Carrot Tissue Cultures Research Articles

Development of STEM tissue culture module in promoting plant biotechnology

Tissue culture is a part of modern biotechnology, which offers a lot of potential in tackling food security, genetic conservation and plant breeding. Thus, it is important to introduce plant biotechnology to the young generation in order to foster their positive attitude, which ultimately in the future, will result in human capital and public awareness in the field itself. This study focused on the development of STEM tissue culture module as a teaching aids to introduce plant biotechnology and exposing the variety of STEM career at school level among primary school students of ages 10-12 years old. This module consists of four activities focusing on the seed identification, water transportation in plants, grass doll and carrot tissue culture. Based on the conducted survey, 80% of the participants exhibited interest in the tissue culture module with 42% reacted positively on the statement that this module triggered their curiosity on plant biotechnology. Hence, this module is a promising teaching aid to promote STEM especially in plant biotechnology.

The frequency of genetic rearrangements during carrot (Daucus carota) somatic embryogenesis is dependent on 2,4-D levels and diminished in its absence

For quantification of genetic variations occurring in plant tissue cultures, DNA sequence alterations and replicon- size changes were monitored through subsequent phases of the model carrot tissue culture system, from the 2,4-D-induced proembryiogenic cell cultures to regenerated plantlets, by RAPD and flow cytometry techniques. Banding patterns of random amplified DNA fragments and ploidy-level distributions of cultured cells were significantly different in the presence and in the absence of 2,4-D. In addition, there were marked differences between the cells induced with lower (1.0 mg/l) and higher (2.5 mg/l) doses of 2,4-D. Among those samples that were cultured in the absence of 2,4-D, the epicotyl (C2), hypocotyl control (H1) and the morphologically normal regenerants (IV2) showed identical banding patterns overlapping with the true-to-type seedling controls (N1, N2). In contrast, treatment of starting explants (H2) with 1.0 mg/l 2,4-D resulted in a marked 82% increase in the number of amplified fragments associated with an appearance of cell lines possessing unusual haploid-like and aneuploid-like DNA contents. The induction with 2.5 mg/l 2,4-D resulted in even greater increase in the number of DNA fragments (~100%) amplified from proembryiogenic cell cultures, while it had no further effect on ploidiy-level distributions compared to the treatment with 1.0 mg/l 2,-D. After the withdrawal of the synthetic auxin, banding patterns and ploidy-level distributions were gradually shifted back to the levels of controls resulting in true-to-type regenerants. Conclusions are in short, the combined use of RAPD and flow cytometry can make quantification of genetic variations typical of dedifferentiated plant cells possible. Quantification opens the window for comprehensive evaluation of different methods, treatments and bioactive compounds.

Extracts from Black Carrot Tissue Culture as Potent Anticancer Agents

Black carrots contain anthocyanins possessing enhanced physiological activities. Explants of young black carrot shoots were cultured in Murashige and Skoog (MS) medium for callus initiation and were transferred to new MS medium supplemented with four different combinations of 2,4-dichlorophenoxyacetic acid and kinetin. Subsequently, the lyophilized calli and black carrot harvested from fields were subjected to ultrasound extraction with ethanol at a ratio of 1:15 (w:v). Obtained extracts were applied to various human cancer cell lines including MCF-7 SK-BR-3 and MDA-MB-231 (human breast adenocarcinomas), HT-29 (human colon adenocarcinoma), PC-3 (human prostate adenocarcinoma), Neuro 2A (Musmusculus neuroblastoma) cancer cell lines and VERO (African green monkey kidney) normal cell line by MTT assay. The highest cytotoxic activity was achieved against Neuro-2A cell lines exhibiting viability of 38-46% at 6.25 μg/ml concentration for all calli and natural extracts. However, a significantly high IC50 value of 170.13 μg/ml was attained in normal cell line VERO indicating that its natural counterpart is an ideal candidate for treatment of brain cancer without causing negative effects to normal healthy cells.

In memoriam Prof. Dr. Karl-Herrmann Neumann (1936–2009)

Prof. Dr. Karl-Herrmann Neumann passed away on October 13, 2009. He was born on a farm in Morgendorf near Leitmeritz, now part of the Czech Republic. In 1945, his family moved to Bernburg in Saxony-Anhalt, which later became part of the German Democratic Republic (GDR). Following some problems he encountered with the communist administration, he moved to the Federal Republic of Germany in 1956 where he finished his schooling and entered Justus-Liebig-University Giessen to study agriculture. This included a 1-year mobility program in Copenhagen, Denmark majoring in agricultural chemistry. He graduated from the Faculty of Agriculture of the Justus-Liebig-University Giessen in 1960. That same year, he received a 1-year-scholarship from Cornell University in Ithaca, New York, USA where he enrolled in their graduate program for 1 year majoring in botany and with minors in biochemistry and physical chemistry. At Cornell, he worked under the supervision of Prof. F.C. Steward focusing on the roles of micronutrients on growth and metabolism of carrot tissue cultures, and was highly inspired by Steward0s ideas and findings on totipotency of plant cells. Later, he returned back to Giessen and completed his PhD under the supervision of Prof. H. Linser, Institute of Plant Nutrition, Faculty of Agriculture in 1962. While working with both Prof. Steward and Prof. Linser, he became proficient in systemic thinking and engaged in multidisciplinary research approaches that subsequently greatly influenced his own scientific career. While pursuing his postdoctoral work with Prof. Linser, he completed his habilitation studies in 1968. During these years, he established one of the first plant cell and tissue culture laboratories in Germany. Among the first critical areas he dealt with was the development of an artificial nutrient medium of defined chemical composition as a replacement for White’s Basal Medium with coconut milk. In 1972, he became a Professor of Biochemistry and Prof. Dr. Karl-Herrmann Neumann

Soil adaptation of transgenic in vitro carrot plantlets

SUMMARYAdapting in vitro transgenic carrots to soil is the most crucial step preceding the field investigation of transgenic carrots. A low proportion of plants, around 0·20, acclimatize to soil (Hardegger & Sturm 1998) and thus prohibit the generation of high-expression carrot lines. In the present paper, a protocol for an efficient soil transfer is presented and the impact of carrot cultivar, soil substrate, tissue culture, and transformation process on transfer process is analysed. Somatic embryo germinants of Daucus carota cv. Rote Riesen 2 and Lobbericher Gelbe Futtermoehre showed a tremendous survival proportion – up to 1·00 – when transferred to their optimal soil substrate: sandy and loamy soil, with low content of macro and micro elements and a pH of 5·8. By optimizing the conditions described here, the proportion of soil acclimatized transgenic carrot plants of D. carota Lobbericher Gelbe Futtermoehre was increased from 0·1 to 0·87, and for the cultivar Rote Riesen from 0·09 to 0·67.

Research Article: N-(indole-3-acetyl) amino acids as auxin sources for somatic embryogenesis in carrot tissue cultures

Abstract The naturally occurring auxin, indole-3-acetic acid (IAA), and IAA-amino acid conjugates were compared to the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) for triggering somatic embryogenesis in carrot cells. Somatic embryos triggered by 2,4-D treatment may be a suitable model system for studying embryogenesis and embryo development. Use of 2,4-D avoids the challenges encountered with the use of IAA in tissue culture, but first it is essential to show that 2,4-D is an accurate substitute for the naturally occurring auxin. IAA or IAA-conjugates were substituted for the 2,4-D normally present in carrot cell culture medium. After 4 weeks the cells were transferred to medium without auxin. The cells were monitored to determine whether they formed somatic embryos exactly as cells do when taken off 2,4-D. Two of the conjugates tested exhibited the same effect on embryogenesis as 2,4-D. Embryos did not form in the presence of the auxin, but did form when the auxin was denied. With one conjugat...

Gamma Irradiation-induced Variation in Carrots (Daucus carota L.)

Carrot tissue cultures, germinating seed, and dry seed were exposed to gamma radiation. Irradiation accelerated germination of carrot seed in the M1 generation at low doses (0.5 and 1 krad), whereas higher doses delayed germination. A high negative correlation was observed between dose and survival of plants after seed irradiation. Plant size and root weight were 20 % to 35% greater than control plants after seeds, but not tissue cultures, were exposed to low doses of gamma irradiation. Higher doses reduced M1 plant size by >50% in germinating seed and tissue culture treatments but less for the dry seed treatment. Seed production decreased while phenotypic variation of M1 plants increased with increasing gamma ray dosage. Root weight and total dissolved solids were highly variable in M2 families. Less variation was observed in total carotene content and none was seen in sugar type (reducing vs. nonreducing sugars). Induced variation in root color and root shape was also observed. Irradiation of germinating seed and tissue cultures yielded more M2 variation than irradiation of dry seed. Putative point mutations were not observed. Unirradiated carrot tissue cultures did not yield significant M2 somaclonal variation. Average root weight of M2 plants increased with increasing gamma ray dosage, especially for the dry seed treatment.

Biochemical and ultrastructural aspects of carrot (Daucus carota L.) tissue culture in different experimental growth conditions

Biochemical and ultrastructural aspects of carrot (Daucus carota L.) tissue culture in different experimental growth conditions

Effects of supplied cinnamic acids and biosynthetic intermediates on the anthocyanins accumulated by wild carrot suspension cultures

Anthocyanins isolated and characterized from the wild carrot suspension cultures used here were 3-O-β-D-glucopyranosyl-(1→6)-[β-D-xylopyranosyl-(1→2)-]β-D<-galactopyranosylcyanidin (1), 3-O-[β-D- xylopyranosyl-(1→2)-β-D-galactopyranosyl]cyanidin (2), 3-O-(6-O-sinapoyl)-β-D-glucopyranosyl-(1→6)-[β-D- xylopyranosyl-(1→2)-]β-D-galactopyranos ylcyanidin (3), 3-O-(6-O-feruoyl)-β-D-glucopyranosyl-(1→6)-[β- D-xylopyranosyl-(1→2)-]β-D-galactopyranosylcyanidin (4), 3-O-(6-O-coumaroyl)-β-D-glucopyranosyl-(1→6)- [β-D-xylopyranosyl-(1→2)-]β-D-galactopyrano sylcyanidin (5), 3-O-[6-O-(3,4,5-trimethoxycinnamoyl)]-β- D-glucopyranosyl-(1→6)-[β-D-xylopyranosyl-(1→2)-]β-D-galactopyranosylcyanidin (6), 3-O-[6-O-(3,4-dime- thoxycinnamoyl)]-β-D-glucopyranosyl-(1→6)-[β-D-xylopyranosyl-(1→2)-]β-D-galactopyranosylcyanidin (7), 3-O-[(6-O-sinapoyl)-β-D-glucopyranosyl-(1→6)-β-D-galactopyranosyl]cyanidin (8), and 3-O-(β-D-galactopyranosyl)cyanidin (9). Except when cinnamic acids were provided in the culture medium, the major anthocyanin present in the two clones examined was 2. When the naturally occurring and some non-naturally occurring cinnamic acids were provided individually in the medium, 1 and 2 were minor components and the anthocyanin acylated with the supplied cinnamic acid, namely 3, 4, 5, 6, or 7 was the major anthocyanin present in the tissue. When caffeic acid was provided the major anthocyanin in the tissue was 4, thereby suggesting that the caffeic acid was methylated before its use in anthocyanin biosynthesis. Other cinnamic acids supplied had limited effects on the anthocyanins accumulated and appeared not to result in the accumulation of new anthocyanins by the tissue. Thus the tissue can use some but not all analogues of sinapic acid to acylate anthocyanins. Additional anthocyanins were detected in extracts of the wild carrot tissue cultures using mass spectrometry (both MS/MS and HPLC/MS). The additional compounds detected have also been found in cultures of black carrot, an Afghan cultivar of Daucus carota ssp. sativa and the flowers of wild carrot giving no evidence for qualitative differences in the anthocyanins synthesized by subspecies, cell cultures from subspecies, or clones from cell cultures. There are major differences in the amounts of individual anthocyanins found in cultures from different subspecies and in different clones from cell cultures. Here anthocyanins without acyl groups were usually found in the tissues and their accumulation is discussed. On the basis of the structures of the isolated anthocyanins, a likely pathway from cyanidin to the accumulated anthocyanins is proposed and discussed.

Simultaneous purification of chloroplast and mitochondrial DNA without isolation of intact organelles from green carrot tissue and suspension cultures

We report here the simultaneous purification of chloroplast (cpDNA) and mitochondrial DNA (mtDNA) from green tissue and suspension cultures of carrot without organelle isolation. This method is based on isolating total nucleic acids from frozen tissue and separating the nuclear, chloroplast and mitochondrial fractions using sequential isopycnic sedimentation in two gradients of cesium chloride containing bisbenzamide. From 10 g of mature carrot leaves, 10 to 30μg of organelle DNA was consistently recovered from mature carrot leaves, while 30 to 50 μg was recovered from suspension cells. The method can be used to isolate chloroplast and mitochondrial DNAs from single plants without sacrificing the individual.

Growth Promotion of Carrot Plantlets In Vitro by Adding Supplements of Phosphate to MS Medium.

In order to promote the growth of redifferentiated carrot (Daucus carota L.) plantlets in vitro in MS medium, changes in the concentration of the medium components and in the quantity of nutrient uptake were investigated. Eight somatic embryos of carrot were placed on medium-supports (made of formed polyester wool) containing 60 ml of MS medium with 1.5% sucrose in a culture vessel (60×60×100 mm) and cultured. From 1 week after placing, growth of the plantlets and concentrations of inorganic and sugar components were measured every week. Phosphate concentration of MS medium and growth rate of the plantlets decreased in the late stage of carrot tissue culture and phosphate uptake by the plantlets also decreased with increase of the dry weight. Phosphate added into the MS medium in the late stage of cultivation increased height and dry weight of the plantlets. It was found that the standard phosphate concentration in MS medium became insufficient in the late stage of cultivation of carrot plantlets, and adding supplement of phosphate into the medium promoted growth.

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.

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.

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.

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.

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 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.

Cucurbitacins in relation to somatic embryonyin vitro

Effects of four cucurbitacins, which occur mostly in seeds of Cucurbitaceae, and of cucurbitaceous tissue extracts on somatic embryony in tissue cultures of carrot,Citrus microcarpa andRanunculus sceleratus are described. Thin layer chromatographic analyses showed that the nucellar extract and the integuments ofLuffa cylindrica contain cucurbitain B. Studies with the extracts on all the three tissue culture systems have corroborated the observed inhibitory effect of cucurbitacins on somatic embryony. The role of cucurbitacins in plant embryogeny, and in the control of zygotic or adventive embryony in crop species is discussed.

Role of bacterial cellulose fibrils in Agrobacterium tumefaciens infection.

During the attachment of Agrobacterium tumefaciens to carrot tissue culture cells, the bacteria synthesize cellulose fibrils. We examined the role of these cellulose fibrils in the attachment process by determining the properties of bacterial mutants unable to synthesize cellulose. Such cellulose-minus bacteria attached to the carrot cell surface, but, in contrast to the parent strain, with which larger clusters of bacteria were seen on the plant cell, cellulose-minus mutant bacteria were attached individually to the plant cell surface. The wild-type bacteria became surrounded by fibrils within 2 h after attachment. No fibrils were seen with the cellulose-minus mutants. Prolonged incubation of wild-type A. tumefaciens with carrot cells resulted in the formation of large aggregates of bacteria, bacterial fibrils, and carrot cells. No such aggregates were formed after the incubation of carrot cells with cellulose-minus A. tumefaciens. The absence of cellulose fibrils also caused an alteration in the kinetics of bacterial attachment to carrot cells. Cellulose synthesis was not required for bacterial virulence; the cellulose-minus mutants were all virulent. However, the ability of the parent bacterial strain to produce tumors was unaffected by washing the inoculation site with water, whereas the ability of the cellulose-minus mutants to form tumors was much reduced by washing the inoculation site with water. Thus, a major role of the cellulose fibrils synthesized by A. tumefaciens appears to be anchoring the bacteria to the host cells, thereby aiding the production of tumors.

Activated Pectin Stained with Heavy Metals

Pectin was visualized in carrot tissue culture with a stain utilizing hydroxylamine and iron. Actively growing cells were fixed in glutaraldehyde or paraformaldehyde. The pectin was activated by an alkaline hydroxylamine treatment. When the so formed pectic hydroxamic acids were reacted with ferric chloride, a reddish-brown color was visible en masse, but the cells were not sufficiently stained for either light or electron microscopy.We hoped to intensify the stain by bridging iron to osmium. Since one Fe (III) complexes three gallocyanins, we tested this dye as a bridge in an attempt to amplify the staining. Thiocarbohydrazide (TCH) was tested as an alternate bridge. After excess iron was washed out, the cells were treated with either 1% gallocyanin for one hour, or 0.5% TCH for one minute. After excess bridge was removed by a second wash, a portion of each treatment was placed in 1% osmium tetroxide for one hour.