Haploid and Doubled Haploid Plant Production in Carrot Using Induced Parthenogenesis and Ovule Excision In Vitro.

Haploid plants have a gametophytic number of chromosomes (n) in the sporophyte. A doubled haploid (DH) plant results from doubling the chromosome set of a haploid plant, as a consequence a homozygosity plant is produced at every locus (true homozygous plant). DH plants are of great significance in breeding programs for the improvement of plants. Here we describe a protocol for the production of doubled haploid plants in carrot (Daucus carota L.) using parthenogenesis induced by wide pollination.

Differences in protodermal cell wall structure in zygotic and somatic embryos of Daucus carota (L.) cultured on solid and in liquid media.

The ultrastructure, cuticle, and distribution of pectic epitopes in outer periclinal walls of protodermal cells of Daucus carota zygotic and somatic embryos from solid and suspension culture were investigated. Lipid substances were present as a continuous layer in zygotic and somatic embryos cultured on solid medium. Somatic embryos from suspension cultures were devoid of cuticle. The ultrastructure of the outer walls of protodermis of embryos was similar in zygotic and somatic embryos from solid culture. Fibrillar material was observed on the surface of somatic embryos. In zygotic embryos, in cotyledons and root pectic epitopes recognised by the antibody JIM5 were observed in all cell walls. In hypocotyls of these embryos, these pectic epitopes were not present in the outer periclinal and anticlinal walls of the protodermis. In somatic embryos from solid media, distribution of pectic epitopes recognised by JIM5 was similar to that described for their zygotic counterparts. In somatic embryos from suspension culture, pectic epitopes recognised by JIM5 were detected in all cell walls. In the cotyledons and hypocotyls, a punctate signal was observed on the outside of the protodermis. Pectic epitopes recognised by JIM7 were present in all cell walls independent of embryo organs. In zygotic embryos, this signal was punctate; in somatic embryos from both cultures, this signal was uniformly distributed. In embryos from suspension cultures, a punctate signal was detected outside the surface of cotyledon and hypocotyl. These data are discussed in light of current models for embryogenesis and the influence of culture conditions on cell wall structure.

Alternative oxidase involvement in Daucus carota somatic embryogenesis.

Plant alternative oxidase (AOX) is a mitochondrial inner membrane enzyme involved in alternative respiration. The critical importance of the enzyme during acclimation upon stress of plant cells is not fully understood and is still an issue of intensive research and discussion. Recently, a role of AOX was suggested for the ability of plant cells to change easily its fate upon stress. In order to get new insights about AOX involvement in cell reprogramming, quantitative real-time polymerase chain reaction (PCR) and inhibitor studies were performed during cell redifferentiation and developmental stages of Daucus carota L. somatic embryogenesis. Transcript level analysis shows that D. carota AOX genes (DcAOX1a and DcAOX2a) are differentially expressed during somatic embryogenesis. DcAOX1a shows lower expression levels, being mainly down-regulated, whereas DcAOX2a presented a large up-regulation during initiation of the realization phase of somatic embryogenesis. However, when globular embryos start to develop, both genes are down-regulated, being this state transient for DcAOX2a. In addition, parallel studies were performed using salicylhydroxamic acid (SHAM) in order to inhibit AOX activity during the realization phase of somatic embryogenesis. Embryogenic cells growing in the presence of the inhibitor were unable to develop embryogenic structures and its growth rate was diminished. This effect was reversible and concentration dependent. The results obtained contribute to the hypothesis that AOX activity supports metabolic reorganization as an essential part of cell reprogramming and, thus, enables restructuring and de novo cell differentiation.

Identification and characterization of bZIP-type transcription factors involved in carrot (Daucus carota L.) somatic embryogenesis.

Seed dormancy is an important adaptive trait that enables seeds of many species to remain quiescent until conditions become favorable for germination. Abscisic acid (ABA) plays an important role in these developmental processes. Like dormancy and germination, the elongation of carrot somatic embryo radicles is retarded by sucrose concentrations at or above 6%, and normal growth resumes at sucrose concentrations below 3%. Using a yeast one-hybrid screening system, we isolated two bZIP-type transcription factors, CAREB1 and CAREB2, from a cDNA library prepared from carrot somatic embryos cultured in a high-sucrose medium. Both CAREB1 and CAREB2 were localized to the nucleus, and specifically bound to the ABA response element (ABRE) in the Dc3 promoter. Expression of CAREB2 was induced in seedlings by drought and exogenous ABA application; whereas expression of CAREB1 increased during late embryogenesis, and reduced dramatically when somatic embryos were treated with fluridone, an inhibitor of ABA synthesis. Overexpression of CAREB1 caused somatic embryos to develop slowly when cultured in low-sucrose medium, and retarded the elongation of the radicles. These results indicate that CAREB1 and CAREB2 have similar DNA-binding activities, but play different roles during carrot development. Our results indicate that CAREB1 functions as an important trans-acting factor in the ABA signal transduction pathway during carrot somatic embryogenesis.

Possible involvement of DNA methylation on expression regulation of carrot LEC1 gene in its 5'-upstream region.

DNA methylation plays important roles in various developmental processes in many organisms. In carrots, the treatment of embryogenic cells (ECs) with DNA methylation inhibitors induces hypomethylation and blocks somatic embryogenesis. CARROT-LEAFY COTYLEDON 1 (C-LEC1) is an important transcription factor for embryo development that shows embryo-specific expression in ECs and somatic and zygotic embryos. However, the regulation of embryo-specific transcription factor genes such as C-LEC1 in plants is not well understood. In this study, we used embryogenic carrot cells (Daucus carota L. cv. US-Harumakigosun) to investigate the DNA methylation status of the embryogenesis-related genes C-LEC1, Carrot ABA INSENSITIVE 3 (C-ABI3), and Daucus carota Embryogenic cell protein 31 (DcECP 31) during the transition from embryogenesis to vegetative growth. The C-LEC1 promoter region showed a reduced level of DNA methylation during somatic embryogenesis followed by an increase during the transition from embryonic to vegetative growth. To test whether the increased level of DNA methylation down-regulates C-LEC1 expression, RNA-directed DNA methylation (RdDM) was used to induce the hypermethylation of two segments of the C-LEC1 5'-upstream region: Regions 1 and 2, corresponding to nucleotides -1,904 to -1,272 and -896 to -251, respectively. When the hypermethylation of Region 1 was induced by RdDM, C-LEC1 expression was reduced in the transgenic ECs, indicating a negative correlation between DNA methylation and C-LEC1 expression. In contrast, the hypermethylation of Region 2 did not greatly affect C-LEC1 expression. Based on these results, we hypothesize that DNA methylation may be involved in the control of C-LEC1 expression during carrot embryogenesis.

Determination of the best temperature and dry condition in carrot primed-seeds.

Seed priming and drying condition effects were investigated immediately after seed-priming and 9 week after the storage. In this experiment, carrot seeds of 'Forto C.V.' were used. These seeds were individually primed for 10 days at 20 degrees C and in PEG (6000) (273 g L(-1)) and KNO3 (200 mmol) solutions. Then they were dried for 1 and 2 h at 15, 25 and 30 degrees C, respectively. One part of the seeds was stored at 5 degrees C in RH/45% For 9 week. Chemical priming effects, drying temperature as well as germination temperature on different traits especially germination percentage were significant. However, drying time had no significant effect on germination percentage after storage period. PEG priming and drying at 25 degrees C for 2 h provided the best condition for germination percentage. Using the best material for pre-priming, along with suitable drying management with appropriate quality and good conditions of the storage is important.

A carrot G-box binding factor-type basic region/leucine zipper factor DcBZ1 is involved in abscisic acid signal transduction in somatic embryogenesis.

Carrot (Daucus carota) somatic embryogenesis has been extensively used as an experimental system for studying embryogenesis. In maturing zygotic embryos, abscisic acid (ABA) is involved in acquisition of desiccation tolerance and dormancy. On the other hand, somatic embryos contain low levels of endogenous ABA and show desiccation intolerance and lack dormancy, but tolerance and dormancy can be induced by exogenous application of ABA. In ABA-treated carrot embryos, some ABA-inducible genes are expressed. We isolated the Daucus carota bZIP1 (DcBZ1) gene encoding a G-box binding factor-type basic region/leucine zipper (GBF-type bZIP) factor from carrot somatic embryos. The expression of DcBZ1 was detected in embryogenic cells, non-embryogenic cells, somatic embryos, developing seeds, seedlings, and true leaves. Notably, higher expression was detected in embryogenic cells, true leaves, and seedlings. The expression of DcBZ1 increased in seedlings and true leaves after ABA treatment, whereas expression was not affected by differences in light conditions. During the development of zygotic and somatic embryos, increased expression of DcBZ1 was commonly detected in the later phase of development. The recombinant DcBZ1 protein showed specific binding activity to the two ABA-responsive element-like motifs (motif X and motif Y) in the promoter region of the carrot ABA-inducible gene according to results from an electrophoretic mobility shift assay. Our findings suggest that the carrot GBF-type bZIP factor, DcBZ1, is involved in ABA signal transduction in embryogenesis and other vegetative tissues.

Isolation of carrot Argonaute1 from subtractive somatic embryogenesis cDNA library.

Carrot Argonaute1 (C-Ago1) was isolated from a subtractive cDNA library to obtain somatic embryogenesis related genes. C-Ago1 has three conserved domains, which are found in all other Argonautes. C-Ago1 has specific expression during somatic embryogenesis, which indicates that microRNA gene expression controlling system is required for somatic embryogenesis.

Identification and characterization of carrot HAP factors that form a complex with the embryo-specific transcription factor C-LEC1.

C-LEC1, an orthologue of Arabidopsis LEC1, is thought to be an essential transcriptional activator required for normal development during the early and late phases of embryogenesis. C-LEC1 is similar in sequence to the HAP3 subunits of other organisms. To understand C-LEC1 function better, a cDNA library of carrot somatic embryos was screened for factors that form complexes with C-LEC1. Two carrot HAP5 homologues and two carrot HAP2 homologues were identified; these factors have significant sequence similarity to the conserved regions of HAP5 and HAP2, respectively. Some of these proteins form heterotrimeric complexes that bind specifically to DNA fragments containing a CCAAT sequence in vitro. The results suggest that C-LEC1 is a component of the CCAAT-box-binding factor and forms a complex with C-HAP2B and C-HAP5A or C-HAP5B that regulates gene expression during carrot embryo development.

KDC2, a functional homomeric potassium channel expressed during carrot embryogenesis.

In Daucus carota, the model system for embryogenesis, it has been demonstrated that potassium and K(+) selective channels are involved in embryo development. Here, we report the isolation and cloning of a new carrot Shaker-like potassium channel, potassium D. carota channel 2 (KDC2), whose expression pattern during somatic embryogenesis proceeds along with the establishment of the polar axes and the settlement of the hypocotyl region. In plants, KDC2 transcript is localized at the shoot level, in the epidermis and guard cells, similarly to its Arabidopsis homolog KAT1. Electrophysiological assays indicated KDC2 as the first carrot subunit able to form homomeric functional channels in Xenopus oocytes, with properties similar to those of Arabidopsis KAT1.

Abscisic acid and stress treatment are essential for the acquisition of embryogenic competence by carrot somatic cells.

Studies of carrot embryogenesis have suggested that abscisic acid (ABA) is involved in somatic embryogenesis. A relationship between endogenous ABA and the induction of somatic embryogenesis was demonstrated using stress-induced system of somatic embryos. The embryonic-specific genes C-ABI3 and embryogenic cell proteins (ECPs) were expressed during stress treatment prior to the formation of somatic embryos. The stress-induction system for embryogenesis was clearly distinguished by two phases: the acquisition of embryogenic competence and the formation of a somatic embryo. Somatic embryo formation was inhibited by the application of fluridone (especially at 10(-4) M), a potent inhibitor of ABA biosynthesis, during stress treatment. The inhibitory effect of fluridone was nullified by the simultaneous application of fluridone and ABA. The level of endogenous ABA increased transiently during stress. However, somatic embryogenesis was not significantly induced by the application of only ABA to the endogenous level, in the absence of stress. These results suggest that the induction of somatic embryogenesis, in particular the acquisition of embryogenic competence, is caused not only by the presence of ABA but also by physiological responses that are directly controlled by stresses.

Static suspension culture of carrot somatic embryos.

Three culture methods of carrot somatic embryos were compared: conventional shake flask culture, static suspension culture using Erlenmeyer flasks, and static suspension culture using petri dishes. Compared with the former two cultures, the last one resulted in an enhanced ratio of torpedo-shaped embryos and suppressed cotyledonary-stage embryo formation.

Construction of a micro-library enriched with genomic replication origins of carrot somatic embryos by laser microdissection.

In this paper, we describe an effective method for constructing a micro-library enriched with chromosomal DNA replication origins. Carrot (Daucus carota L.) somatic embryos at early globular stage were incubated for 15 min in the presence of bromodeoxyuridine (BrdU) to pulse label newly synthesized DNA strands. Nuclei were isolated from the cells, and the DNA was extracted on microscopic slides. DNA fibers spread on slides were visualized using anti-BrdU and FITC-conjugated secondary antibodies. DNA regions where BrdU was incorporated were clearly visualized under a fluorescent microscope as dots on DNA fibers. Regions of DNA fiber containing many fluorescent dots should contain replicons in them. DNA fibers showing many fluorescence dots, or replicons were easily cut and collected using a laser microdissection system equipped with a pulse laser beam. DNA fragments containing many replicons were able to be collected with an efficiency of 20-30 DNA fragments per 1 h. Using degenerate oligonucleotide primed PCR, fragments were randomly amplified from the microdissected fragments, and subcloned to construct a micro-library. This is the first report of the application of a laser microdissection technique for constructing a micro-library enriched with replication origins of chromosomal DNA, although there were some reports on laser microdissection of chromosomes. The simple procedure established here should open up a new application of laser optics.

Possible involvement of abscisic acid in the induction of secondary somatic embryogenesis on seed-coat-derived carrot somatic embryos.

When seed coats (pericarps) were picked from 14-day-old carrot (Daucus carota) seedlings and cultured on agar plates, embryogenic cell clusters were produced very rapidly at a high frequency on the open side edge. Embryo induction progressed without auxin treatment; indeed treatment caused the formation of non-embryogenic callus. The embryogenic tissues (primary embryos) developed normally until the torpedo stage; however, after this a number of secondary somatic embryos were produced in the hypocotyl and root regions. "Tertiary" embryos were formed on some of the secondary embryos, but many developed into normal plantlets. The primary embryos contained significantly higher levels of abscisic acid (ABA) than the hypocotyl-derived normal and seed-coat-derived secondary embryos. Fluridone inhibited the induction of secondary embryogenesis, while exogenously supplied ABA induced not only "tertiary" embryogenesis on the seed-coat-derived secondary embryos, but also secondary embryos on the hypocotyl-derived normal somatic embryos. These results indicate that ABA is one of the important endogenous factors for the induction of secondary embryogenesis on carrot somatic embryos. Higher levels of indole-3-acetic acid (IAA) in primary embryos also suggest the presence of some concerted effect of ABA and IAA on the induction of secondary embryogenesis in primary embryos.

Formation of embryogenic cell clumps from carrot epidermal cells is suppressed by 5-azacytidine, a DNA methylation inhibitor.

Using a direct somatic embryogenesis system in carrot, we examined the role of DNA methylation in the change of cellular differentiation state, from somatic to embryogenic. 5-Azacytidine (aza-C), an inhibitor of DNA methylation suppressed the formation of embryogenic cell clumps from epidermal carrot cells. Aza-C also downregulated the expression of DcLEC1c, a LEC1-like embryonic gene in carrot, during morphogenesis of embryos. A carrot DNA methyltransferase gene, Met1-5 was expressed transiently after the induction of somatic embryogenesis by 2,4-dichlorophenoxyacetic acid (2,4-D), before the formation of embryogenic cell clumps. These findings suggested the significance of DNA methylation in acquiring the embryogenic competence in somatic cells in carrot.

Abscisic acid-inducible nuclear proteins bind to bipartite promoter elements required for ABA response and embryo-regulated expression of the carrot Dc3 gene.

The carrot (Daucus carota L.) lea-class gene Dc3 is expressed in developing seeds and in vegetative tissues subject to drought and treatment with exogenous abscisic acid (ABA). Cis regulatory elements involved in seed-specific expression and in response to ABA were identified in transgenic tobacco (Nicotiana tabacum L.) using beta-glucuronidase (GUS) reporter gene constructs containing a series of deletion and orientation mutants of the Dc3 promoter. These experiments demonstrated that the Dc3 promoter is comprised of a proximal promoter region (PPR) and a distal promoter region (DPR). TCGTGT motifs in the DPR in combination with the PPR comprise a novel, bipartite ABA module in the Dc3 gene promoter. The PPR contains cis-acting elements responsible for the developmental regulation of Dc3 expression in seeds. Five similar sequence motifs with the consensus ACACgtGCa were identified in the PPR. Both DPR and PPR interact with common nuclear proteins that are present in embryos and are inducible by ABA in vegetative tissues.

Potassium and carrot embryogenesis: are K+ channels necessary for development?

The expression pattern of the KDC1 gene, coding for an inwardly-rectifying K(+) channel of Daucus carota , is described in several embryo stages and seedling tissues. Relative quantitative RT-PCR experiments indicated that, during (somatic) embryonic development, the KDC1 transcript appears as early as the globular stage and that the transcript level remains constant throughout the successive heart and torpedo stages. Thereafter, the KDC1 transcript is preferentially expressed in plant roots, but is also present in other tissues, and in particular, in the shoot apical meristem. In situ hybridisation experiments showed that in embryos KDC1 mRNA is detectable preferentially in protoderm cells with a stage dependent expression pattern. At later times, the hybridisation signal is particularly evident in root hairs, root epidermis and endodermis, but is also observed in single cell layers corresponding to L1 of the shoot apical meristem and leaf primordia. Promoter studies with the beta -glucuronidase reporter gene confirm preferential expression of KDC1 in embryo protoderm cells and in plant root epidermis and root hairs. Western blot analysis of embryonic proteins and immunolocalisation experiments on somatic embryos sections revealed the presence of KDC1 during embryo development. Consistent with these observations, patch-clamp experiments performed on protoplasts isolated from embryos at the torpedo stage demonstrated the presence of functional inward rectifying K(+) channels. This is the first report on the expression of a plant ion channel during embryo development.

Isolation of putative glycoprotein gene from early somatic embryo of carrot and its possible involvement in somatic embryo development.

Somatic embryogenesis is a unique process in plant cells. For example, embryogenic cells (EC) of carrot (Daucus carota) maintained in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) regenerate whole plants via somatic embryogenesis after the depletion of 2,4-D. Although some genes such as C-ABI3 and C-LEC1 have been found to be involved in somatic embryogenesis, the critical molecular and cellular mechanisms for somatic embryogenesis are unknown. To characterize the early mechanism in the induction of somatic embryogenesis, we isolated genes expressed during the early stage of somatic embryogenesis after 2,4-D depletion. Subtractive hybridization screening and subsequent RNA gel blot analysis suggested a candidate gene, Carrot Early Somatic Embryogenesis 1 (C-ESE1). C-ESE1 encodes a protein that has agglutinin and S-locus-glycoprotein domains and its expression is highly specific to primordial cells of somatic embryo. Transgenic carrot cells with reduced expression of C-ESE1 had wide intercellular space and decreased polysaccharides on the cell surface and showed delayed development in somatic embryogenesis. The importance of cell-to-cell attachment in somatic embryogenesis is discussed.

Sucrose regulates elongation of carrot somatic embryo radicles as a signal molecule.

Elongation of carrot somatic embryo radicles was inhibited by sucrose at or above 5% (145 mM). This effect would not be released until the sucrose concentration was lowered again. Morphological and cytological studies as well as determination of ABA content and analysis of the expression mode of a Lea gene, all point to its similarity to natural dormancy and germination of seeds. Use of monosaccharides (glucose and fructose), other disaccharide (maltose), and isomolar concentration of osmotica (mannitol and sorbitol), did not show similar regulatory effect. It is thus clear that the regulatory effect is not a result of simple osmotic stress. Hexokinase inhibitors such as glucosamine and N -acetyl-glucosamine did not exert any influence on the regulation-deregulation effects of sucrose. Mannose, which inhibits germination of Arabidopsis seeds, did not prevent carrot somatic embryo radicles from elongating. It is thus inferred that this sucrose-signaling pathway may be independent of hexokinase. As a first step to understand the molecular mechanism of this process, a carrot sucrose transporter gene ( cSUT ) expressed in the embryos and roots specifically was isolated. Studies on transformed yeast mutant with cSUT cDNA identified its sucrose transport activity. Northern hybridization and gel retardation experiment revealed that there is a marked increase in expression of cSUT at the beginning of somatic embryo germination, and this is attributed to regulation on the level of transcription. This suggested the possibility that cSUT has an important role in this sucrose signal regulation system.

Sequential Development of Cysteine Proteinase Activities and Gene Expression during Somatic Embryogenesis in Carrot.

Three bands of proteinase activity (Rf values of 0.5, 0.6, and 0.7) were detected on activity-stained gels after native gel electrophoresis of carrot (Daucus carota L. cv US-Harumakigosun) suspension cells. After the induction of somatic embryogenesis, one activity band (0.7 band) rapidly disappeared; the 0.6 band was absent at the heart-shaped embryo stage. However, the intensity of the 0.5 band increased during embryogenesis. An additional band (0.25 band) appeared after the torpedo-shaped stage. Three bands (0.25, 0.5, and 0.6) were also detected in zygotic seeds. Two activity bands (0.5 and 0.6) were classified as cysteine proteinases based on sensitivities to N-Ethylmaleimide (NEM) or L-3-trans-Carboxyoxirane-2-Carbonyl-L-Leucyl-Agmatine (E-64). To find candidate genes for the cysteine proteinases, we cloned seven cDNAs encoding putative cysteine proteinases from suspension cells and developing somatic embryos. The expression patterns of the seven genes were categorized into three types (Type A, mRNAs increase concomitantly with somatic embryogenesis; Type B, mRNAs decrease quickly in organized cells; Type C, no significant change in transcript level during somatic embryogenesis).