Obtaining carrot (Daucus carota L.) plants in isolated microspore cultures

Microspores were cultured on the modified B₅ liquid medium containing 2.4D (0.1 mg L⁻¹), NAA (0.1 mg L⁻¹), L-glutamine (500 mg L⁻¹), L-serine (100 mg L⁻¹), and sucrose (100 g L⁻¹). The developmental stages of microspores and divisions were observed. Initially, the formation of binuclear and multicellular structures was noticed. Plants regenerated in the cultures in which the tetrad stage of microsporogenesis had predominated. Embryoids were still forming 24 weeks after the cultures were set up. Six weeks after the transfer of androgenetic embryos onto the B₅ regeneration medium, they were converted into complete plants. Out of 90 androgenetic plants planted in a growth chamber, 42 plants adapted to the new conditions. All of those plants proved to be diploids in cytometric analysis.     

High-level expression of human interferon alpha-2b in transgenic carrot (Daucus carota L.) plants

In this study, we report the obtaining of carrot plants expressing human interferon alpha-2b via Agrobacterium-mediated transformation using two vector constructs containing the sequence coding for interferon gene fused with Nicotiana plumbagenifolia calreticulin apoplast targeting signal driven by 35S CaMV promoter and root-specific Mll promoter. The human interferon alpha-2b gene was correctly translated in carrot plants according to Western blot analysis. The recombinant protein exhibited antiviral activity in vitro by inhibition of vesicular stomatitis virus replication in established piglet testicular cells. The results demonstrated the higher activity of interferon accumulated in carrot plants for young leaves (up to 50.7 × 10³ IU/g FW) compared to the mature ones probably due to the degradation-susceptible nature of this protein. The taproot-expressing system could have also provided the sufficient protein amounts (up to 16.5 × 10³ IU/g FW) and could possibly be used for generating interferon alpha-2b protein in planta for preventing and curing infectious diseases.     

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.     

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     

Transgenic herbicide- and disease-tolerant carrot (Daucus carota L.) plants obtained through Agrobacterium-mediated transformation

. Transgenic carrot (Daucus carota L.) plants expressing a rice thaumatin-like protein (tlp), phosphinothricin acetyltransferase (bar) and the hygromycin phosphotransferase (hpt) genes were obtained by Agrobacterium-mediated transformation. Petiole and hypocotyl segments of three carrot cultivars were used as the explant sources. Following infection, selection was achieved on Murashige and Skoog medium with 1 mg/l phosphinothricin or 25 mg/l hygromycin B, which was increased after 2 weeks to 10 mg/l phosphinothricin and 100 mg/l hygromycin B. The presence of the tlp and bar transgenes was confirmed by polymerase chain reaction and Southern blot analyses, and the expression of the thaumatin-like protein was demonstrated by Western blot analysis. Among 45 primary transformants, 13 were selected for assessment of herbicide and/or disease tolerance. The transgenic plants showed varying levels of tolerance to the herbicide phosphinothricin, depending on the transformation events in different lines. Four transgenic lines also showed significantly enhanced tolerance to the foliar and root pathogen Botrytis cinerea or Sclerotinia sclerotiorum when inoculated under controlled environment conditions. Two lines had significantly enhanced tolerance to the herbicide phosphinothricin as well as to both pathogens. These results demonstrate the feasibility of introducing two potentially useful agronomic traits into carrot through genetic engineering.     

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.     

Microspore embryogenesis and production of haploid and doubled haploid plants in carrot (Daucus carota L.)

Protocols were developed for the generation of haploid and doubled haploid plants from isolated microspores of carrot (Daucus carota L.). Forty-seven carrot accessions, including six inbred lines, 11 cultivars, 20 F₁s, two BC₁F₁s, four F₂s, one F₃, and three F₄s, were screened to evaluate the genotype influence on isolated microspore embryogenesis over 4 years. Twenty-eight accessions responded by producing embryos and/or calli. A cytological analysis showed that two modes of carrot microspore embryogenesis exist: an indirect route via calli (C mode), and a direct route via embryos (E mode). Eleven accessions were in the C mode, and 17 were in both modes. The highest production rates were in 10Y25 (a European Nantes cultivar) with 27 calli and 307 embryos, and 100Q6 (a semi-Nantes F₁ hybrid) with 176 calli and 114 embryos. The time period to produce embryos or calli differed significantly between 2 and 6 months. Cold and heat pretreatment generally had a negative impact on the induction of microspore embryogenesis, but a short pretreatment showed a positive influence on some accessions. Twenty-eight lines regenerated plants from the primary individual embryos or calli of three accessions were established to analyze the ploidy level. The percentage of spontaneous diploidization showed very wide differences among the accessions and lines. Differences in leaf color intensity, leaf size, and leaf dissection were found among haploid, doubled haploid, and triploid plants.     

Development of embryoids in carrot root callus culture (Daucus carota L.)

Embryogenesis occurred in carrot root callus (Daucus carota L.) cultivated on simple synthetic medium containing IAA and 2,4-D. Embryoid development continued also during successive years when the tissue was cultivated on the same nutrient medium without those substances. Sometimes production of plants with atypical leaves was also observed. In those plants development of adventive embryoids occurred repeatedly. Result of this work confirmed reports about the organogenic potentiality of this species and about its sensitivity to some chemical substances.     

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.     

Localization of calcium during somatic embryogenesis of carrot (Daucus carota L.)

Summary The distribution of free cytosolic Ca²⁺ was studied during somatic embryogenesis of carrot using confocal scanning laser microscopy with fluo-3 as a fluorescent Ca²⁺ indicator. Chlorotetracycline fluorescence, antimonate precipitation and proton induced X-ray emission analysis were used as additional methods to confirm the results obtained with fluo-3. The process of embryogenesis was found to coincide with a rise in the level of free cytosolic Ca²⁺. The level of Ca²⁺ was low in proembryogenic masses and relatively high in later stages of embryogenesis. The highest signal was found in the protoderm of embryos from the late globular to the torpedo-shaped stage. A gradient in fluorescence intensity was often observed along the longitudinal axis of the embryos. The most conspicuous intracellular signal was found in the nucleus. Other organelles did not take up the dye and were always without fluorescence. The changes in [Ca²⁺]_c are discussed in relation to physiological processes which are known to be important during somatic embryogenesis.This article is dedicated to the memory of the late Dr. Hans-Dieter Reiss.     

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     

Overexpression of a tobacco osmotin gene in carrot (Daucus carota L.) enhances drought tolerance

Osmotin and osmotin-like proteins belong to the PR-5 pathogenesis-related group of proteins and are induced in response to various types of biotic and abiotic stresses in several plant species. Carrot was transformed with a tobacco osmotin gene that encodes a protein lacking the vacuolar-sorting motif that is composed of a 20-amino-acid sequence at the C-terminal end, under the control of the cauliflower mosaic virus 35S promoter, using Agrobacterium-mediated transformation. Transgene integration and expression were confirmed by Southern and western blot analyses, and three selected transgenic lines were evaluated for their ability to tolerate drought stress. Under drought stress conditions, all transformants exhibited slower rates of wilting compared with the wild-type plants and recovered faster when the drought stress was alleviated. Transformants showed lower levels of hydrogen peroxide accumulation, reduced lipid peroxidation and electrolyte leakage, and higher leaf water content under drought stress. Our results provide additional evidence for the protective ability of the osmotin protein against drought stress conditions and suggest a possible means to achieve tolerance against this abiotic stress in plants.     

DNA methylation and Dc8-GUS transgene expression in carrot (Daucus carota L.)

Summary DNA methylation has been associated with gene activity in differentiating and developing plant tissues. The objective of this study was to determine the involvement of methylation in the expression of a gene transferred into carrot (Daucus carota L.) tissues by particle bombardment. Expression of the Dc8-GUS gene construct in response to treatments with 5-azacytidine (S-azaC) and to in vitro methylation by methylases was investigated by histochemical assay of GUS activity. The 5-azaC treatment increased the frequency of Dc8-driven GUS expression in both calli and somatic embryos. The increase occurred with treatment either to E. coli containing the plasmid insert or to the carrot tissues before bombardment. GUS expression, increased by the 5-azaC treatment, was enhanced by ABA treatment of both calli and somatic embryos and was more prominent in the latter. Increased digestion of the 5-azaC-treated plasmid DNA with EcoRII suggested that demethylation had occurred. In vitro methylation of Dc8-GUS by methylases generally resulted in a lower frequency of GUS expression. SssI methylase completely inhibited GUS expression. The level of GUS expression was correlated with the extent of methylation of the plasmid.Abbreviations ABA Abscisic Acid - 5-azaC 5-azacytidine - GUS -glucuronidase - Dc8 carrot promoter     

Seed reserve-dependent growth responses to temperature and water potential in carrot (Daucus carota L.)

Both temperature and soil moisture vary greatly in the surface layers of the soil through which seedlings grow following germination. The work presented studied the impact of these environmental variables on post-germination carrot growth to nominal seedling emergence. The rapid pre-crook downward growth of both the hypocotyl and root was consistent with their requirement for establishment in soil drying from the surface. At all temperatures, both hypocotyl and root growth rates decreased as water stress increased and there was a very distinct temperature optimum that tended to occur at lower temperatures as water stress increased. A model based on the thermodynamics of reversible protein denaturation was adapted to include the effects of water potential in order to describe these growth rate responses. In general, the percentage of seedlings that reached the crook stage (start of upward hypocotyl growth) decreased at the extremes of the temperature range used and was progressively reduced by increasing water stress. A model was developed to describe this response based on the idea that each seedling within a population has lower and upper temperature thresholds and a water potential threshold which define the conditions within which it is able to grow. This threshold modelling approach which applies growth rates within a distribution of temperature and water potential thresholds could be used to simulate seedling growth by dividing time into suitable units.     

胡萝卜(Daucus carota L.)中主要胡萝卜素和番茄红素含量的QTL分析

以高胡萝卜素自交系P50006和HCM A.C.为亲本构建的F2群体为作图群体,对胡萝卜中α-胡萝卜素、α-胡萝卜素、总胡萝卜素和番茄红素含量进行QTL定位及遗传分析。结果表明,α、β-胡萝卜素、总胡萝卜素和番茄红素含量的广义遗传力分别为0.75、0.50、0.31和0.93。遗传图谱包含91个SRAP(Sequence-related amplified polymorphism)标记,分布于9个连锁群,总长度502.9cM,标记间平均距离5.5cM。除α-胡萝卜素含量外,α-胡萝卜素、总胡萝卜素和番茄红素含量分别检测到1个主效QTL,均为加性遗传效应,分别解释表型变异为12.79%、12.87%和14.61%。此外,α-胡萝卜素和番茄红素含量还分别检测到1对上位性QTL,最大遗传效应分别为显性×加性互作和显性×显性互作,分别解释表型变异为15.1%和6.5%。文章中与QTL连锁的分子标记可用于高胡萝卜素、番茄红素的种质筛选和聚合育种。