Carrot Tissue Research Articles

Correlation between rate of cell division and loss of embryogenesis in longterm tissue cultures

In experiments with carrot tissue cultures of different ages (up to 5 years) it was investigated whether relations exist between embryogenesisin vitro and certain growth parameters of the cultures. The normal pattern of embryo-formation on an inductive medium (Ms), with a maximum 20 weeks after the isolation of the cultures and its disappearance after about 60 weeks, could be changed by retardation of growth either by low temperatures or by culture on a non-inductive medium (Mw). In both cases partial inhibition of growth resulted in a prolonged period of competence of the cultures for embryo-formation. It was also found that a close relation existed between the rate of cell division and the percentage of cultures forming embryos; the latter decreased with increasing numbers of cell generations. It is probable that the observed morphogenetic variations depend at least partially upon epigenetic changes which occur during growth and differentiationin vitro.

Further Studies on the Photosynthesis of Carrot Tissue Cultures

The influence of kinetin and sucrose on the photosynthetic activity of carrot (Daucus carota) tissue cultures in relation to growth was investigated. The results showed that light contributes heavily to the growth of tissue cultures measured in terms of fresh and dry weight and cell division activity. In light, the fresh weight, dry weight, and number of cells per explant were about or more than doubled. This indicated that after the development of chloroplasts, carrot tissue cultures can grow autotrophically at least as far as energy and carbon are concerned. Kinetin was shown to have an important role in developing the photosynthetic apparatus and photosynthetic activity of tissue cultures as manifested by the increase of chlorophyll content (60%), Hill activity (about 3-fold), and (14)C-fixation from NaH(14)CO(3) (about 20%). On the other hand, the presence of sucrose in the medium reduced the chlorophyll content by about 30% and (14)C-fixation from NaH(14)CO(3) in the soluble fraction by about 60%. A possible correlation between the influence of kinetin on sugar uptake and the effect of kinetin on (14)C-fixation from NaH(14)CO(3) was discussed.

DNA synthesis during development of adventive embryos of wild carrot

Embryo development in cultured wild carrot tissue was studied by considering the relationship between DNA biosynthesis and embryo morphogenesis. Autoradiographic data of3H-thymirdine incorporation indicated a higherpercentage of cells undergoing DNA synthesis in adventive embryos than in proembryonic masses. A definite pattern of DNA synthesis was observed during the transition from a proembryonic mass to the torpedo stage of embryogenesis. In particular the labeling pattern for both the heart and torpedo stages was found to be coincident with specific morphogenetic events.

Isolation of Thielaviopsis basicola from soil with umbelliferous root tissue as baits

Root tissue of 31 cultivars and varieties representing 12 species and 12 genera of the Umbelliferae were tested and evaluated as baits for Thielaviopsis basicola (Berk. & Br.) Ferr. in soil. The relative ability of root tissue of each species to selectively bait T. basicola from soil was measured as degree of invasion of the root tissues by T. basicola and freedom of the tissue from decay by other organisms. Root tissue of carrot as bait was superior to root tissue of other species tested. The mechanism(s) in carrot which permits selective isolation of T. basicola from soil is not restricted to carrot but can be found in varying degrees in at least five other species in the Umbelliferae.

Toxicity levels of mercury, lead, copper, and zinc in tissue culture systems of cauliflower, lettuce, potato, and carrot

Explanted tissues of cauliflower, carrot, potato, and lettuce have been grown in media containing Zn, Cu, Hg, and Pb. Toxicity levels have been established for each of these elements and a considerable uniformity was noted. Lettuce tissue responds occasionally to high levels of Zn by making extraordinarily prolific growth, a matter that has yet to be explained.

Variations and correlations within and between morphology, pathogenicity, and pectolytic enzyme activity in Sclerotinia from Saskatchewan

One hundred and fourteen isolates of Sclerotinia from 23 different hosts in many parts of Saskatchewan were grouped, according to their morphology, on minimal medium. Two types of seedling pathogenicity tests on six host species were conducted on at least one isolate from each morphological group and one from each host species. A total of 38 isolates was tested. Assays for pectolytic enzyme activities of the same 38 isolates were done using a defined medium, and Swede turnip and carrot tissue as substrates. Polygalacturonase, pectin transeliminase, and pectin methylesterase were all tested. The results showed that an endopolygalacturonase was probably the most prevalent enzyme. Some isolates also produced exopolygalacturonase and pectin methylesterase, but pectin transeliminase was never detected. There was no correlation between pathogenicity of the isolates and their enzyme activities in vitro or in vivo, suggesting that pectolytic enzymes are not responsible alone for pathogenicity. Agglomerative classification was used to demonstrate relationships between the isolates. However, the isolates did not fall into discrete groups based on morphological, pathological, physiological, or even combined characteristics. Neither were there clear host or geographic associations. This continuous variation rather than a segregated population is consistent with Purdy's "broader concept of the species S. sclerotiorum."

Activities of key enzymes of carbohydrate oxidation in disks of carrot storage tissue

In extracts of freshly cut disks (1 × 10 mm) of storage tissue of carrot ( Daucus carota L.) the activities of hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and phosphofructokinase at 30° were 17, 29, 174 and 69 nmoles product formed/min/g fresh wt., respectively. The corresponding values for extracts of disks that had been incubated in aerated water for 24 hr were 67, 174, 261 and 39, respectively. These results are discussed in relation to the control of carbohydrate oxidation.

The Effect of Dissolved Oxygen Concentration on Growth and Differentiation of Carrot (Daucus carota) Tissue

For suspension cultures of carrot tissue there is a critical level of dissolved oxygen concentra tion. Below this level, dry weight increased linearly with time while cell numbers increased exponentially, creating 'meristematic-like ' conditions in the culture. These conditions favoured differentiation into plantlets by embryogenesis while conditions above the critical level, when both cell dry weight and cell numbers increased exponentially, favoured plantlet differentiation by rhizogenesis. Different light treatments affected the yield of structures formed but did not prevent oxygen from controlling the differentiation pathway.

Untersuchungen über den Einfluß des Kinetins und des Eisens auf den Nukleinsäure‐ und Proteinstoffwechsel von Karottengewebekulturen

The Influence of Kinetin and Iron on Nucleic Acid and Protein Metabolism of Carrot Tissue CulturesCarrot‐root tissues cultured in a nutrient solution containing Kinetin had a high rate of cell division, that led to an undifferentiated callus, and a relatively low DNA content per cell. The same tissues growing without Kinetin had a smaller rate of cell division and a higher DNA content, and showed the ability to form roots.By addition of 32P to the nutrient solution, the cultures growing with Kinetin showed a higher specific activity of DNA than those growing without Kinetin.From previous experiments and results of other workers the possibility of the occurence of a metabolic labile DNA fraction in the cultures which grew without Kinetin is discussed.A comparison between the influence of iron and Kinetin on cell division activity, root formation, DNA, RNA and amino acid content of the tissue cultures was made on a cell basis. Iron deficiency or absence of Kinetin in the nutrient solution reduced the cell division activity of carrot tissue cultures and increased the content of DNA, RNA and soluble amino acids. The protein content was reduced by iron deficiency but was increased by the absence of Kinetin.Cultures, growing without Kinetin were able to form roots, while those, growing in an iron deficient medium were unable to show the latter phenomenon.

Suppression of organized growth in cultured wild carrot tissue by 2-chloroethylphosphonic acid

Suppression of organized growth in cultured wild carrot tissue by 2-chloroethylphosphonic acid

Plant Nucleases

Isoenzymes of RNase were detected in plant extracts after polyacrylamide gel electrophoresis with a new buffer system. The gels were incubated in an RNA solution, then dipped for 30 seconds into 0.2% toluidine blue. The method is rapid and is sensitive to very small amounts of RNase. The effects of buffers and ethylenediaminetetraacetate on the different enzymes are illustrated by photographs and scans of the gels.RNase I, from endosperms and roots, was the fastest-moving corn RNase. Two isoenzymes of corn RNase II, from microsomes, were detected in the hybrid WF9 x M14, while each parental inbred had one of the isoenzymes. Three isoenzymes of corn Nuclease I, from crude mitochondria, had about the same mobility as the RNase II isoenzymes, but were inhibited by ethylenediaminetetraacetate. RNases were also detected in tobacco and wild carrot tissue cultures.

Nucleic acid synthesis and induced respiration by disks of carrot storage tissue

Net synthesis of RNA and DNA occured when freshly prepared disks (1 x 10mm) of storage tissue of carrot ( Daucus carota L.) were incubated at 25° under moist conditions. Net synthesis of RNA occured within 8 hr of preparation of the disks and continued for at least 4 days. Net synthesis of DNA was preceded by a lag of 48–72 hr. Incubation in either a simple combination of plant hormones, or in a nutrient medium known to support the growth of carrot tissue cultures, failed to overcome this lag. Incubation in 5-fluorouracil inhibited the net synthesis of RNA and DNA and the development of induced respiration. Incubation in cycloheximede inhibited the net synthesis of RNA and the development of induced respiration. It is suggested that the development of induced respiration is dependent upon the synthesis of RNA and protein.

Effects of cycloheximide on protein synthesis and respiration in disks of carrot storage tissue

This paper describes experiments that were undertaken in order to discover whether, in disks (1 x 10 mm) of storage tissue of carrot ( Daucus carota L.), cycloheximide inhibited protein synthesis directly, or indirectly via an effect on respiration. Freshly cut disks, incubated in water for 4 hr and then transferred for 75 min to a medium that contained [ 14C]-leucine, incorporated [ 14C]-leucine into protein. Cycloheximide (10, gmg/ml) caused a 95 per cent inhibition of this incorporation. In comparable experiments, the effects of cycloheximide on the distribution of 14C from [6- 14C]-glucose were determined. The results indicate very strongly that cycloheximide inhibited protein synthesis directly and not via any effect on respiration. Cycloheximide did not increase the rate of oxygen uptake. It is concluded that protein synthesis occurs when freshly cut dish of carrot are incubated in water and that cycloheximide inhibits this synthesis directly.

Gas hydrates in aqueous-organic systems: VI. Effects of hydrate formers and hydrate crystals in carrot tissue

Carrot samples containing ethylene oxide (EO), tetrahydrofuran (THF), and their hydrates were studied. It was found that: (1) the microscopic appearance of carrot tissue was not altered appreciably by EO or THF (no detectable hydrate crystals), (2) slow formation of EO hydrate (Type I) and THF hydrate (Type II) resulted in extracellular crystalline areas which were large and few in number, and their presence was associated with severe shrinkage and distortion of adjacent tissue (this behavior is similar to that observed during conventional slow freezing of carrot tissue), and (3) crystalline masses of EO hydrate in carrot tissue tended to increase in size and become less numerous as the rate of formation was decreased and the growth period was increased. These results differed from those obtained previously with animal tissue. EO (no detectable hydrate crystals) was much less detrimental to the microscopic appearance of carrot tissue than it was to the microscopic appearance of beef muscle (BM) or rat liver (RL). Furthermore, slow formation of EO hydrate resulted in extracellular crystalline areas which were larger and less numerous in carrot tissue than in BM, or RL, and this in turn resulted in cell shrinkage which was less uniform and more severe (adjacent to crystalline areas) in carrot tissue than in BM or RL.

The Absorption of Boron by Disks of Plant Storage Tissues

Disks prepared from the storage tissues of carrot (Daucus carota) were able to absorb boron from boric acid solutions (0�1 mM). At 25�C, net influx ceased 3-4 hr after the tissue was placed in boric acid solution. At equilibrium the tissues were able to maintain an internal concentration of diffusible boron in excess of that in the external solution. Boron uptake was inhibited by anoxia, 2,4.dinitrophenol, and at 1�C; and, in the presence of these inhibitors, the equilibrium concentration of boron in the tissues only approached that in the external solution. The capacity of the tissues to accumulate boron developed 2-3 days after the beginning of a washing treatment. Boric acid (0�1 mM) in this washing solution inhibited the development of this capacity.

Sucrose suppression of chlorophyll synthesis in carrot tissue cultures: The role of invertase

Free space invertase activities were determined in carrot callus strains CRT1 and CRT2 grown under conditions in which sucrose suppression of chlorophyll synthesis occurred in CRT1 but not CRT2. CRT2 possessed a high free space acid invertase activity (pH optimum 5.0 Km for sucrose 3.1×10(-3)M) while CRT1 lacked this enzyme. [U-(14)C] sucrose introduced into the free space of calluses was rapidly inverted by CRT2, but not by CRT1.Despite their different invertase levels, CRT1 and CRT2 showed similar sucrose uptake rates and took up [U-(14)C-glucosyl] sucrose and [5-T-glucosyl] sucrose from external bathing media essentially without prior inversion.It is concluded that acid invertase in callus tissue relieves the suppression of chlorophyll synthesis caused by sucrose in the free space. The invertase may in some circumstances hydrolyse sucrose before uptake, but is not an essential part of the sucrose uptake mechanism in carrot tissue cultures.

Secretion of photosynthetic products by carrot tissue cultures

Green carrot callus cultures when exposed to (14)CO2 in a liquid medium showed ligh-dependent (14)C-incorporation into sucrose, glutamine and malic acid. About 5% of total (14)C fixed in a 3 h period appeared in these products in the bathing medium; this was not due to tissue damage. Kinetic studies showed that the release occurred from a metabolic and not a storage compartment. The effects of DCMU, temperature and fluoroacetate demonstrated that release from this compartment was under respiratory and not photosynthetic control.

Photosynthesis by carrot tissue cultures

(14)CO2-fixation rates in green carrot callus cultres (about 35 μg chlorophyll/g fresh wt) were determined in gaseous and liquid media using a range of light intensities and CO2 concentrations. Main products of light-dependent CO2-fixation were sucrose, alanine, glutamine, serine/glycine and malic acid. In darkness, glutamine and malic acid were formed.Light CO2-fixation rates were about ten times higher than dark fixation rates and reached 50-90 μmol/mg chlorophyll/h in 10000 lux, 1% CO2 in air. Net O2-evolution by the tissue was demonstrated polarographically under these conditions. Light CO2-fixation rates were linearly related to chlorophyll levels while dark fixation was independent of chlorophyll content. Lowered O2 partial pressures in gaseous conditions increased (14)CO2-fixation rates. Ribulose diphosphate carboxylase and phosphoenol pyruvate carboxylase activities and their distribution in subcellular fractions were examined.When carrot tissue cultures were grown for two or four weeks on agar media lacking a carbohydrate source, in 10000 lux and 1% CO2 in either air or N2, dry weight increases were obtained although chlorophyll levels eventually declined.

Maintenanance of Viability of Carrot Tissue Slices in Washing Solutions After Cutting

In studies of the characteristics of the uptake of boric acid by plant tissues (Wildes and Neales, unpublished data) we used disks cut from carrot roots. Bacon, MacDonald, and Knight (1965) have emphasized the necessity of using storage tissue free of bacteria for such physiological studies. In the course of our experiments we therefore investigated the extent of bacterial contamination in carrot disks immediately after cutting, and also examined the effects of chloramphenicol and calcium chloride in the washing solution on the extent of the development of bacterial contamination in the disks. We also made measurements of various properties that gave evidence of their normal biological activity. The loss of this activity, in our experience, was characterized by a final loss of turgor, browning of both tissue and washing solutions, and a failure to respond in respiration rate to both the addition of salts and 2,4-dinitrophenol.

Sucrose suppression of chlorophyll synthesis in carrot callus cultures

Substrate levels of sucrose were shown to reduce chlorophyll synthesis in carrot tissue culture strain CRT1 but not in strain CRT2. In CRT1 the effect was shown to be a suppression of greening specifically by sucrose rather than a reducing sugar requirement for chlorophyll synthesis. In CRT1 sucrose caused both a reduction in chloroplast numbers per cell and a suppression of lamellar development in plastids. This effect on chloroplast structure was consistent with the observed reduced photosynthetic efficiency (micromoles CO2 per hour per mg chlorophyll) of CRT1 calluses grown on sucrose.