Carrot Tissue Research Articles

REFLECTANCE CHARACTERISTICS OF FRESH-MARKET CARROTS

ABSTRACT Body reflectance was measured for normal, fresh-market carrots and carrots with dry rot, soft rot, black crown and cavity spot defects. The reflection was measured with an integrating spectrophotometer over the wavelengths from 486 to 1043 nm. The resulting data indicated that three of the four defects tested could be identified using radiation with wavelengths between 535 and 722 nm. Cavity spots could not be significantly distinguished from normal carrot tissue.

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by <italic>Agrobacterium tumefaciens</italic> III. Formation of IAA Conjugates in Cultured Carrot Tissues Transformed with Wild-type and Mutant Ti Plasmids

Inactivation of IAA was examined in cultured carrot tissues transformed with Agrobacterium tumefaciens that harbored wild-type, aux− or cyt− Ti plasmids. IAA oxidase activities were similar among the three types of transformed tissue. Metabolites of IAA were examined by following the fate of 3-indolyl-[l-l4C]IAA. IAA conjugates were detected in all transformed tissues as well as in hypocotyl segments of non-transformed carrot seedlings. The rate of formation of IAA conjugates was ten times higher in the tissues transformed with wild-type or cyt− Ti plasmids than in the tissues transformed with aux− Ti plasmids. When the tissues transformed with aux− Ti plasmids were cultured on medium that contained IAA for 6 h, the rate of formation of IAA conjugates in these tissues became as high as that in tissues transformed with wild-type or cyt− Ti plasmids. The tissues transformed with wild-type or cyt− Ti plasmids not only synthesize larger amounts of IAA but also convert a larger amount of free IAA to conjugated IAA than do non-transformed and aux− transformed tissues. Presumably, in carrot, the formation of IAA conjugates decreases the amount of free IAA in the transformed tissues that synthesize large amounts of IAA and, consequently, the level of free IAA can be maintained fairly constant.

Derailment product in NADPH-dependent synthesis of a dihydroisocoumarin 6-hydroxymellein by elicitor-treated carrot cell extracts.

Synthetic activity of 6-hydroxymellein, the immediate precursor of carrot phytoalexin 6-methoxymellein, from acetyl-CoA and malonyl-CoA was induced in carrot cell extracts when the root disks were treated with CuCl2 or oligogalacturonide elicitor. These elicitors showed specific inducing activity of phytoalexin production and did not affect fatty acid synthesis in carrot tissues which may share some common properties with 6-hydroxymellein biosynthesis. 6-Hydroxymellein production was an NADPH-dependent process and, in the absence of the reagent, triacetic acid lactone was produced as a derailment product of the reaction process. This finding suggested that the reduction of the double bond at the 3,4-position of the phytoalexin takes place during the elongation of the poly(oxomethylene) chain. This NADPH-dependent reduction seems to occur at the triacetate stage before the condensation of the third malonyl-CoA as the conversion of carbonyl to hydroxyl group.

Growth effects, uptake and metabolism of trifluralin in tissue cultures.

Growth effects, uptake and metabolism of trifluralin (alpha, alpha, alpha-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) in carrot (Daucus carota L.) and tobacco (Nicotiana tabaccum L.) callus tissue were determined. Carrot callus was initiated from tap root tissue on Murashige and Skoog medium with kinetin and 2,4-dichlorophenoxy acetic acid (2,4-D). Tobacco callus was initiated from pith tissue on Murashige and Skoog medium with indole-3-acetic acid (IAA) and kinetin.

Rasterelektronenmikroskopische untersuchungen zur naßfäuleentwicklung an verschiedenen gemüsearten

Summary 1. The first steps of maceration taking place on carrot, cucumber fruit and onion slices after inoculating Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica were studied by scanning electron microscope. 2. Immediately after inoculation the cells of the pathogen lay on the surface of wound, separately. On carrot slices, 2 h post infectionem (p.i.) the first signs of cell division and development of microcolonies were to observe under anaerobic and aerobic conditions. On cucumber slices the division started under anaerobic conditions 2 h p.i. and under normal O 2 pressure 4 h p.i. On onions tested, the lag-phase lasted 6 h. An intensive multiplication of the pathogen could be proved on carrot and cucumber tissue 24 (anaerobic) resp. 48 h (aerobic) p.i. and on onions 48 (anaerobic) resp. 72 h (aerobic) p.i. 3. Simultaneously with the beginning of multiplication phase a complete slime layer composed from degradated plant cell material on the wound surface was established: Carrots and — only under anaerobic conditions — cucumbers reached this state 2–4 h p.i., however onions needed 6–8 h; under normal O 2 pressure on cucumber slices this process was started 15 h p.i. 4. First signs of lysis of the plant cell walls could be demonstrated on carrot tissue 6–8 h p.i., on onion slices 48 h p.i. 5. The E. carotovora subsp. atroseptica strain inoculated comparingly, induced pathological changes on the plant organs partly earlier than the typical vegetable soft rot pathogen E. carotovora subsp. carotovora under test.

Gene-expression programs in embryogenic and non-embryogenic carrot cultures

Somatic embryogenesis can be synchronized by enriching carrot (Daucus carota L.) suspension cultures for small, dense clusters of cells termed proembryogenic masses (PEMs). Gene-expression programs of PEMs were compared with those of embryonic and mature tissues by in-vitro translation of representative mRNA populations and by nucleic-acid hybridization. Analysis of invitro-translated polypeptides by two-dimensional polyacrylamide gel electrophoresis revealed striking similarities between the mRNA populations of PEM and torpedo-stage embryos; substantial differences, however, were observed when in-vitro translation products of PEMs and torpedo embryos were compared with those of hypocotyls and leaves. Northern blots of RNA isolated from PEMs, staged embryos, and mature carrot tissues were hybridized with cDNA probes for Dc3, Dc5 and Dc13; these cDNA recombinants represent mRNAs that are regulated during carrot somatic embryogenesis. The pattern of expression of these embryo-regulated transcripts was similar in PEMs and somatic embryos but differed in other carrot tissues. These results indicate that many of the molecular processes of embryogenesis are already established in PEMs in the presence of auxin. Additional experiments indicate the utility of Dc3 as a molecular marker for the acquisition of embryogenic potential.

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by <italic>Agrobacterium tumefaciens</italic> II. IAA Biosynthetic Activity of Cultured Carrot Tissues Transformed with Wild-Type and Mutant Ti Plasmids

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by <italic>Agrobacterium tumefaciens</italic> II. IAA Biosynthetic Activity of Cultured Carrot Tissues Transformed with Wild-Type and Mutant Ti Plasmids

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by <italic>Agrobacterium tumefaciens</italic> I. Auxin and Cytokinin Contents of Cultured Tissues Transformed with Wild-Type and Mutant Ti Plasmids

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by <italic>Agrobacterium tumefaciens</italic> I. Auxin and Cytokinin Contents of Cultured Tissues Transformed with Wild-Type and Mutant Ti Plasmids

Production of somatic embryos from carrot tissues in hormone-free medium

Carrot “seed” after the germination of their zygotic embryos, contain cells which can develop in aseptic culture into somatic embros in a reliable and predictable manner without the use of exogenously added growth regulators. In the course of further development beyond the cotyledonary stage, somatic embryos form secondary embryos along their axes. If continually subcultured, they do so in a cyclical manner yielding loosely attached aggregates of embryos and plantlets. Subcellular embryogenesis stops and normal plantlets are recoverable if the medium is allowed to become stale. Excised zygotic embryos of carrot in vitro are also able to form somatic embryos in the same way, but only when broken in half, and at a much lower response level. Component(s) of the basal medium responsible for inducing somatic embryogenesis remain to be elucidated, but a lowered sucrose concentration prevents secondary somatic embryogenesis. Cytological and histological examinations show that the somatic embryos are diploid and are most likely derived both from cells of the fruit wall and the endosperm.

Thioredoxin and NADP-thioredoxin reductase from cultured carrot cells

Dark-grown carrot (Daucus carota L.) tissue cultures were found to contain both protein components of the NADP/thioredoxin system-NADP-thioredoxin reductase and the thioredoxin characteristic of heterotrophic systems, thioredoxin h. Thioredoxin h was purified to apparent homogeneity and, like typical bacterial counterparts, was a 12-kdalton (kDa) acidic protein capable of activating chloroplast NADP-malate dehydrogenase (EC 1.1.1.82) more effectively than fructose-1,6-bisphosphatase (EC 3.1.3.11). NADP-thioredoxin reductase (EC 1.6.4.5) was partially purified and found to be an arsenite-sensitive enzyme composed of two 34-kDa subunits. Carrot NADP-thioredoxin reductase resembled more closely its counterpart from bacteria rather than animal cells in acceptor (thioredoxin) specificity. Upon greening of the cells, the content of NADP-thioredoxin-reductase activity, and, to a lesser extent, thioredoxin h decreased. The results confirm the presence of a heterotrophic-type thioredoxin system in plant cells and raise the question of its physiological function.

Four “necrosis-inducing” components, separated from germination fluid of Botrytis cinerea induce active resistance in carrot root tissue

Each of four groups of fractions, separated from the germination fluid (GF) of Botrytis cinerea by ultrafiltration and Sephacryl S-200 column chromatography, individually caused cell death (detected by Evans Blue) in the 1–2 cell layers below the cut surface of, and induced resistance in, carrot tissue slices 24 ± 1 °C. Induction of resistance was expressed as (a) inhibition of germ-tube growth assessed 16 h after conidia were transferred to carrot slice surfaces that had been pre-treated for 6 h with GF, or its separate fractions, and (b) by a visual disease score, 2 days after inoculation. The four groups of fractions were characterized tentatively as containing two carbohydrate components (< 10 000 D), a component possessing pectolytic activity (49 000 D) and a possible glycoprotein component (63 000 D) respectively. These components were considered to be responsible for the “necrosis-inducing” activity of the germination fluid which, provided damage was only superficial, led to active defence responses in the underlying living carrot tissues.

Transformation of carrot tissues derived from proembryogenic suspension cells: A useful model system for gene expression studies in plants

A method is described for the high frequency transformation of carrot proembryogenic suspension culture cells by a non-oncogenic Ti-plasmid vector (pGV3850::1103) which carried a chimaeric kanamycin resistance gene (nos-NPT-II). Plants were regenerated efficiently from transformed material by somatic embryogenesis in the presence of kanamycin. Transformed tissues expressed readily detectable levels of both NPT-II and nopaline. NPT-II could be detected in total protein extracts by Western blotting. This analysis indicated that NPT-II was produced as a single, full length polypeptide. The T-DNA copy number in individually selected transformants was analysed by Southern blotting and ranged from 1-8 per diploid genome. The copy number and organization of the T-DNA was retained in plants regenerated from these transformants by somatic embryogenesis. These data suggested a clonal origin for the selected kanamycin resistant colonies. NPT-II expression levels appeared to be directly related to gene dosage.

Oxygen Isotope Exchange between Metabolites and Water during Biochemical Reactions Leading to Cellulose Synthesis

Cellulose was produced heterotrophically from different carbon substrates by carrot tissue cultures and Acetobacter xylinum (a cellulose-producing bacterium) and by castor bean seeds germinated in the dark, in each case in the presence of water having known concentration of oxygen-18 ((18)O). We used the relationship between the amount of (18)O in the water and in the cellulose that was synthesized to determine the number and (18)O content of the substrate oxygens that exchanged with water during the reactions leading to cellulose synthesis. Our observations support the hypothesis that oxygen isotope ratios of plant cellulose are determined by isotopic exchange occurring during hydration of carbonyl groups of the intermediates of cellulose synthesis.

The effect of removing cellulase(s) from a commercial pectinase on maceration and liquefaction of carrots

Rohament P, a macerating enzyme preparation from Aspergillus alleaceus containing an endo-polygalacturonase as the major activity but also substantial amounts of cellulase(s), was purified by affinity chromatography on Avicel cellulose. Treating Rohament P with Avicel at different protein:cellulose ratios was more efficient in columns than in batch. An Avicel column, with an enzyme:substrate ratio of 1:80, retained 94% of cellulase from Rohament P in 60 min at 40–45°C, pH 4.4. Treated enzyme containing 6% residual cellulase, when incubated with fresh carrot rasps, released the maximum amount of cells with intact cell walls. Untreated enzyme did not macerate the carrot tissue but liquified under same conditions. Degradation of a washed carrot preparation by treated enzyme was 44% compared with 55% for the untreated enzyme. Gas chromatographic analysis of sugars revealed that treated Rohament P liberated less glucose and others sugars than did untreated enzyme. Enzyme visualization studies of treated and untreated Rohament P reflected a quantitative difference in protein bands in a pH gradient of 4–6 in miniature isoelectric focusing. Application of treated and untreated Rohament P to disks of carrot tissue led to ultrastructural changes. Untreated Rohament P dissolved the middle lamella and disintegrated the fibrillar material predominantly throughout the cell wall, resulting mainly in cell breakage. Treated Rohament P preferentially dissolved the middle lamellar region of the cell wall without touching the fibrillar part. This indicates that pectin is confined to the middle lamellar region.

The production and properties of a crude pectin lyase from Lachnospira multiparas

The production of pectin lyase (PL) was dependent on medium pH with two peaks of production, at pH 61 and 7-2, that at pH 61 showing considerably higher activity. Crude PL activity was stable for at least 3 h at both 30 and 40dC. At 50dC there was a 20%, reduction in activity after 1 h; at 60dC there was a 40% reduction after 30 min yet > 50% activity was retained for a further 2 h. The crude enzyme exhibited macerating ability against carrot tissue commensurate with that of a currently available commercial pectinase enzyme.

Macerating properties of a commercial pectinase on carrot and celery

A technique was developed for measuring the rate of enzymatic maceration in plant tissues. A commercial macerating pectinase (Rohament P, RP), from Aspergillus alleaceus, rich in endopolygalacturonase, softened tissue and released cells from cubes, disksm or rasps of carrot and celery. Cells were counted in a Neubauer counting chamber and residual non-straining tissue was weighed. Cell release dependend on several factors such as surface area of the tissue, temperature, pH, incubation period, and substrate and enzyme concentration. Enzyme concentration greatly influenced the cell number and morphology. The RP enzyme preparation macerated tissue at low concentrations [<0.02% (w/v)]. The carrot or celery tissue was completely liquefied with low levels of RP combined with commercial cellulases from Trichoderma reesei. A similar effect was noticed with the RP enzyme preparation alone at a concentration of 0.1%. Following incubation with the original RP preparation, 50–60% of the cells were liquefield and cell wall thinning was noticed in the residual cells. Liquefaction by the RP enzyme was attributed to cellulases in the preparation. The RP enzyme was modified by depleting its cellulases from the preparation by adsorption on Avicel cellulose. This cellulase-free RP showed only macerating activity and released cells continuously without liquefaction, irrespective of the incubation period or enzyme concentration, cell walls were intact for extended period up to 24–30 h of incubation.

Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33-kDa protein

Extensins are hydroxyproline-rich glycoproteins associated with most dicotyledonous plant cell walls. To isolate cDNA clones encoding extensin, we started by isolating poly(A)(+) RNA from carrot root tissue, and then translating the RNA in vitro, in the presence of tritiated leucine or proline. A 33-kDa peptide was identified in the translation products as a putative extensin precursor because: (i) it is rich in proline and poor in leucine, and (ii) the message appears to be more abundant when carrot tissue is wounded. From a cDNA library constructed with poly(A)(+) RNA from wounded carrots, one cDNA clone (pDC5) was identified that specifically hybridized to poly(A)(+) RNA encoding this 33-kDa peptide. We isolated three cDNA clones (pDC11, pDC12, and pDC16) from another cDNA library using pDC5 as a probe. DNA sequence data, RNA hybridization analysis, and hybrid released in vitro translation indicate that the cDNA clone pDC11 encodes extensin and that cDNA clones pDC12 and pDC16 encode the 33-kDa peptide, which as yet has an unknown identity and function. The assumption that the 33-kDa peptide was an extensin precursor was invalid. RNA hybridization and DNA sequence analysis indicate that pDC5 is a hybrid clone corresponding to two RNA species. RNA hybridization analysis showed that RNA encoded by both clone types is accumulated upon wounding.

Relationship between the concentration of chlorogenic acid in carrot roots and the incidence of carrot fly larval damage

SUMMARYChlorogenic acid (1·24‐3·36 mg/g) was identified as the main phenolic component in the peel of carrots by hplc analysis. The higher the concentration of chlorogenic acid in different cultivars the greater the susceptibility to carrot fly larval damage. Increases in concentration were found both after carrot fly damage and after carrots had overwintered in the field. The presence and location of chlorogenic acid was confirmed in sections of carrot tissues, mounted in 0·05 M ammonia solution by viewing them using a u.v.‐epifluorescent microscope. The importance of phenolic compounds and their function in the production of insect cuticle is discussed in relation to the different concentrations of chlorogenic acid and resistance to carrot fly in carrots.

The influence of phosphorus nutrition on growth, chlorophyll formation and 14CO2 fixation of chlorophyllous carrot callus cultures

AbstractFreshly isolated carrot root sec. phloem explants were cultured in nutrient solution plus inositol, IAA and kinetin and a varied supply of inorganic phosphorus. 14CO2 fixation experiments with the greened cultures were carried out under steady state conditions at the age of 21 days in light and darkness for four minutes. The results show that photosynthetic CO2 fixation by the Calvin cycle is greatly reduced in phosphorus deficient cultures whereas CO2 fixation via PEPC is enhanced. Under phosphorus deficiency the export of primary photosynthetates from the chloroplasts seems to be suppressed while the radioactivity of the labelled insoluble fraction, which represents mainly chloroplastic starch after this short time of 14CO2 fixation, was increased. The results are discussed with respect to the influence of phosphorus nutrition on photosynthetic CO2 fixation and PEP carboxylation in carrot tissue cultures.

Characterization of Sugar Transport in Storage Tissue of Carrot

Abstract Sugars are a major storage carbohydrate and a primary component of carrot root quality. The objective of this study was to determine the characteristics of sugar transport into the storage cells of carrot [Daucus carota (L.)]. Tissue disks were incubated in a buffered solution (pH 6.5) containing various concentrations of sucrose, glucose, or fructose, 1 mm CaCl2, and 100 mm mannitol. Passive uptake was defined as uptake in the presence of 5 μm carbonyl cyanide, - m chlorophenyl hydrazone (CCCP). Active uptake was the difference between total (–CCCP) and passive uptake. Characteristic, biphasic kinetics were observed for all sugars. At sugar concentrations below 10 mm, a saturating active component was operating. Above 10 mm, the influx was a nonsaturating, linear transport system. Active transport was pH dependent, showing high rates of uptake at low pH. Glucose and fructose did not inhibit sucrose influx and vice versa, but they did compete with each other. The kinetics of the hexose competition was noncompetitive inhibition. The competition studies suggested the presence of a separate carrier for each sugar. The evidence indicated that sugar transport across membranes of carrot storage cells is a combination of active and passive transport, consistent with transport kinetics observed in other crops. Active sugar uptake is a significant part of uptake, doubling influx at low apoplastic concentrations. At maturity, sucrose is the major transport and storage sugar. Glucose and fructose however, are transported at considerable rates in vitro, when they are present in the free space.