Shoot histogenesis in tobacco callus cultures

Summary The earliest histological event observed in light-grown shoot-forming tobacco (Nicotiana tabacum L. cv. Wisconsin 38) callus was the deposition of safranin-stainable substances (probably suberin) on cut, exposed cell surfaces. This was followed by the initiation of cell files and the appearance of starch granules. Nodules with lignified tracheary elements also were observed in the upper part of the callus. Pronounced starch accumulation occurred in the lower part of the callus in which protrusions of tissue into the medium occurred. Meristemoids were found in these protrusions as well as elsewhere. In between meristemoids, parenchyma cells with starch granules of varying sizes were observed. Cell strands that connected with the meristemoids also were observed. These strands often terminated at the surface of the protrusion at which point shoot apices originated. The earliest shoots were formed in these protrusions. With time, additiional shoots were formed in other parts of the bottom of the callus and finally in the top part of the callus on prolonged culture. The determination of the loci at which shoot primordia were formed sequentially, was interpreted in relation to the physiological gradient concept. This work was carried out while E. M. was a Visiting Scientist at the University of Calgary under the Scientific Exchange Program between the National Research Council of Canada and the Japan Society for the Promotion of Science. Support for this study was provided by N.R.C. (Canada) Grant A-6467 to T. A. T.     

Sucrose metabolism during tobacco callus growth

Activities of soluble and insoluble invertases and sucrose synthetase in tobacco callus increased significantly within the first 3 days of culture. After this period soluble invertase activity declined, while the activities of the insoluble invertase and the sucrose synthetase were relatively unchanged.     

Starch turnover in shoot-forming tobacco callus

Tobacco callus grown under shoot-forming conditions or in the presence of gibberellic acid, which inhibits shoot formation, was incubated in [¹⁴C]-sucrose at three different periods in culture and then replanted. Evolution of ¹⁴CO₂ occurred during the 10 day post-incubation period. Most of the radioactivity was incorporated into the ethanol-soluble fraction, which lost most of its label after 24 h. Starch was the major ethanol-insoluble component and post-incubation synthesis occurred in this fraction for 24 h or longer. Greater net synthesis of starch occurred in shoot-forming tissue and the loss of label from starch began later than in tissue cultured in the presence of gibbe-rellic acid. Newly synthesized starch was not immediately utilised in the organogenic process, but its utilization could be correlated with the shoot-forming process.     

Transmembrane ferricyanide reduction in tobacco callus cells

Transmembrane ferricyanide reduction in whole cells of normal and of transformed tobacco (Nicotiana tabacum) callus tissue was compared. It was found that low concentrations of indoleacetic acid (IAA, 0.1 μM), gibberellic acid (GA, 0.3 μM), and benzyl adenine (BA, 0.03 μM) stimulate external ferricyanide reduction in normal tobacco callus cells, but inhibit this reaction up to 67% in transformed cells when hormones are applied to cells 10 min prior to assay. Higher concentrations of these growth regulators (1 μM or greater) inhibit transmembrane ferricyanide reduction in both types of cells, with the exception of IAA, giving an initial stimulation of the rate (12%), followed by 24% inhibition after 2 min. The observed external ferricyanide reduction by whole tobacco callus cells may be explained on the basis of a transplasmalemma redox system, which may be associated with the iron metabolism of these cells.     

Modified alkaloid pattern in developing tobacco callus

Developing Nicotiana tabacum L. cv. Wisconsin-38 callus grown on modified Murashige-Skoog (MS) medium with Kao organic acids (pyruvic, citric, malic and fumaric acids) contains abnormally high levels of nornicotine and total alkaloids when compared with the leaves of the donor plant. Nornicotine/nicotine ratios observed during callus development suggest that nicotine is converted into nornicotine in the callus, with subsequent movement of alkaloids into roots formed on the callus and into the agar medium. Addition of Kao organic acids to the medium increases alkaloid levels, but cannot account for the abnormal increase in nicotine demethylation. This study thus reports two new findings: (a) that the total alkaloid content of tobacco callus can be greatly enhanced to 3.75% on a dry weight basis by exogenous organic acids, and (b) that endogenous nornicotine can accumulate in tobacco tissue cultures.     

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.     

14C-metabolism during callus induction in tobacco

Tobacco pith-phloem explants and callus were incubated in ¹⁴C-glucose, ¹⁴C-acetate or ¹⁴C-bicarbonate on different days in culture in the dark. ¹⁴CO₂ production and ¹⁴C incorporation into ethanol-insoluble components were generally greater in the subcultured callus than in the pith-phloem explants during days 0 to 5 in culture. Greatest radioactivity from all substrates was in the ethanol-soluble portion, which was further fractionated into lipids, amino acids, sugars and organic acids. Although incorporation into the different fractions varied with the substrate, the patterns of labelling were relatively similar in the two tissues. The greater wound metabolism in the subcultured callus in comparison to the pith-phloem explant during the induction phase of callus formation was correlated with the earlier visible initiation of cell proliferation in the subcultured tissue.     

Ascorbic acid enhancement of organogenesis in tobacco callus

The effect of ascorbic acid on growth and shoot formation in callus cultures of tobacco (Nicotiana tabacum L.) was investigated, using young (4–12 subcultures) and old (more than 30 subcultures) tissue. It was found that ascorbate, at levels of 4–8×10^-4M, enhanced shoot formation in both young and old callus. Treatment with ascorbate also speeded up the shoot-forming process. In addition, ascorbate completely reversed the inhibition of shoot formation by gibberellic acid in young callus, but was less effective in old callus.     

Photosynthetic characteristics of photoautotrophically grown tobacco callus cells

Haploid callus cells of tobacco (Nicotiana tabacum) were grown photoautotrophically on a solid agar medium in the absence of sucrose in Petri plates in an atmosphere of 1% or 3% CO₂ in air. The averages of dry weight increases for four to five consecutive passages were 2.3- to 3.6-fold per 3-week passage for different subclones. Photosynthetic ¹⁴CO₂ assimilation was maximum at about 1% CO₂ with half-maximal rates obtained at 0.2% CO₂. At saturating CO₂ concentration the average rate of CO₂ fixation was about 5 μmole per gram fresh weight per hour or about 125 μmole per mg of chlorophyll per hour.     

Conditions for strict autotrophic culture of tobacco callus

Organic gelling agents such as agar and agarose provide a heterotrophic substrate for growth of illuminated tobacco callus. When green cells are incubated in CO₂-free air on a medium lacking sucrose but solidified with 1% agar, an increase in relative dry weight is sustained through two passages. Similar results with different inoculum sources, and with three brands of agar and two forms of agarose, suggest this is a general phenomenon. A fully autotrophic culture system was developed employing polyurethane pads to support cells in a liquid medium lacking sucrose. Growth was negligible in two passages in CO₂-free air, and increased with each added increment in CO₂ concentration.     

Variation in nicotine content of tobacco callus cultures

Plants ofNicotiana tabacum L. cv. Burley 21 which showed no difference in nicotine content were used to establish callus cultures. Cultures were initiated from different plants and from different leaves within each plant. The nicotine content of the calli was determined, and the results subjected to an analysis of variance. Differences between plants and differences within plants significantly affected the nicotine content of the cultures. The differences between plants were transmitted sexually and asexually, providing evidence that they are genetically determined. No such differences in nicotine content were found between the plants from which the cultures were established, indicating that nicotine production in vitro involves additional genes to those which are needed for nicotine production in the plant. The differences within plants were further investigated by establishing callus cultures from pith explants taken from different parts of the stem. Explants from apical pith tissue gave calli having far more nicotine and more roots than cultures derived from basal pith explants. This results may reflect the proximity of the apical pith explants to the site of auxin synthesis in the stem apex. Callus cultures derived from pith explants showed greater growth and nicotine production than those derived from leaf explants when the calli were induced on Murashige-Skoog medium containing -naphthalene acetic acid. This result is in conflict with the widely held belief that explants from different parts of the plant give cultures with similar yields of species-specific compounds.Abbreviations HN High nicotine - LN low nicotine     

Effect of some imidazopyrimidines on tobacco callus cultures

Abstract

Several 7-chloro-imidazo(1,2-c)pyrimidines were tested on tobacco callus cultures in order to verify a possible cytokinin and anticytokinin-like activity. These compounds were used alone and in combination with kinetin or zeatin.

The aminofurfuryl derivatives showed a strong inhibitory action on cell multiplication and this effect was enhanced when they were mixed with kinetin or zeatin.

The isopentenyl derivatives on the contrary were able to induce cell division in tobacco callus.     

Manganese toxicity to chlorophyll synthesis in tobacco callus

Tobacco (Nicotiana tabacum) pith explants were grown on manganese containing medium. At moderate concentration (10 millimolar), manganese selectively inhibited chlorophyll synthesis, resulting initially in growth of white callus. Several weeks later the white callus turned brown due to the accumulation of a pigment identified as protoporphyrin IX by its elution profile using high performance liquid chromatography, by its absorption spectrum, and by its fluorescence properties. At a concentration of 100 millimolar manganese the pigment accumulated without growth of the explant.     

Photoautotrophic growth and photosynthesis in tobacco callus cells

Haploid tobacco (Nicotiana tabacum L.) cell cultures derived from quite different cultivars have been grown photoautotrophically in medium lacking sucrose and with 1.6 mum naphthaleneacetic acid and 1.5 mum isopentenylaminopurine. Cells were grown for 5 months on agar medium in Petri plates in air with dry weight increases of 1.5- to 3-fold per month. Callus cells were also grown photoautotrophically for at least three consecutive transfers 3 weeks apart in shallow liquid medium in horizontally placed gas-washing bottles where they were gassed continuously with air or air enriched with CO(2). Raising the CO(2) level in the air surrounding the cells increased the growth rate, and after about 3 weeks in 1% CO(2) the dry weight was approximately 3-fold greater than the inoculum. Growth rates remained about the same after each consecutive transfer. Autotrophic growth with this regime is not restricted to specific clones or cultivars.Photosynthetic measurements in an atmosphere containing (14)CO(2) established that rates of CO(2) assimilation in the callus cells at high CO(2) levels were similar to those of leaves on a chlorophyll basis, but were much slower on a fresh weight basis. Photosynthetic light saturation was achieved at an irradiation of about 125 mueinsteins m(-2) sec(-1) (400-700nm). The availability of photosynthetically dependent haploid cells provides an opportunity to select photosynthetic mutations which can be expressed in plants regenerated from these cells.     

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.     

Effect of glyphosate on ethylene production in tobacco callus

Glyphosate (N-phosphonomethylglycine) caused a significant decrease or a slight increase in ethylene production in tobacco callus (Nicotiana tabacum L.) depending on the concentration of indole-3-acetic acid (IAA) present in the medium. IAA stimulated ethylene production, but a pretreatment with glyphosate greatly reduced the IAA-induced ethylene production. Inasmuch as glyphosate treatment promoted the metabolism of IAA, the decrease in ethylene production induced by glyphosate is attributed to the rapid loss of free IAA in the treated tissue.     

DPX-3778 promotes proliferation of tobacco callus in vitro

DPX-3778, the triethanolamine salt of 3-(p-chlorophenyl)-6-methoxy-s-triazine-2,4(1H,3H) dione, at concentrations of 0.124–2.48 M enhanced ca. 4-5-fold the proliferation of tobacco (Nicotiana tabacum L. cv. Wisconsin 38) callus cultured in the presence of indole-3-acetic acid and kinetin, and retarded its senescence.     

Cytokinin activation of thiamine biosynthesis in tobacco callus cultures

Bioassays of tissue extracts show that high (500-1000 μg/liter) kinetin concentrations which permit growth of tobacco callus cultures on media without added thiamine activate the biosynthesis of this vitamin by the tissues. Although the tissue concentration of thiamine may fall appreciably, it is maintained at a level adequate for survival and slow growth of the cultures, and there is a large net increase in total thiamine content per culture with time. In the second and subsequent passages of tissue on a thiamine free medium, growth is obtained only when high kinetin concentrations are maintained. Effective inhibition of growth by antithiamines suggests that thiamine is utilized by the high-kinetin tissue.

In the presence of low (30-100 μg/liter) kinetin concentrations, which would be optimal for growth in the presence of thiamine, growth only occurs early in the first passage of tissue from a medium with the vitamin to one without it. The thiamine concentration in the tissues falls to low levels, and no net biosynthesis is apparent. The tissues turn dark and die after 2 to 3 weeks. In contrast with this, in the absence of both added thiamine and kinetin no appreciable growth occurs, but the tissues keep their normal appearance, retain their thiamine content, and may stay alive for several weeks.