Cyclic Nucleotides and In Vitro Plant Cultures: I. INDUCTION OF ORGANOGENESIS IN TOBACCO (NICOTIANA TABACUM CALLUS CULTURES

Mangat, B. S. and Janjua, S. 1987. Cyclic nucleotides and invitro plant cultures. I. Induction of organogenesis in tobacco(Nicotiana tabacum) callus cultures.—J. exp. Bot. 38:2059–2067. The possibility that cyclic nucleotides have a mediatory rolesimilar to cytokinins in plant tissue cultures was examined.Calli obtained from tobacco pith tissue were incubated on growthmedia supplemented with either cyclic AMP, cyclic GMP, adenosineor guanosine, in concentrations ranging from (mg dm^–3)0 to 2·0 together with 2·0 mg dm^–3 of IAA.Results were compared with identical calli grown on media containingcomparable amounts of kinetin and IAA. Increase in callus growthwas observed on all media containing cyclic AMP, cyclic GMP,adenosine, guanosine or kinetin. Adenosine or guanosine didnot promote organogenesis. Low concentrations (0·02 and0·05 mg dm^–3) of kinetin stimulated extensive rootdevelopment. Some root formation was also elicited with higheramounts of cyclic AMP (0·1 and 0·2 mg dm^–3)or cyclic GMP (0·2 and 0·5 mg dm^–3). Bothkinetin and cyclic GMP promoted shoot differentiation. However,in contrast to kinetin, cyclic GMP induced organogenesis atlower concentrations (0·02 and 0·1 mg dm^–3).The addition of 2·0 mg dm ^–3 of cyclic AM P toIAA-free growth media elicited shoot differentiation. This wasalso the case with a similar concentration of kinetin or cyclicGMP. Results suggest cytokinin activity for the two cyclic nucleotides. Key words: Tobacco, Nicotiana tabacum, tissue culture, cyclic nucleotides, cyclic AMP, cyclic GMP organogenesis     

Changes in Cytokinins before and during Early Flower Bud Differentiation in Lychee (Litchi chinensis Sonn.)

Lychee (Litchi chinensis) has been analyzed for cytokinins in buds before and after flower bud differentiation, using reversephase high performance liquid chromatography in combination with Amaranthus bioassay and gas chromatography-mass spectrometry-selected ion monitoring. Four cytokinins, zeatin, zeatin riboside, N⁶-(δ²-isopentenyl)adenine, and N⁶-(δ⁶-isopentenyl) adenine riboside, were detected in buds. There was an increase of cytokinin activity in the buds during flower bud differentiation. In dormant buds, the endogenous cytokinin content was low, and the buds did not respond to exogenous cytokinin application. Application of kinetin promotes flower bud differentiation significantly after bud dormancy. These results are interpreted as an indication that the increase in endogenous cytokinin levels during flower bud differentiation may be correlative rather than the cause of flower bud initiation.     

Stimulation of gibberellin-induced germination in lettuce seeds by cyclic 3',5'-adenosine monophosphate

Cyclic 3',5'-adenosine monophosphate and sodium dibutyryl cyclic3',5'-adenosine monophosphate had no effect on dark-germinationof light-sensitive lettuce {Lacluca sativa L., var. Grand Rapids)seeds when given alone. Cyclic 3',5'-adenosine monophosphate,however, synergistically enhanced the 3 µ 10^–5 Mgibberellic acid-induced germination, but showed no such effecton kinetin-stimulated germination. 5'-AMP, in contrast to cyclic3',5'-adenosine monophosphate, had no effect on gibberellicacidinduced germination. (Received August 8, 1971; )     

Accumulation of Cytokinin-Induced Betacyanin in Specific Cells of Amaranthus tricolor Seedlings

The accumulation of betacyanin, in dark-grown Amaranthus tricolorseedlings, in response to cytokinins or red light, occurs mainlyin two specific tissues, the lower epidermal cells of the cotyledons(with the exception of guard cells), and the endodermis of thehypocotyl. The possible significance of this ‘spatialpattern of competence’ is discussed, together with theconcept of target cells in relation to plant hormones. The effect of removing exogenously supplied cytokinin at varioustimes during a 24 h induction period is reported. There is noevidence that cytokinins act by a ‘triggering’ effectwith a long half life, the response in the target cells beingthe same as that expected from the amount of cytokinin and cytokininmetabolite remaining in the tissue at the time of extraction.Either continuous presence of cytokinin is needed or any triggeraction is short lived, and continuous ‘re-triggering’is needed to achieve maximum response. Key words: Amaranthus tricolor, Betacyanin synthesis, Cytokinin action, Target cells     

The Effect of Adenine and Mevalonic acid on the Endogenous Cytokinins of a Cytokinin-dependent Strain of Soya Bean Callus

The combined application of 10^–6 M adenine and 10^–6M mevalonic acid to soya bean callus accelerated its growth.Two biologically active compounds that co-chromatographed withzeatin and isopentenyl adenine were extracted from this callus.Studies with labelled adenine and mevalonic acid indicated thatthe cytokinin-dependent soya bean callus incorporated only avery small amount of the radioactive precursors into the biologically-activecompounds, making it extremely difficult to determine whetherthese compounds were synthesized de novo or whether they aroseas by-products of tRNA turnover. As cytokinins do not accumulatein rapidly-growing cytokinin-dependent soya bean callus culturedon kinetin as a source of cytokinin it seems as if biosynthesisde novo occurs when the callus is supplied with adenine andmevalonic acid. Glycine max (L.) Merrill, soya bean, callus culture, adenine, mevalonic acid, endogenous cytokinins     

Dimethyl sulfoxide: A solvent for cytokinins in theAmaranthus bioassay

Dimethyl sulfoxide present in the agar medium at concentration 0.2 % (v/v) and lower does not inhibit cytokinin-induced betacyanin synthesis in theAmaranthus caudatus seedlings. The activity of kinetin, N⁶-(Δ²-isopentenyl)adenine andtrans- zeatin is the same when these cytokinins are dissolved in either water or dimethyl sulfoxide and incorporated into the medium after autoclaving. A simple method is described which allows the cytokinin activity of slightly water-soluble and thermolabile compounds,e.g. aromatic urea and thiourea derivatives, to be determined in theAmaranthus bioassay.     

Comparison of cytokinin activities of the base,ribonucleoside and 5′- and cyclic-3′,5′-monophosphate ribonucleotides of N6-isopentenyl-, N6-benzyl or 8-bromo-adenine

The cytokinin activities of adenosine 3′,5′-monophosphate, N⁶,O^2″-dibutyryladenosine 3′,5−'monophosphate, 8-bromoadenosine 3′,5′-monophosphate, N⁶-(Δ²-isopentenyl)adenosine 3′,5′-monophosphate, and N⁶-benzyladenosine 3′,5′-monophosphate were determined in the tobacco bioassay and compared with the activities of the corresponding non-cyclic nucleotides, nucleosides and bases of the N⁶-isopentenyl-substituted, N⁶-benzyl-substituted, 8-bromo-substituted, and unsubstituted adenine series. In each of these series the cytokinin activities in decreasing order were: bases ⪢ nucleosides ⪖ nucleotides > cyclic nucleotides. All members of the N⁶-isopentenyl- substituted and N⁶-benzyl-substituted series were highly active cytokinins, reaching maximum activity at concentrations of 1 μM or less, whereas, as expected, all members of the unmodified adenine series were inactive in the tested concentration ranges of up to 180 and 200 μM for adenosine and adenine, and 40 μM for the adenine nucleotides. Members of the 8-bromo-substituted adenine series were much weaker cytokinins than the N⁶-substituted adenine derivatives but showed activity in the same sequence starting at a concentration of about 5 μM. Thus, in the cases of 8-bromoadenosine 3′,5′-monophosphate and N⁶,O^2′-dibutyryl-adenosine 3′,5′-monophosphate, both of which have been reported to promote cell division and growth of plant tissues, the cytokinin activity is related to the 8-bromo substituent and to the N⁶-butyryl substituent, respectively, rather than to the 3′,5′-cyclic monophosphate moiety.     

Effect of Inorganic Phosphate on the Biosynthesis of Purine and Pyrimidine Nucleotides in Suspension-Cultured Cells of Catharanthus roseus

The levels of purine and pyrimidine nucleotides in suspensioncultures of Catharanthus roseus were determined 24 h after stationary-phasecells were transferred to fresh complete (‘+Pi’)or phosphate-deficient (‘–Pi’) Murashige-Skoogmedium. The levels of ATP, GTP, UTP and CTP were from approx.3 to 5-fold greater in the cells grown in ‘+Pi’medium than in the cells grown in ‘–Pi’ medium.The levels of almost all other nucleotides were slightly higherin the cells in ‘+Pi’ medium. The rates of de novoand salvage biosynthesis of purine and pyrimidine nucleotideswere estimated from the rates of incorporation of radioactivityfrom [¹⁴C]formate, [2–¹⁴C]glycine, NaH¹⁴CO₃, [6–¹⁴C]orotate,[8–¹⁴C]adenine, [8–¹⁴C]adenosine, [2–¹⁴C]uraciland [2–¹⁴C]uridine. The results indicated that the activityof both the de novo and the salvage pathway was higher in thecells in ‘+Pi’ medium than in the cells in ‘–Pi’medium. The rate of degradation estimated from the rate of releaseof ¹⁴CO₂ from labelled purines and pyrimidines indicated thatdegradation of uridine was significantly reduced in the cellsin ‘+Pi’ medium, but no significant difference wasfound in the degradation of adenine, adenosine and uracil. Thepossible role of Pi in the control of the biosynthesis of nucleotidesand in the degradation of uridine is discussed. Catharanthus roseus, Madagascar periwinkle, suspension culture, inorganic phosphate, nucleotides, purines, pyrimidines, biosynthesis, degradation     

Effect of a cytokinin antagonist on cytokinin and light-dependent amaranthin synthesis inAmaranthus Tricolor seedlings

InAmaranthus tricolor seedlings, amaranthin synthesis can be induced under the effect either of a cytokinin or of white light. The 3-methyl-7-(n-pentylamino)pyrazolo(4,3-d)pyrimidine (PAMPP), a cytokinin analog that strongly inhibits the growth of tobacco callus, antagonizes the stimulating effect of cytokinin as well as stimulation by light. In dose-response terms, the inhibitory effect of PAMPP was described as competitive with respect to N⁶-benzyladenine (b⁶Ade) or light. The inhibition by PAMPP of the b⁶Ade amaranthin test response or the inhibition by this cytokinin analog of the light amaranthin test response were both reversed by either subsequent light or b⁶Ade treatment.     

Influence of calcium and calcium and calmodulin antagonists on the cytokinin-induced amaranthin accumulation inAmaranthus tricolor

The concentration of kinetin and kinetinriboside plays an essential role in the induction of amaranthin accumulation in cotyledons ofAmaranthus tricolor during germination. The dose/effect ratio shows that kinetin induced 3- to 3.5-fold more amaranthin than kinetinriboside at the same molecular concentration. Various concentrations of exogenous Ca²⁺ did not influence the effects of kinetin on the betacyanin synthesis. However, when Ca²⁺ was applied together with kinetinriboside, the amaranthin production was stimulated. Time-course experiments show a lag phase of 16 h starting from the incubation with kinetin and a distinct increase of amaranthin thereafter. If the seedlings were treated simultaneously with kinetin and Ca²⁺, the increase of amaranthin started after 12 h. At 16 h of incubation in kinetin/Ca²⁺, the amount of amaranthin increased significantly compared to controls incubated with kinetin alone. If Ca²⁺ ions (16 h kinetin/Ca²⁺ incubation) were removed from the medium after 2 h, 4 h, and up to 14 h, the amaranthin content was enhanced compared to controls without Ca²⁺. The stimulating effect was highest in the presence of Ca²⁺ for 8 h. These data show that exogenous Ca²⁺ stimulated the amaranthin synthesis mainly during the first 12 h of incubation. The Ca²⁺ antagonists EGTA, chlorotetracycline, and CoCl₂ reduced the amaranthin content up to 80%. The calmodulin antagonists chloropromazine and trifluoperazine inhibited the betacyanin accumulation up to 97% when applied at the beginning of the incubation. Neither Co²⁺ nor trifluoperazine after 12 h of preincubation in kinetin had inhibiting effects on the amaranthin production. Therefore, we presume that a specific period of competence is required for calmodulin-mediated Ca²⁺ effects on the accumulation of amaranthin induced by cytokinins in the seedlings ofAmaranthus tricolor.     

Effect of a cytokinin antagonist on cytokinin and light-dependent amaranthin synthesis inAmaranthus Tricolor seedlings

InAmaranthus tricolor seedlings, amaranthin synthesis can be induced under the effect either of a cytokinin or of white light. The 3-methyl-7-(n-pentylamino)pyrazolo(4,3-d)pyrimidine (PAMPP), a cytokinin analog that strongly inhibits the growth of tobacco callus, antagonizes the stimulating effect of cytokinin as well as stimulation by light. In dose-response terms, the inhibitory effect of PAMPP was described as competitive with respect to N⁶-benzyladenine (b⁶Ade) or light. The inhibition by PAMPP of the b⁶Ade amaranthin test response or the inhibition by this cytokinin analog of the light amaranthin test response were both reversed by either subsequent light or b⁶Ade treatment.     

Vessel element formation in cultured carrot-root phloem slices

The effects of light, auxin and cytokinin on vessel elementformation in phloem slices of carrot root were examined. When slices of carrot cultivars, ‘Nakamura-senko-futo’and ‘Yamada-hyakunichisenk6- naga’, preculturedin the dark on modified Murashige and Skoog's medium for twodays were cultured on a medium containing 5x10^–6 M 2,4-Din the dark, no vessel element formation occurred. When preculturedslices were cultured in the light with 5x10^–6M 2,4-D,vessel element formation was remarkable. But when 5x10^–7Mkinetin, benzyladenine or zeatin was added, vessel elementswere readily formed even in the dark. When slices were cultured in the light, a cytokinin-like substance(s)that causes vessel element formation was produced in the slices,then was released to the medium. The substance(s) was fairlystable to heat. In slices of carrot cultivars, kuroda-gosun, ‘Kintoki’and ‘Kokubu-senk6-6naga’, a different result forvessel element formation was obtained. When slices of thesecultivars were cultured on a medium containing 5x10^–6M2,4-D in the dark, vessel element formation was remarkable.It seemed, therefore, that these cultivars contain enough ofa cytokinin-like substance(s) to form vessel elements. In fact,vessel element forming activity was found in the alcohol extractof carrot root phloem from these cultivars. (Received June 8, 1971; )     

The Effects of Cytokinins and ABA on Stomatal Behaviour of Maize and Commelina

Epidermal strips and leaf fragments of Commelina and leaf fragmentsof maize were incubated on solutions containing naturally-occurringor synthetic cytokinins and/or ABA. The effects of these treatmentson stomatal behaviour were assessed. Cytokinins alone did notpromote stomatal opening in either species but concentrationsof both zeatin and kinetin from 10^–3 to 10^–1 molm^–3 caused some reversal of ABA-stimulated closure ofmaize stomata. The reversal of the ABA effect increased withincreasing cytokinin concentration. Cytokinins had no effecton ABA-stimulated closure of Commelina stomata. When appliedalone, at high concentration (10^–1 mol m^–3), toCommelina epidermis or leaf pieces both zeatin and kinetin restrictedstomatal opening. Key words: ABA, Cytokinins, Stomata, Maize, Commelina     

Reversal of sucrose inhibition of Lemna flowering by adenine derivatives

Sucrose inhibition of flowering in Lemna perpusilla 6746 wasat least partially reversed by 5'-AMP, cyclic 3',5'-AMP, 5'-ADP,5'-ATP and K₂HPO₄. These results are in contrast to those reportedfor L. gibba in which reversal was effected by cyclic AMP, butnot by other adenine derivatives. ¹ This work was supported by National Science Foundation GrantGB-12955. (Received June 11, 1973; )     

Investigation of the Radish Leaf Bioassay for Kinetins, and Demonstration of Kinetin-like Substances in Algae

This study is part of an investigation into the occurrence ofplant growth substances in marine unicellular algae. Auxinsand gibberellins have previously been detected. This paper reportsthe occurrence of phytokinins in algae, using the radish leaftest. The technique of the test is described and a few modificationsexamined. Radish leaves were shown to be more responsive thanswede leaves. Some requirement for a minimum temperature below7? C during the growing period of the plants appeared to bea factor for maximum response. The size and age of the leafwere shown to influence kinetin activity. Chemicals tested includedbenzyl adenine, a 9-substituted benzyl adenine, and kinetin.Kinetin showed lower activity than the benzyl adenines. Kinetinkept in the solid state at –20? C for 2 years was as activeas freshly prepared kinetin. Gibberellic acid (GA₂) at 10^–5g/ml sometimes showed activity equivalent to 10^–5 g/mlkinetin. Indole-3-yl acetic acid at concentrations of 10^–4to 10^–6 g/ml was inactive. The radish leaf test was successfully used to demonstrate phytokininsin extracts of two species of unicellular marine algae, andin marine phytoplankton samples. Amounts found were within therange 0.1 to 1.0 mg/kg with one exception of 10 mg/kg. Phytokininactivity in these extracts decreased over a period of a fewweeks when stored at –20? C.     

Localization of cytokinin biosynthetic sites in pea plants and carrot roots

The biosynthesis of cytokinins was examined in pea (Pisum sativum L.) plant organs and carrot (Daucus carota L.) root tissues. When pea roots, stems, and leaves were grown separately for three weeks on a culture medium containing [8-¹⁴C]adenine without an exogenous supply of cytokinin and auxin, radioactive cytokinins were synthesized by each of these organs. Incubation of carrot root cambium and noncambium tissues for three days in a liquid culture medium containing [8-¹⁴C]adenine without cytokinin demonstrates that radioactive cytokinins were synthesized in the cambium but not in the noncambium tissue preparation. The radioactive cytokinins extracted from each of these tissues were analyzed by Sephadex LH-20 columns, reverse phase high pressure liquid chromatography, paper chromatography in various solvent systems, and paper electrophoresis. The main species of cytokinins detectable by these methods are N⁶-(Δ²-isopentyl_adenine-5′-monophosphate, 6-(4-hydroxy-3-methyl-2-butenyl-amino)-9-β-ribofuranosylpurine-5′- monophosphate, N⁶-(Δ²-isopentenyl)adenosine, 6-(4-hydroxy-3-methyl-2-butenylamino)-9-β-ribofuranosylpurine, N⁶-(Δ²-isopentenyl)adenine, and 6-(4-hydroxy-3-methyl-2-butenylamino)purine. On the basis of the amounts of cytokinin synthesized per gram fresh tissues, these results indicate that the root is the major site, but not the only site, of cytokinin biosynthesis. Furthermore, cambium and possibly all actively dividing tissues are responsible for the synthesis of this group of plant hormones.     

Sensitivity of oat leaf chlorophyll retention bioassay to natural and synthetic cytokinins

The cytokinin bioassay based on retention of chlorophyll in excised oat leaf pieces is more sensitive to the synthetic cytokinins (6-benzylaminopurine and kinetin) than to the natural cytokinins (trans-zeatin and N⁶-[Δ²isopentenyl]adenine). This difference in sensitivity decreases with increasing length of leaf pieces (from 2 to 10 cm) and with increasing volume of application (from 5 μ1 to 25 μl). Application of the cytokinin solution to the basal and apical part of the 8 cm pieces decreases the sensitivity of the bioassay but has no significant effect on the relative activities of trans-zeatin and 6-benzylaminopurine. Using 7 cm pieces and 20 μl of solution the trans-zeatin and N⁶-(Δ₂-isopentenyl)-adenine can be detected at concentrations of 10^-5 M and 5 x 10^-5 M, respectively.     

Determinate Floral Buds of Plantain (Musa AAB) as a Site of Adventitious Shoot Formation

Floral buds of the ‘False Horn’ plantain clonesMusa (AAB) ‘Harton Verde’, ‘Harton Negra’,and ‘Currare’ terminate in a large single floralstructure. The apices of these floral buds are here designatedas determinate since they have lost the ability to produce additionalfloral initials or buds. Terminal peduncle segments can be culturedin a modified Murashige and Skoog (1962) medium supplementedwith N⁶-benzyl-aminopurine (5 mg I^–1). Under these conditions,this apparent inability to yield buds can be overcome as vegetativeshoot clusters form in the axils of the bracts. Rooted plantletsare obtainable by treating shoots with naphthaleneacetic acid(1 mg I^–1) and activated charcoal (0.025%). The adventitiousorigin of the shoots has been established. Musa cultivars, plantains, floral bud, adventitious buds, tissue culture     

Kinetin inhibition of h-uracil and C-leucine incorporation by tobacco cells in suspension culture

The rate of ¹⁴C-leucine and ³H-uracil incorporation by tobacco cells (Nicotiana tabaccum var. Samsun N.N.) in suspension culture was simultaneously decreased by the addition of kinetin at concentrations above 2.5 × 10⁻⁵m. Ribosomal RNA was the first RNA species affected by kinetin. The purine derivatives, adenine and N⁶-methyl-aminopurine, which exhibit low cytokinin activity overcame the inhibitory effects of kinetin. However, purine derivatives without cytokinin activity, guanine, N^6,6-dimethyl-aminopurine, and 2-aminopurine, did not relieve kinetin inhibition.     

Micropropagation of Pissardi Plum

An efficient medium for multiplication of Prunus cerasifera,J. F. Ehrh. cv. ‘Atropurpurea’, Pissardi plum, consistedof Linsmaier-Skoog basal medium (LS) supplemented with 3% sucrose,10mg 1^–1 N⁴-(²-isopentenyl) adenine (2iP), and 162 mg1^–1 phloroglucinol (Phl). Phl in the medium significantlyenhanced growth (P < 0.1 %) over cultures maintained on LSmedium with 10 mg 1^–1 2iP and no Phl. Plantlets were rootedon a half-strength Murashige-Skoog (MS) medium supplementedwith 0.2 mg 1^–1 indole-3-butyric acid (IBA). Prunus cerasifera, Pissardi plum, micropropagation, phloroglucinol, N⁶-(²-isopentenyl) adenine