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  • 1
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Changes in the amount of mRNA for phenylalanine ammonia-lyase (PAL, EC 4. 3. 1. 5) and chalcone synthase (CHS, EC 2. 3. 1. 74) induced by light were investigated in carrot (Daucus carota L. cv. Kurodagosun) cells cultured in suspension. Cells were cultured for 5 days in 2,4-dichlorophenoxy acetic acid (2,4-D)-free medium and absolute darkness. Transcription of pal and chs genes was induced by irradiation at day 5, resulting in the induction of anthocyanin synthesis. PAL mRNA transcripts were rapidly and transiently up-regulated within 6 h after onset of irradiation, after which they were immediately down-regulated. Then, PAL mRNA increased again slowly, the time course being coordinated with induction of CHS transcription and anthocyanin synthesis. The semi-quantitative polymerase chain reaction (PCR) and primer extension using gene specific oligonucleotides for pal genes revealed that two different pal genes were induced sequentially by light. The rapid and transient induction of the pal gene was not related to anthocyanin synthesis, but was identical to that induced by dilution or transfer effect. This slowly-induced pal gene, which was induced in parallel to the chs gene, corresponded to the pal gene which was induced for anthocyanin synthesis. Anthocyanin synthesis was induced by simultaneous activation of both ant-pal and chs genes, and both mRNAs remained at a high level during anthocyanin synthesis. Expression of ant-pal and chs was partially repressed by either depletion of light or addition of 2,4-D. These genes were completely repressed by addition of 2,4-D in darkness. Therefore, the two signals may act at different points. Trn-pal was not repressed by depletion of light or addition of 2,4-D.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Physiologia plantarum 62 (1984), S. 0 
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Cell elongation occurred when carrot (Daucus carota L. ev. Kurodagosun) cells subcultured through sieving (Y. Ozeki and A. Komamine, Physiol. Plant. 53: 570-577. 1981) were transferred to a medium lacking auxin, while the cells showed no elongation in a medium containing 2, 4-D. Changes in polysaccharides of the cell walls and in their sugar composition during elongation were investigated. All wall components, EDTA-soluble pectic substance, 5 and 24%, KOH-soluble hemicelluloses and cellulose increased markedly during elongation. The increase of hemicelluloses correlated especially with elongation. In the 5% KOH-soluble hemicellulose, galactose and arabinose contents in the walls increased significantly both in amounts (per fresh weight) and relative contents (% in total neutral sugars) during elongation, while the relative contents of glucose and xylose decreased rapidly in the 5 and 24% KOH-soluble hemicelluloses. The methylation analysis tentatively indicated that larger amounts of galactan and/or arabinogalactan and lower amount of xyloglucan were found as components of the two hemicelluloses of elongating cells than those of non-elongating cells. The amounts of total carbohydrate and of uronic acid of extracellular polysaccharides secreted into the medium increased to a larger extent in the elongation culture than in the non-elongation culture. The contents of galactose and arabinose in extracellular polysaccharides increased rapidly in the elongation culture. The biochemical aspects of cell elongation in the absence of auxin were discussed from the viewpoint of the results obtained here.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Changes in the amounts of enzyme proteins and mRNAs for phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and chalcone synthase (CHS, EC 6.-.-.-) were investigated in suspension cultures of carrot (Daucus carola L. cv. Kurodagosun), in which anthocyanin synthesis was induced and repressed in the absence and presence of 2,4-dichrolophenoxyaeetic acid (2,4-D), respectively. The amounts of enzyme proteins were measured using anti-PAL and anti-CHS sera. In a medium lacking 2,4-D, enzyme proteins and mRNAs for both PAL and CHS were induced 5 days after transfer, when the anthocyanin synthesis was induced. They all increased coordinately during days 5-7 after transfer, when anthocyanin was rapidly synthesized, and then they decreased. In contrast, in a medium containing 2,4-D, the amount of enzyme protein and mRNA for CHS were below detectable levels throughout the culture time. As regards PAL, however, whether 2,4-D was present or not, both enzyme protein and mRNA rapidly increased after the transfer of cells to fresh medium, and then decreased and remained at low levels in a medium containing 2,4-D. The addition of 2,4-D to cells cultured for 6 days in a medium lacking 2,4-D caused an immediate decrease in the amounts of enzyme proteins and mRNAs for both PAL and CHS.All the results obtained here indicate that the induction and suppression of the synthesis of PAL and CHS were probably due lo increase and decrease, respectively, in the amounts of their mRNAs. Thus, it is suggested that the induction and suppression of anthocyanin in this system may be regulated at the transcriptional level by 2,4-D.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: A system in which anthocyanin synthesis could be induced under a defined condition, was established in a carrot suspension culture. A cell suspension culture of carrot (Daucus carota L. cv. Kurodagosun) was subcultured for more than a year in a medium containing 5 × 10−7M 2,4-dichlorophenoxyacetic acid (2,4-D). At every subculture the cultures were sieved through nylon screens and the cells and cell clusters collected in the size range of 31–81 μm were transferred to a fresh medium. When the cells were transferred to a medium without auxin, synthesis of anthocyanin was induced. Zeatin promoted anthocyanin synthesis in a medium lacking auxin, with maximum yields of anthocyanin obtained at 10−7 to 10−8M zeatin, 2,4-D at higher concentrations than 10−7M inhibited anthocyanin synthesis completely. The sieved cells were fractionated by Ficoll density gradient centrifugation. Somatic embryos were formed in the fraction of higher density (〉14% of Ficoll) in a medium containing 10−7M zeatin but lacking auxin, while synthesis of anthocyanin was hardly observed. On the other hand, cells in the fraction of lower density (〈12% of Ficoll) synthesized anthocyanin in the same medium, but formed few embryos. Forty to fifty percent of the total cells in this lighter cell fraction synthesized anthocyanin at a maximum. The similarity between anthocyanin synthesis and embryogenesis was observed in the time course as well as in the effects of growth regulators. The correlation between metabolic and morphological differentiation is discussed.
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  • 5
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: When anthocyanin synthesis was induced in cell suspension cultures of carrot (Daucus carota L. cv. Kurodagosun) by transfer to medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D), phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), chalcone synthase (CHS, EC 6.-.-.-), and chalcone-flavanone isomerase (CHFI, EC 5.5.1.6) activities appeared, reaching maxima 6–7 days after transfer. The maximum specific activity of CHS was much lower than that of PAL or CHFI. In a medium containing 2,4-D, no anthocyanin was synthesized, PAL and CHFI activities were suppressed and CHS activity could not be detected at all. The activities of PAL and CHS in cells cultured without 2,4-D for 6 days began to decrease within 3–6 h of 2,4-D addition. CHS activity was completely repressed 24–36 h after the addition, but CHFI activity was almost unchanged at this time. After culture without 2,4-D for 6 days, cell suspensions were transferred to fresh media either lacking or containing 2,4-D. After transfer, PAL increased in both media within 3 h, whereas CHS activity and anthocyanin accumulation were coordinated and both were completely regulated by 2,4-D. Changes in CHS activity rather than PAL activity correlate with changes in anthocyanin accumulation under various culture conditions.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary We have developed an improved artificial seed system by using a hot-water extract from a marine cyanobacterium, Synechococcus sp. NKBG 042902. Carrot somatic embryos (Daucus carota L.) were divided into two size categories (〉 800 μm and 425–800 μm). High frequency germination (91%) was obtained using the large somatic embryos encapsulated in calcium alginate gel containing 400 mg 1−1 of extract. This compares to 35% without addition of the extract. A non-dialysate fraction of the extract showed strong germination-promoting activity compared with a dialysate fraction. The germination frequency of artificial seeds containing 100 mg 1−1 of non-dialysate fraction was more than 90%. Almost all germinating artificial seeds developed into plantlets within 4 days. We also achieved high frequency germination (60%) of artificial seeds encapsulating small somatic embryos (425–800 μm) that contained 100 mg 1−1 of non-dialysate (control 9%). Although the small somatic embryos showed a lower germination frequency than the large embryos, the plantlet development process in these seeds was far more vigorous. Such a high germination frequency has not previously been reported for a carrot artificial seed system.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1432-203X
    Keywords: Cyanobacteria ; Daucus carota L ; Plant Regeneration ; Plant Growth Regulator
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Twenty five strains of marine cyanobacteria were screened for their ability to promote carrot somatic embryogenesis. Hot water extracts prepared from 21 of these strains promoted plantlet formation. Extracts from four strains increased plantlet numbers to an average of over 3.7-fold. Dialysates and nondialysates of each of these extracts also increased plantlet formation. For extracts from filamentous cyanobacteria, Nostoc sp. and Anabaena sp., dialysate was more effective (4.2-fold increase) than nondialysate (3.0-fold increase), whereas for unicellular strains Synechococcus sp. and Xenococcus sp., nondialysate was more effective (5.2-fold increase) than the dialysate (3.2-fold increase). These cyanobacterial extracts also promoted embryolike structure formation from two-year old carrot cell cultures which were unable to produce plantlets using the usual methods. Here, we demonstrate the existence in marine cyanobacterial extracts of low and high molecular weight factors which strongly promote somatic embryogenesis in carrot cell cultures.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1432-203X
    Keywords: Marine Cyanobacteria ; Daucus carota L. ; Plantlet formation ; Chlorophyll content
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Somatic embryos of Daucus carota L. developed into plantlets at high frequency after addition of an extract from a marine cyanobacterium, Synechococcus sp. NKBG 042902. High molecular weight, nondialyzing fraction, separated from the extract, possessed enhanced plantlet formation promoting activity. Plantlet formation frequency was 60 % after addition of nondialysate (100 mg/l) compared to 28 % without addition. Embryos treated with the nondialysate contained five times more chlorophyll than nontreated embryos after 6 days of culture. The chlorophyll a/b ratio of 4-day old treated somatic embryos was found to be similar to that of zygotic embryos. However, the chlorophyll a/b ratio of plantlets induced from nontreated somatic embryos was variable. Nondialysate was fractionated by ultracentrifugation and an active component obtained, which gave a maximum plantlet formation frequency of 71 %, and induced rapid greening of shoots.
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    Molecular genetics and genomics 220 (1990), S. 177-180 
    ISSN: 1617-4623
    Keywords: Genetic tumor ; Agrobacterium rhizogenes ; rol genes ; Auxin sensitivity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Thus far, no evidence has been presented that the rol genes (Ng rol) of Nicotiana glauca (Furner et al. 1986) are expressed in this plant. However, we found that the Ng rol genes were transcribed in genetic tumors formed in hybrids of N. glauca x N. langsdorffii. During the culture of such genetic tumors, the level of transcription of Ng rol B increased while that of Ng rol C decreased in parallel with an increase in the endogenous auxin level in the tissues. Moreover, the transcription of these genes was completely suppressed by the application of exogenous auxin. Since the measured endogenous level of auxin was rather low, the formation of tumors in these hybrids can be tentatively explained as a consequence of the expression of rol genes that is responsible for the increased sensitivity to auxin of the hybrids. This novel finding is discussed in relation to the cause of genetic tumors in Nicotiana.
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  • 10
    ISSN: 1618-0860
    Keywords: Keywords: Anthocyanin, Carrot, Differentiation, myb, Phenylalanine ammonia-lyase (PAL)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: D and is also induced rapidly and transiently by transfer of cells to fresh medium and lowering the cell density. From the carrot genomic library, four clones of PAL genes, gDcPAL1,2,3 and 4, were obtained. Analyses of nucleotide sequences revealed that only the gDcPAL3 gene is responsible for the induction of anthocyanin synthesis by 2,4-D. Several cis-elements, boxes M, P, A, L, and G, exist in the proximal promoter region of gDcPAL3. Transient expression experiments in carrot protoplasts using deletion mutants of the proximal promoter region of gDcPAL3 gene showed that boxes M and L, both of which contain core sequences of the Myb binding sites, might play an important role in gDcPAL3 promoter activity. Four myb cDNAs, Dcmyb8,10,12 and 14 were obtained from a carrot subtracted cDNA library and their structure and expression patterns were analyzed. In addition to the analysis of the proximal region of gDcPAL3 promoter, the possibility of the regulation of gene expression by genomic DNA structure and chromatin modification in metabolic differentiation is discussed.
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