Effects of Auxin Concentration on the Dimensions and Patterns of Tracheary Elements Differentiating in Pith Explants

The optimal concentration of IAA (0.03 mM) for tracheary elementdifferentiation in lettuce pith explants was about ten timesgreater than the optimal concentration for callus proliferation.Related to this, the mean volume per tracheary element increasedwith increasing IAA concentration, 18-fold between 0.001 mMand 0.3 mM IAA. At the highest concentrations, some pith cellsappeared to differentiate directly into tracheary elements,without cell division, resulting in especially large trachearyelements. Tracheary strands developed at intermediate concentrationsof IAA, and led to a small increase in the mean length/breadthratio of tracheary elements. For tracheary elements differentiating from stem cambial derivatives,a reassessment of previous studies indicates that increase inauxin concentration brings greater tracheary element size atconcentrations up to the 0.03 mM optimum. Above this optimum,however, further increase in auxin concentration brings progressivelysmaller tracheary elements, as the high auxin curtails enlargementof the differentiating cells. This contrasts with the pith explants,in which tracheary element size increases with IAA concentrationmost markedly above the optimum concentration. The interpretationof these relations requires an understanding of the effectsof auxin concentration on interacting quantities such as initialsize of cells, rate of enlargement, and rate of differentiation. Lactuca sativa, lettuce, IAA concentration, pith explants, tracheary element dimensions     

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.     

Stimulatory and Inhibitory Effects of Sucrose Concentration on Xylogenesis in Lettuce Pith Explants; Possible Mediation by Ethylene Biosynthesis

Numbers of tracheary elements differentiating in lettuce pithexplants rose with increase in concentration of sucrose in themedium up to an optimal concentration of 0·2%, and fellwith further increase in concentration to about one-tenth maximalat 3% sucrose. Although a few tracheary elements formed withoutexogenous sucrose, a very low concentration of sucrose (0·001%)was sufficient to stimulate additional xylogenesis. Pretreatmentof explants with 3% sucrose caused a persisting inhibition ofxylogenesis, especially in tissue that had been near the siteof sucrose application (sandwich technique). The requirementfor adequate, but not inhibitory, concentrations of sucrosefor xylogenesis may underlie the development of xylem alongsidethe sucrose-rich phloem in normal apical morphogenesis. For callus growth the response to sucrose was different: theoptimal concentration was 3%, with a broad plateau from 1 to4% sucrose. Sucrose concentrations of 2 to 3%, used in manytissue culture media, are thus roughly optimal for callus growth,but ten times the optimum for xylogenesis in lettuce pith explants. It is surprising that 0·001% (0·03 mM) sucrose,applied exogenously, can stimulate xylogenesis: endogenous sugarconcentrations are normally higher. Perhaps the stimulationis mediated by ethylene biosynthesis, which is known to be xylogenic.Rates of ethylene production per explant rose with increasingsucrose concentration from about 0·1 nl h^-1 at 0% sucroseto a slightly (significantly) higher level at 0·004%sucrose and to about 0·5 nl h^-1 at 3% sucrose. D -glucoseresembled sucrose in its effects on xylogenesis and ethyleneproduction, but L-glucose yielded no xylogenesis and littlestimulation of ethylene biosynthesis.Copyright 1994, 1999 AcademicPress Lactuca sativa, Coleus blumei, Nicotiana tabacum, lettuce pith explants, tracheary element differentiation, sucrose, glucose, ethylene     

Patterns of Tracheary Differentiation in Lettuce Pith Explants: Positional Control and Temperature Effects

When lettuce pith explants were cultured for 14 d on a xylogenicmedium, tracheary elements differentiated in greatest numbersbetween 25 and 30 °C. Numbers were depressed at lower temperaturesby slower development and at higher temperatures by adverseprocesses. The data did not support previous suggestions ofa great stimulation of xylogenesis above 30 °C and of aspecial sensitivity to low temperatures. Tracheary elements differentiated in various spatial patterns:as clumps in the peripheral callus, as strands which extendedradially and longitudinally from some of these clumps, as individuallarge tracheids especially at the more extreme temperatures,and as short strands associated with nodules and roots thatformed at favourable temperatures. We suggest that indoleacetic acid (IAA) has various roles inthe positional control of these tracheary patterns: (1) IAAdestruction at the explant surface leads to concentration gradientsthat inhibit tracheary induction close to the surface; (2) IAAtransport from the source in the culture medium to sinks especiallyat the explant surface, coupled with autocatalytic flow facilitation,leads to canalization along pathways that become meristematicand then trachcary strands; (3) the IAA flux (and associatedproton flux) along these pathways tend to orient cortical microtubulesat right angles to the flow, by some mechanism as yet unknown,and hence to control the orientation of tracheary element elongationand secondary wall banding. These suggestions, supported bymorphometric studies of tracheary element dimensions and orientations,and by experiments with localized IAA application, lead to ageneral interpretation of the development of polarity in plants. IAA, Lactuca sativa, lettuce, pith explants, positional control, temperature effects, tracheary element differentiation     

Determination of Tracheary Element Differentiation in Lettuce Pith Explants

Transfer experiments with lettuce pith explants revealed thatprovision of inductive concentrations of both cytokinin andauxin in the culture medium for the first 3 and 5 d respectivelywas sufficient to cause determination of tracheary elementsas shown by their subsequent differentiation in substantialnumbers in explants removed from the inductive hormonal stimuli. Determination, differentiation, tissue culture, IAA, zeatin, tracheary elements, lettuce, Lactuca     

IAA Amino Acid Conjugates Induce Differentiation of Tracheary Strands in Lettuce Pith Explants

Each of four amino acid conjugates of IAA was able to replacethe IAA requirement for xylogenesis in lettuce pith explants,when supplied at concentrations ten to 100 times those optimalfor IAA. Tracheary development induced by these conjugates tendedto be slightly slower and less in amount than with IAA, andthe tracheary strands shorter and less regular. Responses differedsomewhat among the four conjugates: IAA-D, L-aspartate gavedevelopment most like that with free IAA, and IAA-D, L-phenylalanineoften yielded the weakest tracheary development, while responsesto IAA-L-alanine and IAA-glycine were intermediate. The resultsare interpreted in terms of the ‘bound’ IAA conjugatesdiffusing into the pith explants and becoming xylogenic onlyon hydrolysis to ‘free’ IAA. As tracheary strandformation is believed to result from IAA fluxes, it seems thatthe free IAA also moved through the discs, presumably towardsthe surfaces where it degrades rapidly. Tracheary strand formationin these explants can be compared with vascular strand formationin the normal shoot tip, where IAA conjugates (auxin ‘precursors’)move acropetally and are hydrolysed to free IAA especially inthe young leaf primordia, we suggest, yielding local sourcesof IAA which may contribute both to the phyllotactic spacingof primordia and, moving basipetally, to the definition of theauxin pathways that develop as procambial strands behind individualleaf primordia. Lactuca sativa, lettuce, IAA conjugates, tracheary element differentiation, pith explants, xylem strands     

Inhibition of xylogenesis by morphactin in pith parenchyma explants of Lactuca

Pith parenchyma explants of Romaine lettuce (Lactuca salivaLinn. var. Roman?) incubated in the dark for 7 days at 25?Con a nutrient medium containing sucrose, IAA. and kinetin exhibitedextensive differentiation of tracheary elements. The additionof CFL to the medium strongly inhibited tracheary element formation.The lack of tracheary strand formation in the CFL-treated explantssuggests the inhibition of auxin transport. Conclusive evidencethat CFL influences the anatomy of differentiating xylem elementswas lacking. The addition of CFL to various combinations ofxylogenic media was not stimulatory to xylem element formationbeyond the differentiation response observed in the absenceof CFL. Unique patterns of tracheary element formation producedby cytokinin media containing IAA, 2,4-D, and NAA, respectively,were abolished by CFL. As indicated by counts of total trachearyelements formed per explant, the addition of cysteine to a CFL-containingmedium reversed the inhibitory effect of CFL. Tracheary strandformation was not re-established in the explants cultured onthe cysteine+CFL medium. Tracheary element formation was completelysuppressed by TIBA. Cysteine had a slight effect on the inhibitionof differentiation by TIBA. These observations suggest thatCFL inhibits some sulfhydryl- containing system involved eitherin the process of xylem differentiation or in some prerequisiterole necessary for the induction of tracheary element formation. (Received December 27, 1972; )     

Somatic Embryogenesis and Analysis of Peroxidase in Cultured Lettuce (Lactuca sativa L.) Cotyledons

Somatic embryos were induced in lettuce cotyledons culturedon Murashige and Skoog's (MS) medium containing either 2 mgl^–1 6-benzylaminopurine (BA) and 0.2 mg l^–1 naphthaleneaceticacid (NAA) or 0.2 mg l^–1 BA and 2 mg l^–1 NAA. Bothcombinations induced a frequency of over 70%. The explants culturedonly in the presence of 2,4-dichlorphenoxyacetic acid (2,4-D)did not produce somatic embryos. The development of the embryoidswas studied histologically and by scanning electron microscopy.Peroxidase activity was assayed and the isoenzyme pattern ofcalluses was determined by polyacrylamide gel electrophoresis.Callus from an embryogenic line showed a much higher peroxidaseactivity than that from a non-embryogenic line, one extra peroxidaseisozyme band being present and typical of the embryogenic callus.No qualitative differences were detectable between the embryogeniccalluses. Lactuca sativa L, lettuce, somatic embryogenesis, peroxidases, isoenzymes     

Interaction of auxin, cytokinin, and gibberellin on cell division and xylem differentiation in cultured explants of Jerusalem artichoke.

Dark-cultured explants of parenchymatous cells isolated fromJerusalem artichoke tubers were induced to divide and differentiateas tracheary elements on Murashige and Skoog medium containingdifferent combinations of plant growth-hormones such as auxin(IAA), cytokinin (zeatin), and gibberellin (GA₃). Addition ofauxin to the growth-medium induced after a short lag period,very rapid cell division which was followed by differentiationof some of the divided cells as tracheary elements. At the optimallevel of IAA (5.0 mg/liter), the percentage of tracheids differentiatedwith respect to the total number of cell population was 13.54.When the explants were cultured in the presence of both auxin(IAA 5.0 mg/liter) and one cytokinin (zeatin 0.1 mg/liter),not only a strong interaction on cell division and trachearyelement formation was observed but also an increase in the percentageof tracheids differentiated in relation to the total cell population.Auxin-gibberellin and auxin-gibberellin-cytokinin treatmentsalso produced interaction on cell division and cytodifferentiation.In explants treated with the three growth-hormones about 20%of the total cell population differentiated as tracheary elements.Further, all the hormonal treatments gave different patternsof cytodifferentiation which reflected meristematic patterns. ¹ This research was supported by a grant from C. N. R. Italy. (Received April 18, 1973; )     

Effects of auxin on the spatial distribution of cell division and xylogenesis in lettuce pith explants

Summary Cylinders of pith parenchyma were tissue-cultured with their opposite ends on media which differed only in content of the morphogens auxin (IAA), sucrose, or zeatin. A range of concentrations of each of these morphogens applied at one end (none at the other end) resulted in distribution patterns of cell division and xylogenesis that were attributable to interaction between inductive levels and morphogen mobility. Auxin was crucial for tracheary patterns: large tracheary elements formed by direct differentiation of pith cells near the auxin source, smaller but still roughly isodiametric tracheary elements formed after cell division, and tracheary strands developed where, presumably, auxin transport had become polarized and then canalized. Xylogenesis was confined to regions within millimeters of the auxin source, and [¹⁴C]IAA studies showed a steep logarithmic concentration gradient along the cylinder. Patterns of tracheary strands and rings revealed that the pith explants retained some polarity from the stem from which they had been excised. However, the direction of flow of applied auxin was more effective than original polarity in controlling the orientation of tracheary strands and their constituent tracheary elements. It seems that, in tissues with little or no polarity, diffusive flow of auxin gradually induces polar flow in the same direction, together with an associated bioelectric current, and that this orients the cortical microtubules that in turn determine the orientations of cell elongation and of the secondary wall banding in tracheary elements.Abbreviations IAA indoleacetic acid - NAA naphthaleneacetic acid - TIBA triiodobenzoic acid Dedicated to the memory of Professor John G. Torrey     

Genotype-independent transformation of lettuce using Agrobacterium tumefaciens

The genetic manipulation of lettuce (Lactuca sativa) necessitatesa reliable and efficient, genotype-independent method of transformation.Thirteen lettuce cultivars have been assessed for their amenabilityto Agrobacterrum-mediated gene transfer linked to their tissueculture responsiveness, including callus induction and shootregeneration. A reliable protocol has been developed for theroutine production of transgenic plants for all 13 cultivarsinvestigated. Key words: Agrobacterium-mediated transformation, Lactuca sativa genotypes, lettuce     

Differentiation of Shoot Buds in vitro in Tissue 1Sisymbrium irio L.

Shoot bud formation was induced in the stem callus of Sisymbriumirio L., a Cruciferous plant. The callus was established onMurashige and Skoog medium with IAA (1?0 mg l^–1) and kinetin(0?5 mg l^–1). The effect of three purines (kinetin, 6-benzylaminopurine,and 6-methylaminopurine) incorporated singly along with IAAin MS medium was investigated. It was found that kinetin orMAP (3–5 mg l^–1) along with IAA (0?5 mg l^–1)were the most effective in inducing shoot bud formation. Adeninesulphate (10 mg l^–1) with kinetin (1?0 mg l^–1) alsoinduced bud differentiation. The morphogenetic potential of the callus to differentiate shootbuds was seemingly lost in 2 year old callus cultures. However,on successively subculturing on a regeneration medium shootbuds differentiated and the number of buds formed improved onfurther subculture. Two types of meristematic outgrowths were recognized: (i) arisingfrom superficial cells and (ii) arising from deep-seated cellsin the vicinity of tracheidal elements. However, both typesformed meristematic nodules on the surface of which shoot budsdifferentiated. Some embryoids were also recognized arisingsuperficially.     

Somatic Embryogenesis and Its Hormonal Regulation in Tissue Cultures of Freesia refracta

Somatic embryogenesis can be induced in tissue cultures of Freesiarefracta either directly from the epidermal cells of explants,or indirectly via intervening callus. These two pathways ofsomatic embryogenesis can be controlled and regulated by varyingthe combinations and levels of exogenous hormones. When younginflorescence segments were cultured in vitro on modified N₄(MN₄) medium supplemented with 2 mg l^–1 indoleacetic acid(IAA) and 3 mg l^–1 6-benzylaminopurine (BAP), some ofthe epidermal cells began to exhibit the features of embryogeniccells. These cells produced embryoids and developed into newplants through direct somatic embryogenesis. If the same explantswere placed on Murashige and Skoog's (MS) medium containing2 mg l^–1 IAA, 05 mg l^–1 BAP and 05 mg l^–1naphthaleneacetic acid (NAA), pale-yellow translucent nodularcalluses appeared on the surface of the explants. When thiskind of callus was transferred to MN6 medium with 2 mg l^–1IAA and 3 mg l^–1 BAP, embryoids formed which further developedinto plantlets. The regenerated plants were morphologicallynormal and possessed the normal diploid chromosome number of2n = 22. A similar result has also been obtained with youngleaf explants of this plant. The early segmentations of embryogeniccells and the development of embryoids were studied using histologicaland scanning electron microscopic techniques, and the resultshave been discussed in association with the ontogeny and originof the embryoids. Freesia refracta Klatt, somatic embryogenesis, plant regeneration, exogenous hormones     

Gibberellin-mediated synergism of xylogenesis in lettuce pith cultures

Major gibberellins (GAs) in lettuce (Lactuca sativa L. cv Romaine) pith explants have been identified by gas chromatography-mass spectrometry (GC-MS) or GC-selected ion monitoring (GC-SIM) as GA₁, 3-epi-GA₁, GA₈, GA₁₉, and GA₂₀. Treatment of pith explants with indole-3-acetic acid (IAA) (57 micromolar) plus kinetic (0.5 micromolar) induced xylogenesis. In this xylogenic treatment, the concentration of a biologically active, polar GA-like substance(s) increased during the first 2 days of culture, although all of the above GAs decreased (as measured by GC-SIM). In non-xylogenic treatments, where explants were cultured without exogenous hormones, or with IAA or kinetin alone, the concentration of the biologically active, polar GA-like substance(s) decreased during the first two days of culture, as did all of the above GAs (as measured by GC-SIM). Treatment of pith explants with exogenous GA₁ alone did not induce xylogenesis, but GA₁ at very low concentrations (0.0014 and 0.003 micromolar) synergized xylogenesis in the IAA plus kinetin-treated cultures. These results suggest that changes in the concentration of certain endogenous GAs may be involved in xylogenesis mediated by IAA plus kinetin in lettuce pith cultures.     

Ethylene Biosynthesis and Xylogenesis in Lactuca Pith Explants Cultured In Vitro in the Presence of Auxin and Cytokinin: The Effect of Ethylene Precursors and Inhibitors

The possible involvement of ethylene in the induction of xylemdifferentiation was studied in lettuce (Lactuca saliva L. cv.Romaine) pith parenchyma explants. The addition of the ethyleneprecursors L-methionine (0.25 µM), S-adenosylmethionine(25 µM) and 1-aminocyclopropane-l-carboxylic acid (0.01µM), or the ethylene-releasing agent 2-chloroethylphosphonicacid (1.0 µM), to a standard IAA-kinetin-containing mediumenhanced xylogenesis compared to control explants cultured inthe absence of these compounds. In the presence of the ethyleneinhibitors aminoethoxyvinylglycine, Co(NO₃)₂ and AgNO₃, xylogenesiswas inhibited. Inhibition of xylogenesis by aminoethoxyvinylglycine(75 µM), Co(NO₃)₂ (50 µM) and AgNO₃ (6.0 µM)was reversed by exogenous 1-aminocyclopropane-l-carboxylic acid(0.01 µM), 2-chloroethylphosphonic acid (5.0 µM)and L-methionine (0.25 µM), respectively. Ethylene productionby explants cultured on media containing L-methionine or 1-aminocyclopropane-l-carboxylicacid was greater than the biosynthesis of ethylene by explantscultured in the absence of these compounds. The incorporationof 2-chloroethylphosphonic acid into the culture medium resultedin higher rates of ethylene production compared to explantscultured on the IAA-kinetin medium. The presence of either aminoethoxyvinylglycineor Co(NO₃)₂ inhibited ethylene production by explants culturedon the IAA-kinetin medium. The data support the hypothesis thatethylene plays a positive role in the initiation of xylem differentiation. Key words: Xylogenesis, Differentiation, Ethylene, IAA, Kinetin, Lactuca sativa     

Introduction of xylem differentiation in lactuca by ethylene

Evidence was obtained to support the hypothesis that ethylene is involved in xylem differentiation in primary pith explants of Lactuca sativa L. cv Romaine cultured in vitro. Xylem elements differentiated when explants were supplied indole-3-acetic acid (IAA) in combination with either the ethylene biosynthetic precursor 1-aminocyclopropane-1-carboxylic acid (ACC), the ethylene-releasing agent 2-chloroethylphosphonic acid (CEPA), or kinetin. In contrast, no xylem elements differentiated in the presence of IAA, kinetin, ACC, or CEPA alone, or when kinetin was supplied together with ACC or CEPA. These results show that ethylene will substitute qualitatively for cytokinin during auxin-induced xylogenesis, and suggest that both ethylene and auxin are required for xylem differentiation in Lactuca.     

Morphogenic Potential of Cultured Floral Explants of Crocus sativus L. for the In Vitro Production of Saffron

Explants of immature ovaries, stigmas, anthers and petals ofCrocus sativus were cultured on White's media supplemented witheither NAA and zeatin or 2,4-D and BAP in various combinations.The formation of stigma-like structures occurred on the explantsor on the callus derived from the explants, but this was onlyobserved when NAA and zeatin were used. Formation of stigma-likestructures were observed on anthers, petals, stigmas and half-ovaries,with the best result being obtained on explants consisting ofhalf-ovaries cultured on medium containing NAA at 40 mg dm^–3and zeatin at 4.0 mg dm^–3. These stigmas developed anintense orange pigment and grew to 3.0 cm in length and hada strong saffron aroma. A preliminary comparison using thinlayer chromatography of the yellow pigments produced by thestigma-like structures grown in vitro and those grown naturallyshowed the pigment composition to be similar. Key words: Crocus sativus L., explants, NAA, zeatin, saffron     

Somatic Embryogenesis and Plant Regeneration from Inflorescence Culture of Java Citronella: (Cymbopogon winterianus)

Embryogenic callus was induced from immature inflorescence segmentsof Java citronella (Cymbopogon winterianus) and maintained for2 years on Murashige and Skoog's medium supplemented with 2,4-D(l mg l^–1). The callus cells retained the original chromosomenumber of 2n = 20. The somatic embryos germinated into plantletson MS basal medium or medium with IAA, NAA, BAP or KN individually(l mg l^–1). The regenerated plantlets developed a goodroot system on full strength solid MS inorganics medium withIAA (1 mg l^–1). The regenerated plants were similar tothe donor plant in morphology and had the same chromosome number,but showed some variation in the essential oil content. Java citronella, Cymbopogon winterianus, somatic embryogenesis, regeneration, inflorescence culture     

Chemical Regulation of Callus Growth, Organogenesis, Plant Regeneration, and Somatic Embryogenesis in Antirrhinum majus Tissue and Cell Cultures

Excised stem explants of Antirrhinum majus L. var. ‘Kymosyblanc’ were grown in a denned medium to investigate factorsinfluencing bud and root development, callus induction, andsomatic embryogenesis. Auxins such as indoleacetic acid (IAA)and naphthaleneacetie acid (NAA) caused limited callus developmentand abundant root formation, whereas 2,4-dichlorophenoxyaceticacid (2,4-D) promoted soft friable callus with embryos and occasionaldevelopment of thick abnormal roots. 2-Naphthoxyacetic acid(NOA) and coconut milk (CM) used together induced friable greencallus growth and differentiation of small globular embryoswhich eventually developed into plantlets after transfer toauxin-free agar mineral medium containing sucrose. Cytokininssuch as benzyladenine (BA), zeatin, and kinetin induced compactgreen callus but in the absence of auxin failed to promote organogenesis.The interaction of IAA and kinetin resulted in the regenerationof the whole plant from stem explants. When NAA was used withkinetin, shoot development was totally inhibited and abundantroots were formed. Thus, the alternative morphogenetic eventsprobably reflect the biochemical subtleties occurring withinthe callus as a result of differences of actual endogenous levelsof growth substances in the tissues studied. These experimentshave been performed and interpreted on a histological basis.     

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