Inhibition of growth and xylogenesis and promotion of vacuolation in prunus callus by the flavanone prunin

Callus tissue of Prunus avium L. responded to supplied prunin (naringen in 7-glucoside) showing vaculoation and storage of oligomeric proanthocyanidins. In addition, prunin caused restricted callus initiation and/or less callus growth. When prunin was omitted from the medium numerous tracheids and more peroxidases were formed in the callus.Abbreviations DAB diaminobenzidine - DMACA p-diaminocinnamaldehyde - DWG dihydrowogonine 7-glucoside - P prunin - N naringenin     

Initiation and culture of potato tuber callus tissue with picloram

Callus cultures of 7 potato cultivars were initiated from tuber tissue and maintained on Gelrite-solidified media with 1–20 M picloram as the only PGR. Ten M picloram was the optimal concentration for callus induction. By 4–6 weeks after explanting, there was sufficient callus produced for subculture to maintenance media which contained 1–20 M picloram as the only PGR. When grown in the dark at 25°C, subcultured callus typically increased 10-fold in wet weight in 4–5 weeks. The callus produced was friable and a light grey to cream color. Callus cultures were used to establish cell suspension cultures. Callus and cell suspension cultures have been maintained for over 2 years on the picloram containing media.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige-Skoog - NAA naphthaleneacetic acid - PGR plant growth regulator Research paper #9053 of the Idaho Agricultural Experiment Station.     

Studies on some factors affecting solasodine contents in tissue cultures of Solanum nigrum

Tissue cultures of Solanum nigrum L. were initiated from leaf explants on a solid medium containing inorganic salts [Murashige and Skoog (1962), Physiol. Plant. 15: 473–497], vitamins [Gamborg et al. (1968) Exp. Cell Res. 50:151–158], 3% sucrose and combinations of indoleacetic acid and benzyladenine. Solasodine content was determined in differentiated and undifferentiated (callus) tissues by a colorimetric technique and thin layer chromatography. Indoleacetic acid and sucrose in the medium markedly stimulated the production of solasodine in the tissue cultures. In the cultures grown in darkness the differentiated tissues produced significantly more (anywhere from 1.5 to 10 times) solasodine than the callus in several media. When sucrose concentration was increased to 4, 6 and 10% level in the medium which contained 10 μ M benzyladenine as the sole growth regulator, a significant increase of solasodine production in cultures was found.     

The biological activity of cytokinin derivatives in the soybean callus bioassay

The effect of 28 natural and synthetic cytokinins, including cytokinin nucleotides, the growth of soybean cotyledonary callus was investigated. Generally the nucleosides and nucleotides gave a slightly better response than their respective free bases. The differences in response were, however, not significant and there is a distinct possibility that rapid interconversions between these three types of cytokinin occur within the tissue. The O-glucosides of Z and ZR were the most active. Glucosylation in the 3, 7 and 9 positions reduced activity. In the case of BA-derivatives the order of activity of the N-glucosides was 3G > 9G > 7G. Since iso-pentenyl derivatives had little activity they may be very difficult to detect using the soybean callus bioassay.Abbreviations Z zeatin - DHZ dihydrozeatin - IP iso-pentenyladenine - BA benzyladenine - K Kinetin - R riboside - MP monophosphate - OG 0-glucoside - 3G 3-glucoside - 7G 7-glucoside - 9G 9-glucoside - GC-MS gas chromatography—mass spectrometry     

Effect of Hormone Treatment on Growth Bud Formation and Free Amine and Hydroxycinnamoyl Putrescine Levels in Leaf Explant of Nicotiana tabacum Cultivated in Vitro

Foliar explants of Nicotiana tabacum cv Xanthi n.c. were cultured on four different media: a basal medium, basal medium plus benzyladenine, basal medium plus 2,4-dichlorophenoxyacetic acid (2,4-D), and the basal medium containing both hormones. No differentiation or cell division occurred in leaf explants cultured on the basal medium. Addition of benzyladenine caused the formation of buds on the explants, while 2,4-D caused callus formation and proliferation. Likewise, only callus was formed when explants were cultured on medium containing both hormones, but growth was significantly greater than that of callus grown on a medium containing 2,4-D alone. The levels of amines and hydroxycinnamoyl putrescines were determined in the four types of explants. In nongrowing explants, amines (except an aromatic amine, tyramine) and hydroxycinnamoyl putrescines were always at a low level and only small changes in their concentrations were observed. In callus cultures, amine (except an aromatic amine, phenethylamine) and hydroxycinnamoyl putrescine levels were higher than those found in bud cultures. In all the media, transitory accumulation of aromatic amines occurred after a few days of culture. Higher levels of hydroxycinnamoyl putrescines were attained in callus cultures with a slow growth rate (2,4-D alone) than in callus cultures with a fast growth rate (benzyladenine + 2,4-D). The formation of buds was accompanied by significant changes in putrescine and hydroxycinnamoyl putrescine levels. Increasing levels were found during the first 14 days in culture when cell multiplication was rapid, followed by a sharp decline after 20 days in culture as the rate of cell division decreased and differentiation took place. The relationship among amines, hydroxycinnamoyl putrescines, and cell division and bud formation is discussed.     

Regeneration of whole plants from hypocotyl-, root-, and leaf-derived tissue cultures of the pasture legume Stylosanthes guyanensis

Callus cultures were established on Murashige and Skoog medium from seedling hypocotyls and roots of Slylosanlhes guyanensis (Aubl.) Sw. cv. Cook and from leaves of 6-month-old) plants. Shoots developed in primary calli derived from seedling tissue with a number of benzyladenine or kinetin and naphthaleneacetic acid combinations. Shoot formation on primary leaf callus, occurred with 2.0 mg/1 benzyladenine and 2.0 or 1.0 mg/l naphthaieneacetic acid. Undifferentiated callus from all three sources was induced and maintained on medium with 2.0 mg/1 kinetin and 2.0 mg/1 2, 4-dichlorophenoxyacede acid in the dark. Shoot formation and regeneration of whole plants from these calli were achieved at high frequencies. The most successful combination of phytohormones for the induction of shoot development in undifferentiated callus, was 2.0 mg/1 benzyladenine and 1.0 mg/1 naphthaleneacetic acid. The regenerated plants showed no phenotypic abnormalities.     

ANTHOCYANINS OF DIMORPHOTHECA (COMPOSITAE). I. IDENTITY OF PIGMENTS IN FLOWERS,STEMS, AND CALLUS CULTURES

Anthocyanins isolated from ray flowers, disk florets, stem, and callus cultures of Dimorphotheca sinuata were identified and found to be cyanidin-3-glucoside and dephinidin-3-glucoside. The anthocyanins of the callus cultures were of the same identity as those in the whole plant, demonstrating that these glucosides are produced in the same manner under autophytic and heterophytic conditions. The use of this callus culture in experimental studies of anthocyanin production is discussed.     

Promotion by 2,4-D of 7-glucosylation of benzyladenine in seed-derived and shoot apex-derived cell cultures of Dianthus zeyheri

Cell suspension cultures derived from shoot apex or seed callus of the wild carnation Dianthus zeyheri were treated with [8^?14]benzyladenine. The metabolism of applied cytokinin was evaluated with time (30 min. 6 h. 18 h. 48 h) in the presence of both low (2 mg l^?1) and high (4 mg 1^?1) levels of applied 2.4-D. Both culture types metabolised BA to produce a qualitatively similar but quantitatively different array of metabolites. The 7-glucoside of BA was the major metabolite produced by apex-derived cultures, and the second largest product after the riboside in those cultures originating from seed. In both systems higher auxin application promoted 7-glucosylation with a concurrent reduction in radioactivity associated with the‘active’ cytokinins. This suggests that cytokinin-7-glucosyl transferase is stimulated by the auxin 2.4-D. and may, in part, explain the observed antagonistic interaction between these hormone classes.     

Plant regeneration from callus and protoplast cultures of Helianthus giganteus L.

Summary Methods of plant regeneration from callus and protoplasts of Helianthus giganteus L. are described. Embryogenic callus was obtained from leaf explants and plants were regenerated from these calli on MS media with different combinations of benzyladenine and naphtaleneacetic acid. Leaf protoplasts isolated from in vitro grown plants formed somatic embryos when cultured in agarose solidified droplets of V-KM medium containing benzyladenine and naphtaleneacetic acid. Embryos developed into plantlets on media with reduced auxin contents. Regenerated plants were successfully planted in soil.Abbreviations BA benzyladenine - IAA indoleacetic acid - MS Murashige and Skoog medium - NAA naphtaleneacetic acid - V-KM protoplast culture medium of Binding and Nehls     

Callus formation from Malus x domestica cv. ‘Jonathan’ protoplasts

Protoplasts could be successfully isolated and cultured from callus and suspension cultures of Malus xdomestica cv. Jonathan. Protoplast-derived colonies were recovered when the osmoticum (glucose) was gradually reduced in semi-solid 8p medium or by the use of feeder plates. Formation of embryo-like structures was induced from the protoplast-derived callus on media supplemented with IAA and BA. These structures formed roots but plants failed to develop. Protoplasts could be isolated from leaves, but not from stems or petioles. The leaf protoplasts failed to divide.List of abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - ABA abscisic acid - IAA indole acetic acid     

Role of polyamines in the cytokinin-dependent physiological processes. I. Effect of benzyladenine on polyamine levels during chloroplast differentiation in the tissue culture of Dianthus caryophyllus

Tissue culture of Dianthus caryophyllus L. (cv. William Sim.) obligatory requiring N⁶-benzyladenine for greening provides a good system to study the interactions between cytokinins and polyamines. Polyamines were analyzed as dansyl derivatives which are separated by thin layer chromatography and detected by fluorescence spectrophotometry. Green callus growing on benzyladenine — containing medium showed decrease in the contents of free, conjugated and bound putrescine and spermidine in comparison to chlorophyll-less callus (control callus) growing on cytokinin-free medium. The level of spermine free, conjugated and bound forms increased about 6 %, 77 % and 28 % respectively in tissue culture growing in the presence of cytokinin. Spermidine was dominant polyamine bound to chromatin isolated from control callus. Chromatin isolated from green callus was characterized by a lower level of each polyamine in comparison to chlorophyll-less callus. Polyamines were found in plastid membrane fraction isolated from chlorophyll-less and green callus. A significant increase the levels of polyamines (putrescine, spermidine and spermine) bound to plastid membranes in green callus (+ benzyladenine) in comparison to chlorophyll-less callus (− benzyladenine) was observed. Additionaly, methylglyoxal-bis(guanylhydrazone) an inhibitor of S-adenosylmethionine decarboxylase depressed the greening process. Our results suggest that cytokinin-induced chloroplast differentiation in carnation tissue culture may be partly mediated through the polyamines bound to thylakoid membranes. A possible role of polyamines during cytokinin-induced formation of photosynthetic apparatus is discussed.     

Effect of copper on shoot and root regeneration in wheat,triticale, rape and tobacco tissue cultures

CuSO₄ (0.1–100 M) significantly enhanced shoot regeneration from calli of wheat and triticale and of tobacco leaf disc cultures. In cultures of wheat and triticale, CuSO₄ also stimulated root formation. When equal concentrations of CuSO₄ were applied in different media, it was found that the components of the basal media had only modifying effects. CuSO₄ pretreatment promoted plant survival when regenerated wheat plants were transferred directly to potting soil. In contrast with CuSO₄, AgNO₃, which also stimulated shoot regeneration, inhibited rooting in wheat and triticale. In Brassica napus callus cultures, AgNO₃ strongly increased morphogenesis, whereas CuSO₄ had no significant effect.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - KN kinetin - NAA naphthaleneacetic acid     

Proanthocyanidins and potential precursors in needles of douglas fir and in cell suspension cultures derived from seedling shoot tissues

Proanthocyanidins and their potential precursors have been analyzed by paper chromatography and C₁₈ reversed phase columns with high performance liquid chromatography. Total proanthocyanidins on a dry weight basis in cell suspension cultures derived from seedlings of Douglas fir (Pseudotsuga menziesii) were equal to or greater than those found in mature needles of randomly selected outdoor-grown trees. The major monomer and dimer were catechin and epicatechin-catechin, respectively. Although only procyanidins were detectable in cell suspension cultures, mature needles of outdoor-grown trees contained prodelphinidins as well. Immature needles (flush growth) of the same trees contained only trace amounts of prodelphinidins. Eriodictyol-7-glucoside and dihydroquercetin-3′-glucoside were present in all tissues examined. The amount of eriodictyol-7-glucoside was strongly correlated with total proanthocyanidins in immature needles of flush growth (r = 0.89, p = 0.001). The most complex pattern of flavonoids was found in flush growth needles, which contained in addition to the above, naringenin-7-glucoside and five to six flavone glycosides. Chlorogenic acid was detected only in seedlings and in flush growth needles.     

Micropropagation of common ash (Fraxinus excelsior)

Embryos extracted from dried seeds of common ash (Fraxinus excelsior), were germinated on growth regulator-free culture medium. Cotyledonary nodes from these seedlings were placed onto Murashige and Skoog, Woody Plant or Driver and Kuniyuki culture media with 22.2 or 44.4 M benzyladenine, on which they developed into shoot cultures following the outgrowth of axillary buds. With Murashige and Skoog medium, cultures often died. With Woody Plant Medium, survival of the cultures was considerably improved, but large amounts of callus were produced at the cut ends of the explants, and new axillary shoots had long internodes and small leaves. With Driver and Kuniyuki medium, both survival and callus formation were much improved, and the shoots produced were of high quality. Proliferation of axillary shoots was obtained from both shoot tip and nodal explants placed onto Driver and Kuniyuki medium with 22.2 M benzyladenine. Adventitious root formation was best with shoots inserted into half-strength Woody Plant Medium containing 2.45, 4.9 or 9.8 M indolebutyric acid. All of the rooted plantlets tested have successfully established in soil.     

Intracellular concentrations and metabolism of carbon compounds in tobacco callus cultures: effects of light and auxin

Callus cultures derived from pith tissue of Nicotiana tabacum were grown on two media either under continuous illumination or in complete darkness. The first medium limited greening ability of callus grown in the light (3 milligrams per liter naphthalene acetic acid, 0.3 milligram per liter 2-isopentenylaminopurine, Murashige and Skoog salts, and 2% sucrose). The second medium encouraged chlorophyll synthesis (greening) though not shoot formation (0.3 milligram per liter naphthalene acetic acid; 0.3 milligrans per liter 2-isopentylaminopurine). To measure intracellular concentrations, calli were grown for 15 days on these standard media containing [U-¹⁴C]sucrose. The dry weight proportions of the calli (as a fraction of fresh weight) and many metabolite concentrations nearly doubled in light-grown cells compared to dark-grown cells and increased 30 to 40% on low-auxin media relative to high-auxin media. Glutamine concentrations (from 4 to 26 millimolar) were very high, probably due to the NH₃ content of the media. Proline concentrations were 20-fold higher in calli grown on low-auxin media in the light (green cells), possibly a stress response to high osmotic potentials in these cells. To analyze sucrose metabolism, callus cells were allowed to take up 0.2% (weight per volume) [U-¹⁴C]sucrose for up to 90 minutes. In callus tissues and in pith sections from stems of tobacco plants, sucrose was primarily metabolized through invertase activity, producing equal amounts of labeled glucose and fructose. Respiration of ¹⁴CO₂ followed the labeling patterns of tricarboxylic acid cycle intermediates. Photorespiration activity was low.     

Cytokinins and flower bud formation in vitro in tobacco: role of the metabolites

Explants from flower stalks of Nicotiana tabacum L. were cultured on different cytokinins to induce flower bud formation. All cytokinins tested except zeatin and zeatin-riboside induced the same maximal number of flower buds. Benzyladenine, benzyladenosine, and dihydrozeatin were the most active compounds whereas isopentenyladenosine and isopentenyladenine acted at a 20-fold higher concentration. These data suggest that the active cytokinins bind to the same receptor with different affinities. The presence of benzyladenine in the medium was necessary only during the first 2 days of culture (initiation period). The equilibrium between benzyladenine and its conjugates (the riboside, glucoside, and nucleotides) after a 4-day pulse was independent of the benzyladenine concentration whether it was inductive or noninductive for bud formation. The level of all derivatives was proportional to the benzyladenine concentration in the medium. Isopentenyladenine was used as a competitive inhibitor of benzyladenine conjugation. Isopentenyladenine concentrations that were too low for bud formation led to a synergistic increase in bud number when applied together with benzyladenine. Isopentenyladenine decreased benzyladenine uptake and conjugation. In spite of the lower uptake, the concentration of free benzyladenine inside the explants was higher in the presence of isopentenyladenine than in its absence whereas the concentration of the 7-glucoside of benzyladenine was lower. It was concluded that the free cytokinin base is the main active compound.     

Monoclonal antibodies against phloem P-protein from plant tissue cultures. I. Microscopy and biochemical analysis

Most research involving phloem proteins is done with phloem exudates, which are not easily obtained from many plants. We report here on the use of tissue cultures to study phloem proteins. Monoclonal antibodies against the filamentous phloem protein, P-protein, were made by injecting mice with a phloem-enriched fraction isolated from Streptanthus tortuosus callus grown on a medium that stimulates the differentiation of xylem and phloem (phloem[+] cultures). Monoclonal antibodies specific for P-protein were identified by incubating free-hand stem sections of S. tortuosus in hybridoma supernatants, then in a goat anti-mouse antibody conjugated to fluorescein isothiocyanate (FITC), and observing the FITC under an epifluorescence microscope. Antibodies specific for P-protein in stem sections were used to probe nitrocellulose blots of polyacrylamide gels separating proteins isolated from both phloem(+) and phloem(-) tissue cultures. Immunoblots were incubated overnight in hybridoma supernatants followed by a secondary antibody conjugated to alkaline phosphatase. Three monoclonal antibodies—RS21, RS22, and RS23—bound to an 89-kD band in the phloem(+) lanes but failed to bind to any proteins in the phloem(—) lanes. In leaf sections of Arabidopsis thaliana processed by freeze-substitution, a mixture of RS21 and RS22 bound to the P-protein filaments in sieve elements, but not to any proteins in adjacent cells. A control antibody specific for tubulin did not bind to the P-protein filaments.     

Inhibition of ethylene action prevents root penetration through compressed media in tomato (Lycopersicon esculentum) seedlings

In the genus Prunus , so far, somatic embryogenesis has not been reported either from cell suspensions or from their protoplast-derived cells. Rhizogenic cell suspensions of Prunus avium L., initiated from adventitious roots developed from cotyledon-derived callus of mature zygotic embryos, have been subcultured for more than one year without losing their morphogenic potential. A yield of 8 × 10⁵ protoplasts ml⁻¹ of packed cells with a viability of 98% has been routinely obtained. Optimum cell division frequency (around 2.5% at day 10 and 4–6% at day 15) occurs in agarose lenses, with Murashige and Skoog (1962. Physiol. Plant 15: 476–497)-based medium supplemented with 5 μ M naphthalene acetic acid, 1 μ M benzyladenine and 0.25 μ M zeatin. Colony formation has been achieved after 35 days with a plating efficiency of 3–4%. Cell suspensions have been initiated from protoplast-derived callus. While the older cell cultures express a rhizogenic response, the younger ones contain early stages of somatic embryo development. Ultrastructural examination confirms the polarization of these structures.     

Regulation of Phytoalexin Synthesis in Jackbean Callus Cultures: Stimulation of Phenylalanine Ammonia-Lyase and o-Methyltransferase

Jackbean, Canavalia ensiformis (L.), callus tissues synthesized the phytoalexin, medicarpin (3-hydroxy-9-methoxypterocarpan), when treated with spore suspensions of Pithomyces chartarum (Berk. and Curt.) M. B. Ellis, a nonpathogen of jackbean. Medicarpin was isolated from treated callus tissue and identified by its ultraviolet and mass spectra. The minimum spore concentration found to elicit medicarpin synthesis after 26 hours was 1 × 10⁵ spores/ml; levels of medicarpin in callus tissue increased linearly up to 1 × 10⁷ spores/ml, indicating that the recognition sites for presumed elicitors were not saturated. Medicarpin was first detected in callus treated with 1 × 10⁷ spores/ml, 6 to 12 hours after application, and the concentration reached a maximum at 48 hours, slowly declining thereafter to 72 hours. In callus treated with 3.15 mm HgCl₂, medicarpin concentrations were also maximum by 48 hours. Phenylalanine ammonia-lyase (EC 4.3.1.5) activity increased 2-fold in spore-treated callus after 36 hours. Isoliquiritigenin, daidzein, and genistein o-methyltransferase (EC 2.1.1.6) activities were increased 3- to 4-fold in treated callus. Caffeic acid and naringenin were more efficient substrates for o-methyltransferase activity than the other flavonoids or apigenin, but there was no increase in these o-methyltransferase activities in spore-treated callus. The phytoalexin response in this callus tissue culture system compares well with natural plant systems and should be an excellent system for investigating regulation of phytoalexin synthesis.     

American and korean ginseng tissue cultures: Growth,chemical analysis,and plantlet production

Summary Roots, stems, or leaves of American (Panax quinquefolium) and Korean (Panax ginsing) ginseng were grown as callus or supension tissue cultures. Tissue cultures ofP. ginseng would occasionally form plantlets. The fundamental chemical composition, inorganic analysis, and saponin (panaquilin) content of American and Korean ginseng plants and tissue cultures were determined. The crude saponin content is very similar to, but approximately one-half (1.3%, fresh weight) of that present in ginseng roots. Two-dimensional thin layer chromatographic analysis revealed minor differences in the panaquilins present in American and Korean ginseng tissue cultures. The sapogenin, panaxadiol, was isolated from Korean ginseng callus.