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.     

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.     

Auxin Transport in Secondary Tissues1

Auxin transport was investigated in excised stem segments ofNicotiana tabacum L. by the agar block technique using [1-¹⁴C]indol-3yl-acetic acid (IAA). The ability of the stems to transportauxin basipetally increased as secondary development proceeded;by contrast the ability of the pith to transport auxin declinedwith age. By separation of the stem tissues it was shown thatthe great majority of auxin transport took place in cells associatedwith the internal phloem and in cells close to the cambium;in both cases similar velocities of transport were found (c.5.0 mm h^–1 at 22°C). The effects of osmotic gradientson auxin transport through the internal phloem were investigated.IAA was found by chromatography to account for practically allthe radioactivity in receiver blocks and other extracts of stemsegments. The significance of these results is discussed.     

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.     

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.     

Effect of Fusicoccin on Adventitious Root Formation of Birch Shoot Tips (Betula pendula ROTH) Cultured in vitro

The effect of fusicoccin (FC) on adventitious root formationwas investigated using in vitro shoot tip cultures of birch(Betula pendula ROTH) as test system. Treatment with 10^–7–10^–5M FC hastened root appearance as well as 5 ? 10^–6 M IAAdid. Optimal FC concentrations also promoted rooting by increasingthe root number per cutting. FC application during the first48 hours of culture was enough to obtain these effects. Usinginternode segments without any bud it was shown that FC couldnot replace the root inducing activity of endogenous auxin asapplied IAA did, but FC lowered the threshold concentrationof IAA for rooting response and stimulated adventitious rootformation if it was applied with IAA simultanously. Root growthwas enhanced in the early phase but inhibited later by continuoustreatment with FC. Some aspects of possible FC IAA interactionsare discussed. (Received September 4, 1986; Accepted November 24, 1987)     

Effects of Auxin and Phenobarbital on Morphogenesis and Production of Digitoxin in Digitalis Callus

Pieces of callus obtained from seedlings of Digitalis purpureawere grown on solid Murashige-Skoog's medium supplemented with1 mg liter^–1 BA and 0.1 mg liter^–1 IAA or NAA, withor without phenobarbital (40 mg liter^–1). The replacementof the natural auxin IAA by the synthetic auxin NAA increasedcallus growth and inhibited organogenesis, whereas the additionof phenobarbital had the opposite effect. Morphometric measurementsrevealed a high ratio of vacuole to cytoplasm (v/v) in calluscells. This ratio was affected by the different treatments inthe same way as the fresh weight. The activity of mitochondrialcytochrome P450scc (the enzyme that provides the precursor,pregnenolone, for the biosynthesis of cardenolide in foxgloveplants) was detected in the relevant fraction of callus grownunder all experimental conditions, and its activity was increasedby the addition of phenobarbital. The different treatments testedincreased the cardenolide content and quantifiable amounts ofdigitoxin were detected in all callus tissues. It is of specialinterest that phenobarbital added to the culture medium increasedthe accumulation of digitoxin. The mechanism affecting the developmentand production of cardenolide in callus tissues of D. purpureaby phenobarbital and the replacement of IAA by NAA is discussed. (Received July 18, 1994; Accepted December 14, 1994)     

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens III. Formation of IAA Conjugates in Cultured Carrot Tissues Transformed with Wild-type and Mutant Ti Plasmids

Inactivation of IAA was examined in cultured carrot tissuestransformed with Agrobacterium tumefaciens that harbored wild-type,aux^– or cyt^– Ti plasmids. IAA oxidase activities were similar among the three types oftransformed tissue. Metabolites of IAA were examined by followingthe fate of 3-indolyl-[l-^l4C]IAA. IAA conjugates were detectedin all transformed tissues as well as in hypocotyl segmentsof non-transformed carrot seedlings. The rate of formation ofIAA conjugates was ten times higher in the tissues transformedwith wild-type or cyt^– Ti plasmids than in the tissuestransformed with aux^– Ti plasmids. When the tissues transformedwith aux^– Ti plasmids were cultured on medium that containedIAA for 6 h, the rate of formation of IAA conjugates in thesetissues became as high as that in tissues transformed with wild-typeor cyt^– Ti plasmids. The tissues transformed with wild-type or cyt^– Ti plasmidsnot only synthesize larger amounts of IAA but also convert alarger amount of free IAA to conjugated IAA than do non-transformedand aux^– transformed tissues. Presumably, in carrot, theformation of IAA conjugates decreases the amount of free IAAin the transformed tissues that synthesize large amounts ofIAA and, consequently, the level of free IAA can be maintainedfairly constant. (Received June 2, 1989; Accepted May 23, 1990)     

Studies on the Growth and Respiration of Roots: I. THE EFFECTS OF STIMULATORY AND INHIBITORY CONCENTRATIONS OF {beta}-INDOLYLACETIC ACID ON ROOT SECTIONS OF PISUM SATIVUM

Simultaneous observations on extension growth and respirationrate (oxygen consumption) of 2-mm. sections excised from theextension zone of roots of pea (Pisum sativum) growing in distilledwater and 0·5 per cent. sucrose have yielded resultsclosely similar to those of Brown and Sutcliffe (1950). Respirationrate is not obviously correlated with growth rate either inwater or in sucrose, but it is strongly correlated with sectionlength. Respiration rate per unit section length (¬per unitfresh weight) shows a marked downward drift during extensionand is affected little by growth conditions. Tentative suggestionsare advanced to account for the small differences between driftsin o·5 per cent. sucrose and those in distilled water. Medium agitation produces an immediate and sustained stimulationof growth but no stimulation of oxygen uptake until the latergrowth stages. Thus respiration per unit section length is unaffectedby agitation at any stage. A typical growth response to ß-indolylacetic acid(IAA) was obtained, with a maximum stimulation (of about 35per cent.) at 1 part in 10¹¹ and inhibitions increasing progressivelywith concentration beyond the threshold of about i part in 10⁹.Both percentage stimulation and percentage inhibition of growthwere independent of the presence of sucrose. Respiratory responses to ß-indolylacetic acid werecomplex. In water no immediate response could be detected witheither a growth-stimulatory (10^–11) or a growth-inhibitory(10^–-8) concentration, while in 0·5 per cent. sucrosethe inhibitory concentration prevented the small immediate respiratoryrise due to the sucrose, probably by impeding sugar entry. Duringthe subsequent period of rapid growth (up to 36 hours) the smallrespiratory responses observed closely followed the small growthresponses to both concentrations of IAA, suggesting that theformer are the direct result of the differences in section lengthinduced by the auxin. When growth ceases (at 48 hours) sectionswhich have grown considerably in sucrose show respiratory ratesstill closely correlated with section length, whereas in waterboth concentrations of auxin induce marked depressions in respirationrate. It is concluded that ß-indolylacetic acid in bothgrowth-stimulatory and growth- inhibitory concentrations hasno direct effect on the activity of the respiratory enzyme systemof growing root cells. The small respiratory responses are bestexplained as resulting from differential changes in sectionsize and correlated changes in the enzyme complements of thegrowing cell.     

Cooperation of Ethylene and Auxin in the Growth Regulation of Rice Coleoptile Segments

Elongation of coleoptile segments, having or not having a tip,excised from rice (Oryza sativa L. cv. Sasanishiki) seedlingswas promoted by exogenous ethylene above 0.3 µl l^–1as well as by IAA above 0.1 µM. Ethylene production ofdecapitated segments was stimulated by IAA above 1.0µM,and this was strongly inhibited by 1.0 µM AVG. AVG inhibitedthe IAA-stimulated elongation of the decapitated segment witha 4 h lag period, and this was completely recovered by ethyleneapplied at the concentration of 0.03 µl l^–1, whichhad no effect on elongation without exogenous IAA. The effectsof IAA and ethylene on elongation were additive. These factsshow that ethylene produced in response to IAA promotes ricecoleoptile elongation in concert with IAA, probably by prolongingthe possible duration of the IAA-stimulated elongation, butthat they act independently of each other. Moreover, AVG stronglyinhibited the endogenous growth of coleoptile segments withtips and this effect was nullified by the exogenous applicationof 0.03 µl l^–1 ethylene. These data imply that theelongation of intact rice coleoptiles may be regulated cooperativelyby endogenous ethylene and auxin in the same manner as foundin the IAA-stimulated elongation of the decapitated coleoptilesegments. Key words: oryza sativa, Ethylene, Auxin, Coleoptile growth     

ENTRY AND EXIT OF INDOLEACETIC ACID IN CORN COLEOPTILES

The entry and exit phases of radioactive indoleacetic acid transportwere investigated in corn coleoptile sections. Compounds capableof inhibiting auxin transport, particularly p-chloromercuribenzoicacid and N-1-naphthylphthalamic acid, were found to only slightlyblock auxin entry but severely suppress auxin exit. An oxygendeficiency had little effect on auxin entry but was found tostrongly inhibit auxin transport and auxin exit. While indoleaceticacid uptake was proportional to concentration, the exit phasebecame apparently saturated at concentrations above 10^–5M. Both entry and exit were found to have temperature coefficientsof about 2 or more. The low sensitivity of auxin entry to inhibitors,or to oxygen deficiency, and the linearity of entry over a wideconcentration range suggest a diffusion component in entry.The strong sensitivity of exit to inhibitors and to oxygen deficiencyconfirms the involvement of active processes in exit, as expectedof a secretion process. ¹Present address: Research Division, Ontario Water ResourcesCommission, Toronto, Canada. Indiana, U.S.A     

The Polarity of Movement of Endogenously Produced IAA in Relation to a Gravity Perception Mechanism

The polarity of radioactive IAA transport in segments of theleaf sheath base of Avena fatua L. is reversed upon their inversionthrough 180° transport towards the apex being greater thantowards the base. Changes in rates of transport leading to thisreversal can be detected within 10–20 min, correlatingwith the timing of statolith movements from the base to theapex of statocyte cells and conforming to a proposed model forthe gravity control of auxin transport. Transport of H³-trytophan-derived H³-IAA was found to undergosimilar changes in polarity upon re-orientation as did exogenouslyapplied H³ or C¹⁴ IAA. It is concluded that the proposed modelalso relates to the movement of endogenously produced IAA.     

Transport of 14C-lableled indoleacetic acid in Vicia root segments

IAA transport in Vicia root segments was investigated for comparisonwith that in intact roots. Lanolin paste (1-mm-wide ring) oragar blocks (3?3?1.5mm), both containing IAA-2-¹⁴C were appliedto the surface or a cut end of the root segments, respectively;transported ¹⁴C was collected in receiver agar blocks placedon the cut end of the segments. When lanolin paste was appliedto 5-mm segments, basipetal transport of IAA predominated overacropetal transport. When agar blocks were applied to 1- and2-mm segments, the same was true; in longer segments (3 and5 mm long), however, basipetal movement occurred predominantlyat first but was surpassed by acropetal movement after 2–3hr. Among the segments tested (regions 2–4, 4–6and 8–10 mm from the tip), the most apical one showedthe distinctest predominancy of basipetal movement. The velocitiesof the acropetal and basipetal movement of the ¹⁴C were estimatedat 3–3.8 and 8–12 mm/hr, respectively. Autoradiographicstudy and the experiment in which wire was inserted longitudinallythrough the central part of the segments showed that basipetalmovement occurred mainly through the outer part of the rootsand acropetal movement mainly through the central cylinder.The present results were compatible with those obtained previouslywith intact roots. Some properties of polar movement, such asits specificity, inhibition by TIBA, and dependency on terneprature are described. (Received March 22, 1978; )     

Effects of Auxin and Gibberellin on Conidial Germination and Elongation of Young Hyphae in Gibberella fujikuroi and Penicillium notatum

In Gibberella fujikuroi and Penicillium notatum, IAA, 2,4-Dand GA₃ promoted conidial germination and the elongation ofyoung hyphae. The promotive effects of IAA and GA₃ were additive.In both fungi, the concentrations of endogenous auxin and gibberellinin the culture media were 10^–10 to 610^–12M. (Received April 27, 1985; Accepted August 12, 1985)     

Auxin Transport in Roots: V. EFFECTS OF TEMPERATURE ON THE MOVEMENT OF IAA IN ZEA ROOTS

The effects of temperature on the polar movement of IAA through6-mm and 12-mm segments of Zea mays roots have been investigatedover the range from 1 to 50°C. At all temperatures an acropetal polar movement of IAA predominated,although at low temperatures and at 50°C the 6-mm segmentsshowed a transient basipetal polarity, before the persistentacropetal polarity developed. At 1°C the differences betweenacropetal and basipetal movement of IAA were less distinct thanat the other temperatures. There is, however, a marked metabolically-dependentacropetal movement of IAA through the tissues at 1°C, becausewhen the segments were deprived of oxygen the acropetal movementwas severely reduced while the basipetal movement was reducedto a smaller extent. At 1°C and at 5°C there was alwaysa persistent basipetal polarity of IAA movement through 6-mmand 12-mm segments under anaerobic conditions. The velocity of acropetal movement (mm h^–1) was the samethrough the 6-mm and the 12-mm segments and was markedly affectedby temperature. It increased from 1°C to a maximum valueof 8 mm h^–1 at 31°C and then decreased again at 40and 50°C. The velocity of basipetal movement could be assessedonly at 1 and 5°C at which temperatures it was greater thanthe velocity of acropetal movement, and virtually independentof segment length. The acropetal flux of IAA (cpm h^–1) was much less through12-mm segments than through 6-mm segments. For both lengthsof segment, however, the flux showed a complex relationshipwith ambient temperature, increasing from 1°C to a maximumat 10–15°C, declining to a minimum value at 31°Cand then rising again at 40 and 50°C. The basipetal fluxof IAA could be astimated only at 1 and 5°C at which itwas very much smaller than the acropetal flux. The amount of IAA in the receiver blocks increased linearlywith time at the lower temperatures. At temperatures withinthe range 15°C to about 31°C, however, the amount ofIAA in the receiver blocks began to decline if the transportperiods exceeded a certain length. The time at which this declinein the IAA in the receiver block began was related to the ambienttemperature. Chromatographic analysis indicated one radioactive substancein receiver blocks at the apical end of segments supplied withIAA-1-¹⁴C at the basal end after transport periods of 6 h at25°C, and 72 h at 5°C. The Rf of this substance wasclosely similar to that of the radioactive IAA supplied in thedonor blocks.     

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens II. IAA Biosynthetic Activity of Cultured Carrot Tissues Transformed with Wild-Type and Mutant Ti Plasmids

IAA biosynthetic activity was examined in cultured carrot tissuestransformed with Agrobacterium tumefaciens harboring wild-type,aux^– or cyt^– Ti plasmids. In vitro IAAM hydrolaseactivities in tissues transformed with wild-type, and cyt^–Ti plasmids were 3.09 and 19.82 nmol/g proteins/30 min, respectively,but not detectable when aux^– Ti plasmids were used. Theactivity of IAA biosynthesis, determined by the incorporationof radioactivity into IAA in tissues fed with [¹⁴C]-tryptophan,was 34.13, 10.92 and 32.47 pmol/g fr wt/30 min in tissues transformedwith wild type, aux^– and cyt^– Ti plasmids, respectively.The incorporation of radioactivity into the IAAM fraction wasdetected only in the tissues transformed with wild type andcyt^– Ti plasmids. These results showed that the T-DNAencoded pathway of IAA biosynthesis was active in tissues transformedwith wild-type and cyt^h Ti plasmids, and that the activity ofIAA biosynthesis in those tissues was higher than that in tissuestransformed with the aux^– Ti plasmid. (Received March 16, 1988; Accepted July 31, 1988)     

Effect of kinetin on auxin uptake and distribution in normal and tumor-prone Nicotiana shoots

Stem segments of non-tumorous Nicotiana glauca and N. langsdorffiiplants and of their tumor-producing amphidiploid F₁ hybrid weretreated with 6-furfurylaminopurine (kinetin) prior to transporttests with applied labeled indoleacetic acid (IAA-2-¹⁴C). Kinetin-treatmentsincreased the uptake of IAA in non-tumorous shoots; the IAAuptake by N. langsdorffii segments was increased up to 3-fold.The auxin uptake in stem-segments of the tumor-forming hybrid,however, could not be increased significantly by kinetin. Theeven distribution of IAA-¹⁴C in segments of normal and tumorproneNicotiana shoots is stimulated by kinetin. Data are discussedin conjunction with previous results on auxin transport andtumorformation in Nicotiana. (Received August 8, 1972; )     

Calcium Antagonist TMB-8 Inhibits Cell Wall Formation and Growth in Pea

The effects on auxin-stimulated growth and cell wall formationof 8-(N, N-diethylamino)-octyl-3, 4, 5-trimethoxybenzoate.HCI(TMB-8), an intracellular Ca²⁺ antagonist, were investigatedin abraded stem segments from aetiolated seedlings of Pisumsativum L. cv. Alaska. Incubation of segments at pH 6.0 with200 mmol m^–3 TMB-8 resulted in a 50% inhibition of auxin-stimulatedgrowth. Added Ca²⁺ did not restore normal auxin-stimulated growth,presumably because of its well-known stiffening effect on thecell wall. In segments incubated at a pH (7–2) which preventedelongation, auxin promoted the incorporation of [³H]glucoseinto the cell wall relative to total uptake of label. TMB-8abolished about 60% of the total incorporation of label intocell walls in the presence of auxin, but was not effective inthe absence of auxin. Exogenous CaCl₂ reversed the inhibitoryeffect of TMB-8 on relative cell wall incorporation in a parabolicmanner, with a 50% reversal at about 100 mmol m^–3 andcomplete reversal at 1.0 mol m^–3 Ca²⁺. Other ions tested(Mg²⁺, Mn²⁺, Cu²⁺, Zn²⁺) were without substantial effect atconcentrations of 0.5 mol m^–3. Both apparent uptake ofCa²⁺ and consequent reversal of TMB-8 inhibition of cell wallincorporation were blocked by the Ca²⁺ channel blockers verapamiland La³⁺. The data provide further evidence that auxin-stimulatedgrowth is dependent upon continued cell wall incorporation,and suggest that a Ca²⁺ messenger system may be involved inthe promotory actions of auxin on cell wall synthesis and long-termgrowth. Key words: Auxin, calcium, cell wall synthesis     

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