Tobacco Tissue Cultures Research Articles

Isolation and characterization of two isoperoxidases from tobacco tissue cultures

Two isoperoxidases (A f and C n) from the medium of tobacco tissue suspension culture WR-132 grown in darkness have been purified to apparent homogeneity and partially characterized. C n and A f have MWs of ca 30 000 and 54 000, respectively. A f has ca 5.1% carbohydrate, but none could be detected in C n. Both isoperoxidases appear to follow simple Michaelis-Menten kinetics with respect to guaiacol as the substrate. The K ms for guaiacol are 4 and 13.3 mM for A f and C n, respectively, while both isoperoxidases have a pH optimum at 6.5. C n, is dissimilar to other isoperoxidases from tobacco tissue cultures, but A f is very similar to isoperoxidase A 3 from W-38 tobacco tissue culture.

Comparison of tryptic peptide maps of eight isoperoxidases from tobacco tissue cultures

Eight isoperoxidases from tobacco suspension culture WR-132 (termed C n, C 3, C 4, A c, and A f) and tobacco callus culture W-38 (termed A 1, A 2, and A 3) have been subjected to trypsin digestion followed by peptide mapping. The peptide maps of isoperoxidases A f and A 3 are identical. All other isoperoxidases do not appear to be dramatically dissimilar in certain portions of their sequence, since many matching peptides have been found when various isoperoxidases are cross-compared. However, only two, and possibly three, highly homologous peptides are present in all of the isoperoxidases.

The sensitivity of soybean tissue cultures to the thymidine analogue, 5-bromodeoxyuridine

Three lines for soybean cotyledonary callus tissue have been examined for their sensitivity to 5-bromodexyuridine (BUdR) — a cytokinin-dependent, a cytokinin-independent and a cytokinin-dependent, chlorophyllous strain. The cytokinin-requiring line was found to be less sensitive to BUdR than the non-requiring tissue. In addition, the response of this latter strain was not altered by exogenous BAP. Furthermore, no bleaching of the chlorophyllous line occurred in the presence of BUdR. These findings are in direct contrast to some previous studies on tobacco tissue cultures and reinforce the suggestion that the hormonal autonomy of a tissue culture does not necessarily indicate its sensitivity to BUdR.

Cytokinin biosynthesis in a cell-free system from cytokinin-autotrophic tobacco tissue cultures

Cytokinin biosynthesis in a cell-free system from cytokinin-autotrophic tobacco tissue cultures

Substituted Indoleacetic Acids Tested in Tissue Cultures

AbstractMonochloro substituted indole‐3‐acetic acids inhibited shoot induction in tobacco tissue cultures about as much as IAA. Dichloro substituted indole‐3‐acetic acids inhibited shoot formation less. Other substituted indoleacetic acids except 5‐fluoro‐ and 5‐bromoindole‐3‐acetic acid were less active than IAA. Callus growth was quite variable and not correlated with auxin strength measured in the Avena coleoptile test.

Plasmid-dependent attachment of Agrobacterium tumefaciens to plant tissue culture cells.

Kinetic, microscopic, and biochemical studies show that virulent Ti (tumor inducing)-plasmid-containing strains of Agrobacterium attach to normal tobacco and carrot tissue culture cells. Kinetic studies showed that virulent strains of A. tumefaciens attach to the plant tissue culture cells in increasing numbers during the first 1 to 2 h of incubation of the bacteria with the plant cells. Five Ti-plasmid-containing virulent Agrobacterium strains showed greater attachment to tobacco cells than did five avirulent strains. Light and scanning electron microscopic observations confirmed that virulent strains showed little attachment. Bacterial attachment was blocked by prior incubation of the plant cells with lipopolysaccharide extracted from A. tumefaciens, but not from A. radiobacter, suggesting that bacterial lipopolysaccharide is one of the components involved in the attachment process. At least one other bacterial product may be required for attachment in tissue culture because the virulent A. tumefaciens NT1, which lacks the Ti plasmid, does not itself attach to tobacco cells, but its lipopolysaccharide does inhibit the attachment of virulent strains.

Modification of Disease Resistance of Tobacco Callus Tissues by Cytokinins

The effects of differing cytokinin and auxin concentrations on resistance of tobacco (Nicotiana tabacum L.) tissue cultures to race 0 of Phytophthora parasitica var. nicotianae were examined. With 1 micromolar kinetin and either 11.5 micromolar indoleacetic acid or 1 micromolar 2,4-dichlorophen-oxyacetic acid, tissues from resistant cultivars exhibited a "hypersensitive" reaction to zoospores of the fungus and subsequently were colonized only slightly. With susceptible cultivars or with tissues from resistant cultivars supplied with higher cytokinin levels (e.g. 10 micromolar kinetin), this hypersensitive reaction did not occur and tissues were heavily colonized. Benzylaminopurine and kinetin were particularly effective in eliminating both the hypersensitive reaction and disease resistance. Zeatin and 6-(3-methyl-2-butenylamino)purine were less effective. Increases in indoleacetic acid levels reversed the effects of high cytokinin concentrations. The balance of phytohormones apparently controls the host response to the fungus; thus, in this system, resistance or susceptibility can be studied without changing either host or fungal genotype.

Effect of Plant Hormones on Tobacco Mosaic Virus Concentration in Tobacco Tissue Culture

Effect of Plant Hormones on Tobacco Mosaic Virus Concentration in Tobacco Tissue Culture

Acyl lipids in photosynthetically active tissue cultures of tobacco

Lipid mixtures extracted from green leaves, green tissue cultures, etiolated cotyledons and heterotrophic cultures were subjected to quantitative determination of the major phospho- and glycolipids. Fatty acid analyses including positional distribution at the glycerol moiety were carried out for individual lipid components. The results demonstrate that green cultures occupy a position between green leaves and heterotrophic cultures. The major membrane lipids known from photoautotrophic tissues are present in green cultures. Components concentrated in chloroplasts such as monogalactosyl-(MGD) and sulphoquinovosyl diacylglycerol (SQD) and the fatty acids C 16:3 and 3-trans- C 16:1 occur at markedly reduced levels. A general increase in the proportion of C 18:2 at the expense of C 18:3 is observed in tissue cultures. The positional distribution of significant fatty acids is identical in the lipids from green cultures and leaves.

Use of 4‐Amino‐3,5,6‐trichloropicolinic Acid as an Auxin Source in Plant Tissue Cultures1

The auxin‐like properties of 4‐amino‐3,5,6‐trichloropicolinic acid (picloram) were compared with those of other auxins in tissue cultures of wheat (Triticum aestivum), soybeans (Glycine max), tobacco (Nicotiana tabacum), and jackbean (Canavalia ensiformis) explants. Certain problems associated with the use of 2,4‐dichlorophenoxyacetic acid (2,4‐D) in tissue culture were not encountered when picloram was substituted for 2,4‐D in the culture medium of 2,4‐D‐requiring plant materials. Picloram performed as well or better than the auxins to which it was compared when used in plant tissue cultures. In general, picloram was more effective at low concentrations than the other auxins. The data indicate potential uses of picloram in routine callus cultures, in regeneration of plants from calli, and in research concerning the physiological development of plant tissues.

Sur L'Utilisation des Protoplastes en Cytologie: Amelioration D'Une Methode Recente en vue de L'Identification des Chromosomes Mitotiques des GenresNicotianaetPrunus

SUMMARYPlant protoplasts treated in the same way as lymphocytes in animal cytogenetics allowed to point out a chromosomes counting technique in tobacco tissue cultures (MOURAS 1975). The analysis of the factors important to develop this method (pretreatment, osmotic concentration and temperature of the cell-enzyme mixture) leads to an improvement of the method. Hence it becomes possible to consider its application in order to identify not only mitotic chromosomes of tobacco, for which a karyogram is presented (Fig. 1), but also those of species with which the squash technique has only given, till now, poor results: Prunus (Figs. 2 and 3) and Corylus.

Culture conditions for nicotine production in tobacco tissue culture.

A tobacco callus strain, OMT-53, was selected from many cultures as a desirable strain having high nicotine producing capacity. Several culture conditions were examined, aiming to get higher nicotine production with the callus strain, OMT-53. It was revealed that the nicotine production was remarkably enhanced when the callus tissues were cultured at a limited concentration of α-NAA in culture medium. The optimal concentrations of sucrose and nitrogen in the culture medium were 3 % and 840 mg N/L respectively. Some precursors in nicotine biosynthesis were examined, and only ornithine gave a slightly positive effect at 2x10-4m concentration. Cultures at 25°C produced the highest yield for nicotine. Considerable amounts of nicotine (ca. 20% of total nicotine) were also recognized in the culture medium. Under the best culture condition mentioned above, nicotine production in tobacco callus tissues has been elevated to 2.14% on D.W, basis at 4 weeks’ culture. This value is near to that of the intact tobacco p...

Effect of light on nicotine production in tobacco tissue culture.

The effect of light on nicotine production in cultured tobacco callus tissues was investigated. Illumination strikingly inhibited the nicotine production even though the growth of the tissues was slightly stimulated by the light. The inhibitory effect of light increased as the intensity and the length of the illumination increased. No decisive difference in nicotine production was observed between the effects of blue light and red light. The depression of nicotine production by light was restored completely when the tissue was transferred to the dark. Therefore, nicotine production is concluded to be regulated by the light just as in the case of plant growth regulators. The inhibitory effect of light was assumed to becaused rather by the inhibition of nicotine biosynthesis than by the promotion of nicotine decomposition.

Differentiation Ability of the Tobacco Mosaic Virus-Eliminated Tobacco Tissue Cultures

Differentiation Ability of the Tobacco Mosaic Virus-Eliminated Tobacco Tissue Cultures

The role of lectins in cell division of tissue cultures of soybean and tobacco

Summary Lectins, phytohemagglutinins, have been reported to act as mitogenic agents in animal and plant tissue cultures. We have tested the effect of three different lectins (soybean agglutinin, phytohemagglutinin-M, and wheat germ agglutinin) on the growth of tissue cultures of soybean root segments and tobacco with expiants. In no case any significant improvement in growth over controls could be demonstrated, except in the case of wheat germ agglutinin, which appears to enhance the growth of tobacco pith expiants in the presence of an auxin and a cytokinin.

Correlation between Phenylalanine Ammonia Lyase Activity and Phenolic Biosynthesis in p-Fluorophenylalanine-sensitive and -resistant Tobacco and Carrot Tissue Cultures

Phenylalanine ammonia lyase (PAL) activity was measured in p-fluorophenylalanine (PFP)-sensitive and -resistant tobacco (Nicotiana tabacum L.) and carrot (Daucus carota L.) cell lines which are known to oversynthesize phenylalanine. A correlation between phenolic levels and PAL activities was detected. The phenylalanine analog-resistant and -sensitive carrot cells showed no differences in the accumulation of phenolic compounds and PAL activities. The PFP-resistant tobacco cells, however, had 10 times higher levels of phenolics and also 10 to 20 times higher PAL activities than the PFP-sensitive line. The PAL activity in the resistant tobacco line increased dramatically after inoculation of the cells into fresh medium. Conditions affecting this increase were characterized.A comparison of the responses of PAL activity to light treatment and changes in the 2,4-dichlorophenoxyacetic acid concentration in the medium revealed no differences for both tobacco lines. After inoculation of the cells into fresh medium, the rates of nitrate uptake from the medium were similar for both lines as were the rates of protein synthesis. The reason for the increased PAL levels in the resistant tobacco cells remains unknown.

The Action of Fusicoccin Alone and with Some Plant Growth Substances on Tobacco Tissue Cultures

AbstractThe interaction of fusicoccin, a terpenoid glucoside produced by Fusicoccum amygdali Del., and some plant growth regulating substances, i.e., indole‐3‐acetic acid, kinetin, 2,4‐dichlorophenoxy‐acetic acid and abscisic acid, was investigated on pith and sub‐cultured callus cultures of Nicotiana tabacum L. Addition of fusicoccin alone to the basal medium, either in the light or in the dark, caused a fairly good development of tobacco callus. When fusicoccin and kinetin were simultaneously added to the culture medium, the callus growth increased. However, fusicoccin in combination with indole‐3‐acetic acid caused limited callus development: the tissue appeared brown and reduced in volume. Addition of fusicoccin with 2,4‐dichlorophenoxyacetic acid stimulated growth of callus and chlorophyll was formed under light. Finally, abscisic acid did not interfere with the effect of fusicoccin on the callus growth.

Studies on plant growth‐regulating substances

SUMMARYA report of an alkoxypurine showing cytokinin activity is presented. This compound, 6‐benzyloxypurine, has been tested in three tests and in two of these, a wheat leaf senescence and a young coleoptile test, the compound is as active as benzyladenine or kinetin. Although it is much less effective on molar basis than these compounds in a tobacco tissue culture test, it is as active at its optimum concentration. In addition to promoting cell division the presence of 6‐benzyloxypurine leads to morphological differentiation within the tobacco stem tissue, and whole plants have been grown without altering the nutrient medium.

Comparative Growth of Pith Tissue from Haploid and Diploid Plants of Nicotiana tabacum

Summary The fresh-weight yields of tobacco tissue cultures ( N. tabacum cv. Wisconsin-38 and cv. IAC-70), grown on MS-62 media with varying concentrations of IAA and Kinetin were determined and related to the androgenetic or zygotic origin of the explanted tissues. Explants from haploid plants showed larger values than explants from diploid plants, and greater potentiality to differentiate than diploids, suggesting a genetic component in the growth responses due to exogenously added IAA and Kinetin.

Effect of darkness on isoperoxidases in tobacco tissue cultures

In order to study the effect of light on the tobacco tissue culture WR-132, 5 passages (10 days' growth per passage) of these cells were grown in darkness, and 3 passages were separately grown in intense light (16000 lx). All other growth conditions were the same. The resulting isoperoxidase patterns present in these cells and in their growth media were analyzed at 2-day intervals during this period and then compared with the isoperoxidase patterns of cells grown under dim light conditions (10 lx). A new cathodic isoperoxidase (Cn) appeared in the medium within 2 days after the cells were placed in the dark. Cn was present in all media of WR-132 cell cultures analyzed throughout the 5 passages grown in darkness. The fifth passage in darkness produced total cessation of growth (apparent death). Cn increased and new anodic isoperoxidases Aa, Ab, Ad and Ae appeared in the media as the cells approached death in darkness.