Evidence for a Mendelian factor controlling the cytokinin requirement of cultured tobacco cells

Cultured leaf tissues of Nicotiana tabacum L. cv. “Havana 425” normally require an exogenous source of cytokinin for rapid growth; stem-cortex tissues do not—ie, they exhibit the cytokinin-habituated phenotype. We found that plants regenerated from cloned cortex and leaf tissues from one particular plant differed in leaf-tissue phenotype: Leaf tissues derived from leaf cells exhibited the normal, nonhabituated phenotype, whereas leaf tissues derived from cortex cells were cytokinin-habituated. This difference in leaf phenotype was not found using leaf and cortex cells from six other donor plants. The inheritance of the habituated leaf trait was studied in tissues from cortex-derived plants and hybrids between these plants and normal plants. F₁ hybrids were intermediate between the parental types in degree of habituation. No differences were found between reciprocal hybrids. These results suggest that the habituated leaf trait is an incompletely dominant, nuclear trait. Both parental and intermediate phenotypes were recovered in the F₂ progeny. The frequency of habituated leaf progeny in the F₂ and backcross populations provide evidence that the trait is regulated at a single genetic locus.     

The induction of cytokinin habituation in primary pith explants of tobacco

Pith parenchyma tissue ofNicotiana tabacum L. cv. Havana 425 becomes cytokinin habituated when incubated at 35°C on an auxin-containing medium. Under these conditions, habituated, hyperplastic nodules appear on the tissues. We used these nodules to estimate the incidence of habituation by a statistical method. The rate of habituation varied with the season. Tissue isolated from plants in the spring habituated approx. 7 times faster than did tissue isolated from plants in winter. The fact that the average rate, >4×10^–3 per cell generation, was 100–1,000 times faster than the rate of somatic mutation inNicotiana species and depended on the physiological state of the tissue provides further evidence that habituation involves epigenetic changes rather than rare, random genetic mutations. We also found that kinetin (6-furfurylaminopurine) induced habituation and that the concentration required depended on the duration of cytokinin treatment. For long incubation times, approx. 6×10^–10 M kinetin, which is about 1,000-fold lower than the concentration optimal for growth of cytokinin-requiring pith tissue, was sufficient to induce habituation. These results support the hypothesis that the habituated state is maintained by a positive feedback loop in which cytokinins either induce their own synthesis or inhibit their own degradation.     

Biochemical comparisons of habituated and non-habituated callus lines of Glycine max (L.) cv. Acme

A cytokinin habituated non-organogenic callus line of Glycine max L. cv. Acme, was isolated. Cytokinins were detected in both non- and habituated calli. HPLC analysis indicated that the highest biological activity was associated with compounds that cochromatographed with zeatin and derivatives thereof. Cytokinin-like activity in the habituated callus was higher than in the non-habituated callus. No difference was observed in the ability of the two callus types to metabolise trans-zeatin. The habituated callus however, metabolised N⁶(²-isopentenyl)adenine (iP)at a slower rate than the non-habituated line. The regulation of proline and putrescine levels during the growth cycle was similar in both callus types, although less proline was present in the habituated than the non-habituated callus. In both callus types a decrease in putrescine levels was accompanied by an increase in the proline levels on day 21 of the growth cycle. Neither spermine nor spermidine were detected in either the habituated or non-habituated lines. A comparison of the total RNA levels of the habituated and non-habituated material revealed higher RNA levels in the non-habituated than the habituated line. Two-dimensional gel electrophoresis indicated the presence of 3 polypeptides in the habituated callus on day 13 of the growth cycle. These were not detected in the non-habituated callus, of similar age.Abbreviations Ade Adenine - DTT 1,4-dithiothreitol - IEF isoelectrofocussing - iP N⁶(²-isopentenyl)adenine - NAA -Naphthaleneacetic acid - SDS Sodium dodecyl sulphate     

Tissue-Specific Variation in the Cytokinin Habituation of Cultured Tobacco Cells

Cells of tobacco pith parenchyma sometimes lose their requirement for an exogenous supply of a cell division factor usually supplied as the synthetic cytokinin, kinetin. This change in phenotype, known as cytokinin habituation, is inherited by individual cells and appears to result from epigenetic changes rather than from rare, random, genetic mutations. We have found that tissues from different regions of the tobacco plant exhibit different states of habituation in culture. Pith tissues, as reported earlier, are usually cytokinin requiring and rapidly shift to the habituated state in culture. Leaf tissues are very slightly habituated and require kinetin for optimal rates of growth. Tissues from the stem-cortex are initially habituated. Both the leaf and cortex phenotypes are inherited by individual cells and persist for many cell generations in culture. These results show that certain tissue-specific phenotypes persist in culture and provide evidence that a process akin to habituation leading to different stable states of cytokinin requirement occurs in normal development.     

Sterol composition of normal and habituated sugarbeet callus (Beta vulgaris L.Altissima)

Summary A detailed qualitative and quantitative analysis of the sterol content of normal (auxin and cytokinin requiring) and habituated (auxin and cytokinin independent) sugarbeet callus (Beta vulgaris L.altissima) was made using mass spectrometry with gas chromatography. The total sterol content of the two lines did not differ significantly. Δ⁷-Sterols were the most important class of sterols in the two sugarbeet callus lines, as in allChenopodiaceae. Elevated levels of Δ⁸-sterols were found in the habituated callus. These sterols are considered to be badly integrated in the membrane of eucaryotic cells. A partial blocking of Δ⁸-Δ⁷-isomerase is hypothesized in the habituated cell line.     

The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene

The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormone-free media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with ³H and auxin was found to promote conversion of zeatin-type cytokinins to ³H-labelled adenine derivatives. When the very rapid metabolism of exogenous [³H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that trans-hydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.Abbreviations Ade adenine - Ados adenosine - BA 6-benzylaminopurine - C control - Con A concanavallin A - CP cellulose phosphate - IPT isopentenyl transferase - NAA 1-naphthylacetic acid - NP normal phase - NPPU N-(3-nitrophenyl)-N-phenylurea - RIA radioimmunoassay - RP reversed phase We wish to thank Dr. J. Zwar for supplying phenylurea derivitives.     

Hormonal regulation of zeatin-riboside accumulation by cultured tobacco cells

Auxin (11 M -naphthaleneacetic acid) and cytokinin (1.4 M kinetin) regulate cytokinin accumulation by cytokinin-requiring (C^-) and cytokinin-autotrophic (C⁺) lines of Havana 425 tobacco (Nicotiana tabacum L.) tissues. No trans-zeatin riboside (ZR) (<0.5 pmol·g^-1 fresh weight) was detected in six C^- and nine C⁺ lines grown for 14 d on auxin + cytokinin and auxin medium, respectively. C⁺ lines, but not C^- lines accumulated ZR (1.9–5.1 pmol·g^-1 fresh weight) when incubated on hormone-free medium but both lines accumulated ZR when incubated on kinetin medium. Therefore, it appears that kinetin treatment can induce ZR accumulation and that this accumulation is blocked by auxin treatment. Similar effects were obtained with some lines of cells autotrophic for both auxin and cytokinin. Tobacco plants carrying the dominant Habituated leaf-1 allele (Hl-1) differ from wild-type plants in that leaf-derived tissues in culture exhibit a C⁺ phenotype. No differences in ZR content were found in C⁺ leaf tissues from Hl-1/Hl-1 plants and C⁺ tissues that arise epigenetically in wild-type plants. This indicates that the H-1 allele does not act to induce overproduction of ZR. The Hl-1 allele is known to have oncogenic functions similar to the isopentenyl transferase (ipt) locus of the Ti plasmid. Although Hl-1/Hl-1 cells transformed with Ti plasmids defective at the ipt locus are tumorigenic and hormone-autotrophic in culture, they contain low levels of ZR typical of non-transformed Hl-1/Hl-1 cells. Therefore, the high levels of ZR characteristics of cells transformed with wild-type Ti plasmids are not necessary for expression of the tumor phenotype.Abbreviations C^- cytokinin-requiring phenotype - C⁺ cytokinin-autotrophic phenotype - Hl-1 habituated leaf-1 locus - IPA isopentenyladenosine - ipt isopentenyltransferase gene - ZR trans-zeatin riboside     

A cytokinin mutant derived from cultured tobacco cells

Tissues cultured from the leaf lamina of wild-type Nicotiana tabacum L. cv. "Havana 425" plants require an exogenous source of cytokinin for rapid growth. In contrast, leaf tissues of plants heterozygous or homozygous for the partially dominant, monogenic habituated leaf (H1-1) trait, exhibit a cytokinin-autotrophic phenotype in culture. Here we show that the H1 trait can arise in culture. Cytokinin autotrophic variants were obtained by culturing wild-type tissues of leaf lamina successively on media containing reduced concentrations of the cytokinin, kinetin. Plants regenerated from clones of these variants exhibited the H1 phenotype, which segregated in breeding tests as expected for a dominant, monogenic trait. This trait, designated H1-2, is inherited at a different locus than the H1-1 trait described earlier. Our results show that cytokinin mutants can arise in cell culture and that at least two genes regulate the cytokinin requirement of cultured tobacco tissues.     

Stability of the cytokinin requirement in Paul's scarlet rose cells in culture

Summary Cells ofRosa sp. cv Paul's scarlet have been reported to require cytokinin for growth in suspension culture. This report was verified in the present study. However, a rose cell line that was maintained for 2 yr in suspension culture by routine subculturing developed the capacity to grow without exogenous cytokinin. The stability of the cytokinin requirement and the basis for this altered response to cytokinin was investigated. The parental cell line, which has been maintained independently on agar-solidified medium, was subcloned and the cytokinin dependence of the subclones was determined. The subclones were found to exhibit a continuous spectrum of responses, ranging from a high degree of cytokinin dependence for growth to rapid growth upon the initial transfer to cytokinin-deficient medium. The average growth constant (K=1n W/Wo; Wo=initial fresh weight,W=fresh weight after growth for the stated time interval) of 30 subclones grown on medium containing 0.5 μM zeatin was 3.1, with a range of 1.1 to 4.0. The average growth constant of the same subclones when grown on medium lacking a cytokinin was 1.5, with a range of 0 to 3.9. By comparison, the parental cell line exhibited growth constants of 3.5 in the presence of 0.5 μM zeatin and 1.6 in the absence of exogenous cytokinin. Although the growth of some of the subclones after transfer to cytokinin-deficient medium suggested that they were cytokinin autotrophs, this was not the case because none of them grew after a second transfer to medium lacking cytokinin. Culture in medium containing cytokinin conferred upon the cells the capacity for a limited amount of growth after subsequent transfer to medium lacking cytokinin. The extent of this cytokinin-induced growth potential varied from subclone to subclone. Efforts to determine the frequency with which cytokinin autotrophs appeared in a subclone that required cytokinin suggested that it is a rare event and that the cytokinin requirement is a fairly stable phenotypic characteristic of these cells. This research was supported by Grant PCM 7722398 from the National Science Foundation.     

Differential tolerance ofArabidopsis thaliana crown gall tumors and calli to 5-bromo-2′-deoxyuridine

The degree of tolerance of two crown gall tumors and leaf calli ofArabidopsis thaliana to BUdR was compared. The nopaline producing teratoma tumor tolerated BUdR in concentration as high as 2.10^?4 M. The tolerance of octopine producing unorganized crown gall tumor to BUdR was lower, but both exceeded significantly the degree of tolerance to BUdR of untransformedA. thaliana calli, where 10^?5 M BUdR already show some inhibitory effect on the growth rate.     

Evidence for an abundant 33,000-dalton polypeptide regulated by cytokinins in cultured tobacco tissues

When cloned pith and leaf tissues of Nicotiana tabacum L. cv. Havana 425 are subcultured for 3 d on auxin-containing medium and labelled for 18 h with [³⁵S]methionine, up to 10% of the labelled, soluble-protein fraction is found in a single band with an apparent molecular weight of approx. 32,000–34,000 dalton on sodium-dodecylsulfate polyacrylamide-gel electrophoretograms. The labelling of this band, designated P33, is dramatically inhibited by the cytokinin, kinetin, in some cell lines at concentrations as low as 1.4·10^-8 M. P33 is a major component of the protein fraction obtained from non-habituated clones, cytokinin-habituated clones, and revertant subclones of crown-gall-transformed clones, but cannot be detected in clones habituated for both auxin and cytokinin, or crown-gall-transformed clones. The evidence supports the hypothesis that cytokinin in the presence of auxin regulates the production of a specific, major polypeptide in the soluble-protein fraction of the tissue and that this protein is not produced in tissues autotrophic for both auxin and cytokinin.     

Initiation of auxin autonomy in Nicotiana glutinosa cells by the cytokinin-biosynthesis gene from Agrobacterium tumefaciens

Agrobacteria carrying mutations at the auxin-biosynthesizing loci (iaaH and iaaM of the Ti plasmid) induce shoot-forming tumors on many plant species. In some cases, e.g. Nicotiana glutinosa L., tumors induced by such mutant strains exhibit an unorganized and fully autonomous phenotype. These characteristics are stable in culture at both the tissue and cellular level. We demonstrate that the cytokinin-biosynthesis gene (ipt) of the Ti plasmid is responsble for the induction of both auxin and cytokinin autonomy in N. glutinosa. Cloned cell lines carrying an ipt gene but lacking iaaH and iaaM are capable of accumulating indole-3-acetic acid. Interestingly, non-transformed N. glutinosa tissues exhibit an auxin-requiring phenotype when they are grown on medium supplemented with an exogenous supply of cytokinin. These results strongly indicate that exogenously supplied cytokinin does not mimic all the effects of the expression of the ipt gene in causing the auxin-autonomous growth of N. glutinosa cells.Abbreviations FW fresh weight - IAA indole-3-acetic acid - I⁶ Ado isopentenyladenosine - kb kilobase - MS Murashige and Skoog (medium) - NAA -naphthaleneacetic acid - NAM -naphthaleneacetamide - T-DNA transferred DNA     

Cold-sensitive expression of cytokinin habituation by tobacco pith cells in culture

Cloned, cytokinin-habituated tissues of Nicotiana tobacum L. cv. Havana 425 are able to grow in culture at 25° C without added cytokinin. These tissues vary in their expression of the habituated phenotype at 16° C. When cytokinin-requiring pith tissues are converted to the habituated state by 35° C treatment, all of the habituated cells are cold sensitive. After several transfers in culture, some of these habituated cells give rise to stable, cold resistant variants. Both phenotypes are inherited by individual cells. Cold sensitive clones at 16° C and non-habituated clones at 16° C as well as 25° C show the same dose response to the cytokinin, kinetin. This suggests that at the physiological level, cold sensitivity results from a decreased production of cell division factors rather than from a decreased affinity of cellular receptors for these factors.Abbreviations CDF Cell division factor(s) - NAA -naphthalencacetic acid     

Physiology of Hormone Autonomous Tissue Lines Derived From Radiation-Induced Tumors of Arabidopsis thaliana

γ-Radiation-induced tumors of Arabidopsis thaliana L. have been produced as a novel approach to isolation of genes that regulate plant development. Tumors excised from irradiated plants are hormone autonomous in culture and have been maintained on hormone-free medium for up to 4 years. Five tumor tissue lines having different morphologies and growth rates were analyzed for auxin, cytokinin, and 1-aminocyclopropane-1-carboxylic acid (ACC) content, ethylene production, and response to exogenous growth regulators. Normal tissues and two crown gall tissue lines were analyzed for comparison. Rosettes and whole seedlings each contained approximately 30 nanograms· (gram fresh weight)⁻¹ free indoleacetic acid (IAA), 150 nanograms· (gram fresh weight)⁻¹ ester-conjugated IAA, and 10 to 20 micrograms· (gram fresh weight)⁻¹ amide-conjugated IAA. The crown gall lines contained similar amounts of free and ester-conjugated IAA but less amide conjugates. Whereas three of the radiation-induced tumor lines had IAA profiles similar to normal tissues, one line had 10- to 100-fold more free IAA and three- to 10-fold less amide-conjugated IAA. The fifth line had normal free IAA levels but more conjugated IAA than control tissues. Whole seedlings contained approximately 2 nanograms· (gram fresh weight)⁻¹ of both zeatin riboside and isopentenyladenosine. The crown gall lines had 100- to 1000-fold higher levels of each cytokinin. In contrast, the three radiation-induced tumor lines analyzed contained cytokinin levels similar to the control tissue. The radiation-induced tumor tissues produced very little ethylene, although each contained relatively high levels of ACC. Normal callus contained similar amounts of ACC but produced several times more ethylene than the radiation-induced tumor lines. Each of the radiation-induced tumor tissues displayed a unique set of responses to exogenously supplied growth regulators. Only one tumor line showed the same response as normal callus to both auxin and cytokinin feeding. In some cases, one or more tumor lines showed increased sensitivity to certain growth substances. In other cases, growth regulator feeding had no significant effect on tumor tissue growth. Morphology of the radiation-induced tumor tissues generally did not correlate with auxin to cytokinin ratio in the expected manner. The results suggest that a different primary genetic event led to the formation of each tumor and that growth and differentiation in the tumor tissue lines are uncoupled from the normal hormonal controls.     

External calcium requirements for light induction of chlorophyll accumulation and its enhancement by red light and cytokinin pretreatments in excised etiolated cucumber cotyledons

Excised etiolated cucumber (Cucumis sativus L.) cotyledons that were depleted of external Ca²⁺ by equilibration with a Ca²⁺ buffer, which maintained the free Ca²⁺ concentration at 10^–8 M, failed to accumulate chlorophyll upon a 2-h exposure to white light. Increasing amounts of chlorophyll accumulation occurred at increasing external Ca²⁺ concentrations within the range of 10^–7-10^–3 M. Preillumination with red light or pretreatment with benzyladenine, which enhanced the rate of light-induced chlorophyll accumulation in control cotyledons, did not overcome the block to light-induced chlorophyll accumulation caused by the depletion of external Ca²⁺. Etiolated cotyledons that were treated with the Ca²⁺ ionophore, A23187, and then equilibrated with 10^–5 M Ca²⁺, accumulated significantly more chlorophyll during exposure to light than did untreated cotyledons. The enhancing effect of A23187 was approximately equal to that caused by red-light pretreatment. Etiolated cotyledons that were exposed to the Ca²⁺ channel-blocking agent, Nd³⁺ (neodymium), in the presence of 10^–5 M Ca²⁺, did not exhibit an enhancement of chlorophyll accumulation by red-light pretreatment, although they accumulated control levels of chlorophyll upon exposure to light and showed control levels of enhancement of chlorophyll accumulation by cytokinin pretreatment. Conversely, etiolated cotyledons that were equilibrated with 10^–5 M Ca²⁺ in the presence of nifedipine, a blocker of some Ca²⁺ channels, did not exhibit an enhancement of chlorophyll accumulation by cytokinin pretreatment, although they accumulated control levels of chlorophyll upon exposure to light and showed control levels of enhancement of chlorophyll accumulation by red-light pretreatment. These results indicate that external Ca²⁺ is required for chlorophyll accumulation by excised etiolated cucumber cotyledons during the first 2 h of light exposure, and that an influx of external Ca²⁺ is required for the enhancing effect of redlight and cytokinin. The differential abilities of Nd³⁺ and nifedipine to block the effects of red-light and cytokinin pretreatments suggests that enhancement of chlorophyll accumulation by red-light and cytokinin may involve different classes of Ca²⁺ channels.Abbreviations A23187 antibiotic 23187 calcium ionophore - Chl chlorophyll - nifedipine 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester We thank Randy Wayne for advice and encouragement.     

NMR natural abundance estimation of13C-metabolic solutes in normal and habituated sugarbeet cell lines

Summary NMR (nuclear magnetic resonance) spectroscopy was used to identify metabolic solutes in one normal and two habituated sugarbeet cell lines (Beta vulgaris L.altissima) obtained from the same mother strain. This technique was applied to investigate the intracellular naturally occurring¹³C isotopes (1.1% of total natural carbon) in living sugarbeet suspension cells and perchloric cell extracts. A combination of¹H,¹³C, double-quantum filter correlation spectroscopy, heteronuclear multiple-bond correlation, and heteronuclear multiple-quantum coherence spectra from perchloric cell extracts enabled us to identify the main compounds in the different extract solutions. This was verified by spiking the solutions with small amounts of reference compounds to exclude the influence exerted by pH on the chemical shifts of the different compounds in the¹H and¹³C spectra. The comparison of the three sugarbeet cell lines' NMR spectra showed the presence of sucrose, glucose, and fructose in the three strains. On the other hand, it revealed a strong discrepancy between metabolic solutes. Spectra from the habituated lines showed the presence of glutamine. Some amino acids such as alanine or valine, and unidentified signals corresponding to aromatic rings were only characterized in the habituated nonorganogenic cells. On the basis of these¹³C NMR data we assumed that the discrepancy between the different sugarbeet cell lines could be due to an increase in the metabolic activity of the habituated cell lines in relation to their autonomous growth.Abbreviations DQF-COSY double-quantum filter correlation spectroscopy - HO habituated organogenous - HNO habituated nonorganogenous - HMBC heteronuclear multiple-bond correlation - HMQC heteronuclear multiple-quantum coherence - N normal - NMR nuclear magnetic resonance - TSP sodium tetradeutero-3-(trimethylsilyl)-propionate     

Regulation of Cytokinin Oxidase Activity in Callus Tissues of Phaseolus vulgaris L. cv Great Northern

The regulation of cytokinin oxidase activity in callus tissues of Phaseolus vulgaris L. cv Great Northern has been examined using an assay based on the oxidation of N⁶-(Δ²-isopentenyl)adenine-8-¹⁴C (i⁶ Ade-8-¹⁴C) to adenine. Solutions of exogenous cytokinins applied directly to the surface of the callus tissues induced relatively rapid increases in cytokinin oxidase activity. The increase in activity was detectable after 1 hour and continued for about 8 hours, reaching values two- to three-fold higher than the controls. The cytokinin-induced increase in cytokinin oxidase activity was inhibited in tissues pretreated with cordycepin or cycloheximide, suggesting that RNA and protein synthesis may be required for the response. Rifampicin and chloramphenicol, at concentrations that inhibited the growth of Great Northern callus tissues, were ineffective in inhibiting the increase in activity. All cytokinin-active compounds tested, including both substrates and nonsubstrates of cytokinin oxidase, were effective in inducing elevated levels of the enzyme in Great Northern callus tissue. The cytokinin-active urea derivative, Thidiazuron, was as effective as any adenine derivative in inducing this response. The addition of Thidiazuron to the reaction volumes used to assay cytokinin oxidase activity resulted in a marked inhibition of the degradation of the labeled i⁶ Ade-8-¹⁴C substrate. On the basis of this result, it is possible that Thidiazuron may serve as a substrate for cytokinin oxidase, but other mechanisms of inhibition have not yet been excluded.     

Mass-spectrometric quantification of cytokinin nucleotides and glycosides in tobacco crown-gall tissue

the cytokinins of tobacco crown-gall tissue have been analysed by quantitative mass spectrometry using ²H₂-labelled cytokinin riboside 5-monophosphates and ¹⁵N₄-labelled cytokinin glycosides as internal standards. The principal endogenous cytokinin of this tissue is zeatin riboside 5-monophosphate. The biologically inactive 7-glucoside of zeatin is the most abundant basic cytokinin in the tissue. These findings expose the limitations of previously reported analyses of similar tissues, which were restricted to biologically active basic cytokinins. The present study demonstrates that the endogenous cytokinins of tobacco crowngall tissue show a clear correspondence to the range of metabolites formed when exogenous cytokinins are supplied to nontumorous tobacco cells.Abbreviations DHZ dihydrozeatin - DHZ7G dihydrozeatin 7-glucoside - DHZMP dihydrozeatin 9-riboside 5-monophosphate - DHZR dihydrozeatin 9-riboside - GC-MS coupled gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - Z7G zeatin 7-glucoside - Z9G zeatin 9-glucoside - ZOG zeatin O-glucoside - ZMP zeatin 9-riboside 5-monophosphate - ZR zeatin 9-riboside - ZROG zeatin 9-riboside O-glucoside     

Effects of light and growth regulators on adventitious bud formation in horseradish (Armoracia rusticana)

Summary To clarify that the presence of Ri T-DNA genes are not prerequisite for the light-induced bud formation in horseradish (Armoracia rusticana) hairy roots, leaf and root segments of nontransformed horseradish plants were used as explants. Bud formation from nontransformed tissues was observed in hormone-free medium under 16 h daylight conditions, but not under continuous darkness. To investigate the effects of growth regulators on bud formation, leaf and root explants were treated with auxin (1-naphthaleneacetic acid; NAA) and / or cytokinin (6-benzyl-aminopurine; BA). The most effective treatment in the dark to stimulate bud formation was BA at 1 mg·1^-1. These results show that adventitious bud formation in horseradish can be induced by light and growth regulators, and especially cytokinin, may be involved in bud formation, irrespective of whether the tissues were transformed with Ri T-DNA.Abbreviations BA 6-benzyl-aminopurine - NAA 1-Naphthaleneacetic acid - MS Murashige & Skoog (1962) medium