utility of Dc3 as a molecular marker for the acquisition of embryogenic potential.

A Large-scale Isolation of Vacuoles from Protoplasts of Mature Carrot Taproots

Summary A simple large-scale method was developed for the isolation of viable protoplasts and then vacuoles from mature taproots of carrot ( Daucus carota L.). Digestion of phloem parenchyma with Cellulase Y-C and Pectolyase Y-23 for 3 h yielded 2 to 3 × 10 6 protoplasts/g fwt. Vacuoles were obtained from these protoplasts by a combination of osmotic and pH shock. Distribution of marker enzymes showed a yield of 50 % of the vacuoles with extravacuolar contamination of less than 1 % malate dehydrogenase and 4 % glucose-6-phosphate isomerase. In an application of the method it was demonstrated that the sugars accumulated by mature carrot taproots (fructose, glucose, and sucrose) are stored in the vacuole.