Shikimate Pathway Activity during Shoot Initiation in Tobacco Callus Cultures

The activity of the shikimic acid pathway during shoot initiation in tobacco (Nicotiana tabacum L. Wisconsin 38) callus was examined. Enhancement of the activities of 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthase, shikimate kinase, chorismate mutase, and anthranilate synthase was observed during culture of tobacco callus under shootforming conditions in comparison to tissue cultured under non-organforming conditions. Confirmation of these findings was obtained by examining the incorporation of d-[¹⁴C]glucose into quinic and shikimic acids and of [¹⁴C]shikimic acid into tyrosine, phenylalanine, and tryptophan.     

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.     

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.     

Carbohydrate Availability and Shoot Formation in Tobacco Callus Cultures

Tobacco callus grown on a shoot-forming medium containing sorbitol or no carbon source survived, but did not produce shoots. Transfer of tissue from a sucrose medium to carbohydrate-deficient media and vice versa suggested that the growth of the tissue was a function of a total period in contact with available carbohydrate. Both starch and free sugars in the tissue were utilized during shoot initiation. Furthermore, it appeared that the continuous availability of carbohydrate was required for shoot primordium growth and/or their development into leafy vegetative shoots in dark-grown cultures.     

Adenosine Phosphate and Nicotinamide Adenine Dinucleotide Pool Sizes during Shoot Initiation in Tobacco Callus

Shoot-forming tobacco callus is found to have high levels of adenosine phosphates and NAD⁺, and a low energy charge during meristemoid and shoot primordium formation. NADH levels are low and show little change during this period. There is a decline in the content of NADPH to nondetectable levels during the process, and a transient increase of NADP⁺ is observed early in culture. These patterns are indicative of a shift to a more intensive rate of metabolism during meristemoid and shoot primordium formation and apparently reflect the requirement for energy and reducing power during organ initiation.     

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.     

Effects of Gibberellic Acid and Abscisic Acid on Shoot Formation in Tobacco Callus Cultures

Tobacco (Nicotiana tabacum L. cv. W. 38) callus grown on a shoot-forming medium was exposed to gibberellic acid (GA₃) and abscisic acid (ABA) for varying lengths of time and at different periods during culture. The results suggest that if the tissue accumulated sufficient GA₃ prior to the initiation of meristemoids and shoot primordia, repression of shoot formation occurred. This repression was not reversed by increasing the levels of auxin or cytokinin in the medium, but ABA could partially overcome the GA₃ repression of shoot formation.     

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.     

Shoot Differentiation in Callus Cultures of Datura innoxia

Datura innoxia Mill. callus cultures formed shoots in 2–4 weeks on media containing; a) gibberellic acid, b) indoleacetic acid, c) low concentrations of naphthylacetic acid, d) low concentrations of 2,4-dichlorophenoxyacetic acid, e) benzylaminopurine, f) no growth substance. Benzylaminopurine promoted shoot differentiation. Gibberellic acid inhibited shoot formation weakly, but inhibited proper leaf blade formation. Root differentiation was rare. The callus cultures of Datura innoxia grew rapidly (100-fold in 4 weeks) on a slightly modified Murashige and Skoog medium (0.5 mg/l thiamin · HCl, pH 5.5, no glycine) in light at 30°C. Callus grew well on any single one of the growth substances NAA (10^?5M), 2,4-D (10^?6M) or BAP (3 × 10^?6M). Growth was less and more erratic on GA or IAA. The callus cultures did not grow significantly better when BAP was combined with one of the auxins or with GA.     

Osmotic Requirement for Shoot Formation in Tobacco Callus

Tobacco callus grown on medium containing 3% sucrose (w/v) shows optimum growth and produces the highest number of shoots; whereas cultures grown on medium with lower or higher sucrose levels show a reduced growth rate and capacity to form shoots. Cultures grown on a low sucrose containing medium produce a high number of shoots only if the medium is supplemented with mannitol to give the same water potential as that of the 3% sucrose medium. Mannitol cannot replace the sucrose requirement for growth. Increased levels of Bacto Agar, tricarboxylic acid cycle intermediates and amino acids added to the medium do not promote shoot formation or replace the carbohydrate requirement for that process. The success in partially replacing the sucrose requirement for shoot formation with mannitol supports the view that part of the tissue carbohydrate is acting in an osmoregulatory role. This finding is further interpreted in terms of the hypothesis of turgor-driven growth and cell expansion.     

Endogenous Gibberellins and Growth of Tobacco Callus Cultures

Tobacco callus grown under a range of conditions for different lengths of time contained various levels of gibberellin-like substances. Culture conditions, viz: light versus darkness and the quantity of cytokinin in the medium, affected the amount of gibberellins found in the tissue. These culture conditions were also important in controlling growth rate of the callus and modified the ability of the tissue to respond to exogenous gibberellins. Furthermore, substances which are known to inhibit gibberellin biosynthesis and also thought to block gibberellin action in some cases, were found to reduce the rate of growth. These data support the idea that endogenous gibberellins may be important in the control of the normal growth of tobacco cells in culture.     

Cytokinin-controlled Indoleacetic Acid Oxidase Isoenzymes in Tobacco Callus Cultures

Indoleacetic acid oxidase in tobacco callus tissues (Nicotiana tabacum L., cultivar White Gold) was resolved into seven anionic isoenzymes by polyacrylamide gel disc electrophoresis. Different concentrations of kinetin and zeatin in the presence of indoleacetic acid affected the level of this enzyme, particularly two fast-moving isoenzymes, A₅ and A₆. The optimal concentration of kinetin was 0.2 μm; increasing concentrations above this level progressively lowered the total activity of indoleacetic acid oxidase and repressed the development of isoenzymes A₅ and A₆. Actinomycin D and cycloheximide inhibited the development of these two isoenzymes under the influence of 0.2 μm kinetin, suggesting a requirement for RNA and protein synthesis. The cytokinin-promoted indoleacetic acid oxidase isoenzymes A₅ and A₆ increased with time and paralleled the dry weight increase of tobacco callus tissues, but the total activity of indoleacetic acid oxidase per unit dry weight of tobacco callus varied with time depending on the stage of plant growth.     

Ozone Induced Carbon Dioxide Evolution in Tobacco Callus Cultures

Callus derived from Bel–W3 and Bel–B tobacco plants when exposed to ozone turned brown as a consequence of surface cell destruction. Ozone fumigations above a threshold concentration of 0.10 μl/1 for two hoars caused an increase in the rate of tissue carbon dioxide (CO₂) evolution. The maximum increase in CO₂ evolution was about 65 percent for both the ozone sensitive Bel–W3 and resistant Bel–B callus. However, the ozone dosage required to attain maximum increase in CO₂ evolution was approximately two times greater for the resistant variety. Callus cultures that grew roots were observed to be more resistant to ozone. The addition of the antioxidant N,N'dipnenyl–p–phenylenediamine (DPPD) m the nutrient medium retarded ozone induced CO₂ evolution.     

Changes in Some Enzyme Activities during Growth and Organogenesis in Dark-Grown Tobacco Callus Cultures

Enzymes involved in malate metabolism, viz., glutamic-oxalacetictransaminase, malate dehydrogenase, malic enzyme and phosphoenolpyruvatecarboxylase, had severalfold higher specific activities in organ-formingcallus cultures of tobacco compared to non-organ-forming cultures.These activities increased considerably during the days precedingshoot and root differentiation. While malate accumulated untilday 15 in non-organ-forming callus, it accumulated up to day6 in shoot-and root-forming callus. Total and reducing sugarsaccumulated until day 3 and declined thereafter in all the cultures.Thus, tobacco callus may utilize this pathway for deriving reducingpower which is required for organogenetic processes. (Received April 30, 1987; Accepted December 1, 1987)     

Rooting and the Metabolism of Nicotine in Tobacco Callus Cultures

The usefulness of exogenous nicotine as a factor in the induction of morphogenesis in a tobacco tissue culture medium has been demonstrated. Nicotiana rustica callus cell cultures were grown on a modified Murashige and Skoog medium with 2 mg/l indoleacetic acid (IAA) and 0.2 mg/l kinetin (MMS). Root morphogenesis was induced in roller tube callus cell cultures and solid callus cell cultures grown on MMS without kinetin supplemented with 10–100 mg/l nicotine. Optimal nicotine concentration for root induction was 50 mg/l. Other tests using varying combinations of IAA, kinetin and nicotine produced no obvious morphogenesis, although some changes in the amount of callus growth and endogenous protein concentration did correlate with nicotine concentration relative to the presence of IAA and/or kinetin. In liquid MMS medium, ¹⁴C-nicotine was primarily incorporated into the protein fraction of cultured cells while primarily incorporated into the cell wall and/or cell membrane fraction of cells cultured on MMS without kinetin in the medium. In MMS without IAA and MMS without both IAA and kinetin, there was incorporation, but to a lesser extent in both the protein and the cell wall and/or cell membrane fractions.     

Shoot and Plantlet Formation in Organ and Callus Cultures of Albizzia lebbek Benth

Plantlets were produced in vitro from root and hypocotyl explantstaken from seedlings of the tree legume, Albizzia lebbek. Theseexplants formed shoots when cultured with 5.0 mg l^–1 kinetinand 1.0 mg l^–1 IAA in MS medium. Shoots were also inducedin large numbers from callus treated with benzylaminopurine.About 20 per cent of the shoots rooted and were grown into plants. Albizzia lebbek Benth, tree legume, hypocotyl, root, in vitro cultures, shoot-plantlet induction     

Cytokinin Activation of de novo Thiamine Biosynthesis in Tobacco Callus Cultures

The effect of kinetin on the de novo formation of thiamine in tobacco callus cultures was measured by following the isotope dilution of previously introduced (14)C-thiamine. Thiamine was determined by the thiochrome fluorescence assay after chromatographic purification.Morphological effects induced by high kinetin concentrations were visible within a week after tissue transfer, but thiamine synthesis was insignificant for 2 weeks both in cultures with high (1000 mug/l) and low (30 mug/l) kinetin treatments. Thiamine synthesis during the third week was observed at both kinetin levels, the high kinetin treatment supporting 2.5 times the thiamine synthesis of the low kinetin treatment. The kinetin induced increases in thiamine observed earlier by Digby and Skoog apparently resulted from stimulation of thiamine synthesis rather than from sparing its destruction. Thiamine synthesis is initiated when thiamine concentration reaches a minimum in the callus tissue. This suggests that kinetin is required for the synthesis, but that the activation of synthesis is under feedback control sensitive to the level of thiamine in the tissue.