Modification of logarithmic growth rates of tobacco callus tissue by gibberellic Acid

Logarithmic growth rates (either fresh or dry weight basis) of tobacco callus tissues grown on 10⁻⁴ to 10⁻¹ μm cytokinin are increased if gibberellic acid (10⁻³-2 μm) is incorporated into the medium. At higher (1-10 μm) cytokinin concentrations gibberellic acid has little effect on growth rate but extends the duration of logarithmic growth. The gibberellic acid effect is noticeable only after one weight doubling, is dependent on concentration, and occurs when either glucose or sucrose is used as carbon source. The gibberellic acid response includes a decrease in percentage of dry weight relative to control tissues. The maximum dry weight yield, although achieved sooner than controls, does not differ appreciably from yields of tissue not treated with gibberellic acid.     

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 μm will decrease growth rates; a concentration of 0.081 μm 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 μm). 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 μm.     

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.     

Incorporation of 3H-adenine into free cytokinins by cytokinin-autonomous tobacco callus tissue

Cytokinin-autonomous tobacco callus was incubated in defined mineral medium containing ³H-adenine for 60 minutes. Radioactivity was incorporated into the four predominant free cytokinins, ribosyl-$\text{trans}$-zeatin, $\text{trans}$-zeatin, N⁶-(Δ²-isopentenyl) adenosine and N⁶-(Δ²-isopentenyl) adenine. The bases were more abundant than their respective ribosides, N⁶-(Δ²-isopentenyl) adenine being the most abundant cytokinin. No discrete peaks of radioactivity could be detected on the HPLC column eluate corresponding to the elution volumes of $\text{cis}$-zeatin and ribosyl-$\text{cis}$-zeatin.     

Modification of disease resistance of tobacco callus tissues by cytokinins

The effects of differing cytokinin and auxin concentrations on resistance of tobacco (Nicotiana tabacum L.) tissue cultures to race 0 of Phytophthora parasitica var. nicotianae were examined. With 1 micromolar kinetin and either 11.5 micromolar indoleacetic acid or 1 micromolar 2,4-dichlorophen-oxyacetic acid, tissues from resistant cultivars exhibited a “hypersensitive” reaction to zoospores of the fungus and subsequently were colonized only slightly. With susceptible cultivars or with tissues from resistant cultivars supplied with higher cytokinin levels (e.g. 10 micromolar kinetin), this hypersensitive reaction did not occur and tissues were heavily colonized. Benzylaminopurine and kinetin were particularly effective in eliminating both the hypersensitive reaction and disease resistance. Zeatin and 6-(3-methyl-2-butenylamino)purine were less effective. Increases in indoleacetic acid levels reversed the effects of high cytokinin concentrations. The balance of phytohormones apparently controls the host response to the fungus; thus, in this system, resistance or susceptibility can be studied without changing either host or fungal genotype.     

Effects of cytokinins on the respiration of soybean callus tissue

A technique which incorporates a brief blending step to disperse callus tissue into small clumps of cells was developed, and the effects of cytokinins on respiration of soybean (Glycine max [L.] Merrill var. Acme) callus tissue prepared in this way were studied. Adenine alone did not affect respiration, but kinetin and zeatin showed effects correlating with their reported effects on growth of this tissue; after about 3 hours both hormones promoted respiration at concentrations which promote growth, while kinetin, but not zeatin, also exhibited inhibition at higher concentrations. Studies with 2,4-dinitrophenol led to the suggestion that although the respiration of this tissue is largely under the control of ATP levels, kinetin does not exert its control on respiration through effects on ATP levels or oxidative phosphorylation during the monitoring period. Further inhibitor and substrate studies provided evidence that the promotion of respiration by kinetin results from an increase in substrate entering the tricarboxylic acid cycle, perhaps by an effect on pyruvate metabolism.     

Distribution of incorporated, synthetic cytokinins in ribosomal RNA preparations from tobacco callus

The distribution of incorporated synthetic cytokinins (N⁶-[8-¹⁴C]benzyladenine ([8-¹⁴C]bzl⁶Ade) and N⁶[8-¹⁴C]furfuryladenine ([8-¹⁴C]fr⁶Ade) in ribosomal RNA prepared from tobacco callus (Nicotiana tabacum L. var. Wis. No. 38) grown in the presence of one of these for 25 or 26 days has been studied. The rRNA of tissue supplied with [8-¹⁴C]bzl⁶Ade or [8-¹⁴C]fr⁶Ade was fractionated by methylated albumin-Kieselguhr column chromatography and preparative gel electrophoresis, respectively. In each case about 80% of the incorporated cytokinin was recovered as the ribonucleoside [8-¹⁴C]bzl⁶A or [8-¹⁴C]fr⁶A in the rRNA peak after the fractionations. [8-¹⁴C]fr⁶A was found associated with both the 18S and 25S rRNA components in quantities roughly proportional to their 260 nm absorbance. This pattern of apparently nonspecific association was not affected by prior denaturation of the RNA with formamide.     

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.     

Biosynthesis of cytokinins by tobacco cell cultures

In synchronously cultured tobacco cells (Nicotiana tabacum cv.Xanthi), the incorporation of U-¹⁴C-adenosine into butanol-solublecytokinins in vivo was studied. The radioactivity was incorporatedinto zeatin, ribosylzeatin, isopentenyladenosine and glucosylzeatinafter 20 min. The radioactive cytokinins were identified bythin-layer chromatography and high performance liquid chromatography.From the short time course of the incorporation of ¹⁴C-adenosineinto butanol-soluble cytokinins, the presence of the followingbiosynthetic pathway in vivo was suggested: adenosine is converedinto isopentenyladenosine and then into zeatin via ribosylzeatin.The biosynthetic pathway of free cytokinins in vivo is comparedwith that in vitro. (Received June 20, 1980; )     

Biosynthesis of cytokinins by tobacco cell cultures

In synchronously cultured tobacco cells (Nicotiana tabacum cv.Xanthi), the incorporation of U-¹⁴C-adenosine into butanol-solublecytokinins in vivo was studied. The radioactivity was incorporatedinto zeatin, ribosylzeatin, isopentenyladenosine and glucosylzeatinafter 20 min. The radioactive cytokinins were identified bythin-layer chromatography and high performance liquid chromatography.From the short time course of the incorporation of ¹⁴C-adenosineinto butanol-soluble cytokinins, the presence of the followingbiosynthetic pathway in vivo was suggested: adenosine is converedinto isopentenyladenosine and then into zeatin via ribosylzeatin.The biosynthetic pathway of free cytokinins in vivo is comparedwith that in vitro. (Received June 20, 1980; )     

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.     

Properties of glutamate dehydrogenase purified from green tobacco callus tissue

Glutamate dehydrogenase [L-glutamate : NAD(P) oxidoreductase(deaminating) EC 1.4.1.3 [EC] .] has been purified from the mitochondrialfraction of green tobacco callus tissue. The enzyme was stableat –20?C for several months. The pH optimum for the aminationreaction was 7.8. But the optimum for the deamination reactionwas indistinct because it was in an extremely alkaline domain.Relative activities of the enzyme for amination were 50 withNADH and 10 with NADPH, and those for deamination were 5 withNAD and 1 with NADP at pH 7.9. The enzyme was inactivated by EDTA, but its activity partiallyrestored by the addition of divalent cations such as Ca²⁺, Mn²⁺,Zn²⁺, Cu²⁺ and Mg²⁺. Ca²⁺, Mn²⁺ and Zn²⁺ activated the reductiveamination 141, 122 and 39% respectively, but these divalentcations scarcely affected the oxidative deamination. Citrate and fumarate acted as inhibitors for reductive amination,and oxaloacetate for oxidative deamination of the enzyme reaction.These inhibitions were counteracted by the addition of Ca²⁺.ATP and ADP exerted an inhibitory effect on both directionsof the enzyme reaction. The inhibitory effect was hardly preventedby the addition of AMP. Ca²⁺ caused considerable recovery fromthe inhibition of ATP and ADP. Amino acids scarcely affectedthe enzyme activity. Michaelis constants were 0.28 mM for NAD, 0.065 mM for NADH,2.19 mM for a-ketoglutarate, 43.6 mM for ammonium chloride and4.24 mM for L-glutamate. ¹To whom requests for reprints should be addressed. (Received June 25, 1980; )     

Different effects of two brassinosteroids on growth,auxin and cytokinin content in tobacco callus tissue

Two synthetic brassinosteroids, 24-epibrassinolide (24-epiBR) and 2,3, 17-trihydroxy-5-androstan-6-one (THA-BR), exhibit different effects on growth of tobacco callus tissue. When added to a culture medium containing growth-limiting amounts of auxin, 24-epiBR reduced and THA-BR increased the fresh weight yield of tissue up to 53% and 207%, respectively, after 6 weeks of cultivation. The stimulatory and inhibitory effects of the two brassinosteroids on tissue growth occurred over a broad range of concentrations without a pronounced maximum corresponding to the yes or no type of response. Different effects of 24-epiBR and THA-BR on tissue growth were inversely proportional to the content of endogenous cytokinins. Maximum contents of predominant cytokinins N⁶-(²-isopentenyl)adenine (iP) and trans-zeatin (Z) in tissues supplied with 24-epiBR in growth-inhibiting concentrations were up to 3.7 fold and 3.4 fold higher, respectively, as compared to tissues grown on media containing growth-stimulating concentrations of THA-BR. Stimulation of tissue growth by THA-BR correlated with content of endogenous IAA and an inverse correlation was found between the content of endogenous IAA and cytokinins in tissues supplied with 24-epiBR. THA-BR exhibited weak cytokinin-like activity in a bioassay based on stimulation of growth of lateral buds of pea while 24-epiBR was inactive. Results indicate that the qualitatively different effects of the two brassinosteroids on growth of tobacco tissue may reflect their different influence on content of endogenous cytokinin.Abbreviations BR(s) brassinosteroid(s) - 24-epiBR 24-epibrassinolide - THA-BR 2,33, 17-trihydroxy-5-androstan-6-one - CK(s) cytokinin(s) - iP N⁶-(²-isopentenyl)adenine - [9R]iP N⁶-(²-isopentenyl)adenosine - Z trans-zeatin - [9R]Z ribosyl-trans-zeatin - ABA abscisic acid - IAA indole-3-acetic acid - NAA naphtalene-1-acetic acid - DEAE cellulose diethylaminoethyl cellulose - HPLC high performance liquid chromatography - ELISA enzyme linked immuno-sorbent assay     

Coniferyl alcohol, sinapyl alcohol and scopoletin in tobacco callus tissue during growth of a subculture

Coniferyl and sinapyl alcohols were isolated, identified and quantitatively determined as unbound (or weakly bound) phenylpropanoids in neutral hot-water extracts of Nicotiana tabacum L. callus tissue. This is the first identification of these alcohols in cultured tobacco callus. Scopoletin was also detected in these extracts, and it was the most abundant of these three phenylpropanoids with concentrations that ranged from 50–119 μg/g dry wt. Coniferyl alcohol (17–34 μg/g dry wt.) and sinapyl alcohol (23–35 μg/g dry wt.) were present in nearly equimolar concentration ratios and at levels which were about half those determined for unbound (or weakly bound) scopoletin. The amount of scopoletin extracted increased about 10 times when 1 M HCl-50% methanol - 0.3% ascorbic acid was used as the extractant. This indicated that most scopoletin moieties were strongly bound, perhaps by acid-hydrolyzable linkage. Coniferyl alcohol and sinapyl alcohol were not found in the acid extracts, presumably because they were acid-labile. In general, the concentration of each endogenous unbound (or weakly bound) phenylpropanoid appeared to remain relatively constant throughout the growth phase of the subculture. The only exceptions to this were the relatively higher concentrations of scopoletin and coniferyl alcohol present during the initial 0–2 weeks of subculture.     

A device for comparing callus growth rates in vitro

A device to compare the kinetics of callus growth in vitro is described. Changes in volumes of callus grown in scintillation vials were monitored photometrically without removing the sample from the solid support and medium. It is shown that a fiberglass-paper solid support is superior to a plastic foam solid support for the growth of American chestnut callus.     

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.