Effect of cycloheximide and cordycepin on auxin-induced elongation and {beta}-glucan degradation of noncellulosic polysaccharides of Avena coleoptile cell wall

The effect of cycloheximide (10^–5 M) and cordycepin (10^–4M) used as protein and RNA synthesis inhibitors, respectively,on auxin action in noncellulosic ß-glucan degradationof Avena coleoptile cell wall was investigated. Both depressedauxin-induced ßglucan degradation of the cell wallas well as auxin-induced elongation and cell wall loosening,suggesting that the process of ß-glucan degradationof the cell wall is closely associated with cell wall looseningand that auxin enhances the activity of an enzyme for ß-glucandegradation through de novo synthesis of RNA and protein butnot through activation of the enzyme in situ. Kinetic studywith the inhibitors showed that RNA metabolism involved in ß-glucandegradation was stimulated by auxin treatment of only 15 minwhile a longer lag phase (about 1 hr) existed for the synthesisof the enzyme. (Received December 16, 1978; )     

Relation of special RNA to sensitivity of yeast strains to auxin

RNA functional in auxin action was studied in the followingfour strains of yeasts differing in their sensitivity to auxin:KV2, diploid strain of Saccharomyces ellipsoideus irresponsiveto auxin but made responsive by gibberellic acid treatment;N55, auxinresponsive mutant derived from KV2; A2-0, diploidstrain of 5. cerevisiae irresponsive even when gibberellic acidis given; and A2–N102, responsive mutant derived fromA2–0. The RNA fraction extracted from N55 and partitionedin a phenol layer sensitized KV2 to auxin. This kind of RNAwas not detected in KV2 cells. The functional RNA from N55 wasof low molecular weight, as was functional RNA isolated fromgibberellic acid-treated Jerusalem artichoke tuber. Gibberellicacid-treated KV2 cells have been known to contain RNA whichfunctions similarly to the RNA mentioned above. Both A2–0and A2–N102 contained RNA which made KV2 cells responsiveto auxin. Some factors other than functional RNA may determinethe difference in the sensitivity to auxin between A2–0and A2–N102. (Received August 20, 1968; )     

Auxin and Ethylene Control of Growth in Seedlings of Zea mays L. and Avena sativa L

The effects of applied ethylene on the growth of coleoptilesand mesocotyls of etiolated monocot seedlings (oat and maize)have been compared with those on the epicotyl of a dicot seedling(the etiolated pea). Significant inhibition of elongation by ethylene (10 µll^–1for 24 h) was found in intact seedlings of all three species,but lateral expansion growth was observed only in the pea internodeand oat mesocotyl tissue. The sensitivity of the growth of seedlingparts to ethylene is in the decreasing order pea internode,oat coleoptile and oat mesocotyl, with maize exhibiting theleast growth response. Although excised segments of mesocotyland coleoptile or pea internode all exhibit enhanced elongationgrowth in IAA solutions (10^–6–2 ? 10^–5 moll^–1), no consistent effects were found in ethylene. Ethyleneproduction in segments was significantly enhanced by applicationof auxin (IAA, 10^–5 mol l^–6 or less) in all tissuesexcept those of the eat mesocotyl. Segments of maize show a slow rate of metabolism of applied[2-¹⁴C]IAA (30 per cent converted to other metabolites within9 h) and a high capacity for polar auxin transport. Ethylene(10 µl l^–1 for 24 h) has little effect on eitherof these processes. The oat has a smaller capacity for polartransport than maize and the rate ef metabolism of auxin isas fast as in the pea (90 per cent metabolized in 6 h). Althoughethylene pretreatment does not change the rate of auxin metabolismin oat, there is a marked reduction in auxin transport. It is proposed that the insensitivity of maize seedlings toethylene is related to the supply and persistence of auxin whichcould protect the seedling against the effects of applied orendogenously produced ethylene. Although the mesocotyl of oatis sensitive to applied ethylene it may be in part protectedagainst ethylene in vivo by the absence of an auxin-enhancedethylene production system. The results are discussed in relationto a model for the auxin and ethylene control of cell growthin the pea.     

Analysis of the Promoter of the Auxin-Inducible Gene, parC, of Tobacco

The auxin-responsive region (AuxRR) in the promoter of the parCgene was analyzed in transgenic tobacco plants in which the5' flanking region of the parC promoter was placed upstreamof the gene for rß-glucuronidase (GUS). The AuxRRwas located between nucleotides (nt) –226 and –54.Detailed dissection of this segment revealed that the presenceof the non-contiguous sequences from nt –226 to –151and from nt –84 to –54 was required for the expressionof the auxin responsiveness of the parC promoter. The sequencefrom nt –226 to –151 was found to contain a sequencewhich resembles the as-1 element in the 35S promoter of cauliflowermosaic virus (CaMV). Although it has been reported that theas-1 element is involved in auxin responsiveness [Liu and Lam(1994) J. Biol. Chem. 269: 668], we showed that introductionof a point mutation into the as-1-like sequence completely eliminatedauxin responsiveness, a result that suggests that the sequenceis indispensable for auxin responsiveness. However, the presenceof the as-1-like sequence alone was not sufficient for auxinresponsiveness, since the segment (nt –226 to –84)that included the as-1-like sequence failed to confer auxinresponsiveness on the core promoter. It is possible that thetwo separately located sequences play specific roles in interactionswith trans-factors that are required for the expression of theauxin responsiveness of the parC promoter. (Received March 11, 1996; Accepted July 9, 1996)     

Studies on the Geotropism of Roots: III. EFFECTS OF GEOTROPIC STIMULATION ON GROWTH-SUBSTANCE CONCENTRATIONS IN VICIA FABA ROOT TIPS

The auxins contained in 5-mm. tips of horizontal Vicia fabaroots have been compared with those in tips of vertical rootsafter cold ethanol extraction, paper-chromatographic separation,and Avena mesocotyl bioassay. At about the time curvature commencesin horizontal roots there is a marked increase in the contentof an auxin corresponding to ‘AP(ii)’ of pea roots(Rf 0.35–0.65 in isobutanol/methanol/water). There areindications that this is not due to its release from an inactivebound state but that it is either synthesized de novo or maybe converted from another auxin corresponding to ‘AP(iii)’of pea roots (Rf 0.75–1.0). The literature dealing with the auxins of geotropically stimulatedorgans is reassessed and it is concluded that, with the exceptionof the Avena coleoptile, there is very little evidence favouringa simple transport redistribution of auxin under gravity; themajority of the data favour an effect of gravity on auxin metabolism.     

The Elongation Response of Watermelon Hypocotyls to Indole-3-Acetic Acid: A Comparative Study of Excised Segments and Intact Plants

Carrington, C. M. S. and Esnard, J. 1988. The elongation responseof watermelon hypocotyls to indole-3-acetic acid: a comparativestudy of excised segments and intact plants.—J. exp. Bot39: 441–450. The auxin-growth response along the hypocotyl of Citrullus lanatus(Thumb.) Mansf. seedlings was studied. In excised segments,promotion of elongation was seen in all zones at the concentrationsof IAA used (10–4–10–2 mol m-3). In intactplants, only the most basal zone showed unequivocal IAA-extensionwhile in the most apical zone elongation was inhibited by auxin.This difference between segments and intact plants for apicalzones suggests a modifying effect of the apex and cotyledonson the growth response. Indeed, removal of the apex and colyledonsonly affected elongation in the zones adjacent to the excisionbut only in buffer-treated plants, not auxin-treated plants.Auxin supplied apically to the intact plant only resulted ina short-lived promotion of elongation whereas basally suppliedauxin gave a longer-lasting effect Zonal differences betweenauxin-promoted growth of excised segments suggests that sensitivityto auxin varies in the hypocotyl. The response of intact plantsto auxin was shown to be more complex than in segments. Thus,responses given by segments are poor indicators of auxin activityin intact plants. Key words: IAA, Citrullus lanatus, growth, plant hormone sensitivity     

Hormonal Control of Xylogenesis in Pith Parenchyma Explants of Lactuca

The time course of xylem differentiation was determined in explantsof lettuce pith parenchyma (Lactuca sativa L. cv. Romana) culturedon Murashige and Skoog (1962) medium using different concentrationsof auxin (IAA) and one cytokinin (zeatin or kinetin). Increasinglevels of auxin from I mg 1^–1 to 15 mg 1^–1 in thepresence of a constant level of a cytokinin (zeatin or kinetin)yielded up to 10 mg 1^–1 IAA, an increase in the numberof tracheary element formations. Cytokinin concentrations aboveand below o.1 mg 1^–1 interacting with an optimal xylogenicamount of auxin inhibited xylogenesis. The IAA (10 mg 1^–1)-zeatin(0.1 mg ^1–1) treatment produced the greatest number oftracheids, while kinetin compared to zeatin did not producesuch an effect. The different effectiveness of zeatin and kinetinin inducing tracheary element formations was not due to a differentcapacity of the two cytokinins to stimulate cell division butit seems likely that zeatin, because of interaction with IAA,is more active than kinetin in the determination of the dividingcells in a specific type of cytodifferentiation. The IAA (10mg 1^–1)-zeatin (0.1 mg 1^–1) treatment produced about6.9 per cent tracheids with respect to cell division while IAA(10 mg 1^–1)-kinetin (0.1 mg 1^–1) produced 4.2 percent. These results are discussed with reference to the problemsof hormonal control of xylem differentiation.     

Effect of {alpha}-Tomatine on the Response of Wheat Coleoptile Segments to Exogenous Indole-3-acetic Acid

A concentration of 10^–5 M tomatine had no effect on leakagefrom, or elongation of, wheat coleoptile segments, but consistentlyreduced IAA-enhanced extension growth by c. 50 per cent. Therewas no evidence of chemical interaction between the alkaloidand the auxin in solution, and IAA action was not affected bypre-treatment for up to 3 h with 10^–5 M tomatine. Studieswith [2-¹⁴C]IAA revealed that 10^–5 M tomatine did notinhibit uptake of auxin into segments. The effect of pre-treatingsegments for up to 3 h with IAA could be virtually nullifiedby 10^–5 M tomatine, as could also IAA-induced changesin properties of coleoptile cell walls. Results are discussedin relation to the ability of tomatine to disrupt membrane functionand to current hypotheses implicating membranes in the primaryaction of auxin.     

Mineral nutrition and auxin-induced growth of Triticum coleoptile sections

A revised method has been described for assaying auxin by thegrowth of Triticum coleoptile sections. With additions of Ca(NO₃)₂10^–4 and MgSO₄ 10^–5 mol liter^–1 the sensitivityand accuracy have been increased. This is mainly due to Ca.The coleoptiles obviously suffer from Ca-deficiency. The importanceof a strict time schedule for manipulations is emphasiced. Theduration of the tests is limited to 6 hr.Indole-3-acetic acidcan be determined in concentrations down to 10^–9mol liter^–1;from 10^–10 to 10^–9mol liter^–1 the log-logrelation between concentration and growth is linear. Above 10^–7mol liter^–1 elongation takes place under an abnormal increasein elastic extension, indicating that growth is limited by someunknown wallstabilizing factor. The interrelation between auxin,an anti-auxin and Ca are discussed. (Received May 8, 1973; )     

Changes in Auxins in Expanding and Senescent Primary Leaves of Dwarf French Bean (Phaseolus vulgaris)

A bound, unextractable auxin, or auxin precursor, in the primaryleaves of dwarf French bean was converted to a form, probablyindol-3yl-acetic acid, extractable in ethyl acetate, by maceratingleaves in pH 7.5 phosphate buffer and incubating them at 37–38°C for 2 days in the presence of toluene or hibitane. Boilingthe leaves before macerating and incubating them prevented therelease of bound auxin. This bound auxin was also released whenmacerated leaves were hydrolysed with normal potassium hydroxideat 100° C for 2 hours. The amounts of free auxin and tryptophanin senescent primary leaves increased as their bound auxin andchlorophyll decreased. Removing the apical growing region immediatelyabove the node bearing the primary leaves delayed their senescenceand the conversion of their bound to free auxin.     

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     

Relationship between hormone content and autonomy in various autonomous tobacco cells cultured in suspension

Various autonomous cultured tobacco cells including crown gallwere examined for their contents of growth regulators by meansof Avena curvature test, cell-division induction test, and tobaccopith callus test. The crown gall cells derived from cv. Hicks produced auxin andcytokinin in the high levels of 300–500 µg IAA equivalentsand 40–80 µg kinetin equivalents per kg, respectively.The major auxin was identified as indole-3-acetic acid basedon mass spectrometry and gas chromatography. These cells alsoproduced methyl indole-3-acetate as a minor component. One ofthe cytokinins was identified as ribosyl-trans-zeatin by meansof both gas chromatography-mass spectrometry and high performanceliquid chromatography. Auxin and cytokinin activities were not detected in the followingthree suspension cultured tobacco cells: cells requiring neithercytokinin nor auxin derived from the callus of N. tabacum cv.Bright Yellow and cells requiring auxin but not cytokinin derivedfrom the calluses of cv. Bright Yellow and cv. Hicks. Theirauxin and cytokinin contents per kg were less than 1 µgIAA equivalent and less than 0.1 µg kinetin equivalent,respectively. The results obtained in this study indicate that enhanced hormonalcontent is not the only reason for autonomous growth. (Received August 16, 1979; )     

A Possible Double Role for Brassinolide in the Reorientation of Cortical Microtubules in the Epidermal Cells of Azuki Bean Epicotyls

Brassinolide, at 10^–8M or higher, enhanced the elongationof epicotyl segments from azuki bean seedlings that was inducedby IAA, but it did not enhance the increase in fresh weightof the segments, an indication that brassinolide suppressedthe lateral expansion of the segments. The additional elongationcaused by brassinolide was completely prevented in the presenceof 10^–5 M cremart, which disrupted the cortical microtubules(MTs) in epidermal cells in the segments, and in the presenceof 10^–6M 2,6-dichlorobenzonitrile, an inhibitor of thesynthesis of cellulose. Brassinolide at 10^–7M, appliedtogether with IAA, increased the percentage of epidermal cellswith transversely oriented cortical MTs. Brassinolide appearsto enhance the longitudinal expansion and suppress the lateralexpansion of epicotyl cells by organizing cortical MTs transverselyto the cell axis and, thereby, causing the deposition of cellulosemicrofibrils in the same orientation. Brassinolide by itself, at 10^–8M or higher, induced theelongation of epicotyl segments and the elongation caused bybrassinolide was partially prevented by 10^–5M cremart,results that suggest that brassinolide regulates cell expansionvia at least two processes, an MT-dependent process and an MT-independentprocess. Brassinolide by itself increased the percentage ofepidermal cells with transversely oriented cortical MTs. Since,in azuki bean epicotyls, the percentage of cells with transverseMTs is increased only by the combination of auxin and gibberellinbut not by either alone, brassinolide applied alone seems toplay a double role, similar to that of auxin and of gibberellin,in organizing cortical MTs. (Received September 2, 1994; Accepted November 16, 1994)     

The Effect of Triacontanol on Peroxidase, IAA, and Plant Growth in Pisum sativum var.'Alaska' and 'Little Marvel'

The present study investigates the effects of triacontanol (CH₃(CH₂)₂₈CH₂OH),on plant growth (root and stem), peroxidase activity (apicalmeristem tissue), and auxin destruction (apical meristem tissue)in ‘Little Marvel’ dwarf (LM) and ‘Alaska’peas (AP). Triacontanol inhibited root growth in LM comparedto untreated controls. However, root growth in AP tissue wasenhanced by 1.0 mg I^–1 triacontanol and inhibited by allother treatments, in comparison to untreated controls. Wateruptake in triacontanol-treated AP plants was greater than inuntreated controls, with the converse being the case for LM.Triacontanol treatment caused an increase in peroxidase activityin both LM and AP plants compared to untreated controls. Interms of (1–¹⁴C)IAA destruction, GA₃ + 0.01 mg 1^–1triacontanol caused appreciable auxin breakdown (40%) in LMtissue, with GA₃ + 0.1 mg 1^–1 triacontanol giving a 43%decrease compared to untreated controls. In AP tissue, 10 µMGA3 increased auxin destruction by 188% whereas 0.1 mg I^–1triacontanol caused a 20% decrease compared to untreated controls.The effects of triacontanol on root and stem growth, peroxidaseactivity, and auxin destruction appear to be cultivar-specific,with respect to LM and AP varieties of peas.     

Effects of Sequential Exposure to Auxin and Cytokinin on Xylogenesis in Cultured Explants of Jerusalem Artichoke (Helianthus tuberosus L.)

During the course of a 4-d culture period, explants of Jerusalemartichoke tuber were exposed to auxin (0.2 mg 1^–1 2, 4-dichlorophenoxyaceticacid), and cytokinin (5.0 mg 1^–1 benzyl-amino purine),under a range of sequential regimes, to study the influenceof each hormone on tracheary element formation. The resultsindicate that auxin was necessary early in the culture periodand was primarily involved in cell proliferation. Cytokininstimulated xylogenesis when present late in the culture period,concomitant with the phase of cytodifferentiation, but not whenrestricted to the early period. The implications for a sustainedperiod of commitment to differentiation are discussed. Xylem differentiation, Jerusalem artichoke, auxin, cytokinin, tissue culture     

Regulation of Tracheary Element Differentiation by Exogenous L-Methionine in Callus of Soya Bean Cultivars

The relationship between the induction of tracheary elementdifferentiation and exogenous L-methionine was examined in agar-growncultures of soya bean callus initiated from Glycine max L. ‘Wayne’and ‘Clark 63’. Although Wayne is a normal cultivarsoya bean, seedlings of Clark 63 exhibit abnormal growth at25 °C due to exessive ethylene biosynthesis at this temperature.Wayne callus showed increased xylogenesis in the presence ofexogenous L-methionine (3.7 µg 1^–1) in comparisonto IAA–KN controls at both 20 and 25 °C. Clark 63callus produced greater numbers of tracheary elements in responseto exogenous L-methionine only at 25 °C. The induction ofxylem differentiation was independent of the maintenance temperatureof the stock cultures of both cultivars. Xylogenesis initiatedbyan IAA–KN medium was inhibited by the addition of AgNO₃(20 mg 1^–1) to the extent of 76.5 per cent in cv. Wayneand 6 per cent in cv. Clark 63. The inhibitory effect was partiallyreversed by the addition of L-methionine (3.7 µg 1^–1)to the IAA–KN–AgNO₂ medium. These data support thehypothesis that xylogenesis in vitro involves auxin, cytokininand ethylene. differentiation, xylogenesis, L-methionine, ethylene, Glycine max L., soya bean, callus culture, auxin, kinetin     

Regulation of lycopene formation in cell suspension culture of VFNT tomato (Lycopersicon esculentum) by CPTA, growth regulators, sucrose, and temperature

The onium compound 2–(4-chlorophenylthio)-triethylamine(CPTA) was used to increase lycopene formation to levels approximatingthose in field–or glasshousegrown fruit, and then growthregulators, sucrose and temperature were used to regulate lycopeneaccumulation. It was found that the native auxin indole–3–acetic acid (IAA) was substantially more effective than 2,4-dichlorophenoxyaceticacid (2,4–D) in promoting lycopene formation, sucroseinhibited lycopene formation (cell basis), and temperature produceda pattern similar to that observed in the field with a temperatureoptimum between 18 and 26 C. Suggestions for further improvementsin technique are included. Key words: Suspension culture, tomato, lycopene     

Growth Substances in the Roots of Vicia faba

An investigation has been made into the growth regulators presentin ethanol extracts of the seedling roots of Vicia faba afterseparation on paper partition chromatograms, using segmentsof Avena coleoptiles and mesocotyls and of Pisum sativum.rootsas biological assay material. Acetonitrile purification shows the presence of at least threeauxins running in isobutanol: methanol: water, at Rfs of 0–0·25,0·4–0·6, and 0·65–0·95;the latter may represent two different auxins. A similar, butclearer, picture is shown by the ether-soluble acid fraction.Here an auxin at Rf 0–0·25 also stimulates rootgrowth and could be ‘accelerator ’. A second atRf 0–0·25 is an indole compound which inhibitsroot growth and does not seem to be be IAA. A third at Rf 0·8–1·0is also a root-growth inhibitor and gives no indole reaction.The ‘inhibitor ß’ complex was demonstrated(Rf 0·65–0·85) together with a number ofother inhibitors at lower Rf value. The ether-soluble neutral component also contains auxins orauxin precursors. The water-soluble, ether-insoluble fraction contains four readilyinterconvertible substances with auxin properties. They allappear to inhibit root growth and give no indole reaction.     

The Effect of Morphactin (Methyl 2-Chloro-9-hydroxyfluorene-9-carboxylate) on Basipetal Transport of Indol-3-ylacetic Acid in Hypocotyl Sections of Phaseolus vulgaris L.

The effect of morphactin (methyl 2-chloro-9-hydroxyfluorene-9-carboxylate)on basipetal transport of auxin (Indol-3-ylacetic acid-2-¹⁴C)was studied in bean (Phaseolus vulgaris) hypocotyl with thedonor-receiver block method. Morphactin (5 x 10^–6m) reduced IAA (5 x 10^–6m) transportintensity by an average of 83 per cent and auxin transport capacityby 90 per cent, but transport velocity was not affected. Morphactin did not inhibit uptake of IAA into hypocotyl tissue,but it did prevent transfer of IAA from the tissue into receiverblocks. Chromatographic analysis of the tissue after 4 h IAA-2-¹⁴Ctransport showed that 54 per cent of the total activity wasin the form of IAA in the control and 42 per cent in the morphactintreated tissue. No difference was found in the rate of decarboxylationof IAA-1-¹⁴C between control and morphactin treated tissue sections.Nor could any difference between control and morphactin be shownin the radioactivity associated with a TCA ppt fraction. Ina study of the transportable auxin pool, morphactin decreasedthe size of the pool and increased the half-life of decay ofauxin transport from 1•22 h to 3•85 h. In a kineticanalysis of the reversal of morphactin (5 x 10^–6m) inhibitionby increasing concentration of IAA-2-14C (5 x 10^–6m to2 x 10^–5m), it was shown that IAA transport resemblesMichaelis-Menten enzyme reaction kinetics, and that inhibitionby morphactin fitted a ‘mixed type’ model. IAA hada dissociation constant of 8•5 x 10^–6m and morphactinthat of 4•3 x 10^–7m with a K_m for the transport processof 8•5 x 10^–6m.     

Timing of the phases in adventitous root formation in apple microcuttings

As with other regeneration processes, adventitious root formationmay be divided into three phases, namely dedifferentiation,induction and differentiation. We assumed that the appropriatehormonal conditions for rooting, in particular a high levelof auxin and a low level of cytokinin, are required only duringthe induction phase. Hence, the effect of 24 h pulses with indolebutyricacid (IBA) or benzylaminopurine (BAP) should be maximal duringthis phase. On this assumption, the timing of the three phaseswas determined in microcuttings of Malus ‘Jork 9’.The promotion of rooting was largest for 24 h IBA-pulses given24–48 h or 48–72 h after the onset of culture. Duringculture of shoots on IBA-containing medium 24 h BAP-pulses weregiven. Inhibition of rooting was maximal for the BAP-pulsesgiven between 24 and 96 h. An analysis of the kinetics of rootemergence also indicated that added IBA acted from 24 h onwards:emergence of roots from the tissue occurred simultaneously forthe IBA pulses at 0–24 h and 24–48 h, but lagged1 d behind for the 48–72 h pulse and 2 d for the 72–96h pulse. We concluded that dedifferentiation occurred from 0to 24 h, induction from 24 to 72 or 96 h, and after that differentiation.Histological observations showed that after 24 h, cells withswollen nuclei and dense cytoplasm had appeared in the regionsof the stem where the roots were formed. The first cell divisionswere observed after 48 h. After 96 h, meristemoids of c. 30cells had been formed. After the BAP-pulses at 24–48 hor 48–72 h, these meristematic cells formed callus. Key words: Adventitious rooting, apple, auxin, cytokinin, Malus, regeneration.