Interaction of a Brassinosteroid with IAA and GA3 in the Elongation of Cucumber Hypocotyl Sections

A synthetic brassinosteroid, 22,23(S,S)-homobrassinolide (hBR),was examined for its interaction with IAA and GA₃ in the elongationof hypocotyl sections of light-grown cucumber (Cucumis salivusL. cv. Aonagajibai) seedlings. hBR alone was less active thanIAA. Its optimal concentration was around 10 µM and thelowest effective concentration between 10 and 100 µM,which is more than 100 times higher than that of brassinolide.hBR was more active in sections from younger seedlings. Itsgrowth-promoting effect was negated or greatly reduced by inhibitorsof auxin-induced elongation such as p-chlorophenoxyisobutyricacid and kinetin. hBR acted synergistically with IAA and 2,4-Dbut not with GA₃ showing only an additive effect. Sequentialtreatment of sections with hBR and then with IAA also resultedin synergistic enhancement of auxininduced elongation, but whenthe order of treatment was reversed, hBR was inactive. The synergisticeffect was obtained with 1 h pretreatment with hBR and couldbe reduced by subsequent washing with water. There was no sequentialinteraction between hBR and GA₃. The synergistic pretreatmenteffects of hBR and GA₃ were simply additive to each other. Amembrane-bound ATPase inhibitor, dicyclohexylcarbodiimide, inhibitedthe hBR-induced elongation, but did not affect GA₃-induced elongation.The findings led to the conclusion that brassinosteroids enhanceauxin action and possess growth-promoting activity which isindependent of that of gibberellin. (Received November 9, 1984; Accepted February 18, 1985)     

IAA Oxidase preparations from fresh and aged Ipomoea batatas tuber discs

IAA oxidase preparations from fresh sweet potato tuber discs oxidized IAA only in the presence of added phenolic cofactors, and the pH optimum for enzyme activity depended on the cofactor used. Ageing of tuber discs, either by aeration in distilled water or by incubation on moist filter paper, resulted in increased peroxidase and phenol-stimulated IAA oxidase activities, as well as the development of IAA oxidase activity in the absence of added cofactors. High phenolase activity of fresh tuber discs decreased considerably with ageing. Phenol-stimulated IAA oxidase activity reached maximal levels before IAA oxidase activity in the absence of added cofactors. Enzyme preparations from aged tuber discs had double pH optima, similar to those previously described for sweet potato root IAA oxidase preparations. IAA in the concentration range 10^?4 to 10^?2 M inhibited the increase in peroxidase and IAA oxidase activities with ageing. DCP-stimulated IAA oxidase activities in preparations from both fresh and aged sweet potato tuber discs were inhibited by manganous ion.     

Differentiation of Photorespiratory Activity between Mesophyll and Bundle Sheath Cells of C4 Plants II. Peroxisomes of Panicum miliaceum L.

Peroxisomes were isolated by sucrose density gradient centrifugationfrom mesophyll and bundle sheath protoplasts of a C₄ plant,Panicum miliaceum L. The equilibrium density in the gradientwas 1.25 for bundle sheath peroxisomes and 1.23 for mesophyllperoxisomes, the former density being similar to that of peroxisomesof wheat mesophyll protoplasts. Photorespiratory and other microbody enzymes were assayed forthe peroxisomes of P. miliaceum to detect possible differentiationat an enzyme level. The specific activities of photorespiratoryenzymes, except for hydroxypyruvate reductase, in bundle sheathperoxisomes were 40–60% of those in wheat peroxisomes,when compared on a protein basis, and only 20–30% in mesophyllperoxisomes. However, peroxisomes from both cell types containedsignificant levels of all the enzymes involved in the photorespiratoryglycolate pathway, when compared with castor bean glyoxysomes.The activity of hydroxypyruvate reductase in the peroxisomesof P. miliaceum was comparable to or higher than that in wheatperoxisomes. Two ß-oxidation enzymes and urate oxidasewere detected in the peroxisomes in a similar level to thatin wheat peroxisomes. These results suggest that the peroxisomes of mesophyll andbundle sheath cells of P. miliaceum are essentially similarto those of C₃ plants, and that they cannot be differentiatedexcept for a difference in equilibrium density in a sucrosegradient. (Received December 24, 1984; Accepted April 9, 1985)     

Properties of peroxidases from tobacco cell suspension culture

An enzyme preparation from suspension cultured tobacco cells oxidized IAA only in the presence of added cofactors, Mn²⁺ and 2,4-dichlorophenol, and showed two pH optima for the oxidation at pH 4·5 and 5·5. Effects of various phenolic compounds and metal ions on IAA oxidase activity were examined. The properties of seven peroxidase fractions separated by column chromatography on DEAE-cellulose and CM-Sephadex, were compared. The peroxidases were different in relative activity toward o-dianisidine and guaiacol. All the peroxidases catalysed IAA oxidation in the presence of added cofactors. The pH optima for guaiacol peroxidation were very similar among the seven isozymes, but the optima for IAA oxidation were different. The anionic and neutral fractions showed pH optima near pH 5·5, but the cationic isozymes showed optima near pH 4·5. With guaiacol as hydrogen donor, an anionic peroxidase (A-1) and a cationic peroxidase (C-4) were very different in H₂O₂ concentration requirements for their activity. Peroxidase A-1 was active at a wide range of H₂O₂ concentrations, while peroxidase C-4 showed a more restricted H₂O₂ requirement. Gel filtration and polyacrylamide gel studies indicated that the three cationic peroxidases have the same molecular weight.     

Indolepyruvate Pathway for Indole-3-Acetic Acid Biosynthesis in Bradyrhizobium elkanii

Indole-3-acetaldehyde (IAAId) was detected in the culture supernatantof Bradyrhizobium elkanii. Deuteriumlabelled L-tryptophan (Trp)was incorporated into IAAId and indole-3-acetic acid (IAA),suggesting that B. elkanii produces IAA via IAAId from Trp.In B. elkanii cell suspension, indole-3-pyruvic acid (IPyA)was converted to IAAId, and exogenously added IAAId was rapidlyconverted to IAA. Furthermore, the activity of indolepyruvatedecarboxylase (IPDC), which catalyzes the decarboxylation ofIPyA to produce IAAId and is a key enzyme for IPyA pathway,was detected in B. elkanii cell-free extract. The IPDC activitydepended on Mg²⁺ and thiamine pyrophosphate, cofactors of decarboxylation.This mounting evidence strongly suggests that IAA synthesisoccurs via IPyA pathway (Trp IPyA p IAAId IAA) in B. elkanii. (Received December 11, 1995; Accepted March 4, 1996)     

Natural Occurrence of Indoleacetamide and Amidohydrolase Activity in Etiolated Aseptically-Grown Squash Seedlings

Etiolated seedling tissues of aseptically grown squash (Cucurbitamaxima Duch) contain indole-3-acetamide (IAM) as a natural endogenouscompound, conclusively identified by gas chromatography-massspectrometry (GC-MS). Roots of aseptically raised seedlingsalso contain amide hydrolysing activity, which converts IAMto IAA, indoleacetonitrile (IAN) to IAM and IAA, and 1-naphthaleneacetamideto 1-naphthaleneacetic acid. This activity was enriched 48-foldby fractional precipitation with ammonium sulphate, Sephadexgel nitration and anion exchange chromatography. Being hydrolytic,it works equally well in air and in vacuo, without added cofactors.The partially purified enzyme works optimally between pH 7 and7.5, and a K_m value of 80 µM was calculated with IAM asthe substrate. The product of this reaction was definitivelyidentified as IAA by GC-MS. The temperature optimum of thisamidohydrolase lies around 45°C, and it is stable to freezing.A comparison of its properties with the amidohydrolase of Agrobacteriumor crown gall tissue, shows it to be different. In view of thenatural occurrence of both IAM and the amidohydrolase, it issuggested that the IAM pathway of IAA biogenesis is feasiblein etiolated squash seedlings. ⁴Deceased 2/2-1993.     

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)     

Paraquat Resistant Tobacco Calluses with Enhanced Superoxide Dismutase Activity

Seven paraquat resistant calluses of tobacco (Nicotiana tabacumL. cv. Samsun) were obtained by three successive screeningsof protoplast-derived calluses on a paraquat containing medium.Superoxide dismutase (SOD) activity of the resistant calluseswas 14- to 159-fold that of the leaf cells on protein basis.Paraquat-resistant calluses, however, showed little increasein catalase and peroxidase activities. More than 90% of SODactivity in the resistant calluses was inhibited by KCN, aswas the SOD activity in leaves, indicating that the major SODin the callus appears to be the Cu, Zn containing enzyme. Thecallus cells, however, expressed the immunologically distinguishedSOD isozyme from the enzyme in the leaves. (Received April 23, 1984; Accepted August 6, 1984)     

Phytochemical studies on tobacco alkaloids XIV. The occurrence and properties of putrescine N-methyltransferase in tobacco roots

Putrescine N-methyltransferase, a new enzyme catalyzing theformation of N-methylputrescine from putrescine and S-adenosyl-L-methioninewas found in roots of tobacco plants. The enzyme was purified30-fold from crude extracts of tobacco roots. NMethylputrescinewas identified as the reaction product by comparison with theauthentic compound. The enzyme had a pH optimum between pH 8and 9, and a molecular weight of about 60,000, as determinedby gel filtration. Km values for putrescine and 5-adenosyl-L-methioninewere 4.0 x 10^–4 M and 1.1 x 10^–4 M, respectively.Enzyme activity was inhibited by N-chloromercuribenzoate andAg⁺. No cofactors were required. Of the various substrates tested,only putrescine served as a methyl acceptor. The enzyme waslocalized exclusively in the roots and its activity was greadyenhanced by decapitation. The presence of putrescine N-methyltransferase in tobacco rootsstrongly suggests that N-methylputrescine participates as anintermediate in nicotine biosynthesis. (Received March 2, 1971; )     

Effects of Indol-3yl Acetic Acid on the Citrate-Condensing Reaction by Preparations of Root and Shoot Tissue

Preparations of citrate condensing enzyme (citrate oxaloacetate-lyase(CoA-acetylating) E. C. 4. 1. 3. 7) from root and shoot tissueof 5-day-old bean seedlings (Phaseolus vulgaris L., var. Burpee'sStringless Greenpod) had different activities, expressed asreaction rate per unit of fresh tissue. Activity per mg proteinwas increased when protein concentrations of the preparationswere reduced by dilution. Addition of indol-3yl acetic acid(IAA) enhanced activity of both root and shoot preparations.The effect was optimal at a concentration of 1.25x10^-4 M andthe enzyme was inhibited at 1.25x10^-3 M. Enhanement was greaterin root than in shoot preparations and in mixtures of equalamounts of each prepartion activity was intermediate betweenthose of the separte enzymes in absence of IAA but in its presenceapproached that of the shoot preparation. Apparent citrate synthesis in vivi was increased in shoots by application of IAA but therewas no such effect in roots.     

Auxin-Induced Changes in the Molecular Weight Distribution of Cell Wall Xyloglucans in Avena Coleoptiles

The effect of auxin on the molecular weight (Mw) distributionof cell wall xyloglucans was investigated by gel permeationchromatography using coleoptile segments of Avena sativa L.cv. Victory, and the following results were obtained.

1. The water-insoluble hemicellulose (HC-A) mainly consisted ofxyloglucans. Iodine staining method revealed that relativelylarge amounts of xyloglucans were present in the water-solublehemicellulose (HC-B) and water-soluble polysaccharide (WS) fractions.
2. IAA did not cause remarkable changes in xyloglucan contentsin the hemicellulose, but significantly increased the xyloglucancontent in the WS fraction.
3. IAA substantially decreased theweight-average Mw of HC-A. Thiseffect became apparent within30 min of the incubation period,and was not affected by the0.15 M mannitol or 2% sucrose applied.Hydrogen ions also causeda decrease in the weight-average Mwof HC-A; its effect beingreversible.
4. Neither IAA nor hydrogen ions caused any remarkablechangesin the weightaverage Mw of water-soluble xyloglucansin theHC-B.

These results suggest that cell wall xyloglucans have an importantrole in auxininduced cell wall loosening in oat coleoptile cells. (Received May 10, 1984; Accepted August 20, 1984)     

Carbonic Anhydrase from the Blue-Green Alga (Cyanobacterium) Anabaena variabilis

Carbonic anhydrase (CA) activity was detected in homogenatesfrom Anabaena variabilis ATCC 29413, M-2 and M-3, but not inthe suspension of the intact cells. Activity was higher in cellsgrown in ordinary air (low-CO₂ cells) than in those grown inair enriched with 2–4% CO₂ (high-CO₂ cells). Fractionationby centrifugation indicated that the CA from A. variabilis ATCC29413 is soluble, whereas both soluble and insoluble forms existin A. variabilis M-2 and M-3. The addition of dithiothreitoland Mg^{2 $} greatly decreased the CA activity of A. variabilisATCC 29413. The specific activity of the CA from A. variabilis ATCC 29413was increased ca. 200 times by purification with ammonium sulfate,DEAE-Sephadex A-50 and Sephadex G-100. Major and minor CA peaksin Sephadex G-100 chromatography showed respective molecularweights of 48,000 and 25,000. The molecular weight of the CAdetermined by polyacrylamide disc gel electrophoresis was 42,000?5,000.The activity of CA was inhibited by ethoxyzolamide (I₅₀=2.8?10^-9M), acetazolamide (I₅₀=2.5?10^-7 M) and sulfanilamide (I₅₀=2.9?10^-6M). (Received January 5, 1984; Accepted April 26, 1984)     

In Vitro Phosphorylation of Proteins in IAA-Treated Mesocotyls in Zea mays

Five-mm sections of elongation zones of Zea mesocotyls wereincubated for designated periods with various concentrationsof IAA. In vitro protein phosphorylation in the soluble fraction(85,000 x g supernatant) prepared from the sections was analyzedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The phosphorylation of proteins in the soluble fraction thathad been prepared from sections incubated for 20 min in thepresence of 10{small tilde}^s M IAA was greater than that inthe sections incubated for 20 min without IAA. The amount ofphosphorylation of proteins per protein became higher when higherconcentrations increased (10{small tilde}⁸—10{small tilde}⁵M).The growth of sections incubated in the presence of 10{smalltilde}⁸ M IAA or higher concentrations was greater than thatof sections incubated in the absence of IAA. The promotion ofgrowth by IAA was greater at higher concentrations of IAA. Proteinsin the soluble fraction, prepared from sections incubated for20 min in the presence of 10{small tilde}⁵ M IAA, were phosphorylatedin the presence of either 10 fM cAMP, 10 µM cGMP, 100µM W-7, 100 µM W-5, 20 µM H-7 or 20 µMHA1004. The calmodulin antagonist, W-7, and the inhibitor ofprotein kinase C, H-7, inhibited the phosphorylation of proteinsstimulated by incubation with IAA. These results suggest thatIAA promotes cell elongation via protein phosphorylation thatdepends on calmodulin-dependent protein kinase and protein kinaseC. (Received November 29, 1995; Accepted May 20, 1996)     

Production of Indole-3-Acetic Acid by Bradyrhizobium japonicum: A Correlation with Genotype Grouping and Rhizobitoxine Production

Bioassays show that rhizobitoxine-producing strains of Bradyrhizobiumjaponicum excreted another phytotoxic compound into their culturefluid. This compound was purified and identified by HPLC andmass spectrometry as indole-3-acetic acid (IAA). The levelsof IAA produced by the different strains of B. japonicum, forwhich the genotype groups have been determined with respectto the degree of base substitution in and around nifDKE, werequantified using gas chromatography/mass spectrometry and adeuterated internal standard. Genotype II strains, which producerhizobitoxine, excreted more than 20µof IAA into theirculture fluid. However, no IAA was detected in the culture supernatantsof genotype I strains, which do not produce rhizobitoxine. Thiswas true even when tryptophan was added to the medium. Moreover,cells of genotypes I and II strains, which were grown underour culture conditions, did not show IAA degradation activity.These results suggest that, in wild-type B. japonicum strains,complete IAA biosynthesis is confined exclusively to genotypeII strains that produce rhizobitoxine. (Received April 9, 1990; Accepted October 6, 1990)     

The Role of Benzoic Acid and Plant Hormones in Flowering of Lemna gibba G3

The level of benzoic acid was measured in Lemna gibba G3 grownon M and E media under inductive and non-inductive daylengths.Benzoic acid was slightly higher in plants grown on M mediumbut there was no difference in the benzoic acid levels in floweringand vegetative plants. When L. gibba G3 was grown under continuouslight on 1/10 M medium or 1/2 H medium there was virtually noflowering, but addition of benzoic acid to either medium ledto a substantial flowering response. In both cases this floweringresponse was inhibited by the plant hormones IAA, GA₃, ABA andzeatin, with IAA and GA₃ being the least inhibitory and ABAbeing the most inhibitory. This same pattern of inhibition wasseen when L. gibba G3 was grown on M medium under continuouslight, conditions that lead to photoinduction of flowering.These results leave open the possibility that endogenous benzoicacid may interact with other factors to influence the floweringresponse in L. gibba G3. (Received November 13, 1984; Accepted February 27, 1985)     

INVESTIGATIONS ON AUXIN-GIBBERELLIN INTERACTION ON THE PHOTOTROPISM OF HELIANTHUS ANNUUS HYPOCOTYL

On applying IAA-GB and IAA-NAA mixtures to the hypocotyls ofHelianthus annuus, the plants showed an increase and a decrease,respectively, in their phototropic response compared with thecontrol treated with IAA-H₂O only. In the in vitro experimentsit was observed that the illumination (with 1,500 lux light,lasting for 24 hr) caused photoinactivation of IAA in the mixturesof IAA-H₂O, IAA-GB and IAA-NAA by 50%, 24% and 0%, respectively.In the presence of 0.01% riboflavin as a photosensitizer inthe same mixtures, the degree of photodestruction of IAA causedby the same illumination was 92%, 97% and 86%, respectively. The stronger positive phototropism in the IAA-GB treated hypocotylwas accounted for by the situation that on the illuminated sidethe photo-inactivation of IAA was accelerated by GB, while onthe shaded side IAA and GB acted synergistically. On the otherhand, the weaker phototropism in the IAA-NAA treated hypocotylwas explained as due to a partial inhibition of photoinactivationof IAA by NAA. (Received October 6, 1962; )     

Auxin-Binding Protein in Etiolated Mung Bean Seedlings: Purification and Properties of Auxin-Bindmg Protein-II

An auxin-binding protein (ABP-II) was purified from the extractof etiolated mung bean seedlings by affinity chromatographyon 2,4-D-linked Sepharose 4B and by gel filtration on Sepharose4B and Sephacryl S-200. The molecular weight was estimated tobe about 190,000 by gel filtration on Sephacryl S-200. ABP-IIgave a single band corresponding to a molecular weight of about48,000 on SDS-polyacrylamide gel electrophoresis. The dissociationconstants of ABP-II for 2,4-D determined by amrnonium sulfateprecipitation and equilibrium dialysis were 9.5?10^–6 Mand 1.1?10^–5 M, respectively. ¹⁴C-2,4-D-binding to ABP-IIwas reversible and inhibited by addition of IAA, naphthalene-1-aceticacid, 2,4,5-trichlorophenoxyacetic acid or p-chlorophenoxyisobutylicacid to the assay mixture. (Received September 5, 1984; Accepted November 5, 1984)     

Effects of Some Growth Regulators on Polyamine Biosynthetic Enzymes in Etiolated Pea Seedlings

This study was designed to examine possible links between polyaminebiosynthesis and effects of growth regulatory compounds. Anauxin (IAA), a cytokinin [benzyladenine; benzylaminopurine (BAP)],an ethylene source (ethephon) and abscisic acid (ABA) were individuallyapplied to terminal buds of excised etiolated or red light (R)-exposedpea epicotyls. Effects were noted on bud fresh weight and onthe two main enzymes of putrescine biosynthesis, arginine decarboxylase(ADC; EC 4.1.1.19 [EC] ) and ornithine decarboxylase (ODC; EC 4.1.1.17 [EC] ).As previously reported [Dai and Galston (1981) Plant Physiol.67: 266], both bud growth and ADC activity are increased byR light. In such buds, ADC is raised further by 1–10 µMBAP or ABA and inhibited by 1–10 µM IAA or ethylene(50 mg/liter or more of ethephon). In all cases, effects ofR-irradiation plus 1 mM growth regulators on ODC activity wasthe inverse of their effects on ADC, indicating independentcontrol of these pathways. These results do not support theview that putrescine biosynthetic activity is correlated withgrowth in etiolated pea seedlings. ¹Supported by a grant from NSF to A.W.G. ²Supported by a grant from the Turkish Government. Permanentaddress: Department of General Botany, University of Istanbul,S?leymaniye, Istanbul, Turkey. ³On sabbatical leave from the Department of Horticulture, HebrewUniversity of Jerusalem, Rehovot, Israel. (Received September 22, 1983; Accepted February 28, 1984)     

Influence of Axis Removal on Amino-, Carboxy- and Endopeptidase Activities in Cotyledons of Germinating Vigna mungo Seeds

Endopeptidase (azocaseolytic enzyme) and carboxypeptidase activitiesin cotyledons of germinating Vigna mungo seeds increased until3 days after the onset of imbibition and decreased thereafter.In detached and incubated cotyledons, the endopeptidase activityincreased only a little while the carboxypeptidase activitycontinued increasing even after 3 days of incubation. The activitiesof leucine-aminopeptidase and alanine-aminopeptidase, exceptfor that of one leucine-aminopeptidase isoenzyme relativelyabundantly present in unimbibed dry cotyledons, increased slightlyon the first day and declined during germination. In detachedcotyledons, the activities maintained their initial levels throughoutthe incubation period. When cotyledons were detached from germinatingseedlings on days 2 and 4 then incubated, the endopeptidaseactivity started to decrease just after removal of the axisbut the carboxypeptidase activity increased more markedly thanwhen the axis remained attached. Exogenously supplied GA₃, kinetin,IAA, or their combinations, showed no significant effect onthe developmental patterns of the endopeptidase and carboxypeptidaseactivities in cotyledons. These results are discussed in relationto the role of the axis in controlling peptidase formation incotyledons of germinating V. mungo seeds. (Received November 18, 1983; Accepted February 28, 1984)     

Tracheary Element Differentiation Induced in Isolated Cylinders of Lettuce Pith: a Bipolar Gradient Technique

To study the influence of morphogenetic gradients on vasculardifferentiation patterns, a new technique was developed whichallows different substances to be applied at opposite ends ofa tissue block. It yielded information on the mobility of particularmorphogens and on the dependence of callus formation and trachearyelement differentiation on their presence. Application of indol-3ylacetic acid (1AA) (10 mg l^–1), zeatin (0.1 mg l^–1)and sucrose (3 per cent, w/v) in various combinations to theends of cylindrical explants of lettuce pith (Lactuca sativaL.) showed that (a) callus formation was stimulated by IAA,whereas induction of tracheary elements required both IAA andzeatin; (b) callus was confined to a few millimetres at theends of the explants, and tracheary elements occurred mainlywithin the callus; (c) sucrose or its metabolic products diffusedthe 10 mm length of the explants, while IAA and zeatin wereeffective only close to the application site; and (d) some callusand tracheary elements formed when no sucrose was applied, butboth increased with sucrose application, though inhibition oftracheary elements formation occurred with high sucrose concentrations. differentiation, pith explant, tissue culture, xylogenesis, indol-3yl acetic acid, sucrose, zeatin, lettuce, Lactuca sativa