Pith Callus Research Articles

Differential exhibition of photoreversible phytochrome in callus from two woody species, Actinidia deliciosa and Olea europaea

As part of an investigation of the photomorphogenetic control of organogenesis in woody species, the levels of phytochrome in callus tissues of Actinidia deliciosa (kiwi fruit) and Olea europaea (olive) has been studied by in vivo spectrophotometry. Photoreversible signals were detected in Actinidia leaf callus, irrespective of whether it had been grown in red light, far-red light or darkness. In contrast, phytochrome could not be detected in callus generated from Actinidia pith tissues. Analysis of callus derived from olive leaf tissues showed the presence of phytochrome. The results indicate that callus derived from leaf tissues of these two woody plants contains a light stable (Type II) phytochrome. The absence of detectable phytochrome in Actinidia pith callus indicates that biochemical differences in the original tissues are partially retained in the callus derived from them.

IN VITRO NITROGEN METABOLISM AND ORGANOGENESIS OF NICOTIANA TABACUM

The role of N metabolism in organogenesis and growth was studied using tobacco pith callus. Callus was cultured on a solid medium containing 10 μM (1.75 mg/l) IAA and 2 μM (0.43 mg/l) kinetin for 56 days. In the growth experiment, ratios of NH4+-N to NO3--N (0:60, 20:40, 30:30, 40:20 and 60:0 mM) were supplied by (NH4)2 SO4 and KNO3. Callus and media were analyzed for inorganic N. Callus supported by 30:30 and 40:20 media removed the highest amounts of NH4+-N and NO3--N from the media and exhibited organogenesis. Final dry weight was greatest in callus supported by the 30:30 medium. In the organogenesis experiment, the transfer history of the inoculum source affected N uptake, organogenesis and growth. Inorganic N was supplied by NH4NO3 and KNO3-. The net uptake of NH4+-N and NO3--N was lower in shoot-forming than in root-forming and non-organogenic callus subculture from 7-day-old stock cultures. The final pH of the medium supporting shoot-forming callus was lowest. Growth, on a dry weight basis, was lowest in shoot-forming callus. Callus subculture from 60-day-old stock cultures formed no shoots.

Cytokinin Habituation in Juvenile and Flowering Tobacco

Abstract The possible link between cytokinin and flowering was examine in tobacco. The degree of cytokinin autotrophy and the competence for cytokinin habitution were measured in callus derived from pith tissue of Nicotiana tabacum cvs. H425 an W38. Explants were taken from internodes at all positions up the stem in juvenile and mature plants. To test whether the competence of cells to form flowers was linked with crtokinin habituation, thin cell layer explants from comparable internodes were tested for their ability to form floral buds. Callus derived from the upper parts of plants showed cytokinin autotrophy whether or not the plants were flowering. Flower buds were formed only on thin cell layer explants from the upper part of plants which were already flowering. Cytokinin habituation and competence to flower are therefore not directly linked although cytokinin habituation could be a prerequisite for meristematic activity and for flowering. Pith from internodes in the lower half of mature pants formed callus which was cytokinin-dependent, although these same internodes in juvenile plants were cytokinin-autotrophic. The ability to form cytokinin-autotrophic callus was therefore greatest in the meristematic regions and was lost as the pith cells aged. Competence to habituate after 35 °C treatment was also shown by pith callus from a few internodes in the middle of the plant below those already forming cytokinin-autotropic callus.

Bound Auxin Metabolism in Cultured Crown-Gall Tissues of Tobacco

Bound auxin metabolism in cultured crown-gall tumor cells and pith callus of tobacco was examined by feeding radiolabeled auxins and auxin conjugates. In all tissues fed [(14)C]indoleacetic acid (IAA), at least one-third of the IAA was decarboxylated, and most of the remaining radiolabel occurred in a compound(s) which did not release IAA with alkaline hydrolysis. In cells transformed by the A6 strain of Agrobacterium tumefaciens, the only detectable IAA conjugate was indole-3-acetylaspartic acid (IAAsp), whereas cells transformed by the gene 2 mutant strain A66 produced an unidentified amide conjugate but no IAAsp. By contrast, cells fed [(14)C]naphthaleneacetic acid (NAA) accumulated several amide and ester conjugates. The major NAA metabolite in A6-transformed cells was naphthaleneacetylaspartic acid (NAAsp), whereas the major metabolites in A66-transformed cells were NAA esters. In addition, A66-transformed cells produced an amide conjugate of NAA which was not found in A6-transformed cells and which showed chromatographic properties similar to the unknown IAA conjugate. Pith callus fed [(14)C] NAA differed from both tumor lines in that it preferentially accumulated amide conjugates other than NAAsp. Differences in the accumulation of IAA and NAA conjugates were attributed in part to the high capacity of tobacco cells to oxidize IAA and in part to the specificity of bound auxin hydrolases. All tissues readily metabolized IAAsp and indole-3-acetyl-myo-inositol, but hydrolyzed NAAsp very slowly. Indirect evidence is provided which suggests that ester conjugates of NAA are poorly hydrolyzed as well. Analysis of tissues fed [(14)C]NAA together with high concentrations of unlabeled IAA or NAA indicates that tissue-specific differences in NAA metabolism were not the result of variation in endogenous auxin levels. Our results support the view that bound auxin hydrolysis is highly specific and an important factor controlling bound auxin accumulation.

Regulators of cell division in plant tissues XXIX. The activities of cytokinin glucosides and alanine conjugates in cytokinin bioassays

In a number of cytokinin bioassays, the activities of the following compounds were compared: 3-, 7-, and 9-glucosides of 6-benzylaminopurine (BAP); 7- and 9-glucosides of zeatin; O-glucosides of zeatin, dihydrozeatin, and their ribosides; 9-alanine conjugates of zeatin, and BAP. The bioassays included the radish cotyledon, theAmaranthus betacyanin, the oat leaf senescence, and the tobacco pith callus. Cytokinin activity was markedly reduced by 7- and 9-glucosylation in nearly all bioassays, but 3-glucosylation of BAP and O-glucosylation of the zeatin sidechain usually had little effect on activity. However, there were two notable exceptions to this generalization: the activity of O-glucosylzeatin markedly exceeded that of zeatin in the oat leaf senescence assay; 9-glucosyl-BAP and free BAP were similarly active in retarding the senescence of radish leaf discs. The 9-alanine conjugate of zeatin (lupinic acid) and of BAP were markedly less active than zeatin and BAP, respectively, in all bioassays, but the responses evoked by these conjugates at high concentrations in theAmaranthus bioassay approached those caused by the corresponding base. The activities of several new compounds related to the alanine conjugate of BAP were also assessed. To serve as a guide in the selection of the most suitable bioassay for detection of the above-mentioned cytokinin conjugates, the lowest detectable amounts in selected bioassays have been compared.

Organogenesis in tissue culture of Solanum melongena cv. Florida Market

Cell suspension cultures were initiated from pith callus of eggplant, Solanum melongena cv. Florida Market on Murashige and Skoog (MS) medium supplemented with 13.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The effects of mineral salts (MgSO 4, KNO 3, CaCl 2), sucrose and 2,4-D were studied on aggregate formation in suspension cultures. Aggregates from a cell suspension when cultured on an MS agar medium supplemented with various concentrations and combinations of indole-3-acetic acid (IAA) and γ,γ-isopentenyl adenine (2iP) differentiated into green nodules, but they failed to develop into organized shoots on the same medium. Upon transfer of these green nodules to MS medium supplemented with 0.1 μM ascorbic acid (autoclaved with the medium) alone or in combination with 49 μM 2iP, developed further into organized leafy shoots. This suggests that some degradation product of ascorbic acid might be responsible for inducing leafy shoots. Based on leaf morphology and chromosome analysis, regenerated plants were identified as tetraploids.

Relationship between hormone content and autonomy in various autonomous tobacco cells cultured in suspension

Various autonomous cultured tobacco cells including crown gall were examined for their contents of growth regulators by means of Avena curvature test, cell-division induction test, and tobacco pith callus test. The crown gall cells derived from cv. Hicks produced auxin and cytokinin in the high levels of 300–500 μg IAA equivalents and 40–80 μg kinetin equivalents per kg, respectively. The major auxin was identified as indole-3-acetic acid based on mass spectrometry and gas chromatography. These cells also produced methyl indole-3-acetate as a minor component. One of the cytokinins was identified as ribosyl-trans-zeatin by means of both gas chromatography-mass spectrometry and high performance liquid chromatography. Auxin and cytokinin activities were not detected in the following three suspension cultured tobacco cells: cells requiring neither cytokinin nor auxin derived from the callus of N. tabacum cv. Bright Yellow and cells requiring auxin but not cytokinin derived from the calluses of cv. Bright Yellow and cv. Hicks. Their auxin and cytokinin contents per kg were less than 1 μg IAA equivalent and less than 0.1 μg kinetin equivalent, respectively. The results obtained in this study indicate that enhanced hormonal content is not the only reason for autonomous growth.

Trifluralin effects on tobacco callus tissue: Mitosis and selected metabolic effects

Inhibition of growth of pith callus of tobacco ( Nicotiana tabacum, var. S-73) by the herbicide trifluralin (α,α,α - trifluoro - 2,6 - dinitro - N,N- dipropyl - p - toluidine) was previously observed. Inhibition of cell division in callus tissue of varying age by this herbicide was investigated using the Feulgen reagent and light microscopy. Upon staining and counting the number of cells in each phase of mitosis, a decrease in the number of cells in metaphase, anaphase, and telophase in the treated tissues was found. In addition to this reduction, arrested metaphases and multinucleated cells were observed. Similar results were observed with 10 −4 M colchicine. The effects of trifluralin on incorporation of 14C-precursors into callus RNA, DNA, and protein were also investigated. Apparent RNA, DNA, and protein synthesis in callus were inhibited by trifluralin (5 × 10 −6 M) treatment. The inhibition, however, was not expressed until 5–7 days after initiating treatment. Colchicine also affected apparent RNA, DNA, and protein synthesis; however, these effects were different than those observed with trifluralin. Incorporation of 14C-amino acids into protein was most severely inhibited by colchicine.

A particle-bound auxin receptor from tobacco pith callus

The binding of NAA to particulate fractions isolated from tobacco pith callus was studied. The presence of a specific auxin receptor with high affinity was demonstrated. NAA-binding was maximal after 30 min of incubation at 36°C (pH 5). The affinity constant for NAA was approx. 3 × 10 6/mol and the concentration of binding-sites was approx. 75 pmol/g fresh weight (60 pmol/mg protein). The affinities for various auxin analogues were roughly correlated with their activities in auxin bio-assays. The same type of receptor was found in freshly excised tobacco pith, but at a lower concentration.

Transcription in nuclei isolated from tobacco tissues

An advantage of using isolated nuclei for the study of transcription is the presence of endogenous RNA polymerases. Moreover, in the nuclei, the native state of the chromatin, including regulatory proteins, is maintained. Transcription in nuclei from various plant sources, e.g., soybean hypocotyls [ 1 ] and tobacco callus [2] has been studied. Using tobacco callus nuclei as an in vitro system we did not find any effect of hormones on transcription [3]. However, one should be aware that nuclei isolated from different tissues of the same plant species may behave differently. Therefore, we have compared the characteristics of nuclei isolated from tobacco stem pith, pith callus and leaves. Clear differences between the properties of the nuclei were found in the amounts of RNA synthesized per Irg DNA, in the contents of free RNA polymerases, and in the stimulation of RNA synthesis by heparin. Exogenous DNA of various origins was transcribed to a different extent by the nuclei studied.

Spontaneous 5-Bromodeoxyuridine Tolerance of Nicotiana Genetic Tumours

Summary Hormone independent Nicotiana glauca × N. langsdorffii genetic tumours are insensitive to cytotoxic effects of 5-bromodeoxyuridine up to the concentrations of 10–4 M. N. glauca pith callus, Constitutively cytokinin independent, exhibits significantly lower BUdR tolerance and hormone dependent N. langsdorffii pith callus is BUdR sensitive. Thus the degree of BUdR tolerance parallels the degree of hormonal autonomy. These results further substantiate our previour hypothesis that BUdR tolerance of plant cells may be connected with cytokinin-auxin autonomy.

Expression of Disease Reaction Types in Soybean Callus from Resistant and Susceptible Plants

Expression of Disease Reaction Types in Soybean Callus from Resistant and Susceptible Plants

RNA synthesis in isolated tobacco callus nuclei, and the influence of phytohormones

By an improved method nuclei were isolated from tobacco pith callus tissues and their RNA polymerase (nucleoside triphosphate: RNA nucleotidyltransferase (DNA-dependent), EC 2.7.7.6) activity was studied. The α-amanitin-sensitive polymerase activity in the nuclei was highest at high ionic strength (175 mM(NH 4) 2SO 4) and was stimulated more by Mg 2+ than by Mn 2+. In the presence of 1 μg/ml of α-amanitin the assumed RNA polymerase I activity in the nuclei was maximal without (NH 4) 2SO 4 and showed a preference for Mn 2+ over Mg 2+. Using [ γ- 32P] GTP as a label we proved that the isolated nuclei were able to initiate the synthesis of new RNA chains. Phytohormones (auxin and/or cytokinin) did not increase the incorporation of radioactive UMP in the nuclei, whether these were isolated in the presence or absence of the hormones.

Nicotiana tabacum Callus Studies III. Size and Shape of Vascular Elements

Summary Tracheary and sieve elements induced in vitro in Nicotiana pith callus are as wide as but about five times shorter than those in the internodes of the plants from which the expiants derive. Shape, especially in the tracheary elements, is extremely irregular. Giant tracheidal cells differentiate in the presence of indoleacetic acid and gibberellin in explants which otherwise have undergone limited amounts of callus development. These tracheids are more regular in shape and are considered to be the direct redifferentiation products of the pith parenchyma cells. Although the sieve elements show much less variation in shape than do the tracheary elements, the slopes of their end walls are more varied than those of sieve elements in the intact plant.

Effect of N-phenyl-N′-(4-pyridyl)urea on shoot formation in tobacco pith disk and callus cultured in vitro

Effect of N-phenyl-N′-(4-pyridyl)urea on shoot formation in tobacco pith disk and callus cultured in vitro

T-2 Toxin Decreases Logarithmic Growth Rates of Tobacco Callus Tissues

T-2 toxin, a mycotoxin produced by Fusarium tricinctum, decreases logarithmic growth rates of tobacco (Nicotiana tabacum L.) pith callus tissues. Toxin concentrations as low as 0.003 mum will decrease growth rates; a concentration of 0.081 mum will halt growth completely. Additional exogenous cytokinin will reduce the inhibition by toxin only when the initial cytokinin and toxin concentrations are quite low (about 0.01 mum). When inhibited tissues are transferred to media lacking toxin, they assume the faster, control rates almost immediately. Maximal yields of tissue (yields at the point at which no sugar was detected in the medium) are not affected by toxin concentrations of 0.01 to 0.036 mum.

日本ナシの果実の発育に関する研究 (第1報)

Immature fruits of Japanese pear, Pyrus serotina, were picked and homogenized with 70% ethanol. The homogenate was extracted with 70% ethanol three times and filtrated. The filtrate was concentrated, the residue adjusted to pH 8.4 with 1N NaOH and then extracted with n-butanol. The butanol phase was evaporated and aqueous extract of the residue was added to a Dowex-50(H+ form) column and eluted with water, 1N NH4OH and 5N NH4OH successively. Cytokinin activities in the three fractions were tested by tobacco(Wisconsin No. 38) pith callus assay. Cytokinin activities were detected in both 1N and 5N NH4OH fractions, but not in the water. Based on these results, the NH4OH fractions (Am-fraction) were combined and analysed by microcrystalline cellulose thin layer and gas liquid chromatographies. The fraction separated by TLC for the bioassay of cytokinin revealed activities at Rf values 0.5 and 0.7. Analyses of TMS derivative of Am-fraction by GLC gave four peaks. Mixture of the TMS derivative with that of authentic zeatin analyzed on GLC revealed that peak 2 coincides with zeatin, and peak 1 was considered to be dihydrozeatin.

N6-(Δ2-Isopentenyl)adenosine: Its Occurrence as a Free Nucleoside in an Autonomous Strain of Tobacco Tissue

Cytokinins from both the free nucleoside pool and the transfer RNA have been isolated and identified in a habituated strain of tobacco pith callus (Nicotiana tabacum [L] var. Wisconsin 38). The transfer RNA of this strain contains both N(6)-(Delta(2)-isopentenyl) adenosine and N(6)-(4-hydroxy-3-methylbut-2-cis-enyl) adenosine. The trans-hydroxylated derivative is absent from the transfer RNA of this dark-grown tissue. N(6)-(Delta(2)-Isopentenyl)-adenosine was identified as a component of the free nucleoside pool in concentrations of about 10 micrograms per kilogram of tissue.The cytokinin-requiring tissue grown in the presence of N(6)-furfurylaminopurine contains less than 85 nanograms per kilogram of N(6)-(Delta(2)-isopentenyl) adenosine. This is the detectable limit under the conditions of the experiment.

Effects of Sucrose and Kinetin on Growth and Chlorophyll Synthesis in Tobacco Tissue Cultures

Investigations were carried out on the effects of various combinations of sucrose and kinetin concentrations on growth and chlorophyll production in a green and a nongreen clone of pith callus of Nicotiana tabacum L. It was found that 2 milligrams per liter or higher amounts of kinetin induced greening in the nongreen tissue. The observations suggested that growth of the callus and synthesis of chlorophyll and soluble protein are controlled by relative concentrations of sucrose and kinetin in the medium. Kinetin was found to be inhibitory for chlorophyll synthesis in the green callus.

Limitation of Tobacco Callus Tissue Growth by Carbohydrate Availability

Growth rate, maximum dry weight yield, and carbohydrate utilization were measured with pith callus derived from Nicotiana tabacum L. var. Wisconsin No. 38. Maximum tissue dry weights increased as carbohydrate (either glucose or sucrose) in the medium was increased. The time at which maximum growth was obtained coincided with the time at which carbohydrate was exhausted from the medium. The addition of carbohydrate to the medium before the end of log phase growth supported that amount of additional growth which would have been obtained if all of the carbohydrate had been added to the medium prior to planting the tissue. Maximum obtainable dry weights at logarithmic growth rates greater than 0.16 doubling per day depended on the amount of carbohydrate in the medium and not on a particular hormonal regime.