Carrot Tissue Research Articles

Retarding Vegetable Softening by Cold Alkaline Pectin Deesterification Before Cooking

ABSTRACTSnap beans and carrot tissues, deesterified at 1°C and pH 12.5, soften much more slowly than control tissues when cooked at neutral or slightly alkaline pH. High pH or NaCl during cooking had little effect on the degree of softening. Soaking in 0.05 M EDTA led to an 86% loss in firmness. The results were consistent with a reduction in the β‐elimination depolymerization of pectin during heating as a result of deesterification in the alkali. Deesterified tissues remained susceptible to pronounced softening during cooking at pH 3.5. Potatoes, cauliflower and apples showed similar effects. Peas gave a reduced response. Beets and dry beans did not show a retardation of softening after an alkali soak.

An investigation of the antibacterial effect of carrot on Listeria monocytogenes.

Carrot slices immersed in a potassium phosphate buffer (0.1 mol/l, pH 7.0) or carrot tissue macerated in the buffer had a lethal effect on Listeria monocytogenes. This antilisterial activity was suppressed by anaerobiosis, thiol compounds (1 mmol/l) and bovine serum albumin (0.05%) but was not affected by sodium ascorbate (200 mmol/l), propyl gallate (25 mmol/l), catalase (1100 U/ml), superoxide dismutase (357 U/ml), or chelating agents (10 mmol/l). Free-radical scavengers had no effect at 10 mmol or 50 mmol/l but histidine and diazabenzocyclooctane at 100 mmol/l reduced the antilisterial activity. The addition of Tween 20, 0.05% (v/v), to carrot macerates improved the recovery of the activity in the supernatant liquid after centrifugation at 10,000 g for 2 min. The addition of higher concentrations of the detergent to the macerate reduced the antilisterial activity.

β-fructosidases and in vitro dedifferentiation-redifferentiation of carrot cells

Marked changes in the activity of acid and alkaline β-fructosidases, as well as in their distribution between cell fractions, appear to be intimately related to the process of cell differentiation in carrot tissue cultures. Somatic embryogenesis was characterized by an increase in alkaline β-fructosidase and a decrease in the acid enzyme. Non-embryogenic cell lines contrast with embryogenic cell lines in maintaining very high acid β-fructosidase activities. The concentration of calcium in the embryogenic culture had a marked effect on embryogenesis. The development of the β-fructosidases was distinctly influenced by calcium. While the acid β-fructosidase production was inhibited by increasing calcium concentrations, either in the presence or absence of auxin, the alkaline enzyme reached the highest activity only in the auxin-free medium and at calcium concentrations already inhibitory to the production of the acid enzyme.

Electrical impedance analysis in plant tissues: The effect of freeze-thaw injury on the electrical properties of potato tuber and carrot root tissues

Electrical impedances in the range 100 Hz to 800 KHz were measured in potato tubers and carrot root cortex both before and after freeze-thaw cycles. These impedance data were analyzed using equivalent circuit modelling based on complex nonlinear least squares (CNLS) (Zhang and Willison 1991a). After freezing to and thawing from −3, −6, −9 and −12 °C, carrot root tissues were best characterized by a double shell model composed of extracellular resistance, cytoplasmic resistance, plasma membrance capacitance, vacuole interior resistance, and tonoplast capacitance. Although freeze–thaw cycles to −3 or −6 °C did not kill the carrot tissues, extracellular resistance and vacuole interior resistance were halved relative to control tissues, and cytoplasmic resistance decreased to a third of the control value. Plasma membrane capacitance and tonoplast capacitance were not affected by the −3 and −6 °C noninjurious stresses. After carrot root or potato tuber tissues had been killed by freezing (−3 °C for potato tuber and −18 °C for carrot), the tissues were best represented by a single-shell model composed of extravesicular resistance, intravesicular resistance, and vesicle membrane. Compared with unfrozen controls, freeze-killed potato tubers were characterized by a 30-fold decrease in extracellular resistance, a 7-fold decrease in intracellular resistance, and a 10-fold decrease in plasma membrane capacitance. Freeze-killed carrot roots were characterized by a 30-fold decrease in extracellular resistance, 3-fold decrease in intracellular resistance, and 3.5-fold decrease in plasma membrane capacitance. These results are compatible with massive rupturing of protoplasts during lethal freeze–thaw injury.Key words: Solanum tuberosum L., Daucus carota L., freeze–thaw injury, electrical impedance

The textural analysis of cooked potato. 1. Physical principles of the separate measurement of softness and dryness

The texture of cooked potatoes was described by two physical parameters: softness, resulting from both cell cleavage and cell separation, and dryness, depending on the ratio of cell separation to cell cleavage. The softness of potato tissue was measured by determining its resistance to cutting with a thin stainless steel wire. Softness increased rapidly with cooking time, particularly when the cultivar was predicted tobe soft in texture by published taste panel trials. Raw potato tissue released intercellular fluid from fractured surfaces due to cell cleavage. After cooking less fluid was released, i.e. the texture became drier, as cell separation increased. The erosion of cells from cooked potato tissue by a high-pressure jet of water was used to measure dryness quantitatively. Erosion increased during cooking. Carrot tissue, whose cells do not separate on cooking, did not erode, but anomalous results with apple tissue suggested that measurement of dryness in this way might not be fully independent of softness.

LITERATURE ABSTRACTS

LITERATURE ABSTRACTS

Phytotoxic properties of Pseudomonas syringae pv. syringae toxins

Syringomycin E, syringomycin G and syringopeptin 25A, the main components of the Pseudomonas syringae pv. syringae toxin mixture, were assayed for their phytotoxicity, determined as electrolyte leakage from carrot tissues, necrosis of tobacco leaves, and death of potato tissues, and for their antimicrobial activity on Rhodotorula pilimanae. p]In the antimicrobial assay, syringomycins were 30 times more active than syringopeptin 25A, but, in the electrolyte leakage assay, they proved to be 40 times less active than the former since a significant effect required concentrations of 16 and 0·4 μ m respectively. A statistically identical effect on electrolyte leakage was obtained with syringopeptin 25A or with an unfractionated toxin mixture equimolar for syringopeptin 25A. A similar pattern of activity was observed in the tobacco leaf and potato disc assays. After a short incubation at pH 10, syringomycins completely lost their antimicrobial and phytotoxic activities, while syringopeptin 25A retained all its antimicrobial activity and most of its phytotoxicity. These findings indicate that syringopeptin 25A and syringomycins are mainly responsible for, respectively, the phytotoxic activity and the antimicrobial activity on R. pilimanae, characteristic of the unfractionated toxin mixture of P. s. pv. syringae. The high phytotoxicity of syringopeptin 25A is a new finding which prompts a careful examination of the role played by the individual metabolites in the disease caused by P. s. pv. syringae ecotypes.

Influence of pH treatment on pectic substances and firmness of blanched carrots

The influence of blanching and the gradual reduction of pH within carrot tissue from 6.2 to 3.9 by acetic acid infiltration was studied. Maximum firmness of about 86 N was observed at pH 4.4, while at higher or lower pH firmness was about 58 N. Galacturonic acid content, and neutral sugars of the water-soluble pectic, EDTA and alkaline-soluble pectin fractions were assessed after the pH treatments. EDTA-soluble pectin increased after blanching at all pH values, but less so at pH 4.4. Total pectin decreased to a lesser extent at pH 4.4 than at either higher (6.2) or lower (3.9) pH values. Lower amounts of soluble pectin were found at pH 4.4 than in the control. A significant positive correlation was found between firmness and the alkali-soluble pectin content.

Measurement of firmness in carrot tissue during cooking using dynamic, static and sensory tests

AbstractThe suitability of an oscillatory shear technique to distinguish the rigidity of carrot tissue of different maturity and morphological origin has been evaluated. The rigidity was related to apparent G' (G' = shear modulus) of tissues and growing regions showed greater rigidity than mature tissue. Despite these differences in raw tissue, all samples exhibited a rapid decline in apparent G' around 55–60°C when small discs of these tissues (2 mm thick × 13 mm dia) were heated from 20 to 80°C. Force‐compression studies on carrot tissue were performed using a TA.XT2 texture analyser. Carrot discs (3 mm thick × 20.6 mm dia) showed a linear elastic region up to 37% of compression at 20°C. When discs of carrot tissue were heated in the range 20–80°C, they showed a gradual decline in modulus of elasticity (E) unlike apparent G'.Heated‐stage microscopic studies were carried out to examine the reasons for rapid changes in G' of carrot tissue during heating. As the temperature increased from 20 to 60°C, thickening of the cell wall was observed with disruption of plasma membrane and loss of cellular integrity evident around 60°C.The relationship between sensory analysis and the rheological parameters was also studied. There was a gradual decline of both the apparent G' and E values as well as the sensory score as carrots were cooked in boiling water but, after 20 min. plateau values were observed. There was a linear relationship between sensory score and apparent G' and E values (r = 0.86 to 0.93) suggesting the instrumental methods used in this investigation were able to measure the same intensity of textural characteristics as perceived by a taste panel.

Regulation of IAA—Amino Acid Conjugate Formation in Carrot Tissues11Agrobacterium tumefaciens

Regulation of IAA-amino acid conjugate formation was examined in cultured carrot tissues transformed with Agrobacterium tumefaciens harbouring aux− Ti plasmids. IAA-conjugate formation in carrot tissues transformed with aux− Ti plasmids was induced by exogenously-applied IAA. When these tissues were treated with an inhibitor of protein synthesis (cycloheximide) before IAA treatment, the induction of IAA-conjugate formation was completely inhibited. By contrast, pre-treatment with an inhibitor of RNA synthesis (actinomycin D) before IAA treatment reduced induction of IAA-conjugate formation. These results suggest that IAA-inducible IAA-conjugate is catabolized by enzyme(s) and the formation of conjugated IAA is regulated at the transcriptional level in carrot. Presumably, in carrot tissues transformed with wild-type or cyt− Ti plasmids, the same mechanism(s) exists and the free IAA content in the carrot tissues is maintained at a low level.

ニンジンの培養組織のクロロフィル蛍光動画像解析

Rapid changes (CFI) in intensity of chlorophyll a fluorescence during dark-light transient reflect the various reactions of photosynthesis. The image analysis of CFI gives information not only about the localized differences in photosynthetic activity on whole leaves in situ but also on the development of photosynthetic system.Development of the photosynthetic system in cultured tissues is very complex. Therefore, the development of carrot (Daucus carota L. cv US-Harumakigosun) tissues was studied by the image analysis of CFI. The nodular callus, which had been formed by culturing 1-weekold hypocotyl segments on MS agar medium containing 2, 4-D (1 mg⋅l-1), was used for the experiments. After the nodular callus was transplanted to MS agar medium without 2, 4-D, the embryogenic cells appeared within 2 to 3 weeks. The tissues grew under 25°C air temperature and 80 μmol photons⋅ m-2. sec-1. At 3 days after the transplanting, the appearance of chlorophyll a in the nodular callus was detected by the CFI images. The CFI patterns changed in the development of photosynthetic apparatus of developing leaves. The changes in each developing stage were very complex but the progress in the photosynthetic system was diagnosed by the CFI curves. The CFI pattern of the developing leaves at 60 days after the transplanting were similar to the typical pattern of mature leaves.

Calcium-Pumping ATPases in Vesicles from Carrot Cells

Ca(2+)-ATPases keep cytoplasmic [Ca(2+)] low by pumping Ca(2+) into intracellular compartments or out of the cell. The transport properties of Ca(2+)-pumping ATPases from carrot (Daucus carota cv Danvers) tissue culture cells were studied. ATP-dependent Ca(2+) transport in vesicles that comigrated with an endoplasmic reticulum marker, was stimulated three- to fourfold by calmodulin. Cyclopiazonic acid (a specific inhibitor of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase) partially inhibited oxalate-stimulated Ca(2+) transport activity; however, it had no effect on calmodulin-stimulated Ca(2+) uptake driven by ATP or GTP. The results would suggest the presence of two types of Ca(2+)-ATPases, an endoplasmic reticulum- and a plasma membrane-type. Interestingly, incubation of membranes with [gamma(32)P]ATP resulted in the formation of a single acyl [(32)P]phosphoprotein of 120 kilodaltons. Formation of this phosphoprotein was dependent on Ca(2+), but independent of Mg(2+). Its enhancement by La(3+) is characteristic of a phosphorylated enzyme intermediate of a plasma membrane-type Ca-ATPase. Calmodulin stimulated Ca(2+) transport was decreased by W-7 (a calmodulin antagonist), ML-7 (myosin light chain kinase inhibitor) or thyroxine. Acidic phospholipids, like phosphatidylserine, stimulated Ca(2+) transport, similar to their effect on the erythrocyte plasma membrane Ca(2+)-ATPase. These results would indicate that the calmodulin-stimulated Ca(2+) transport originated in large part from a plasma membrane-type Ca(2+) pump of 120 kilodaltons. The possibility of calmodulin-stimulated Ca(2+)-ATPases on endomembranes, such as the endoplasmic reticulum and secretory vesicles, as well as the plasma membrane is suggested.

Indole-3-acetic acid and indole-3-butyric acid in tissues of carrot inoculated withAgrobacterium rhizogenes

The role of auxins in induction of roots byAgrobacterium rhizogenes was studied in carrot root disks. Transformed roots were produced on root disks by inoculation withA. rhizogenes, A4. Measurement of indole-3-acetic acid (IAA) by gas chromatography-mass spectrometry (GC-MS) indicated that there was a significant increase in the concentration of IAA in transformed callus and induced roots compared with initial IAA concentrations in carrot disks. Indole-3-butyric acid (IBA) was found to occur naturally in carrot roots. The presence of IBA, a potent root inducer, must be taken into account when assessing the role of auxin during transformation and induction of roots byA. rhizogenes.

Abstracts of Papers Presented at the Annual Meeting of the Canadian Phytopathological Society Banff, Alberta June 1991

Abstracts of Papers Presented at the Annual Meeting of the Canadian Phytopathological Society Banff, Alberta June 1991

ULTRASTRUCTURAL AND TEXTURAL CHANGES IN PROCESSED CARROT TISSUE1

ABSTRACTProcessed carrot tissues were studied using a transmission electron microscope (TEM) and an Instron Universal Testing Instrument. Carrot slices were evaluated raw, after thermal processing and after freezing and thawing. Cell walls of carrot tissues, frozen and thawed, were intact and dense. The middle lamella lost pectic materials. Overall, frozen and thawed cells were not so closely pressed as those of raw tissues. In thermally processed carrots the cell wall and the middle lamella were less densely stained compared to raw or frozen‐thawed carrots. Thermally processed carrots exhibited significantly smaller modulus of deformability and stiffness and toughness values than raw carrots. There were no significant differences between the modulus of deformability and stiffness values for frozen‐thawed and thermally processed carrots. Toughness values were, however, significantly lower for the thermally processed in comparison with the frozen‐thawed carrots. Instrumental texture measurements indicated that different areas of carrot tissue behave differently to thermal processing.

The effects of gamma irradiation on the growth and cytology of carrot ( Daucus carota L.) tissue culture

Carrot ( Daucus carota L.) tissue suspension cultures were subjected to 0.5–40 krad of gamma irradiation. Callus fresh weight was significantly increased and dry weight slightly increased by low doses (0.5 and 1 krad). Cell size increased at all doses. Cell number and colony-forming ability decreased. There was a negative correlation between radiation dose and mitotic index, and a positive correlation between dose and mitotic abnormalities (multipolar separations, lagging chromosomes, and bridges). Frequency of prophase was increased, and anaphase and telophase were reduced. Tissue culture conditions increased the incidence of aneuploidy and polyploidy in comparison to carrot root tips. Aneuploidy in tissue cultures was decreased and polyploidy was increased by gamma irradiation. Gamma irradiation stimulated shoot formation a 1 krad, and inhibited shoot formation at high doses (20–40 krads). The frequency of abnormal plants (albinos, and plants with no roots or deformation of leaves) regenerated from carrot cell cultures was increased by gamma irradiation.

Plant Uptake of Pentachlorophenol from Sludge‐Amended Soils

AbstractA greenhouse study was conducted to determine the effects of sludge on plant uptake of 14C‐pentachlorophenol (PCP). Plants included tall fescue (Festuca arundinacea Schreb.), lettuce (Latuca sativa L.), carrot (Daucus carota L.), and chile pepper (Capsicum annum L.). Minimal intact PCP was detected in the fescue and lettuce by gas chromatography/mass spectrometry (GC/MS) analysis. No intact PCP was detected in the carrot tissue extracts. Chile pepper was not analyzed for intact PCP because methylene chloride extracts contained minimal 14C. The GC/MS analysis of soil extracts at harvest suggests a half‐life of PCP of about 10 d independent of sludge rate or PCP loading rate. Rapid degradation of PCP in the soil apparently limited PCP availability to the plant. Bioconcentration factors (dry plant wt./initial soil PCP concentration) based on intact PCP were <0.01 for all crops, suggesting little PCP uptake. Thus, food‐chain crop PCP uptake in these alkaline soils should not limit land application of sludge.

The roles of aspartic proteinase and endo-pectin lyase enzymes in the primary stages of infection and pathogenesis of various host tissues by different isolates of Botrytis cinerea Pers ex. Pers

A single spore isolate of Botrytis cinerea, obtained from carrot tissue, produced an aspartic proteinase and an endo-pectin lyase both in vitro in a liquid medium containing 0.4% gelatin as protein source and in infected carrot tissue. Both enzymes caused cell death and induced resistance to B. cinerea in surface cell layers of carrot root slices when applied at relatively low concentrations. At higher concentrations, both caused extensive cell death in carrot tissue and of carrot suspension cells. Neither enzyme lysed osmotically balanced carrot protoplasts unless carrot cell wall preparations were also present. Plasmolysed carrot cells were protected from the phytotoxic activity of the endo-pectin lyase but not from proteinase-induced damage. It is suggested that the phytotoxic activity of both the endo-pectin lyase and aspartic proteinase is indirectly caused by the toxic effect of cell wall components released by each enzyme. Accumulation of 6-methoxymellein was induced in carrot root slices by soluble, non-phytotoxic, elicitors released as a result of the action of aspartic proteinase or endo-pectin lyase on carrot cell walls. Proteinase production was an early event in infection of carrot, strawberry, raspberry, cabbage, grape and broad bean by eight different isolates of the pathogen. The level of infection in these hosts was markedly reduced when spores were pre-treated with pepstatin, a specific inhibitor of the aspartic proteinase, implying a primary role for this enzyme in pathogenesis.

Purification and characterization of an aspartic proteinase secreted by Botrytis cinerea Pers ex. Pers in culture and in infected carrots

A single spore isolate of Botrytis cinerea, obtained from carrot tissue, when grown in a liquid medium containing gelatin as sole nitrogen source, secreted an aspartic proteinase (E.C. 3.4.23) with many similarities to the one extracted from B. cinerea-infected carrot tissue. This enzyme was not detected in healthy tissue. The enzyme from both culture and infected tissue had a pH optimum of 3·5 and a pI of 4·0; its action was inhibited by pepstatin and diazoacetyl-norleucine methyl ester, known inhibitors of aspartic proteinases, but not by compounds known to inhibit serine-, thiol- or metalloproteinases. The enzyme existed as a single unit with a molecular mass of 38–39 kD on sodium dodecylsulphate polyacrylamide gels. It was present in ungerminated spores and was produced during germination before pectic enzyme components, both in culture and in host tissue. Cell death was detected in B. cinerea-infected carrot root tissue before pectic enzyme or necrosis-inducing glycoprotein or polysaccharide production, supporting the view that the aspartic proteinase may be primarily responsible for phytotoxicity.

Molecular analysis of a phylogenetically conserved carrot gene: Developmental and environmental regulation

Extensive studies of gene expression programs in carrot somatic embryos identified a gene, designated Dc3, that serves as a reliable molecular marker for the acquisition of embryogenic potential by carrot cells in culture. The complete sequence of a carrot genomic region, DcG3, encoding a Dc3-like mRNA, was determined. The DcG3 transcription unit contains a single intron and encodes mRNA that is expressed at high levels in embryonic tissue but is undetectable in somatic tissue of carrot. The predicted protein sequence of DcG3 is 163 amino acids and includes two approximately 50 amino acid direct repeats which in turn include additional repetitive elements with an unusual distribution of charged amino acids. Dc3 and Dc3-like mRNAs are encoded by a small divergent gene family. Furthermore, similarities of the Dc3 gene family with genes from other plant species that are expressed in response to environmental and developmental cues suggest a possible role in seed desiccation and possibly in more general water-stress responses in plants. Analysis of transgenic tobacco containing a beta-glucuronidase (GUS) reporter gene fused to a 1.7 kb 5' upstream element of DcG3 defined a promoter/enhancer complex that confers developmentally and environmentally regulated expression of GUS activity. Thus, DcG3 is phylogenetically conserved together with the trans-acting factors required for its regulated expression in transgenic tobacco.