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)     

Regulation of Stem Abscission and Callus Growth in Shoot Explants of Sweet Orange [Citrus sinensis (L.) Osbeck]

Two-node explants from Sweet Orange cv. St Ives Valencia orangeshoots produced prolific callus and formed secondary abscissionzones within internodes when cultured in vitro with abscisicacid (ABA, 5 µM) or -naphthaleneacetic acid (NAA, 5 µM).Benzyladenine (BA, 1 µm) induced callus but had littleeffect on abscission. Secondary abscission zone formation wasassociated with ABA-induced and auxin-induced ethylene formation.Treatment of explants with inhibitors of ethylene synthesis[aminoethoxyvinyl glycine (AVG), Co²⁺, PO₄^3–] preventedformation of secondary abscission zones but had variable effectson callus formation. Newly made explants contained high concentrationsof endogenous ABA (up to 6000 ng g^–1 f.wt), as measuredby GC/MS/SIM. Long-term subculture of explants (two years) inmedia containing BA (1 µm) led to a reduction in endogenousABA level (40 ng g^–1 f. wt) and to loss of capacity toform extensive callus and secondary abscission zones. Citrus sinensis (L.) Osbeck cv. St Ives Valencia, sweet orange, secondary abscission zones, in vitro, ethylene, endogenous ABA, endogenous IAA     

Chemical Factors Controlling Floral Bud Formation of Torenia Stem Segments Cultured in Vitro II. Effects of Growth Regulators

Correlative effects between growth regulators added to a mediumand different physiological states of explants on adventitiousbud formation and flowering were investigated using Toreniastem segments cultured in vitro. Indoleacetic acid stimulatedfloral bud formation and its development in explants taken fromreproductive plants. These stimulative effects were clearlyseen in explants taken from plants in which flower abscissionwas taking place, but insignificant when explants were preparedfrom younger materials. Abscisic acid acted in a reverse wayto auxin, greatly promoting floral bud initiation and floweringof originally vegetative explnts. Zeatin at a concentrationof 1 mg/liter inhibited floral bud formation, and at a low concentrationsit was generally ineffective. However, floral bud formationand flowering of explants taken either from basal parts of stemsor from 18- to 20-week-old plants were promoted by zeatin treatment.The action of gibberellic acid seemed rather indirect: at aconcentration of 0.01 mg/liter, it generally stimulated floralbud formation but at a concentration of 1 mg/liter, it was ofteninhibitory. (Received January 17, 1981; Accepted February 21, 1981)     

Inhibition of Cytokinin-Induced Adventitious Bud Initiation in Torenia Stem Segments by Inhibitors of Serine Proteases

Application of di-isopropyl fluorophosphate (DFP), a highlysensitive inhibitor for serine enzymes, strongly inhibited cytokinin-inducedadventitious bud initiation in Torenia stem segments culturedin vitro. The inhibitory effect was not evident when DFP wasapplied after 3 days of culture. Amount of DFP-binding proteinsremarkably increased in superficial tissues of explants culturedfor 3 and 4 days on a medium containing benzyladenine. At least14 kinds of DFP-binding polypeptides were detected by SDS-polyacrylamidegel electrophoresis and fluorography. DFP-binding to some ofthese polypeptides was inhibited by a prior treatment with phenylmethylsulfonylfluoride and N-p-tosyl-L-lysine chloromethyl ketone. From theseresults, it was suggested that some serine proteases might berelated with biochemical events occurring during the initialstage of adventitious bud differentiation in Torenia stem segments. (Received May 8, 1984; Accepted July 5, 1984)     

Significance of Spermidine in the Initiation of Adventitious Buds in Stem Segments of Torenia

When stem segments of Torenia fournieri Lind. were culturedin vitro, the initiation of adventitious buds, which is usuallyinduced by cytokinin, was induced by application of polyamines,such as putrescine and spermidine. Addition of arginine hada slight inductive effect. When cyclohexylamine, an inhibitorof spermidine synthase, was added simultaneously with putrescine,induction of the initiation of buds by putrescine was stronglyinhibited. However, application of the inhibitor together withspermidine had no effect on the spermidine-induced initiationof buds. The induction of initiation of buds by a cytokinin,by a calcium ionophore, by cyclic AMP, and by a phorbol ester,which was accompanied in each case by elevation of the levelsof endogenous spermidine, was also inhibited by cyclohexylamine.These results suggest the involvement of spermidine in the initiationof adventitious buds in stem segments of Torenia. ²Present address: Radiation Effects Research Foundation, Hijiyamakouen5-2, Minami-ku, Hiroshima, 732 Japan.     

Influence of explant source, plant growth regulators and culture environment on culture initiation and establishment of Quercus robur L. in vitro

Suitable cytokinin supplements and culture environments havebeen determined for the initiation and establishment of shootcultures of Quercus robur seedling tissue. Initiation of axillaryshoot development from nodal explants required culture mediumsupplemented with BA (6-benzylamminopurine). The greatest numbersof stem segments for culture proliferation were obtained using1.0 mg I^-1 BA after 56 d culture. The frequency of shoot developmentand subsequent formation of multiple shoots at initiation wasinfluenced by the position of the nodal explant in the seedlingshoot, incubation temperature and daylength. Explants from basaland apical regions, which contained multiple axillary buds,produced the lowest frequencies of axillary shoot developmentand multiple shoot formation, many remained quiescent. Axillaryshoot development was greatest in single nodal explants excisedfrom the midstem positions, elongated regions of the shoot wherenodes were formerly associated with a leaf. Higher temperaturesstimulated shoot formation with greater numbers of stem segmentsfor culture multiplication being obtained from nodal explantsincubated at 25C. Axillary shoot development was promoted innodal explants maintained under daylengths of 16 h or more.Stem segments cut from axillary shoots which developed fromnodal explants were used to establish shoot multiplication cultureson medium supplemented with 0.4 mg I^-1 BA. Shoot formation fromstem segments was greater at higher incubation temperaturesof 25C and 30C. Multiplication coefficients for stem segmentsincreased after one subculture. Key words: Quercus robur, oak, micropropagation, cytokinin, temperature, daylength, rest, quiescence     

In Vitro Culture and Plant Regeneration in Vicia faba subsp. Equina (var. Spring Blaze)

Explants of stem, leaves, roots, and cotyledons from etiolatedaxenically grown Vicia faba seedlings were cultured on a rangeof media. Shoot organogenesis was only obtained with nodal stemand cotyledonary node explants when cultured on MS medium with3% sucrose, 2.0 mg 1^–1 BAP and 02 mg 1^–1 NAA. Callusproliferation accompanied shoot organogenesis from nodal stemexplants. Successive subculture of nodal stem callus resultedin proliferation of regenerative callus which contained severalshoot bud initials. The capacity for shoot regeneration fromthis callus was maintained for 9 months. Histological studiesreveal de novo formation of meristematic centres in callus andtheir further development into bud primordia. High frequencyrooting of these adventitious shoots was obtained on half-strengthMS medium with 1.5% sucrose, 0.1 mg 1^–1 NAA and 0.5 mg1^–1 kinetin. Key words: Vicia faba, adventitious shoots, axillary shoots, de novomeristem formation, organogenesis, tissue culture     

The Role of Basipetal Auxin Transport in the Positional Control of Abscission Sites Induced in Impatiens sultani Stem Explants

If segments of Impatiens sultani stem are explanted and incubated,separation layers often form across them and lead to abscission.To test the suggested role of auxin concentration in controllingthe position of abscission sites, explants were labelled byapplying [¹⁴C]IAA to the shoot tip 4 h prior to explanting;transport of auxin applied in this way seems to resemble thatof endogenous auxin. During subsequent incubation of explantsfor 20 h, basipetal transport resulted in ¹⁴C accumulating justabove the base of the explants (nearly 80 % in the bottom 4mm of 24 mm explants). In internodal explants that had beenwounded at explanting by incising one side so as to sever avascular bundle, and in nodal explants with the leaf removed,the ¹⁴C also accumulated just above the wound or node to abouttwice the concentration otherwise expected; this accumulationwas probably due to basipetal transport being impeded by vasculardiscontinuity at the wound or node. Accumulation just abovethe base, or above a wound or node, resulted in gradients of¹⁴C concentration (presumably reflecting endogenous auxin concentration)decreasing in the morphologically upward direction at each ofthese three positions where abscission sites tend to occur. Impatiens sultani, abscission, auxin, IAA, node, polarized transport, positional control, separation layer, wounding     

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     

Involvement of the Accumulation of Glutamine in the Initiation of Adventitious Buds in Stem Segments of Torenia

Active meristematic divisions in stem segments of Torenia culturedin vitro can be induced in the epidermis by application of cytokininor the calcium ionophore A23187 [GenBank] , resulting in the differentiationof adventitious buds. Endogenous free glutamine accumulatedat a high concentration in the epidermal tissues during theearly stages of such cultures. The accumulation of glutaminewas caused by an increase in glutamine synthetase (GS) activity,and the increase of GS activity was suppressed by the applicationof some inhibitors of GS activity, mRNA synthesis, protein synthesis,or calmodulin. Incorporation of these inhibitors into the culturemedium also inhibited initiation of adventitious buds. The inhibitoryeffect of an inhibitor of GS, methionine sulfoximine (MSX),was apparent only at the very begining of the culture, and theeffect could be overcome by the simultaneous addition of glutamine.The inhibitory action of MSX on initiation of buds seemed tobe caused by an accumulation of ammonium ions. Reduction inlevels of NH₄NO₃ in or its elimination from the culture mediumstimulated the initiation of adventitious buds. Therefore, boththe accumulation of glutamine and the reduction in levels ofammonium ions seem to play a role in the initiation of adventitiousbuds in stem., segments of Torenia. ¹Present address: Faculty of Agriculture, University of Saga,Honjo-cho, Saga, Saga, 840 Japan. (Received October 3, 1988; Accepted March 9, 1989)     

Effects of IAA, Zeatin, Ammonium Nitrate and Sucrose on the Initiation and Development of Floral Buds in Torenia Stem Segments Cultured In Vitro

In Torenia stem segments cultured on a defined medium from whichammonium nitrate and growth regulators were omitted, adventitiousbuds were readily formed from epidermal tissue, with subsequentdifferentiation of floral buds. Using this plant material, thecorrelation between the time of application of various chemicalsand the time-course of floral bud differentiation was investigated.Histological examination showed that adventitious buds werevegetative during the first two weeks of the culture, and floralprimordia appeared after about three to four weeks of culture.We divided the flowering process in Torenia stem segments intothe following 3 phases: the first phase (first 2 weeks) duringwhich adventitious buds are formed, the second phase (3rd and4th weeks) during which floral buds are initiated and the thirdphase (5th to 12th weeks) during which floral buds develop.Then we added IAA, zeatin, ammonium nitrate or a high concentrationof sucrose to the medium during one, two or three of these phases.Ammonium nitrate added during the third phase suppressed floralbud development, but the high concentration of sucrose givenduring this phase stimulated it. These two chemicals influencedonly the development of floral buds previously initiated. Theapplication of IAA during the first phase promoted both theinitiation and development of floral buds. However, its applicationafter 2 weeks of culture failed to promote floral bud formation.Zeatin inhibited floral bud formation in a manner similar toammonium nitrate, but if it was added to the medium only duringthe first phase, it slightly promoted the initiation and developmentof floral buds. (Received July 7, 1981; Accepted October 12, 1981)     

Involvement of Cyclic AMP in Adventitious Bud Initiation of Torenia Stem Segments

Adventitous bud initiation in epidermis of Torenia stem segmentscultured in vitro, which is usually induced by cytokinin, couldbe induced by application of dibutyryl cyclic AMP in the absenceof cytokinin. Similar stimulatory effects on bud initiationwere observed when various promoting reagents to accumulateendogenous cyclic AMP were added, such as activators for adenylatecyclase, forskolin and prostaglandin E₁, and inhibitors of cyclicnucleotide phospho-diesterase, theophylline and isobutyl methylxanthine. Endogenous content of cyclic AMP in Torenia stem segmentswere increased by the application of the above chemicals, calciumionophore or cytokinin. These results suggested that endogenousconcentrations of cyclic AMP were involved in adventitious budinitiation of Torenia stem segments. Furthermore, some inhibitorsof protein kinases inhibited bud initiation induced by cyclicAMP-accumulating reagents. (Received August 15, 1989; Accepted November 4, 1989)     

Transport and Compartmentation of Abscisic Acid in Roots of Hordeum distichon under Osmotic Stress

Using modified compartmental analysis the unidirectional fluxesof abscisic acid (ABA) and their cytoplasmic and vacuolar contentsin ³H-ABA preloaded barley root segments (Hordeum distichoncv. Aura) have been studied. When root segments were stressedosmotically with sorbitol (osmotic potential of the media ⁰= 0.2 MPa) cytoplasmic and vacuolar contents of ABA were enhanced.Under increased stress cytoplasmic and vacuolar contents weremuch lower than in the unstressed controls. ABA fluxes werevery sensitive to osmotic stress and ABA transport from thecytoplasm of the xylem parenchyma to the xylem vessels (_cx)was rapidly inhibited. The cultivar Aura has higher cytoplasmicand vacuolar ABA contents than the barley cultivar Kocherperle.This correlates well with the higher stress tolerance of theAura cultivar. Key words: Abscisic acid, Compartmentation, Osmotic stress     

Functional aspects of Abscisic Acid Metabolism in Cotyledons of Phaseolus vulgaris L. Seedlings

During the early stages of germination and vegetative development,cotyledons of intact bean (Phaseolus vulgaris L.) seedlingsshowed active ABA catabolism causing a low endogenous ABA content.At the end of the substrate mobilizing phase, when the cotyledonsbecame senescent, a drastic increase of the endogenous ABA contentlinked with a decrease of the ABA catabolic activity was observed.This indicates that a close connection exists between senescenceand endogenous ABA content and metabolism in bean cotyledons. Removal of the apical growth region induced in the cotyledonsactivation of the ABA catabolism and the endogenous ABA concentrationdecreased below the detection limit (0.1 ng/g fr wt) at theonset of the outgrowth of the axillary buds. From then, apicaldominance was restored and the cotyledons returned to the senescentstate, which was correlated with a drastic increase of theirendogenous ABA content. ¹ Bevoegd verklaard navorser N. F. W. O. ² Wetenschappelijk medewerker F. K. F. O. (Received November 25, 1980; Accepted February 13, 1981)     

Salt Tolerance in the Halophyte Suaeda maritima L.Dum.: Abscisic Acid Concentrations in Response to Constant and Altered Salinity

Endogenous abscisic acid contents were measured by gas-liquidchromatography in shoots of Suaeda maritima growing both inthe steady state over a range of salinities and over a time-coursefollowing an increase in the culture solution salinity of betweenapproximately 100 and 400 mol m^–3 NaCl. In steady-stateplants, the ABA content was maximal in the absence of salt at41 ng g^–1 fr. wt., declining to a minimum at 200 mol m^–3NaCl of 24 ng g^–1 fr. wt. Increase of culture solutionsalinity resulted in a marked increase in shoot ABA which wasmaximal after 6 h or 24 h in plants previously growing at 200mol m^–3 NaCl and in the absence of salt, respectively.Additionally, culture solution water potentials were loweredby 1.0 MPa (equivalent to raising the salt concentration byaround 200 mol m^–3); this resulted in a similar increasein endogenous ABA content to that brought about by an iso-osmoticsalt increase. Results are discussed in relation to the possiblerole of ABA in halophyte salt tolerance mechanisms. Key words: Suaeda, halophyte, abscisic acid, salt tolerance     

Effects of ABA on Synthesis of Cell-Wall Polysaccharides in Segments of Etiolated Squash Hypocotyl I. Changes in Incorporation of Glucose and myo-Inositol into Cell-Wall Components

Externally supplied [³H]myo-inositol and [¹⁴C]glucose were incorporatedin cell-wall fractions of segments of etiolated squash hypocotyl.The extent of incorporation of [¹⁴C]glucose into cell-wall fractionswas very much greater than that of [³H]myo-inositol. Radioactivityfrom [¹⁴C]-glucose was effectively incorporated into hemicelluloseB and cellulose fractions and was incorporated uniformly intohexose, pentose and uronic acid residues, but radioactivityfrom [³H]myo-inositol was incorporated predominantly into uronicacid and pentose residues in the pectin and hemicellulose Bfractions. Exogenously applied ABA significantly suppressed the elongationof segments of squash hypocotyl and the incorporation of radioactivityfrom [^l4C]glucose and [³H]myo-inositol into the segments. Furthermore,ABA significantly inhibited the distribution of incorporatedradioactivity from [¹⁴C]glucose into the cellulose fraction,but did not affect distribution into the pectic fraction. Bycontrast, ABA only slightly inhibited the distribution of theincorporated radioactivity from [³H]myo-inositol into the pecticfraction. These results suggest that most of the cell-wall polysaccharidesin segments of squash hypocotyl are synthesized via the UDP-sugarpathway, and that ABA significantly inhibits the synthesis ofcellulose but not the synthesis of pectic polysaccharides whenABA suppresses the elongation of the segments. (Received March 25, 1988; Accepted November 15, 1988)     

Callus Culture of Sainfoin (Onobrychis viciifolia) and Plant Regeneration through Somatic Embryogenesis

Callus of sainfoin (Onobrychis viciifolia Scop.) was initiatedfrom stem and root explants which were obtained from seedlingsgrowing in vitro, on Linsmaier Skoog (LS) medium supplementedwith 1 mg l^–1 2, 4-D and 1 mg l^–1 BA or only 1 mgl^–1 BA, and the Vacin and Went medium without hormones.Somatic embryos were formed on LS medium containing 1 m l^–1BA. Embryos developed into complete plants on filter paper saturatedwith hormone-free LS medium. Onobrychis viciifolia, somatic embryogenesis, callus culture, plant regeneration     

Effects of Abscisic Acid on the Synthesis of Cell-Wall Polysaccharides in Segments of Etiolated Squash Hypocotyl II. Levels of UDP-Neutral Sugars

The effects of abscisic acid (ABA) on the formation of UDP-sugarsin segments of squash (Cucurbita maxima Duch.) hypocotyls wereexamined with [³H]glucose. After a 3-h incubation, 74% of totalradioactivity incorporated into neutral sugar residues of UDP-sugarswas found in glucose residues, 19% in galactose residues andless than 7% in other sugar residues. Exogenously applied ABAincreased the extent of incorporation of [³H]glucose into UDP-glucose,UDP-galactose and UDP-xylose by 30%, 30%, and 20%, respectively.However, ABA neither affected the total incorporation of radioactivityinto segments nor the incorporation into free glucose and sucrosein the segments. Since ABA inhibits the synthesis of celluloseand hemicellulosic polysaccharides in cell walls of segmentsof squash hypocotyls prior to the suppression of elongationof segments (Wakabayashi et al. 1989a, b), the present resultsindicate that ABA inhibits cell-wall synthesis by affectingsome step(s) beyond the formation of UDP-sugars and not by decreasingthe synthesis of UDP-sugars in the hypocotyl segments. ¹Present address: Biological Laboratory, Faculty of Education,Kagawa University, Saiwai-cho 1-1, Takamatsu, 760 Japan (Received May 30, 1990; Accepted February 5, 1991)     

Bl?tenbildung bei Lemna paucicostata 6746 durch kombinierte Anwendung von Abscisins?ure und GCG

To a certain extent the flowering of Lemna paucicostata 6746is evoked in continuous light by application of abscisic acid(ABA) and CCC. Moreover, the action of the combined substancesappears in two separate concentration ranges. In the range ofABA 2?10^–9 M/CCC 10^–7–10^–6 M floweringis initiated without inhibition of vegetative growth and proceedsonly in the presence of high intensity light and sucrose. Acombination of ABA 2?10^–5 M/CCC 10^–3M simultaneouslycauses a strong inhibition of frond multiplication. Here theeffect can be observed also under low intensity light conditionsand without sucrose in the medium. A range with flower inhibitingactivity lies between the two flower promoting concentrationranges. (Received November 16, 1972; )     

Transport and Accumulation of IAA-14C in Tumour-forming Nicotiana Hybrids

The polar transport of indol-3yl-acetic acid (IAA-2-¹⁴C) instem explants and decapitated shoots of tumour-prone Nicotianahybrids (2n, 3n, and 4n) was compared with that in the normal,non-tumorous parent species N. glauca and N. langsdorffii. Thetotal uptake of the auxin from donor blocks was greatest inthe hybrids and N. glauca. The velocity of the basipetal movementof IAA-¹⁴C was the same in all species tested, i.e. 8 mm/h.The transport capacity for the hormone, however, was decreasedin the three tumour-prone hybrids. Gas chromatography showedthat between 70 and 90 per cent of the transported auxin waspresent in the form of IAA, between 10 and 30 per cent in theform of indol-3yl-aldehyde (IAld). The basipetal transport exceeded the acropetal transport inyoung (third) intemodes of all plants studied, whereas in olderstem segments (tenth intenodes) the reverse was found. The polarity of auxin transport was less well expressed in thetumorous hybrids. Blocking the active transport by pre-treatment of stem cuttingswith 2,4-dinitrophenol (2,4-DNP) caused a drastic reductionin the polar IAA-¹⁴C movement; in all plants tested the auxintransport was reduced to the same low level. The accumulation of auxin at the base of cuttings was higherin N. glauca and the 2n hybrid than in N. langsdorffii, i.e.about seven times higher after 1-h and three times higher after12-h transport experiments. The release of ¹⁴C from the cuttinginto an agar receiver block, however, was markedly reduced inthe 2n hybrid, whereas in N. glauca the labelled substancesmoved more freely into the receiver blocks. Differences in the capacity for the accumulation and the releaseof IAA-¹⁴C in hybrid and N. glauca stem tissues were studiedusing decapitated greenhouse plants wounded by incision abovethe fourth internode. Accumulation of the auxin occurred onlyabove the wound-cut in hybrid plants. This observation is consistentwith the view that tumour formation on hybrid stems occurs atsites of wounding. Our data suggest an elevated auxin levelto be present during tumour initiation at these sites. These results on polar transport and accumulation of IAA-¹⁴Cin tumorous Nicotiana plants together with our previous dataon various endogenous auxins suggest that the induction of neoplasticgrowth in tobacco plants is correlated with increased auxinlevels and an accumulation of the hormone at sites of wounding.