Combined Effects of Auxin Transport Inhibitors and Cytokinin: Alterations of Organ Development in Tobacco

We have examined the effects of the auxin transport inhibitors1-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid(TIBA) on leaf morphogenesis of transgenic Nicotiana tabacum(cv. Xanthi) plants expressing the Agrobacterium tumefacienscytokinin biosynthetic gene, ipt. We have observed the formationof saucer-shaped leaf-like organs at the shoot apex and at lateralbuds. The formation of apical saucer-shaped leaf-like organscan be duplicated by the application of exogenous NPA and cytokininto wild-type tobacco seedlings. We have also observed adventitiousleaf-like organs with altered petiole and blade morphology inthe transgenic plants treated with auxin transport inhibitors.These results suggest that the combination of diminished auxintransport and elevated cytokinin can lead to alterations inleaf development in tobacco. ⁴Present address: Genesis Research and Development Corporation,P.O. Box 50, Auckland, New Zealand     

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens I. Auxin and Cytokinin Contents of Cultured Tissues Transformed with Wild-Type and Mutant Ti Plasmids

Carrot and tobacco plants were transformed with Agrobacteriumtumefaciens harboring wild-type, aux^– or cyt^– Tiplasmids. In tobacco, these wild and mutant Ti plasmids inducedthe formation of non-morphogenic galls, galls with shoots, andgalls with roots, respectively. In carrot, however, transformationwith any of these plasmids resulted in only the formation ofamorphous tumors. Determination of IAA and cytokinin contentsshowed that in tobacco, significantly high amounts of cytokininsor IAA are present in the cells transformed with Ti plasmidspossessing cytokinin or IAA biosynthetic genes, respectively.In carrot, cytokinin contents were also high in the cells transformedwith Ti plasmids having cytokinin biosynthetic genes, whereasIAA contents of the cells were similar regardless of the plasmidsused for transformation. These results suggest that the mechanism regulating IAA metabolismmay be different in tobacco and carrot. (Received June 25, 1987; Accepted February 1, 1988)     

Hormonal regulation of genetic tumor induction: Cytokinin and abscisic acid

Young seedlings of the tumor-prone amphiploid Nicotiana suaveolens? N. langsdorjfii were grown aseptically on nutrient mediumin a controlled environment chamber. At regular intervals theincidence of tumor formation was scored and plants were harvested.Total cytokinin activity was determined by means of the cucumbercotyledon bioassay, while ABA activity was measured by radioimmunoassay.A close correlation between onset of tumor formation and elevationin endogenous cytokinin activity was demonstrated, but no correlationwas observed between onset of tumorigenesis and change in thelevel of ABA. In addition, exposure of plants to exogenous ABAdid not alter therate of tumor formation. These results arediscussed in relation to the trigger mechanism for tumor inductionin the Nicotiana system. ¹A preliminary report of some of this work was presented atthe American Society of Plant Physiologists meeting in June1978. ³Present address: Department of Biology, West Virginia University,Morgantown, West Virginia 26506, U.S.A. (Received March 10, 1979; )     

Inhibition of Cell Division and DNA Synthesis by Gibberellin in Isolated Zinnia Mesophyll Cells

A culture system of isolated mesophyll cells of Zinnia eleganswas used to examine the action of gibberellic acid (GA) on celldivision. Isolated Zinnia mesophyll cells cultured in a mediumcontaining auxin and cytokinin reinitiated cell division ina partly synchronized manner. When mesophyll cells isolatedfrom 21-day-old seedlings were used, GA added to the culturemedium at concentrations of 1 x 10^–6 M or higher suppressedthe initial rise in the number of divided cells. Tracer experimentswith [³H]-dThd revealed that GA treatment inhibited the incorporationof [³H]-dThd into DNA in the nucleus without inhibiting theuptake of [³H]-dThd into the cells, indicating that GA inhibitedDNA synthesis. GA applied at 48 h inhibited the incorporationof [³H]-dThd into DNA during the following 24 h, but GA appliedat 72 h did not inhibit the incorporation during the subsequent24 h. This suggests that GA affects the process of reinitiationof DNA synthesis, but does not affect DNA synthesis once cellshave become proliferative. (Received January 14, 1986; Accepted March 31, 1986)     

Involvement of Poly(ADP-Ribose) Synthesis in Transdifferentiation of Isolated Mesophyll Cells of Zinnia elegans into Tracheary Elements

The relationship between poly(ADP-ribose) synthesis and cytodifferentiationwas studied in the well characterized Zinnia system, in whichisolated mesophyll cells of Zinnia elegans transdifferentiateinto tracheary elements (TE) in a suspension culture in thepresence of both auxin and cytokinin. The rate of poly(ADP-ribose)synthesis was measured in nuclei isolated from cells that hadbeen induced to undergo transdifferentiation, and activationof such synthesis was observed before the appearance of TE duringculture. In cultures without auxin or cytokinin, poly-(ADP-ribose)synthesis appeared to proceed much more slowly. Treatment of cells with a potent inhibitor of poly-(ADP-ribose)polymerase, namely, 6(5H)-phenanthridinone (PT), resulted inthe blockage of TE formation and a decrease in the frequencyof cell division. PT was very effective in interfering withtransdifferentiation, in particular, when supplied between the24th hour and the 36th hour of culture. Repair-type DNA synthesis,which has been proposed to participate in transdifferentiation,was suppressed by the treatment with PT. These results suggestthat poIy(ADP-ribose) synthesis and subsequent repair-type DNAsynthesis might play a critical role in the transdifferentiationof Zinnia cells. ³Present address: Botanical Gardens, Faculty of Science, Universityof Tokyo, Hakusan, Bunkyo-ku, Tokyo, 112 Japan. ⁴Present address: Department of Chemical and Biological Sciences,Faculty of Science, Japan Women's University, Mejirodai, Bunkyo-ku,Tokyo, 112 Japan.     

Cytokinin-Mediated Translational Control of Protein Synthesis in Cultured Cells of Glycine max

Polyribosome formation was stimulated by cytokinin treatmentof cultured cells of Glycine max cv. Funk Delicious. When suspensioncultures were given 0·5 µM zeatin after 24 h inculture in medium lacking a cytokinin, a nearly 2-fold increasein the polyribosome/monoribosome ratio occurred over the subsequent3 h. The effect of actinomycin D and of 5-fluorouridine on RNAsynthesis and on the polyribosome/monoribosome ratios of thesecells was examined. Actinomycin D at 5 and 20 µg/ml^–1inhibitedtotal RNA synthesis by 39 and 60%, respectively, as measuredby [³H]uridine incorporation into acid-precipitable material.The degree of inhibition of precursor incorporation into polyribosomalRNA was similar. At 0·1 mM, 5-fluorouridine inhibited[³H]uridine incorporation by 76%, and [³H]guanosine incorporationby 66% into polyribosomal RNA after 3 h of treatment. Fractionationof the polyribosomal RNA by oligo(dT)-cellulose chromatographydemonstrated that low concentrations of both actinomycin D (5µg ml^–1) and 5-fluorouridine (0·1 mM) inhibitedthe synthesis of ribosomal RNA to a greater extent than thepoly(A)-containing fraction of the messenger RNA. Synthesisof the poly(A)-containing RNA was inhibited by 24% with 5µgml^–1 actinomycin D and by 30% with 0·1 mM 5-fluorouridine.At the above concentrations, these two inhibitors reduced thepolyribosome/monoribosome ratio of the cytokinin-deprived cellsover a 3 h period, but they did not prevent cytokinin-inducedpolyribosome formation. These results provide further evidencethat cytokinin regulates polyribosome levels through an effecton protein synthesis at the translational level     

Involvement of Calcium in Adventitious Bud Initiation in Torenia Stem Segments

In Torenia stem segments cultured in vitro, active meristematicdivisions are induced in the epidermis by treatment with cytokinin,resulting in the formation of adventitious buds. Applicationof the calcium ionophore A23187 [GenBank] was found to induce meristematicdivisions in the absence of cytokinin. The induction by A23187 [GenBank] was inhibited by simultaneous addition of auxin, but not byanti-cytokinin. A two hour pre-treatment with A23187 [GenBank] was alsoeffective, but only when it was applied to the explants justafter their excision from mother plants. The A23187 [GenBank] -inducedmeristematic zones developed into dome-shaped structures, butnot into complete adventitious buds. Complete elimination ofcalcium from the culture medium caused 50% inhibition of A23187 [GenBank] -and/or cytokinin-induced initiation of meristematic divisions.When the explants were preincubated with EGTA and then culturedon a Ca-free medium containing EGTA, cytokinin failed to inducebud initiation. Similar inhibition was also obtained by lanthanum,a calcium antagonist, by verapamil, a calcium channel inhibitor,and by trifluoperazine and chlorpromazine, calmodulin inhibitors.These results support the idea that adventitious bud initiationinduced by cytokinin in Torenia stem segments may be mediated,at least partially, by an increase in the level of intracellularCa²⁺. ¹Bioscience Research Center, Mitsui Petrochemical IndustriesLtd., Waki-cho, Kuga-gun, Yamaguchi 740, Japan. (Received May 9, 1985; Accepted October 5, 1985)     

Gas Chromatography-Flame Thermionic Detector Analysis of Cytokinins in Etiolated Squash Seedlings using 14C-BA as an Internal Standard

Cytokinin contents in cotyledon, hypocotyl and root of etiolatedsquash (Cucurbita maxima Duch.) seedlings were determined byinstrumental analysis using ¹⁴C-benzyladenine (¹⁴C-BA) as aninternal standard. Crude extracts were purified using insolublepolyvinylpyrrolidone, cellulose-phosphate column and SEP-PAKC18 cartridge, then applied to a Sephadex LH-20 column to separatezeatin riboside (ZR), isopentenyl adenosine, isopentenyl adenine,¹⁴C-BA and a mixture of zeatin (Z) and dihydrozeatin (DHZ).The recovery rate for the cytokinin fractions after LH-20 wascorrected by ¹⁴C-BA. Each cytokinin fraction was further purifiedby HPLC which also separated Z and DHZ in the LH-20 fraction.Before permethylation, ¹⁴C-BA was added to each of the cytokininfractions to correct the methylation rates. Each methylatedcytokinin fraction was again purified by HPLC, then subjectedto gas chromatography with a capillary column and flame thermionicdetector. The detection limit of cytokinins by this system was0.1 ng. cis-ZK was the most abundant cytokinin in all tissues of theetiolated squash seedlings. Active cytokinins such as trans-ZRand trans-Z were mostly found in cotyledons with lesser amountsin the roots. DHZ was most abundant in the cotyledon. All cytokininsisolated by this procedure were confirmed by gas chromatographyselectedion monitoring. (Received December 26, 1986; Accepted June 1, 1987)     

Metabolism and Biosynthesis of Cytokinins in Tobacco Crown Gall Cells

The metabolism of ribosylzeatin (RZ) was studied using tobaccocrown gall cells which produce RZ as one of the major endogenouscytokinins. When [8-¹⁴C]RZ was fed to the cells, it was convertedinto its phosphate (which was rigorously determined to be the5'-monophosphate), RZ-O-glucoside, inosine (or its phosphate),adenosine and adenosine-O-glucoside. When [8-¹⁴C]N⁶-(²-isopentenyl)adenosine(i⁶Ado), a probable precursor of RZ, was fed to the cells, itwas converted into (i⁶Ado)-O-glucoside, inosine (or its phosphate),adenosine, adenosine-O-glucoside and adenosine phosphate, butno incorporation of radioactivity into RZ was observed. Thepresent study led to the following conclusions: i) i⁶Ado isnot a precursor of RZ in the cells, ii) both deaminase and cytokininoxidase are involved in the catabolism of cytokinin, and iii)the metabolism of RZ is quite different from that of i⁶Ado. (Received December 24, 1985; Accepted April 1, 1986)     

Increase of cytokinin activity in detached etiolated cotyledons of squash after illumination

Cytokinins nullified the lag period of the formation of chlorophyllin detached etiolated cotyledons of squash (Cucurbita moschataDuch. var. melonaeformis Makino cv. Tokyo). One hour after illumination,cytokinin activity in detached cotyledons rapidly increasedand maintained a certain level for another hour. ¹ Present address: Department of Enviromental Studies, Collegeof Integrated Arts and Sciences, Hiroshima University, Hiroshima730, Japan. (Received November 25, 1976; )     

Kinetics of Endogenous Cytokinin, IAA and ABA Levels in Relation to the Growth and Morphology of Tobacco Crown Gall Tissue

The kinetics of endogenous cytokinin, IAA and ABA levels duringthe growth cycle of a wild-type tobacco crown gall (W₃₈-B₆S₃)were compared with that of a shoot-inducing (Shi^-) mutant. Inboth tumor types, high IAA and cytokinin (essentially ribosyl-trans-zeatinand its corresponding glucoside) levels were built up by theend of the linear growth phase and maintained during the greaterpart of the exponential growth period. The stationary phasewas preceded by a very drastic decrease in the endogenous levelof both hormones. Quantitatively, the wild-type tumour showed a higher IAA leveland a reduced cytokinin level compared with the Shi^- mutant.No significantly different endogenous ABA pattern was observed.The reduced cytokinin level might correspond to the ratio oftransformed/untransformed cells in the wild-type tumour whereasthe reduced IAA level in the Shi^- mutant may be correlated withthe deletion of gene 2 in the T-DNA of the pGV 2206 Ti plasmid. The elevated cytokinin/IAA ratio induced shooting mainly ofthe untransformed cells in the Shi^- mutant tissue whereas inthe wild-type, the shoot suppression was compatible with thereduced cytokinin/IAA ratio. ⁴Senior Research Associate Nationaal Fonds WetenschappelijkOnderzoek (N.F.W.O.). ⁵Research Associate N.F.W.O. ⁶Recipient of an Instituut voor Wetenschappelijk Onderzoek inNijverheid en Landbouw grant. (Received February 23, 1984; Accepted June 19, 1984)     

In Vitro Regeneration from Seedling Organs of Brassica oleracea var. italica Plenck cv. Green Comet I. Effect of Plant Growth Regulators

The calabrese cultivar Brassica oleracea var. italica cv. GreenComet was used in a study of the effects of exogenous hormoneson the growth and differentiation of seedling organs in vitro.Four types of explants were tested: hypocotyl segments, rootsegments, primary leaf discs and cotyledon discs. These explantswere incubated on media containing factorial combinations ofBAP x IBA, BAP x NAA, KN x IBA and KN x NAA (all at 0, 0.1,10 and 10.0mg l^–1). Hypocotyls were the most regenerativeexplants; shoot production was favoured by cytokinin: auxinratios greater than one and was decreased by IBA at 10 mg l^–1when callus was produced. Shoot formation from root explantsoccurred either in the absence of hormones or with low concentrations;no shoot was produced when any hormone was present at 10 mgl^–1. In contrast, shoot production from primary leaf diseswas favoured by high concentrations of both auxin and cytokininwith the combination of BAP and IBA the most effective. Shootproduction from cotyledon discs was sporadic with no consistentresponse on any auxin/cytokinin combination. After further experimentson the optimization of hormone concentration, the followingcombinations were chosen as allowing reliable regeneration:0.1 mg l^–1 BAP+0.1mg l^–1 IBA for hypocotyl segments,0.075 mg l^–1 KN +0.025 mg l^–1 IBA for root segments,and 5.0 mg l^–1 BAP+5.0 mg l^–1 IBA for leaf discs. Brassica oleracea var. italica, calabrese, tissue culture, seedling, auxin, cytokinin     

Functional Correlation between Endogenous Phytohormone Levels and Hormone Autotrophy of Transformed and Habituated Soybean Cell Lines

We analysed the time course of the endogenous free IAA and cytokininlevels in hormone requiring and hormone autotrophic (both transformedand untransformed) Glycine max. L. Merr. cv. Mandarin tissuecultures. The auxin habituated line showed an enhanced endogenous IAAlevel, whereas the IAA as well as the cytokinin concentrationsin the cytokinin habituated line differed not significantlyfrom the non-habituated hormone requiring soybean callus. It were only the auxin habituated cells that could be inducedto fully habituated cells, from which a pale and a green typewas isolated. The phytohormone autotrophic growth of the paletype was sustained by enhanced IAA levels, whereas the greentype was characterised by elevated cytokinin concentrations. These results on the phytohormone content of partially and fullyhabituated soybean calli were compared with soybean crown galllines and discussed in view of the positive effect of exogenouslyapplied cytokinins on the endogenous IAA levels. ³Recipient of an Instituut voor Wetenschappelijk Onderzoek inNijverheid en Landbouw (I.W.O.N.L.) grant. ⁴Senior Research Associate Nationaal Fonds voor wetenschappelijkOnderzoek (N.F.W.O.). (Received March 25, 1988; Accepted July 7, 1988)     

Uptake, transport and metabolism of cytokinin in moss protonema

Uptake, transport and metabolism of cytokinin in the protonemaof Funaria hygrometrica were studied using labelled kinetin(6-furfurylamino [8-¹⁴C]-purine). All cells of the protonema,chloronema and caulonema, were able to take up kinetin, whichwas carried in the symplastic transport system from cell tocell. Radioactivity was especially accumulated in growing cellsof the protonema. Kinetin was metabolized immediately afteruptake. While only very little kinetin (less than 1%) remainedas free kinetin and one part was immobilized in chromatographicseparation [e.g. attached to proteins and incorporated intonucleic acids (17)], most of the remaining kinetin was metabolizedto adenine derivatives. Exogenously supplied adenosine changedthe metabolism of kinetin. In the caulonema, adenosine reducedthe turnover of kinetin to other adenine derivatives and enhancedthe content of labelling in the start fraction. Thus adenosinecan stimulate cytokinin-dependent bud formation in moss protonema. (Received November 24, 1977; )     

Dynamics of Endogenous IAA and Cytokinins during the Growth Cycle of Soybean Crown Gall and Untransformed Callus

The dynamics of the endogenous IAA and cytokinin levels duringthe growth cycle of two soybean crown gall lines (green andpale) induced by a nop⁺ Agrobacterium tumefaciens C58 strain,were compared with an untransformed soybean callus line. In both transformed tumor lines maximum cytokinin (essentiallyglucosyl-trans-zeatin) levels were attained in the beginningof the exponential growth phase, followed by a drastic decreasejust before the stationary phase was reached. Quantitativelythe green tumor line showed a 2–3 times higher cytokinincontent compared with the pale line. In the untransformed soybeancallus hardly any significant levels of cytokinin could be detected. Analysis of endogenous IAA levels showed no difference betweenthe two tumor lines and the untransformed callus tissue, allshowing a low and constant level throughout the entire growthcycle. The relevance of the endogenous accumulation of phytohormonesin relation to the hormone autotrophic growth of transformedsoybean tissue is discussed. ³ Senior Research Associate Nationaal Fonds WetenschappelijkOnderzoek (N.F.W.O.). ⁴ Recipient of an Instituut voor Wetenschappelijk Onderzoekin Nijverheid en Landbouw (I.W.O.N.L.) grant.     

In Vitro Floral Development of Oilseed Rape (Brassica napus L.): The Effects of pH and Plant Growth Regulators

The effects of various plant growth regulators and that of pHon the in vitro growth and development of young inflorescencesof Brassica napus L. cv. Westar were examined. A cytokinin wasrequired for normal maturation of floral buds, including thecompletion of microsporogenesis, and it stimulated the initiationof additional buds on the inflorescence axis. Benzylaminopurine(BAP) was the most effective of the cytokinins tested. Gibberellicacid (GA₃) and naphthaleneacetic acid (NAA) alone were ineffective.In combination with BAP, both reduced the positive influenceof the cytokinin but GA₃ was more inhibitory than NAA. At alow initial pH (3.9–4.6), the percentage of cultures whichproduced normal buds was significantly higher, especially inthe presence of 10^-7 M or 5 ? 10^-7 M BAP, in comparison to cultureswith a pH of 5.3-6.0, the standard range for plant tissue culture.     

Role of Cytokinin in Vessel Regeneration in Wounded Coleus Internodes

Cytokinin was found to be a controlling or limiting factor invessel regeneration around a wound in internodes of Coleus blumeiBenth. in which the endogenous cytokinin level was minimized.The cytokinin was applied in aqueous solution to the base ofexcised, mature internodes that had an active vascular cambium.Each internode also received IAA in lanolin at its apical end.Under low (0.1 %, w/w) or high (10%, w/w) auxin concentrations,the control internodes (without exogenous cytokinin) exhibitedsmall amounts of vessel regeneration. At appropriate concentrationszeatin, kinetin and 6-benzylamino-purine (BAP) induced a significantincrease in vessel regeneration around the wound. The threecytokinins also induced novel patterns of supplementary regenerationfurther from the wound surface. Kinetin and BAP showed the strongestpromoting effect at 5 and 10 µg ml^–1, while zeatinwas most effective at 20 µg ml^–1. At a low (0.1%) auxin level zeatin was the most effective cytokinin, whereaskinetin was the most effective one at high (1 %) auxin. An inhibitoryeffect on vessel regeneration was observed at the highest kinetinconcentration tested (50 µg ml^–1). The regenerationof vessels induced by cytokinin was very polar. Many more regeneratedvessel members differentiated below the wound than above it,and the regeneration process proceeded acropetally from thebase of the internode to its upper parts. Our results implya basipetal polar increase in cambium responsiveness along thestem axis from internode 5 to 7. The possible significance ofsuch a basipetal increase in cambium sensitivity in wood formationin trees is discussed. Auxin, Coleus blumei, cytokinin, vascular differentiation, vessel regeneration, wound xylem     

Endogenous Cytokinins in Flower Bud Forming Explants of Tobacco

The accumulation of endogenous cytokinins was studied in pedicelexplants of tobacco (Nicotiana tabacumL.) during regenerationof flower buds in vitro. Maximal bud formation was induced onmedia containing 1.0 mmol m^–3 of benzyladenine or dihydrozeatin.No buds were formed in the absence of cytokinin. The levelsof dihydrozeatin, zeatin, and the corresponding ribosides weredetermined in explants cultured in the presence or absence ofcytokinin by means of a competitive ELISA technique. In explantsincubated without a cytokinin, only the dihydrozeatin concentrationincreased significantly during the first day of incubation anddecreased during the second day. No increase was observed inexplants incubated in the presence of benzyladenine. The concentrationof dihydrozeatin in these bud-forming explants was only 10 to15% of the concentration built up in explants cultured on dihydrozeatininstead of benzyladenine. This suggests that the endogenouscytokinins only play a minor role in the regeneration of flowerbuds in vitro. Key words: cytokinin, flower bud development, tissue culture, tobacco     

Reversible Structural Changes Associated With Callus Formation and Plantlet Development from Aseptically Cultured Shoots of Taro (Colocasia esculenta var antiquorum)

Taro callus maintained on Knop's medium with 2, 0·2 or0·02 mg l^–1 2,4,5-trichiorophenoxyacetic acid (2,4,5-T)or Linsmaier-Skoog (LS) containing 1 mgl^–1 of the cytokininadenine-N-benzyl-9-tetrahydro-2H- pyran-2-yl (SD8339) or 6 dimethylaininopurineand 0·1 mgl^–1 -naphthaleneacetic acid underwenta transition to a stable organized growth form which is referredto as a calloid. On transfer to LS medium th 0·2 mgl^–12,4,5-T in the absence of cytokinin the calloid reverts backto callus. Colocasia esculenta(L.)Schott, taro, callus, calloid, in vitro selection, histology, micropropagation, tissue culture, cytokinin     

Biosynthesis of Cytokinin in Germinating Seeds of Zea mays

The embryos of germinating Zea mays seed were supplied with[¹⁴C]-adenine Following incubation, the tissue was extractedand extensively purified by non-exchange chromatography andthin layer chromatography. Radioactivity was found to be incorporatedinto zeatin nucleotide indicating that the embryo in the germinatingseed is capable of cytokinin biosynthesis. Key words: Zea mays cytokinin, zeatin nucleotide, biosynthesis, seed