Nitrate assimulation in shoot-forming tobacco callus cultures

Summary The contents of nitrogen-containing compounds and the activities of nitrate assimilating enzymes were determined in tobacco callus cultured under shoot-forming and non-shoot-forming conditions. Whole tissue and tissues cut into top and bottom portions were examined. Highes levels of total-N, protein-N, nitrate and ammonium-N, as well as higher activities of nitrate and nitrite reductases were found in shoot-forming whole tissue and in the shoot-forming bottom portion of tobacco callus in comparison to the non-shoot-forming proliferating tissues throughout the culture period. These findings indicate that enhanced nitrogen assimilation occurs during de, de novo shoot organogenesis. This work was supported by NSERC of Canada Grant No. A-6467 to T.A.T.     

Turnover of Shikimate Pathway Metabolites during Shoot Initiation in Tobacco Callus Cultures

The turnover of shikimate pathway intermediates and end productswas examined in tobacco (Nicotiana tabacum L. Wisconsin 38)callus cultured under shoot-forming and non-shoot-forming conditions.In shoot-forming tissue there was a higher rate of net synthesisof quinic and shikimic acids than in proliferating callus. Post-incubation,there was a decrease in labeled quinate and an increase in shikimate.The changes in activity of quinate:NAD^$ oxidoreductase werein agreement with the above. The aromatic amino acids, tyrosine,phenylalanine and tryptophan, showed little turnover in theproliferating tissues. On the other hand, higher rates of netsynthesis and degradation, mainly of tyrosine, were observedin shoot-forming tissues. These findings are discussed in relationto the shoot-initiation process. (Received October 14, 1983; Accepted June 4, 1984)     

Starch Metabolism, Respiration, and Shoot Formation in Tobacco Callus Cultures

The starch content of shoot-forming and non-shoot-forming tobacco callus cultured in light and darkness was determined. A variety of carbohydrates and cytokinins incorporated into the culture medium were effective in bringing about starch accumulation and shoot formation in the tissue. In addition, the respiratory activity of the callus, grown in the presence or absence of gibberellic acid, was measured. A strong correlation between the starch content of the tissue, its rate of respiration, and shoot formation was observed.     

Modulation of shoot formation in tobacco callus by tissue transfer between permissive and inhibitory media

Summary In an attempt to understand events involved in the cellular regulation of in vitro plant organogenesis, experiments were performed in which tobacco (Nicotiana tabacum L.) callus was transferred at different days in culture from a shoot-forming medium to a non-shoot-forming medium and vice versa. The transfers were made at key histologic stages of the shoot-forming process and known biochemical and biophysical correlates were examined. The changes in starch accumulation and disappearance supported the previously assigned functions, and could be correlated with the histologic changes that occurred in the callus after transfer at the different culture times. In contrast, the changes in respiration could not be correlated with these events. The changes in osmotic and turgor potentials after transfer showed that osmotic adjustment preceded both shoot initiation and development. This suggests that osmotic adjustment might play an important role in in vitro organogenesis. This research was supported by the Natural Sciences and Engineering Research Council of Canada grant A-6467 to T. A. T.     

Non-autotrophic CO2 Fixation during Shoot Formation in Tobacco Callus

Non-autotrophic carbon fixation has been studied during growthof tobacco callus cultured in dark under shoot-forming (SF)and non-shoot-forming (NSF) conditions. The enzymes involvedin malate metabolism—phosphoenolpyruvate carboxylase,malic dehydrogenase, glutamic-oxalacetic transaminase, and malicenzyme—increased sharply during the first 4 d of cultureparticularly in SF tissue. The activities of the enzymes studiedwere considerably greater in SF than in NSF tissue. There wasa dramatic increase in malate content in SF tissue during thefirst 4 d of culture. Subsequently malate was rapidly depletedduring the time of organogenesis. In NSF tissue there was acontinuous build-up of malate content throughout the cultureperiod. We suggest that malate derived from dark fixation ofCO₂ plays differing roles in NSF (callus) and SF tissues. Inthe former, malate acts primarily as an osmotic solute regulating,at least in part, cell expansion between successive cell divisions.In shoot-forming tissue, on the other hand, malate preferentiallyprovides NADPH for reductive biosynthesis.     

Optimizing plant regeneration from seed-derived callus cultures of Kentucky bluegrass. The effect of benzyladenine

The effect of benzyladenine (BA) on the production of shoot-forming callus from seeds of two Poa pratensis cultivars was studied. Addition of low concentrations (0.1–0.3 mg l^-1) of BA to Murashige & Skoog (MS) callus induction medium containing 1 or 2 mg l^-1 2,4-dichlorophenoxyacetic acid (2,4-d) stimulated somatic embryogenesis and strongly increased the percentage of seeds producing shoot-forming callus in both cultivars.     

Shoot initiation in light- and dark-grown tobacco callus: the role of ethylene

Shoot-forming tobacco ( Nicotiana tabacum L. cv. Wisconsin 38) callus produces less endogenous ethylene than non-shoot-forming tissue cultured in the light (16 h photoperiod) or the dark. In shoot-forming tissue more ethylene is produced early in culture (days 0–5) than later. Also dark-grown tissue produces much more ethylene than light-grown. On the basis of experiments in which (1) gaseous ethylene was added to or (2) CO₂ removed from the flasks, (3) Ethrel (an ethylene releasing agent) and (4) 1-aminocyclopropane 1-carboxylic acid (an ethylene precursor) were added to the medium, it was determined that this gaseous phytohormone had two contrary effects on shoot initiation (shoot primordium formation). Early in culture (days 0–5) endogenous or exogenous ethylene inhibited organogenesis, but later (days 5–10) exogenous ethylene or increased endogenous ethylene production speeded up primordium formation.     

Starch Metabolism in Shoot-forming Tobacco Callus

Starch, free sugars and protein contents, and the specific activitiesof enzymes of starch metabolism were determined in tobacco calluscultured under shoot-forming and non-shoot-forming conditions.Shoot-forming cultures contained higher levels of starch, freesugars and protein. Shoot-forming cultures had higher specificactivities for starch-synthesising enzymes throughout culture.On the other hand, higher levels of activity for starch-degradingenzymes in shoot-forming tissues were only observed during organizeddevelopment. The role of phosphorylase in the cultured tissuewas not clear.     

Alternative and cytochrome pathway respiration during shoot bud formation in cultured Pinus radiata cotyledons

Respiration rates for excised cotyledons of Pinus radiata cultured in the presence (shoot-forming) and absence (non-shoot-forming) of N⁶-benzyladenine (BA) over a 21-day period were measured using a Clark-type oxygen electrode. The capacities and activities of cytochrome and alternative pathways of respiration were determined from titrations with KCN (1-10 m M ) and salicylhydroxamic acid (2–20 m M ) individually and in combination. Respiration accounted for by alternative (AP) and cytochrome (CP) pathways varied with both culture treatment and age in culture. Rates of total respiration, CP respiration and AP activity rose concurrent with key developmental events of shoot bud formation. The greatest AP capacity was measured at day 3 in shoot-forming tissue. In contrast, for cotyledons cultured under non-shoot-forming conditions, no AP activity was observed after day 3 despite relatively constant AP capacity throughout the culture period. Although initial increases in cotyledon respiration during the culture period may be related to wounding and introduction to a tissue culture environment, later differences in respiratory patterns between shoot-forming and non-shoot-forming cotyledons appear to be associated with the cytokinin-induced developmental changes which give rise to shoot primordia in cultured radiata pine cotyledons.     

Plant regeneration from embryogenic suspension cultures of dune reed

Embryogenic callus, derived from mature seeds of dune reed (Phragmites communisTrinius) was used to establish suspension culture. Green shoot-forming type and albino shoot-forming type embryogenic callus of dune reed were selected carefully by the difference of shape and color of callus growing under light and mechanically dispersed before suspending in liquid MS medium supplemented with 1.0 mg l^–12,4-D. They were subcultured every 5 days to remove mucilaginous material in the early culture stage. Both fine albino and green shoot-forming cell suspension lines of dune reed were composed of rapidly growing small cell aggregates that were densely cytoplasmic and potentially embryogenic. Globular somatic embryos were continuously produced in each liquid medium containing 1.0 mg l^–1 2,4-D. The cell aggregates in fine albino cell suspension line (size below 300 m) were smaller than that of green shoot-forming cell suspension line (size between 300 and 800 m). Following transfer to a differentiation medium, both suspension cultures formed regenerating plants with normal roots and albinotic or green shoots, respectively.     

Mitochondrial activity during shoot formation and growth in tobacco callus

Mitochondria isolated from tobacco ( Nicotiana tabacum L. cv. Wisconsin 38) callus growing on either shoot-forming or non-shoot forming medium show an increase in state 3 and state 4 respiration and a drop in respiratory control and ADP/O ratios after subculture. the protein content of the mitochondria fraction and the activity of succinate dehydrogenase, malate dehydrogenase, cytochrome c oxidase and catalase also increase after subculture but there is no apparent difference between shoot-forming and non-shoot-forming tissue. For mitochondria assayed at their native osmolarities, a trend of higher respiration rates and respiratory control as well as lower levels of cyanide-resistant respiration was observed for shoot-forming tissue. Generally, differences were greatest after day 9 in culture, the time during which primordia formation occurred in the shoot-forming callus. These patterns are in concert with the view that the shoot-forming process has a high energy requirement which must be realized during the time of primordia formation.     

Glucose Oxidation during Shoot Initiation in Tobacco Callus Cultures

Involvement of the Embden-Meyerhof Parnas and the pentose phosphatepathways in glucose oxidation in glucose oxidation in tobaccocallus was examined. Marked changes in the activities of glucokinase,aldolase, glucose-6-phosphate dehydrogenase, and phosphogluconatedehydrogenase were observed during culture of tobacco callusunder shoot-forming and non-shoot-forming conditions. Activitiesof these enzymes were higher in shoot-forming tissue than innon-shoot-forming tissue. Furthermore, the activities of thepentose phosphate pathway enzymes showed greater differencesthan those of the Embden-Meyerhof-Parnas pathway. Confirmationof these findings was obtained by investigating the contributionsof ¹⁴C from [14C-1]- and [¹⁴C–6]-glucose to CO2 released.The significance of these findings on glucose oxidation in relationto the shoot-initiation process are discussed.     

A simple and efficient method for obtaining transgenic soybean callus tissues

In the present study, a simple and efficient method for obtaining transgenic callus tissues of soybean [Glycine max (L.) Merr.] was developed based on Agrobacterium-mediated transformation. Hypocotyl segments of soybean were used as the starting material. Several factors such as soybean genotype, Agrobacterium concentration, inoculation time, co-cultivation period and addition of antioxidants in co-cultivation medium affecting the transformation efficiency were examined. The explants were cultured on callus induction medium containing 0.5 mg L^?1 6-benzylaminopurine and 2.0 mg L^?1, 2,4-Dichlorophenoxyacetic acid for callus induction. Callus tissues were induced at both the acropetal and basipetal ends. CaMV35S::GUS and CaMV35S::GFP transgenic callus tissues were obtained using the optimized protocol. The average transformation efficiency reached up to 87.7 % based on GUS detection. From inoculation with Agrobacterium to obtaining transgenic soybean callus will take about 3 weeks. In order to validate this method for gene function investigation, GVG::GmSARK transgenic soybean callus tissues were obtained and their senescence-associated phenotypes were assessed. To our knowledge, this is the first report using hypocotyl segments as starting materials to obtain transgenic callus, and this system provides a method for high-throughput screening of functional genes of interest in transformed soybean callus.     

Tropane Alkaloids from Callus Cultures, Differentiated and Shoots of Hyoscyamus muticus L.

Alkaloid production has been observed in cotyledonary leaf derived callus tissues, and also in in vitro differentiated shoots, and roots of Hyoscyamus muticus. The callus tissue was developed form cotyledonary leaf explants on Murashige and Skoog medium enriched with 2 mg 1^-1 2, 4-D and 0.5 mg 1^-1 BAP. Cotyledonary leaf derived callus was proliferated in the same medium for 2 passages (1 passage 28-30 days). Green and compact callus was used for alkaloid extraction. Shoots and roots formed on MS medium containing 0.05 mg 1^-1 NAA and 0.5 mg 1^-1 BAP, and also compact, nodular and embryogenic calli from which these shoots and roots differentiated, were used for alkaloid extraction. Chromatographic studies performed with TLC showed the presence of hyoscyamine as the major alkaloid present in the callus tissues, differentiated shoots and roots. However, alkaloid content varied in different tissues. Differentiated roots were found to contain maximum amount of hyoscyamine.     

Studies on the Production of Digitalis Cardenolides by Plant Tissue Culture: II. EFFECT OF LIGHT AND PLANT GROWTH SUBSTANCES ON DIGITOXIN FORMATION BY UNDIFFERENTIATED CELLS AND SHOOT-FORMING CULTURES OF DIGITALIS PURPUREA L. GROWN IN LIQUID MEDIA

Undifferentiated, highly chlorophyllous cell cultures; undifferentiated white cell cultures; green, shoot-forming cultures; and white, shoot-forming cultures of Digitalis purpurea L. were established and subcultured every 3 weeks in liquid media in the light or in the dark. The digitoxin content, the chlorophyll content, and the ribulose bisphosphate carboxylase activity of these cultures were assayed. The light-grown, green, shoot-forming cultures accumulated considerable amounts of digitoxin (about 20 to 40 micrograms per gram dry weight), and the white, shoot-forming cultures without chloroplasts accumulated about one-third that amount of digitoxin. The chlorophyll content and the ribulose bisphosphate carboxylase activity of the undifferentiated green cells were about the same as they were in the green, shoot-forming cultures, but the digitoxin content of the former was extremely low (about 0.05 to 0.2 microgram per gram dry weight), which is about the same as that in undifferentiated white cells without chloroplasts. Thus, it was concluded that the chloroplasts are not essential for the synthesis of digitoxin in Digitalis cells. The optimum concentrations of the tested compounds for accumulation of digitoxin were: benzyladenine, 0.01 to 1 milligram per liter; indoleacetic acid, 0.1 to 1 milligram per liter; α-naphthaleneacetic acid; 0.1 milligram per liter; and 2,4-dichlorophenoxyacetic acid, 0.01 milligram per liter.     

Morphological differentiation in callus cultures of lavandin: a role of ethylene

The involvement of ethylene in shoot formation in vitro was studied in one year old lavandin (Lavandula officinalis Chaix x Lavandula latifoliaVillars) callus. A peak in ethylene evolution characterized thenon-regenerating leaf callus line, as compared to the shoot-forming calyxcallus line, on the growth medium provided with 2,4-dichlorophenoxyacetic acid (1 mg dm-3) and kinetin (0.5 mg dm-3). After one year in culture, calyxcallus attained the capacity to grow on auxin-reduced media, showing decreased ethylene production and faster shoot bud emergence, when transferred onto the regeneration medium, supplemented with 10 mg dm^-3 benzyladenine. Shoot formation was also inhibited by addition of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid, indicating an involvement of ethylene in the failure of regeneration. This revised version was published online in July 2006 with corrections to the Cover Date.     

Differential alcohol dehydrogenase and malate dehydrogenase isozyme expression in long-term callus tissue cultures ofCereus peruvianus (Cactaceae)

Alcohol dehydrogenase (ADH) and mitochondrial malate dehydrogenase (mMDH) isozymes were tested as markers to study the effect of a high kinetin concentration on isozyme phenotypes and on the development ofCereus peruvianus callus tissue culture. Three-year-old callus tissues were used as samples. Callus tissue samples grown on 4.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and on 4.0 and 8.0 mg/LN-(2-furanylmethyl)-1H-purine-6 amine (kinetin) were cut and transferred to fresh medium containing 4.0 mg/L 2,4-D and 4.0, 8.0, 16.0, and 32 mg/L kinetin combinations. The pattern of changes observed in the ADH and mMDH isozymes as well as the growth of callus tissues was independent of the concentrations tested. The various ADH and mMDH isozymes seem to be products of differential association of subunits of the twoAdh and twomMdh genes. Both genes are active throughout callus tissue development; however, gene expression changed with various callus culture conditions. This study addresses how long-term callus culture conditions affect constitutive and differential gene expression of theAdh andmMdh genes inC. peruvianus.     

Effect of sodium sulfate on in vitro organogenesis of tobacco callus

Callus of tobacco (Nicotiana tabacum L. cv. Wisconsin 38) was grown on callus-proliferating (CP) and shoot-forming (SF) media with elevated sodium sulfate (Na₂SO₄) concentrations either in the light or dark for more than one year. An increase in Na₂SO₄ concentration resulted in a decrease in callus growth index, an increase in percent dry weight of callus tissues grown on both media, and a decrease in both number of calli forming shoots and number of shoots per callus in SF medium. The CP callus grown in the light spontaneously began to form shoots after the 5th monthly transfer, and spontaneous root formation occured after the 16th transfer in the presence of 0.75 and 1.0% Na₂SO₄. Both water () and osmotic (_s) potentials of the callus increased with increasing Na₂SO₄ concentration; and callus exhibited greater and _s in the light than dark for both CP and SF media.     

Regeneration of plantlets from cell suspension culture derived callus of white poplar (Populus alba L.)

Friable calli derived from the stem tissues of Populus alba were used to establish cell suspension cultures which were characterized for in vitro growth and regeneration capacity. Suspended cells and callus recovered from these cells were maximal on a fresh weight basis using MS liquid medium containing 0.44 M BAP and 4.52 M 2,4-D. Shoot regeneration from the recovered callus was observed within 30 to 40 days of culture. The number of shoots was increased by subculturing the shoot-forming callus 2 to 3 times on MS medium supplemented with 19.7 M 2iP and 0.05 M IBA. Regenerated shoots were easily rooted on half-strength MS medium lacking growth regulators, and the plantlets were transferred to pots containing vermiculite for greenhouse growth.Abbreviations BAP 6-benzylaminopurine - 2iP 2-isopentenyladenine - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - PCV packed cell volume - MS medium Murashige and Skoog medium (1962)     

Tyrosine and Phenylalanine Ammonia Lyase Activities during Shoot Initiation in Tobacco Callus Cultures

Both phenylalanine ammonia lyase and tyrosine ammonia lyase were detected in tobacco (Nicotiana tabacum L. Wisconsin 38) callus. The enzymes were separated from each other by Sephadex G-200 column chromatography. Increased activity of tyrosine ammonia lyase was observed during culture of tobacco callus under shoot-forming conditions, while activity of phenylalanine ammonia lyase increased during culture under non-organ-forming conditions. Confirmation of these findings was obtained by examining the incorporation of [¹⁴C]tyrosine and [¹⁴C]phenylalanine into p-coumarate and trans-cinnamate, respectively.