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1.
Takahata K Takeuchi M Fujita M Azuma J Kamada H Sato F 《Plant & cell physiology》2004,45(11):1658-1668
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. 相似文献
2.
Yamamoto N Kobayashi H Togashi T Mori Y Kikuchi K Kuriyama K Tokuji Y 《Journal of plant physiology》2005,162(1):2493-54
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. 相似文献
3.
Summary Various aspects of somatic embryogenesis in carrot suspension cultures were reviewed on the basis of results obtained in our
laboratory. We have established high-frequency and synchronous somatic embryogenesis systems needed for biochemical and molecular
analysis. Using these systems, four phases of somatic embryogenesis were identified. The importance of expression of polarities
in these phases, particularly from single cells to embryogenic cell clusters, in determining somatic embryogenesis, is emphasized.
At the molecular level, genes expressed during somatic embryogenesis were described, and they were classified into three categories:
(1) genes involved in cell division, (2) genes involved in organ formation and (3) genes specific for the process of somatic
embryogenesis. From the results obtained, it is concluded that discrete developmental phases in carrot somatic embryogenesis
are characterized by distinct biochemical and molecular events, but much remains to be understood. 相似文献
4.
Effect of 2,4-dichlorophenoxyacetic Acid on the expression of embryogenic program in carrot 总被引:3,自引:1,他引:2
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Embryogenesis in a wild carrot cell line, W001C, can start and progress up to the first morphogenetic stage (the globular-stage embryo) in 2,4-dichloropenoxyacetic acid (2,4-D). To clarify the quantitative effect of 2,4-D on this cell line, morphological and biochemical criteria have been used to monitor embryogenesis in the presence of increasing concentrations of 2,4-D. The biochemical criteria are the ability to inactivate cycloheximide and the expression of an embryogenic polypeptide E1. The results show that 2,4-D can affect embryogenesis in a quantitative manner but never fully suppresses embryogenesis unless it is coupled with high cell density. 相似文献
5.
K Sankara Rao 《Journal of biosciences》1996,21(6):827-841
The overall architectural pattern of the mature plant is established during embryogenesis. Very little is known about the
molecular processes that underlie embryo morphogenesis. Last decade has, nevertheless, seen a burst of information on the
subject. The synchronous somatic embryogenesis system of carrot is largely being used as the experimental system. Information
on the molecular regulation of embryogenesis obtained with carrot somatic embryos as well as observations on sandalwood embryogenic
system developed in our laboratory are summarized in this review. The basic experimental strategy of molecular analysis mostly
relied on a comparison between genes and proteins being expressed in embryogenic and non-embryogenic cells as well as in the
different stages of embryogenesis. Events such as expression of totipotency of cells and establishment of polarity which are
so critical for embryo development have been characterized using the strategy. Several genes have been identified and cloned
from the carrot system. These include sequences that encode certain extracellular proteins (EPs) that influence cell proliferation
and embryogenesis in specific ways and sequences of the abscisic acid (ABA) inducible late embryogenesis abundant (LEA) proteins
which are most abundant and differentially expressed mRNAs in somatic embryos. That LEAs are expressed in the somatic embryos
of a tree flora also is evidenced from studies on sandalwood. Several undescribed or novel sequences that are enhanced in
embryos were identified. A sequence of this nature exists in sandalwood embryos was demonstrated using aCuscuta haustorial (organ-specific) cDNA probe. Somatic embryogenesis systems have been used to assess the expression of genes isolated
from non-embryogenic tissues. Particular attention has been focused on both cell cycle and histone genes 相似文献
6.
Developmental regulation of polyamine metabolism in growth and differentiation of carrot culture 总被引:5,自引:0,他引:5
Polyamine levels and the activities of two polyamine biosynthetic enzymes, arginine decarboxylase (EC 4.1.1.19) and S-adenosylmethionine decarboxylase (EC 4.1.1.50), were determined during somatic embryogenesis of carrot (Daucus carota L.) cell cultures. Embryogenic cultures showed severalfold increases in polyamine levels over nondifferentiating controls. A mutant cell line that failed to form embryos but grew at the same rate as the wild-type line also failed to show increases in polyamine levels, thus providing evidence that this increased polyamine content was in fact associated with the development of embryos. Furthermore, inhibition of these increases in polyamines caused by drugs inhibited embryogenesis and the effect was reversible with spermidine. The activities of arginine decarboxylase and Sadenosylmethionine decarboxylase were found to be suppressed by auxin; however, the specific effects differed between exogenous 2,4-dichlorophenoxyacetic acid and endogenous indole-3-acetic acid. The results indicate that increased polyamine levels are required for cellular differentiation and development occurring during somatic embryogenesis in carrot cell cultures.Abbreviations ADC
arginine decarboxylase
- 2,4-D
2,4-dichlorophenoxyacetic acid
- DFMA
difluoromethylarginine
- DCHAS
dicyclohexylammonium sulfate
- SAMDC
S-adenosylmethionine decarboxylase 相似文献
7.
8.
Induction of anthocyanin synthesis occurs during metabolic differentiation in carrot suspension cultured cells grown in medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D), and is closely correlated with embryogenesis. Anthocyanin synthesis may also be induced by light-irradiation under different culture conditions. The phenylalanine ammonia-lyase (PAL) gene (TRN-PAL), which was transiently induced by the transfer effect, was also rapidly induced after light-irradiation. However, TRN-PAL was not involved in anthocyanin synthesis. A second PAL gene, ANT-PAL, was involved in anthocyanin synthesis. ANT-PAL was induced during metabolic differentiation in medium lacking 2,4-D parallel with the induction of chalcone synthase (CHS). PAL genes in the carrot genome are expressed differentially depending on the nature of the environmental stimulus, e.g. transfer effect and light, and other parameters which also affect anthocyanin synthesis.Abbreviations CHS
chalcone synthase
- 2,4-D
2,4-dichlorophenoxyacetic acid
- GUS
-glucuronidase
- Luc
firefly luciferase
- PAL
phenylalanine ammonia-lyase
- UV
ultraviolet 相似文献
9.
Inhibition by 2,4-D of somatic embryogenesis in carrot as explored by its reversal by difluoromethylornithine 总被引:1,自引:0,他引:1
The development of somatic embryos is, in many plants, inhibited by 2,4-dichlorophenoxyacetic acid (2,4-D) and other auxins. The finding that difluoromethylornithine (DFMO) can counteract this inhibition has been used to test some of the hypotheses for the mechanism of inhibition.
Inhibition of somatic embryogenesis in carrot ( Daucus carota L.) by exogenous ethylene (from ethephon), antioxidants (ascorbic acid and glutathione), ethanol/acetaldehyde and abscisic acid was not counteracted by DFMO, indicating that the inhibitory effect of 2,4-D is not manifest through the formation of these compounds. Embryogenesis was abolished by micromolar concentrations of the polar auxin transport inhibitors 2, 3, 5-triiodobenzoic acid (TIBA), N-1-naphthylphthalamic acid (NPA) and 9-hydroxyfluorene-9-carboxylic acid (HFCA). This inhibition was counteracted to a considerable extent by DFMO. Inhibition by relatively high concentrations of the antiauxin 2-( p -chlorophenoxy)-isobutyric acid (CPIB), which does not affect polar auxin transport, was in contrast not counteracted by DFMO. These findings indicate that exogenous auxins may inhibit embryogenesis by interfering with the ability of postglobular embryos to set up internal auxin gradients necessary for polarized growth. 相似文献
Inhibition of somatic embryogenesis in carrot ( Daucus carota L.) by exogenous ethylene (from ethephon), antioxidants (ascorbic acid and glutathione), ethanol/acetaldehyde and abscisic acid was not counteracted by DFMO, indicating that the inhibitory effect of 2,4-D is not manifest through the formation of these compounds. Embryogenesis was abolished by micromolar concentrations of the polar auxin transport inhibitors 2, 3, 5-triiodobenzoic acid (TIBA), N-1-naphthylphthalamic acid (NPA) and 9-hydroxyfluorene-9-carboxylic acid (HFCA). This inhibition was counteracted to a considerable extent by DFMO. Inhibition by relatively high concentrations of the antiauxin 2-( p -chlorophenoxy)-isobutyric acid (CPIB), which does not affect polar auxin transport, was in contrast not counteracted by DFMO. These findings indicate that exogenous auxins may inhibit embryogenesis by interfering with the ability of postglobular embryos to set up internal auxin gradients necessary for polarized growth. 相似文献
10.
Analysis of the rolC promoter region involved in somatic embryogenesis-related activation in carrot cell cultures. 总被引:2,自引:0,他引:2
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In cell cultures of carrot (Daucus carota L.), somatic embryogenesis can be induced by transferring cells from a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) to one devoid of 2,4-D. Previous analysis of transgenic carrot cells containing the 5' non-coding sequence of the Ri plasmid rolC and a structural gene for bacterial beta-glucuronidase (uidA) has shown that the chimeric gene is actively expressed after induction of somatic embryogenesis. In this study, we demonstrate that activation of the rolC promoter is dependent on the process of embryo development but not on the duration of the cell culture in 2,4-D-free medium. We also analyzed the cis region of the rolC promoter that is responsible for somatic embryogenesis-related activation (SERA), namely relatively low beta-glucuronidase (GUS) activity in calli and proembryogenic masses (PEM) and high GUS activity in heart- and torpedo-stage embryos. When the -255-bp region of the rolC gene was used, SERA was retained. Internal deletions within this -255-bp region did not alter SERA by the rolC promoter. Furthermore, when a rolC promoter fragment (-848 to -94 bp) was fused to the cauliflower mosaic virus (CaMV) 35S core region (-90 to +6 bp), it conferred relatively low GUS activity in calli and PEM but high GUS activity in heart and torpedo embryos. When -848 to -255-bp or -255- to -94-bp fragments of the rolC promoter were fused to the same CaMV 35S core region, GUS activity patterns were not related to somatic embryogenesis. These results suggest that the combination of several regulatory regions in the rolC promoter may be required for SERA in carrot cell cultures. 相似文献
11.
Summary To date it has been accepted that preprophase bands of microtubules (PPBs) either do not precede cell division or do so inconsistently in suspension cultures, the assumption being that such cultures proliferate in an unorganized state in which placement of cell plates is not regulated by the PPB system that is widespread in organized tissues. Using indirect immunofluorescence microscopy with antitubulin, the relative frequency of occurrence of PPBs in enzymatically separated cells from root tips and suspension cultures of carrot and tobacco, was quantified by taking the ratio of the number of PPBs: phragmoplast. This ratio was termed the PPB index.One carrot suspension culture proliferated in a medium containing 2,4-Dichlorophenoxyacetic acid (2,4-D), and recognizable stages in somatic embryogenesis formed when 2,4-D was removed from the medium. Another carrot suspension culture was nonembryogenic and removal of 2,4-D resulted in a reduction of cell division and increase in cell elongation. The tobacco culture was a cytokinin habituated cell line and also required 2,4-D to maintain cell division. It ceased proliferation, and cell elongation took place if 2,4-D was removed.The PPB index in the root tips from both species, and in both types of carrot suspension culture was approximately the same but was approx. 15-fold lower in the tobacco suspension. PPBs in the tobacco suspension were atypical in structure as well as sparse in numbers. The PPB index allows quantitative comparisons between different tissues to be made. The low PPB index and the irregular PPBs in the tobacco suspension correlates with its inability to undergo organized morphogenesis and generate spatially defined cell lineages upon 2,4-D removal. In contrast, the high PPB index in the carrot suspension cultures correlates with their potential for organized embryo formation, whether or not that potential is realized by withdrawal of 2,4-D. However, their high PPB index is not obligatorily coupled to embryogenesis. 相似文献
12.
Somatic embryogenesis induced by the simple application of abscisic acid to carrot (Daucus carota L.) seedlings in culture 总被引:3,自引:0,他引:3
Seedlings of carrot (Daucus carota L. cv. Red Cored Chantenay) formed somatic embryos when cultured on medium containing abscisic acid (ABA) as the sole source
of growth regulator. The number of embryos per number of seedlings changed depending on the concentration of ABA added to
the medium, with a maximum embryo number at 1 × 10−4 M ABA. Seedling age was critical for response to exogenous ABA; no seedling with a hypocotyl longer than 3.0 cm was able to
form an embryo. Removal of shoot apices from seedlings completely inhibited the embryogenesis induced by application of exogenous
ABA, suggesting that the action of ABA requires some substance(s) that is translocated basipetally from shoot apices through
hypocotyls. Histologically, somatic embryos shared common epidermal cells and differentiated not through the formation of
embryogenic cell clumps, but directly from epidermal cells. These morphological traits are distinct from those of embryogenesis
via formation of embryogenic cell clumps, which has been found in embryogenic carrot cultures established using 2,4-dichlorophenoxyacetic
acid or other auxins. These results suggest that ABA acts as a signal substance in stress-induced carrot seedling somatic
embryogenesis.
Received: 22 April 2000 / Accepted: 8 June 2000 相似文献
13.
The process of carrot (Daucus carota L.) somatic embryogenesis is highly sensitive to exogenously added ethanol, since 5 mM ethanol inhibits this process by 50%, whereas the growth of proliferating carrot cells is inhibited to the same extent by 20 mM ethanol. This is consistent with the fact that proliferating cultures produce ethanol and release it into the medium at concentrations up to 20 mM, whereas embryogenic culture medium contains less than 1 mM ethanol. Data are presented showing the influence of cell density and 2,4-dichlorophenoxyacetic acid on ethanol production and on the presence of an alcohol-dehydrogenase (EC 1.1.1.1.) inactivator in carrot embryos.Abbreviations ADH
alcohol dehydrogenase
- 6-BAP
6-benzylaminopurine
- 2,4-D
2,4-dichlorophenoxyacetic acid
- DTT
dithiothreitol
- FW
fresh weight 相似文献
14.
Phytosulphokine-{alpha}, a peptidyl plant growth factor, stimulates somatic embryogenesis in carrot 总被引:1,自引:0,他引:1
Kobayashi T; Eun C; Hanai H; Matsubayashi Y; Sakagami Y; Kamada H 《Journal of experimental botany》1999,50(336):1123-1128
Phytosulphokine- (PSK-) is the first chemically characterized peptide
that acts as a plant growth factor. It stimulates the proliferation of
asparagus and rice cells, but no information is yet available on its
effects on plant morphogenesis. The effects of PSK- on somatic
embryogenesis in carrot (Daucus carota L.) were
examined. PSK-, when added to the induction medium for somatic
embryogenesis, increased the number of somatic embryos. The chemical
analogues [2-5]PSK- and tyrosine sulphate ester (Tyr-SO3 H), which
have been used as negative controls in other systems, had no effect.
Moreover the proliferation of cells during somatic embryogenesis was also
enhanced by PSK- these results indicate that PSK-
enhanced cell division and, as a consequence, stimulated carrot somatic
embryogenesis. PSK- also stimulated the proliferation of
embryogenic cells in medium that contained 2,4-dichlorophenoxyacetic acid
(2,4-D), in which somatic embryos did not form, as well as the
proliferation of non-embryogenic cells (cells that had lost the ability to
form somatic embryos) in medium without 2,4-D. These results indicate that
PSK- has a stimulatory effect on cell division generally in carrot
cell cultures.Key words: Daucus carota, plant growth
factor, somatic embryogenesis, sulphated peptide.
相似文献
15.
Isolation and characterization of a diverse set of genes from carrot somatic embryos. 总被引:4,自引:0,他引:4
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The early events in plant embryogenesis are critical for pattern formation, since it is during this process that the primary apical meristems and the embryo polarity axis are established. However, little is known about the molecular events that are unique to the early stages of embryogenesis. This study of gene expression during plant embryogenesis is focused on identifying molecular markers from carrot (Daucus carota) somatic embryos and characterizing the expression and regulation of these genes through embryo development. A cDNA library, prepared from polysomal mRNA of globular embryos, was screened using a subtracted probe; 49 clones were isolated and preliminarily characterized. Sequence analysis revealed a large set of genes, including many new genes, that are expressed in a variety of patterns during embryogenesis and may be regulated by different molecular mechanisms. To our knowledge, this group of clones represents the largest collection of embryo-enhanced genes isolated thus far, and demonstrates the utility of the subtracted-probe approach to the somatic embryo system. It is anticipated that many of these genes may serve as useful molecular markers for early embryo development. 相似文献
16.
Summary Electrophoretic analysis of isozymal differences was performed with extracts of wild carrot (Daucus carota L.) cells, grown in the presence and absence of 2,4-dichlorophenoxyacetic acid (2,4-D). There were no differences in the
patterns of malate dehydrogenase, acid phosphatase, aspartate aminotransferase, and γ-glutamyl transferase. Quantitative differences
in peroxidase isozymes were detected, the plus 2,4-D cultures having lower activities. Esterase patterns were similar, but
there were differences in individual isozyme activities and an additional form present in the minus 2,4-D cells. the greatest
differences were in patterns of glutamate dehydrogenase with the minus 2,4-D cultures containing only the slowly migrating
isozymes. The changes in glutamate dehydrogenase, as revealed by isozyme changes, together with the requirement for ammonia
in embryogenesis, suggests that this enzyme may be associated with differentiation in wild carrot cells. 相似文献
17.
Synchronization of somatic embryogenesis at high frequency is a useful system for the mass production of embryos. Many attempts have been carried out, however, it was difficult to obtain the system in which most of the initial embryogenic cells or cell clusters synchronously differentiate to embryos. In carrot suspension cultures, high frequency, synchronous embryogenesis systems (following three systems) have been established.(1) Small spherical single cells from suspension cultures obtained by sieving and density gradient centrifugation in Percoll solutions differentiated to embryogenic cell clusters at high frequency when they were cultured in a medium containing 2,4-dichlorophenoxyacetic acid (0.05 micromolar), zeatin (1 micromolar) and mannitol (0.2 molar). (2) Embryogenic cell clusters from suspension cultures obtained by sieving, density gradient centrifugation in Ficoll solutions, and subsequent centrifugation at a low speed for a short time synchronously differentiated to embryos, especially globular embryos at high frequency, when they were cultured in a medium containing zeatin (0.1 micromolar) but no auxin. (3) Embryogenic cell clusters obtained by above method are cultured at cell densities of 2×103 cell clusters ml-1. Globular embryos which were sieved from embryos induced synchronously differentiated to torpedo-shaped embryos at high frequency when they were cultured at densities below 150 globular embryos ml-1.Using these systems, the whole process of embryogenesis from single cells to whole plants could be synchronously induced at high frequency.Abbreviations ABA
abscissic acid
- 2,4-d
2,4-dichlorophenoxyacetic acid
- GA3
gibberellin A3
- IAA
indoleacetic acid
- NAA
naphthylacetic acid 相似文献
18.
Patterns of ionic currents accompanying shape and surface changes during growth of cell clusters from carrot suspension cultures were examined using a vibrating probe. Electrical polarity was established in clusters undergoing apparently disorganized proliferation in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D). This electrical polarity is similar to that found in organized somatic embryos which form upon removal of 2,4-D, and implies that the proliferating clusters are actually suppressed embryos. This implication is further supported by observations using scanning electron microscopy. We conclude that the potential to undergo embryogenesis is present even before the 2,4-D is removed, but cannot be realized in the presence of the auxin. 相似文献
19.
By means of co-culture in growth regulator-free medium we analysed whether factors secreted into the medium of Daucus carota (carrot) somatic embryo cultures would be able to overcome the developmental arrest of globular Arabidopsis thaliana somatic embryos. Instead of Arabidopsis embryogenesis being promoted the development of carrot somatic embryos was inhibited at the globular stage in the presence
of Arabidopsis suspension culture aggregates with attached globular embryos. Several experiments showed that this was due to the release
of previously accumulated 2,4-D by the Arabidopsis cultures. (1) In addition to arresting carrot embryogenesis, co-culture with Arabidopsis cell suspensions also induced callus formation on Arabidopsis root segments. (2) Both effects only occurred with Arabidopsis suspensions grown in the presence of 2,4-D and not with those grown in the presence of NAA, demonstrating that Arabidopsis is not segregating a “general” inhibiting factor. (3) Both effects could be prevented by either binding 2,4-D to active charcoal
or by washing it away by changing the medium daily. (4) Uptake of 2,4-D into Arabidopsis cells during culture in 2,4-D containing medium and subsequent release of 2,4-D after transfer to growth regulator-free medium
was measured. (5) These low levels of released 2,4-D (0.2– 0.5 μm) could mimic the observed effects. Taken together these data suggest that the high intracellular 2,4-D content of Arabidopsis cultures may interfere with Arabidopsis somatic embryo development beyond the globular stage.
Received: 13 November 1997 / Revision received: 2 February 1998 / Accepted: 16 November 1998 相似文献