Cavity spot of carrot (Daucus carota)

Cavity spot disease of carrot (Daucus carota) has been one of the intractable problems for both growers and scientists. Carrots are rejected at grading with one or two visible lesions, and when disease incidence passes a relatively low threshold it becomes uneconomic to harvest crops. For the scientist, there has been considerable pressure to produce both information on the cause of the disease and a cure. Many putative causes have been advanced over the years, but these were almost always contradicted by subsequent work. The first solid indication of involvement of a pathogen was when three different fungicides with activity against Oomycete fungi all reduced disease. Very quickly the causal agents Pythium violae and Pythium sulcatum were isolated from cavity spot lesions and Koch's postulates satisfied. The species are not typical of the more common pythia, having slow growth at normal temperatures, which means that in the context of isolation work, plates may be overgrown by other species before they are seen. Metalaxyl fungicide was identified as the most effective in controlling cavity spot caused by P. violae, but P. sulcatum is naturally tolerant of the fungicide. Recently, metalaxyl has been shown to be subject to enhanced microbial degradation. This phenomenon has been associated with failure to control cavity spot. No other fungicide has been shown to be consistently effective in the field, and none has been registered for disease control. For the future, this means that control of cavity spot can not be based solely on fungicidal control. Other, complementary strategies are necessary for reducing disease. Calcium carbonate is known to have significant effects on cavity spot, probably by inducing a soil microflora inhibitory to filamentous fungi. Management of agronomic aspects such as irrigation, soil cultivation and the length of time for which crops are grown may all be used, while carrot cultivars with some field resistance may be beneficial. However, one of the most significant factors is disease avoidance by not selecting fields with high inoculum levels. One serology‐based risk assessment test has been produced and commercialised, and molecular probes which could be the basis of more sensitive tests are available for both pathogens. The potential for disease reduction via a management strategy combining several key components is discussed.     

Historical and contemporary gene dispersal in wild carrot (Daucus carota ssp. carota) populations

Background and Aims

Wild carrot is the ancestor of cultivated carrot and is the most important gene pool for carrot breeding. Transgenic carrot may be released into the environment in the future. The aim of the present study was to determine how far a gene can disperse in wild carrot populations, facilitating risk assessment and management of transgene introgression from cultivated to wild carrots and helping to design sampling strategies for germplasm collections.

Methods

Wild carrots were sampled from Meijendel and Alkmaar in The Netherlands and genotyped with 12 microsatellite markers. Spatial autocorrelation analyses were used to detect spatial genetic structures (SGSs). Historical gene dispersal estimates were based on an isolation by distance model. Mating system and contemporary pollen dispersal were estimated using 437 offspring of 20 mothers with different spatial distances and a correlated paternity analysis in the Meijendel population.

Key Results

Significant SGSs are found in both populations and they are not significantly different from each other. Combined SGS analysis indicated significant positive genetic correlations up to 27 m. Historical gene dispersal σ_g and neighbourhood size N_b were estimated to be 4–12 m [95 % confidence interval (CI): 3–25] and 42–73 plants (95 % CI: 28–322) in Meijendel and 10–31 m (95 % CI: 7–∞) and 57–198 plants (95 % CI: 28–∞) in Alkmaar with longer gene dispersal in lower density populations. Contemporary pollen dispersal follows a fat-tailed exponential-power distribution, implying pollen of wild carrots could be dispersed by insects over long distance. The estimated outcrossing rate was 96 %.

Conclusions

SGSs in wild carrots may be the result of high outcrossing, restricted seed dispersal and long-distance pollen dispersal. High outcrossing and long-distance pollen dispersal suggest high frequency of transgene flow might occur from cultivated to wild carrots and that they could easily spread within and between populations.     

Transformation and regeneration of carrot (Daucus carota L.)

A protocol is presented for the efficient transformation of carrot (Daucus carota L. cv. Nantaise) by Agrobacterium tumefaciens. The binary vector contained the marker gene -glucuronidase (GUS), driven by the 35S promoter of cauliflower mosaic virus, and the nptII gene, which confers kanamycin resistance. Highest T-DNA transfer rates were obtained by co-cultivating bacteria with hypocotyl segments of dark-grown seedlings on solidified B5 medium containing naphthaleneacetic acid and 6-benzylaminopurine. After 2 days, bacterial growth was stopped with antibiotics. Two weeks later, the explants were placed on agar containing the kanamycin derivate geneticin; antibiotic-resistant calli developed during the following 4 weeks. Suspension cultures were obtained from resistant calli and plants regenerated via somatic embryogenesis in liquid culture. The majority of plants were phenotypically normal and, depending on the Agrobacterium strain used, harbored single or multiple copies of the T-DNA. About equal levels of GUS activity were found in different organs of young plants up to 6 weeks after embryogenesis. In leaves of older plants, GUS activity was markedly reduced, whereas the activities in phloem and xylem parenchyma cells of developing tap roots were still high and fairly uniform. Thus, the 35S promoter may be a useful tool to drive the expression of transgenes in developing carrot storage roots.     

Involvement of gibberellin and cytokinin in the formation of embryogenic cell clumps in carrot (Daucus carota)

Somatic embryogenesis of carrots is a typical example of the totipotency of plant cells. However, little is known about the process of change from somatic cells to embryogenic cells. To test the involvement of plant hormones in the acquisition process of embryogenic potency, we investigated the effects of plant growth regulators and their inhibitors on auxin-induced direct somatic embryogenesis of carrots. Gibberellin (GA) inhibited the early stage of embryogenic cell differentiation/development to the globular stage and uniconazole, an inhibitor of GA synthesis, promoted the secondary embryogenesis from the primary embryo. Purine riboside, an anticytokinin, inhibited direct somatic embryogenesis, and this effect was nullified by the application of cytokinin (CK). These results show that GA and CK regulate the early stage of auxin-induced somatic embryogenesis in carrots.     

Crotonic acid as a bioactive factor in carrot seeds (Daucus carota L.)

Water extracts from the carrot seed (Daucus carota L.) var. Perfekcja exhibit plant growth inhibitory properties against cress, cucumber, onion and carrot in a dose-dependant manner. This property results from the action of low-and high-molecular components of the extract. The low-molecular component was identified as crotonic acid ((E)-2-butenoic acid). Its presence was also confirmed in other late varieties of carrot. The determined strong herbicidal properties of crotonic acid and its availability after release to soil combined with its high level in seeds suggest that it might be considered as an allelopathic and autotoxic factor in the seeds.     

Different arabinogalactan proteins are present in carrot (Daucus carota) cell culture medium and in seeds

Arabinogalactan proteins (AGPs) were isolated by Yariv phenylglycoside precipitation from the medium of carrot ( Daucus carota L.) cell cultures and from carrot seeds. The isolates showed a different composition of AGPs. The medium AGPs contained an arabinose poor AGP fraction that had relatively high levels of glucuronic acid and rhamnose. In contrast the seed AGPs only contained arabinose and galactose-rich AGP fractions that had low levels of glucuronic acid. Linkage analysis on all fractions showed that most of the arabinose residues were terminally linked and that almost all galactose was present in the 1,3-, 1,6- and 1,3,6- form. The strongly branched type II arabinogalactans are characteristic of the carbohydrate part of AGPs. AGP characteristic amino acid residues as Hyp, Pro, Glx, Ser, Gly, Asx, Ala, Leu and Thr were detected in three different fractions.     

Occurrence of petaloid stamens in wild carrot (Daucus carota) from Sweden

Sterile petal-like stamens, reported by others in North American wild carrot, are here reported and illustrated from a wild carrot accession from Sweden. Although the North American material has been utilized already in carrot breeding, a more fundamental study of stamen petaloidy in botanical research dealing with flower differentiation is urged.     

The effects of excess boron with niacin on Daucus carota L. (carrot) root callus

Niacin (Nicotinic acid, B3 vitamin) may be involved in reduction of toxic effects of boron by regulating growth metabolism. This study was designed to examine whether external niacin treatment would improve the boron mobility in carrot callus cells or not. The results showed that excess boron caused tracheary inversions in meristematic root tissue, and also a shortage was seen in tracheary lengths with boric acid treatment. Boron excess induced the plant tolerance to water stress inverting the tracheary cells. This shortage converted nearly to normal size with niacin and boron treatment together. The results showed that boron mobility induced by niacin could reduce significantly the fresh and dry weight of carrot root cells, protein and ABA content was reduced also, in contrary, external boron and boron with niacin treatment considerable increased the two factors after one month stress. Fresh weight reduction and ABA content reduction indicated that niacin treatment caused water stress on the root cells of carrot, but boron treatment and boron with niacin treatment increased drought tolerance in carrot cells by increasing the both factors. In addition, turning the conversion of the length of the trachearies to their normal size proved that niacin treatment ended the polarizing effects of boron on cell walls.     

Mitochondrial gene diversity associated with the atp9 stop codon in natural populations of wild carrot (Daucus carota ssp. carota)

Mitochondrial genomes extracted from the wild populations of Daucus carota have been used as a genetic resource by breeders of cultivated carrot, yet little is known concerning the extent of their diversity in nature. Of special interest is an SNP in the putative stop codon of the mitochondrial gene atp9 that has been associated previously with male-sterile and male-fertile phenotypic variants. In this study, either the sequence or PCR/RFLP genotypes were obtained from the mitochondrial genes atp1, atp9, and cox1 found in D. carota individuals collected from 24 populations in the eastern United States. More than half of the 128 individuals surveyed had a CAA or AAA, rather than TAA, genotype at the position usually thought to function as an atp9 stop codon in this species. We also found no evidence for mitochondrial RNA editing (Cytosine to Uridine) of the CAA stop codon in either floral or leaf tissue. Evidence for intragenic recombination, as opposed to the more common intergenic recombination in plant mitochondrial genomes, in our data set is presented. Indel and SNP variants elsewhere in atp9, and in the other 2 genes surveyed, were nonrandomly associated with the 3 atp9 stop codon variants, though further analysis suggested that multilocus genotypic diversity had been enhanced by recombination. Overall the mitochondrial genetic diversity was only modestly structured among populations with an F(ST) of 0.34.     

Suboptimal temperature favors reserve formation in biennial carrot (Daucus carota) plants

In response to suboptimal temperatures, temperate annual plants often increase root:shoot ratios, build-up carbohydrates and display typical morphological and anatomical changes. We know less about the responses of biennials such as carrot. As a model plant, carrot has the additional feature of two functionally and morphologically distinct root parts: the taproot, which stores carbohydrate and other compounds, and the fibrous root system involved in acquisition of water and nutrients. Here, we analyze the effects of temperature (12 vs 25°C) on growth, carbohydrate accumulation and whole-plant morphology in two carrot cultivars. Our working hypothesis is that suboptimal temperature favors active formation of reserve structures, rather than passive accumulation of storage carbohydrates. In comparison with plants grown at 25°C, plants grown at 12°C had: (1) higher fibrous root:shoot ratio (13%) , (2) thicker (10–15%) and smaller (up to two- to three-fold) leaves, (3) lower leaf cuticular permeance (two- to four-fold), (4) higher taproot:shoot ratio (two-fold), (5) higher phloem:xylem ratios in taproot (two- to six-fold), (6) unchanged percentage dry matter content (%DMC) in leaves, petioles or fibrous roots and (7) higher %DMC in taproot (20%). However, %DMC of individual taproot tissues (phloem and xylem) was unaffected by temperatures and was consistently higher in the phloem (up to 30%). Therefore, the higher %DMC of whole taproots at 12°C was attributed solely to the increased development of phloem tissue. Carrot, therefore, shares many of the most conspicuous elements of temperate plant responses to low temperatures. Consistently with our hypothesis, however, carrots grown at suboptimal temperature promoted reserve structures, rather than the increase in carbohydrate concentration typical of most temperate annual species and woody perennials.     

Methylation of mitochondrial DNA in carrot (Daucus carota L.)

The methylation status of carrot (Daucus carota L.) mitochondrial DNA (mtDNA) was studied using isoschizomeric restriction enzymes MspI/HpaII (CCGG) and MvaI/EcoRII [CC(A/T)GG]. Southern hybridisations with probes for mitochondrial genes coxII and atpA were performed. MtDNAs isolated from non-embryogenic cell suspensions and roots were analysed. No differences were found using MspI/HpaII but after digesting the mtDNA with MvaI and EcoRII, some qualitative and quantitative differences between the restriction patterns appeared. Distinction was also revealed after Southern hybridisation with the coxII probe. These data indicate that the mtDNA of carrot is methylated in CNG trinucleotides and unmethylated in CG dinucleotides in CCGG sequences. The results were reproducible for cell suspensions of various genotypes and even cultivars but the extent of methylation was different in the root. The possible role of methylation in the mitochondrial genome of higher plants is discussed. Received: 16 April 1997 / Revision received: 4 July 1997 / Accepted: 30 July 1997     

Factors influencing the Agrobacterium tumefaciens-mediated transformation of carrot (Daucus carota L.)

To develop an efficient procedure for Agrobacterium tumefaciens-mediated genetic transformation of carrot (Daucus carota L.) the effects of several factors were studied. Parameters which significantly affected the transformation frequency were the variety, the explant type, and the co-cultivation period. Under optimal conditions, using the A. tumefaciens C58C1 containing either pGSTRN943 or pGSGluc1 and 3 days of co-cultivation, the frequency of transformation of petiole explants of the variety Nanco was greater than 45%. This procedure does not require acetosyringone or prolonged precultivation period. Using kanamycin (100 mg l^-1) for selection, a large number of transgenic plantlets developed from the embryogenic calli within 8–10 weeks of culture on hormone-free medium. Transformation was confirmed by histochemical detection of -glucuronidase activity in the transformed cells, by the ability of petiole segments to produce embryogenic calli in presence of kanamycin, and by Southern hybridization analyses.     

Isolation of development-related genes in somatic embryo radicle of carrot (Daucus carota L.) using modified cDNA RDA

Using modified cDNA RDA capitalizing on the high affinity of streptavidin for biotin and magnetic-absorption-based separation, we have obtained four bands of specifically expressed cDNA in the carrot somatic embryo deregulated for 12 h, which were designated as NR-1, NR-2, NR-3 and NR-4, respectively. As revealed by homology analysis of their DNA sequences after cloning them into pBS, remarkable homology was demonstrated in NR-2, NR-3 and NR-4 with the genes coding for LEA (late embryogenesis abundant protein), Dna J and xyloglucan endo-trans-glycosylase in plants. On the other hand, NR-1 showing no homology with any known sequence may have come from unknown genes. Using 32P-labeled NR-1 as probe, hybridization with cDNA fragment population has shown that we have actually cloned a new gene fragment related to radicle development. As shown by further Southern hybridization, these genes may be present in carrot genome in the form of single or low copies.     

Effect of brassinosteroid on cell division and enlargement in cultured carrot (Daucus carota L.) cells

When added at 3.10^–6 mM to auxin-starved suspension cultured carrot (Daucus carota L.) cells, the steroid hormone 24-epibrassinolide (BR) induces cell enlargement but not cell division.This is at variance from the effect of 2,4-D which, in the same experimental conditions, restores cell division without having any effect on cell enlargement.Furthermore, in the tested experimental conditions, the effect of BR is dominant over the effect of 2,4-D when the two hormones are simultaneously supplied to the auxin-starved culture.Abbreviations BR brassinosteroid, 24-epibrassinolide - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - GA₃ gibberellic acid     

The effects of ripening and drying of carrot seed (Daucus carota) crops on germination

Carrot seeds taken from the parent plant were capable of germinating before the stage when maximum seed dry weight was reached but even after this stage, when the seed moisture content had fallen below 20%, improvement in seed germination characteristics continued. In the latter stages of seed growth losses by shedding were 12–20 kg/ha/day. For low density crops (10 plants/m²) the yield of viable seed was at a maximum in crops whose seed was harvested with a moisture content of between 20 and 40% but no consistent relationship could be established for high density crops (80 plants/m²). There were no effects of umbel order or plant density on mean germination time or spread of germination. At any early harvest, percentage germination was highest for primary-umbel-seeds and seeds from low density crops but the differences between the seed origins diminished with later harvests. Drying the seeds on the umbels improved the percentage germination, reduced the mean germination time and the spread of germination particularly at the early harvests compared with seeds removed from the umbels and germinated immediately without drying.     

Effect of ABA and slow drying on DNA replication in carrot (Daucus carota) embryoids

Addition of abscisic acid (ABA) at the torpedo-shaped stage of development and slow dehydration are two parameters necessary to produce completely desiccation-tolerant carrot ( Daucus carota L.) embryoids. The mode of action of these parameters is still largely unidentified. Employing flow cytometry we investigated their effect on DNA replication and cell cycle activity of the developing embryoids. DNA replication was determined as percentage of 4C nuclei. Addition of ABA did not alter DNA replication and cell cycle during embryoid development in vitro, in spite of the putative quiescent state of the torpedo-shaped embryoids. In contrast, during slow drying the nuclei were preferentially arrested in the presynthesis G₀/G_1-phase and the amount of G₂ nuclei decreased. Dry zygotic carrot embryos do not contain any G₂ nuclei and are completely desiccation tolerant. The decline of G₂ nuclei in dry somatic embryoids seems to coincide with the increase in desiccation tolerance, which is incomplete compared to zygotic embryos. Our results suggest that in order to withstand anhydrobiosis, DNA replication may be controlled during the embryoid developmental program and slow dehydration, but not by the plant growth regulator ABA.     

Elicitation of anthocyanin production in cell cultures of carrot (Daucus carota L) by using elicitors and abiotic stress

Summary A cell line of carrot (Daucus carota L) which produces anthocyanin was subjected to various elicitors and abiotic stresses: The elicitors tested were culture filtrates (CF) and cell extracts (CE) of certain bacteria and yeasts. The abiotic stresses were salts of certain metal ions. The production increase obtained with cell extracts of Bacillus cereus. Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus were 49, 72, 45 and 41% respectively over the control. Maximum elicitation was obtained with elicitor derived from cell extract of the yeast Rhodotorula rubra where it enhanced anthocyanin production by two fold. The abiotic stress agents Ca, Mn, Zn, Co, Fe & V enhanced anthocyanin production. Of all the metal ions tested Ca was the most effective. The elicitation process was governed by the type and level of elicitor.     

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