The Uptake of Growth Substances: XI. VARIATIONS IN THE ACCUMULATION OF SUBSTITUTED PHENOXYACETIC ACIDS OF DIFFERING PHYSIOLOGICAL ACTIVITY BY SEGMENTS OF AVENA MESOCOTYL

An examination has been made of the phase of continuous accumulationof phenoxyacetic acid (POA) and the 2-, 4-, 2,4-, 2,6-, and2,4,5-chloro-derivatives, containing carbon-14 in the carboxylgroup, by segments of the Avena mesocotyl. On the basis of previousfindings to eliminate the initial transient components of uptake(Type I processes) the segments were pretreated for 13 to 18h in buffer or buffer containing the respective non-radioactivecompound. For five of the compounds the relationship between the rateof uptake and the external concentration takes the form of arectangular hyperbola, but for the sixth, 2,4,5-T, this relationshipdoes not hold. The data, except those for 2,4,5-T, have beenevaluated as linear regressions of rate of uptake against thequotient of rate over concentration. From each regression equationtwo constants have been derived: the point ‘B’ wherethe line intersects the rate axis (the theoretically maximumrate) and the slope of the regression ‘K’, whichcan alternatively be expressed as the concentration at whichthe observed rate equals half the value of ‘B’. The calculated values of B and K for POA are approximately twiceas great as the corresponding values for 2-CPA, and about 25times greater than for 4-CPA. The values for 2,4-D are closeto those for 4-CPA, and 2,6-D is intermediate between 2-CPAand 4-CPA. Although the constants for 2,4,5-T could not be calculatedprecisely, the rates of accumulation are about one-fourth ofthose measured for 2,4-D at equivalent concentrations. The uptake of radioactive 2,4-D is slightly depressed in thepresence of nonradioactive POA. Greater reductions are causedby 2-CPA or 2,6-D, and 2,4-D or 2,4,5-T are even more inhibitory.The pattern of inhibition caused by 2,4,5-T indicates competitionfor common sites of uptake, while POA appears not to be competitive.In corresponding experiments with POA, the presence of the otherregulators only caused small inhibitions and the order was different. Earlier work showed that in Avena accumulation is accompaniedby the conversion to a varying degree of the individual substitutedphenoxyacetic acids to conjugated derivatives. It is suggestedthat the variation between compounds in their rates of accumulationis in part due to differences in the stability of the conjugatedderivatives, and that the facility of conversion is a factorin determining physiological activity.     

The Uptake of Growth Substances: V. VARIATION IN THE UPTAKE OF A SERIES OF CHLORINATED PHENOXYACETIC ACIDS BY STEM TISSUES4GOSSYPIUM HIRSUTUM AND ITS RELATIONSHIP TO DIFFERENCES IN AUXIN ACTIVITY

When segments excised from the etiolated hypocotyls of Gossypiumhirsutum are pretreated in buffer, the subsequent uptake ofradioactive 2,4-dichlorophenoxyacetic acid (2,4-D-1-¹⁴C) isdepressed and the net loss of radioactivity which normally followsa phase of positive uptake by freshly excised segments doesnot take place. Uptake by fresh segments, in contrast with uptakeafter pretreatment, has a high Q₁₀ and is markedly depressedby both 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 3-indolylaceticacid. On these grounds it is proposed that net loss resultsfrom the release of material accumulated by a specific mechanismwhich, with time, becomes inoperative. Additional experimentssuggest that part of the 2,4-D taken up by stem segments ofTriticum vulgare and Avena sativa is accumulated by a similarmechanism. For 1-cm segments, entry is most rapid through the cut ends,and the effects of pretreatment exert their maximal effectsin the tissue near the ends. Therefore very short segments havebeen used to compare the courses of uptake of phenoxyaceticacid (POA) and its 2-, 4-, 2,6-, 2,4- and 2,4,5- chloro- derivatives.The patterns observed are similar to those previously reportedfor 1 -cm segments, although the differences between compoundsare greater. The courses of uptake of 2,4-D and 2,4,5-T, bothterminate in a phase when there is a net loss. POA and the 2-chloro-substitutedacid (2-CPA) are both continuously accumulated. No net lossis found with either the 2,6- (2,6-D) or the 4- chloro (4-CPA)compounds but the rates of uptake progressively decrease toa low level. It is proposed that the processes which determine the patternof uptake of chlorinated phenoxyacetic acids include two typesof accumulation. With Type I accumulation the mechanisms involvedrapidly become disorganized after tissues are excised from theplant. Type 2 accumulation, on the other hand, is stable. Theavailable data indicate that Type I accumulation is peculiarto compounds with marked auxin-like properties.     

Factors Affecting Growth and Aggregate Dissociation in Batch Suspension Cultures of Datura innoxia (Miller)

Growth kinetics of Datura innoxia batch suspension cultureswen monitored by a Klett-turbidimetric technique. While cultured. wt varied linearly with Klett units, f. wt and packed cellvolume did not. Turbidimetrically determined doubling timeswere highly reproducible. The method proved to be useful inthe determination of acutely lethal conantrations of a seriesof anti-metabolites. In certain circumstances, aggregate dissociation in batch suspensioncultures of D. innoxia was found to be coupled to growth rate.Suspensions maintained with 10^–5 M 2,4-D exhibited a relativelyslow growth rate with a high degree of aggregate dissociation:10^–4 M 2,4-D promoted a maximum growth rate, but dramaticallysuppressed aggregate dissociation. At 10^–5 M 2,4-D, themitotic index of smaller-aggregate fractions was greater thanthe mitotic index of the large-aggregate fraction. At 10^–5M 2,4-D the converse was observed. Supraoptimal 2,4-D concentrationsthus enhanced both aggregate dissociation and the growth ofsmaller aggregates. When present in concentrations promoting optimal growth. malicand succinic acids caused a decrease in aggregate dissociation.Casein hydrolysate dramatically enhanced growth, but did notaffect aggregate dissociation to the same degree as 2,4-D orthe Krebs cycle organic acids. Suggestions are made concerningmedium composition to be used in future mutant selection schemesusing D. innoxia. Datura innoxia (Miller), suspension culture, growth kinetics, mitotic index, 2,4-dichorophenoxy acetic acid     

Ethylene and Ethane Production in 2,4-D Treated and Salt Treated Tobacco Tissue Cultures

Gas chromatography was used to measure ethylene (ethene) andethane production by tobacco (Nicotiana tabacum cv. Wisconsinno. 38) callus tissues grown on media containing inorganic saltsaccording to Murashige and Skoog (1962), sucrose, myo-inositol,thiamine-HCl kinetic according to Linsmaier and Skoog (1965),and either 2,4-dichiorophenoxyacetic acid (2,4-D) in the range0–100 mgl^–1 or 2 mgl^–1 indoi-3-ylacetic acidplus NaCl in the range 0–200 Meq l^–1. Ethylene productionrates were high (> 500 nl h^–1 g^1– fresh weight)initially in all treatments. Subsequently, ethylene productiondeclined in rapidly growing cultures but remained high in moderatelyand severely 2,4-D (> 0·5 mgl^–1) stressed andin severely NaCl (150 Meql^–1) stressed cultures. Highinitial rates of ethane production (> 200 nl h^–1 g^–1fresh weight) were obtained under conditions of severe stresscaused by 2,4-D or NaCl but not in control or moderately inhibitedcultures. With further incubation ethane production declinedin the severely stressed cultures. It is concluded that ethyleneproduction can be used as an index of moderate 2,4-D stressand severe NaCl stress by virtue of the high persisting ratesof ethylene production in stressed cultures. Ethane productioncan be used as an early index of severe stress caused by either2,4-D or NaCl in vitro. Nicotiana tabacum L., tobacco, ethylene, ethenen, ethane, 2,4-dichlorophenoxyacetic acid, auxin, stress, callus tissue     

Haploid Plantlet Formation from Female Gametophytes of Ephedra foliata Boiss. in vitro

Female gametophytes (at the archegonial stage) excised fromyoung ovules of Ephedra foliata Boiss, were cultured on a basalmedium (Murnshige and Skoog's combinations of major and minorsalts, Iron source, vitamins, myo-inositol along with 2 percent sucrose and 10 per cent coconut milk) under aseptic conditions.Growth and morphogenetic responses of the explants to auxinswere compared at different concentrations and a study of theirinteractions with cytokinins has also been made. At 2 mg 1^–1,2, 4-D induced profuse callusing which subsequently producedroots. NAA at 4 mg 1^–1 was optimal for callus growth androoting. Combinations of 2,4-D and kinetin were more effectivein inducing roots and shoot buds than those of 2,4-D and benzylamino-purine (BAP). Addition of BAP (0.05 mg ^1–1) to themedium containing optimal concentrations of NAA resulted information of a large number of roots. Kinetin induced only rootingin the presence of 4 mg 1^–1 NAA. A high concentrationof BAP (8 mg 1^–1), stimulated shoot bud formation. Forthe further development of shoot buds, neither auxin nor cytokininwas needed. Cytological observations revealed the presence ofhaploid number of chromosomes, i.e. seven. Ephedra foliata, tissue culture, callus, regeneration, 2,4-dichlorophenoxyacetic acid, naphthalene acetic acid, kinetin, benzyl amino-purine     

Analogues of Phenoxyacetic Acid and the Generation of Calluses from Seeds of Indica Rice

In a search for callus-inducing compounds for indica rice thatare superior in this regard to 2,4-dichlorophenoxyacetic acid(2,4-D), we tested 13 derivatives of phenoxyacetic acids, namely,mono- and di- chloro-, mononitro- and 4-halogeno-substitutedphenoxyacetic acids for their ability to stimulate the inductionof calluses from mature seeds of rice (Oryza sativa L. cv. Chiemchanh). Five analogues of benzoic acid were also tested. Thecallus-forming ability and the optimal concentration for inductionof calluses were differed from compound to compound. The optimal concentration for callus induction of 4-fluorophenoxyaceticacid (4FPA) was relatively high (250 µM), but its callus-formingactivity was stronger than that of 2,4-D, even when comparedwith 2,4-D at its optimal concentration (50 µM). 4FPA-inducedcalluses retained their regenerative ability for at least 15months with 9 subcultures, while calluses induced by 2,4-D losttheir regenerative ability after 3 subcultures. 4FPA-induced calluses showed higher regenerative ability thandid calluses induced by 2,4-D, in almost all of 20 varietiesof indica tested, including three indica-japonica hybrids. ³Present address: Kibun Co.Ltd., Ginza, Tokyo, 104 Japan ⁴Present address: Hyogo Agricultural Research Institute, Kasai,Hyogo, 679-01 Japan (Received October 25, 1989; Accepted May 17, 1990)     

Some Physiological Responses of Chlorella pyrenoidosa to 2,4-Dichlorophenoxyacetic Acid

An 18-h treatment of synchronously-grown Chlorella pyrenoidosawith 2,4-D did not significantly alter the size, dry weight,degree of synchrony, or pigment content of the cells, nor weredetectable quantities of ethylene produced. When Chlorella pyrenoidosawas treated with 5?10^–4 M 2,4-D, there was a statisticallysignificant stimulation of both net oxygen uptake and productionwhile 5?10 M 2,4-D inhibited both processes. When Chlorellapyrenoidosa was treated with 5?10^–4 M and 5?10^–3M 2,4-D, significantly greater amounts of glycollate were presentin the culture medium, even though an assay for glycollate dehydrogenaseshowed that the activity of this enzyme from 2,4-D-treated Chlorellapyrenoidosa was three times greater than in control cells. Looselybound 2,4-D was partitioned from a nonaqueously isolated chloroplastfraction, while other cell fractions failed to show detectablequantities of 2,4-D. It is postulated that in Chlorella pyrenoidosathe chloroplast is a target for 2,4-D action and that interferencein photorespiratory processes may underlie the observed responses.     

Somatic Embryogenesis from Clonal Leaf Tissues of Cassava

Leaf lobes were isolated from palmate leaves of clonal cassava(Manihot esculenta Crantz) material growing in vitro or in glasshouseconditions and subjected to a two-stage culture procedure involvingincubation on Murashige and Skoog (MS2) basal medium supplementedwith 2–12 mg l^–1 2,4-D for 20 d (Stage I) beforetransfer to MS2 basal medium supplemented with 0.01 mg l^–12,4-D and 0.1 mg l^–1 6-benzylamino purine (BAP) (StageII medium). Embryogenetic tissues, foliose structures and somatic embryosdeveloped from leaf lobes at all Stage I 2,4-D concentrations,except on those explants isolated from shoot-tip cultures incubatedon MS2 basal medium supplemented with 0.1 mg l^–1 NAA and1.0 mg l^–1 BAP. Leaf lobes isolated directly from glasshouse plants showed optimalembryogenetic competence when subjected to a Stage I cultureperiod of 17 d, although foliose structure initiation was optimalwith shorter Stage I durations. Leaf lobes of 2–4 mm lengthand those isolated from phyllotaxic leaf numbers 4 and 5 showedthe greatest embryogenetic competence. Manihot esculenta, cassava, somatic embryogenesis, tissue culture, morphogenetic competence     

Purification and Properties of an Auxin-Binding Protein from the Shoot Apex of Peach Tree

A soluble auxin-binding protein was purified from the shootapices of peach trees by chromatography on columns of CM-Toyopearl,Sephacryl S-200, 2,4-D-linked-Sepharose 4B and ConA-Sepharose.The molecular mass of the purified protein was estimated tobe about 100 kDa. After electrophoresis on a denaturing gel,the protein gave a single band with a molecular mass of 20 kDa.From Scatchard analyses, the dissociation constant for 2,4-Dwas calculated to be 4.1 10^–5 M and the specific bindingof 2,4-D at saturating concentration was 42 nmol (mg protein)^–1.The binding of [¹⁴C]-2,4-D to the protein was reversible andwas inhibited by IAA, 1-naphthylacetic acid and p-chlorophenoxyisobutyricacid. (Received June 25, 1992; Accepted October 20, 1992)     

Regeneration of Lasiurus scindicus from Tissue Culture

Embryogenic callus cultures were initiated from mature embryosof Lasiurus scindicus on Murashige and Skoog's medium supplementedwith 6 mg l^–1 2,4-Dichlorophenoxyacetic acid (2,4-D).These cultures were maintained on 2 mg l^–1 2,4-D. Plantletswere regenerated via somatic embryogenesis when the calli weretransferred onto hormone-free MS basal medium. Young plantswere successfully transplanted to pots and grown to maturityin a greenhouse. Grass, Lasiurus scindicus, Thar Desert, drought tolerant, somatic embryogenesis, plant regeneration     

Studies on the Growth in Culture of Plant Cells: XX. UTILIZATION OF 2,4-DICHLOROPHENOXYACETIC ACID BY STEADY-STATE CELL CULTURES OF ACER PSEUDOPLATANUS L

Omission of 2,4-dichlorophenoxyacetic acid (2,4-D) from batchcultures of sycamore produced an immediate reduction in ratesof cell division and eventually in rates of biomass accumulation.The sequential responses of a chemostat and of turbidostat culturessubjected to gradual withdrawal of 2,4-P were: (i) a transientincrease in biomass accumulation, (ii) increased accumulationof p-coumaric acid, flavonoids, and lignin, (iii) increasedcell aggregation, (iv) reduced rates of cell division, and (v)death. During stepwise reduction of 2,4-D supplied to turbidostatcultures, rates of 2,4-D uptake were reduced when the spentmedium concentration fell to 3?5–1?0 ? 10^–7 M. Underthese conditions the 2,4-D concentration in soluble and insolublecell fractions declined. The growth responses were correlatedwith the spent-medium 2,4-D concentration but not with its concentrationin the intracellular fractions.     

Morphological and physiological characterization of IAA-less-sensitive and IAA-sensitive phases in rooting of Azukia cuttings

In disbudded epicotyl cuttings taken from light grown 5-dayold Azukia angularis Phaseolus angularis) seedlings, all adventitiousrootlets appeared on the second day of incubation. No root primordiawere observed within the first 24 hr and no increase in thenumber of roots occurred after 48 hr. Puromycin (5.5?10^–5M), p-fluorophenylalanine (1?10^–3M),2-thiouracil (2.3?10^–4M) and 2,6-diaminopurine (2?10^–5M)inhibited rooting when applied to cuttings on the second day,but showed no inhibition when applied on the first day. Unlike these inhibitors, pyrithiamine (7.2?10^–5M) inhibitedrooting when it was applied to cuttings on the first day. A rooting promoting effect was observed with actinomycin D (2.4?10^–6M),2,4-dinitrophenol (3?10^–5M) and p-fluorophenylalanine(1?10^–4M) applied to the cuttings on the first day, whereasindoleacetic acid (1.7?10^–4M) showed its promoting effectmost effectively on the second day. ¹Contribution No. 17 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Tokyo, Japan. (Received June 4, 1969; )     

Somatic Embryogenesis and Plant Regeneration from Cultured Leaf Explants of Zea mays

Young leaf segments of Zea mays L. seedlings were cultured onMurashige and Skoog's basal nutrient medium supplemented with2 mg l^–1 2, 4-D and sub-cultured on medium containing8 mg l^–1 2,4-D. Two types of callus tissues appeared—embryogenicand non-embryogenic. The embryogenic callus tissue producednumerous somatic embryos which on transfer to media containinglow amounts of 2,4-D or ABA produced plantlets. Callus tissuesexhibited embryogenic potential for more than 1 year. Zea mays L. cv. Ageti-76, Zea mays L. cv. N-L-D-Comp., maize, leaf, callus, somatic embryogenesis, regeneration     

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)     

The Translocation of 3,5-Dichlorophenoxyacetic Acid in Relation to its Effect on Potato Common Scab

The translocation of 3,5-dichlorophenoxyacetic acid (3,5-D)in the potato plant was studied to clarify the mode of actionagainst potato common scab. 3,5-D was translocated to the tuberfaster and metabolised less than 2,4-D. Translocation to tuberswas proportional to growth and a concentration of 10–25nmol 3,5-D g^–1 fr. wt was attained 8 d after a foliarapplication. Previous evidence indicates this concentrationwas insufficient to be directly toxic to the pathogen Streptomycesscabies and the results suggest that 3,5-D acts in the tuberby altering the host's response to infection.     

The Uptake of Growth Substances: XV. THE DIFFERENTIAL EFFECTS OF PROGRESSIVE CHLORINATION OF PHENOXYACETIC ACID ON ENTRY INTO THE EPIDERMAL AND CUT SURFACES OF STEM SEGMENTS

The comparative patterns of entry into segments with sealedand open ends, excised from etiolated internodes of Pisum sativum,have been examined for phenoxyacetic acid (POA) and its 2-,4-, 2,4-, 2,6-, 3,5-, 2,4,5- and 2,4, 6-chloro derivatives,each containing ¹⁴C in the carboxyl group. Sealing the ends greatly depresses the level of entry, on averagean eight-fold reduction at 9 h. Likewise, the interrelationsbetween the degree of chlorination and uptake potential aredisparate. For segments with exposed cut surfaces the finalcontent is maximal for POA and the 2-chloro compound and minimalfor the 3,5-dichloro derivative (3,5-D) with an eight-fold difference.With sealed ends this difference is reduced to two-fold butwhile 3,5-D accumulates least uptake is now highest for POAand 2,4-D. There are also changes in the order with time. Initially,2,4,5-T penetrates fastest into sealed segments but for segmentswith open ends entry is most rapid for the 4- and 2,4,6-chloroderivatives. Additions of streptomycin and cetyltrimethylammoniumbromide(CTAB) induce differential changes in the patterns of uptake.Where uptake is promoted the enhancement is not restricted toactive auxins. Sealing the ends may alter the nature of theresponse. The likely physico-chemical and metabolic processes concernedin the two routes of entry are discussed and the results comparedwith previous divergent findings on the relationship betweenchemical structure and uptake by Lemna minor and penetrationinto leaves of Phaseolus vulgaris.     

Biochemical Changes Associated with the Growth of Root Tips of Vicia faba in vitro, and the Effect of 2,4-Dichlorophenoxyacetic Acid

Root tips of Vicia faba were cultured for 3 weeks on a semi-solidmedium containing 40 g/l sucrose ±10^–5 M 2:4-dichlorophenoxyaceticacid (2,4-D). Inhibition of root elongation and the stimulationof an actively dividing meristematic zone in the pericycle regionwere observed in the 2,4-D-treated tissues. Biochemical dataon the DNA content and respiration of these root tips correlatewell with the morphological observations. 2,4-D also induceda marked decrease in the -cellulose content of the cell wallsand analyses of the carbohydrate content of the ethanol-solublepool and starch content of the cultured root tips have providedtentative evidence for some of the controlling factors exertedin the presence and absence of 2,4-D.     

The Uptake of Growth Substances: X. THE ACCUMULATION OF PHENOXYACETIC ACID AND 2,4-DICHLOROPHENOXYACETIC ACID BY SEGMENTS OF AVENA MESOCOTYL

Segments of Avena mesocotyl were placed in buffered solutionsof phenoxyacetic acid (POA) or 2,4-dichlorophebnoxyacetic acid(2,4-D), containing carbon-14 in the carboxyl group, and thequantities of radioactivity taken up by the tissues measured.With freshly cut segments in solutions of 2,4-D there is accumulationof carbon-14, but the course of uptake is interrupted by a temporaryphase when some of the accumulated 2,4-D is released to theexternal solution. If after cutting the segments are first pretreatedby placing them for about 15 h in buffer, and then transferredto 2,4-D, there is progressive accumulation with no phase ofnet loss. Pretreated segments absorb greater quantities of either 2,4-Dor POA than freshly excised tissues. Following pretreatmentin buffer the course of uptake of POA is linear but for 2,4-Dthe course is curvilinear. However, after pretreatment withnon-radioactive 2,4-D the subsequent rate of uptake of radioactive2,4-D is constant over long periods. The uptake of radioactive2,4-D is largely independent of the concentration of non-radioactive2,4-D given during pretreatment. When segments which have absorbed 2,4-D-1-¹⁴C are transferredto buffer, a relatively small proportion of the carbon-14, the‘mobile fraction’, is released. The amount releasedfollowing different periods of uptake is constant whereas thelevel of non-mobile carbon-14, the ‘residual fraction’,rises progressively in step with accumulation. The uptake of POA and 2,4-D is accompained by the formationwithin the tissues of other radioactive substances. It is concludedthat the residual fraction is composed, at least in part, ofthese metabolic products and that accumulation and metabolicconversion are inter-connected. Dinitrophenol (DNP) slowly and progressively depresses the uptakeof POA whereas the uptake of 2,4-D is very rapidly arrested.However, after about 2 h, in the continued presence of DNP,uptake of 2,4-D restarts but the rate never attains that ofthe control. These divergent effects of DNP indicate that POAand 2,4-D are accumulated by different pathways.     

Effects of Auxin and Gibberellin on Elongation of Young Hyphae in Neurospora crassa

IAA, 2,4-D and GA₃ promoted the elongation of young hyphae inNeurospora crassa at the optimum concentrations of 10^–6,10^–6 and 10^–4 M, respectively. The effects of IAAand GA₃ were additive. (Received June 17, 1983; Accepted December 22, 1983)     

Studies on the Growth in Culture of Plant Cells: XXI FINE STRUCTURAL FEATURES OF ACER PSEUDOPLATANULS L. CELLS RESPONDING TO 2,4-DICHLOROPHENOXYACETIC ACID WITHLDRAWAL IN TURBIDOSTAT CULTURE

A culture of Acer pseudoplatanus L. grown in the presence ofan equilibrium level of 2,4-dichlorophenoxyacetic acid (2,4-D)of 1?5 ? 10^–7 M (state IV culture) showed, in comparisonwith one of a similar specific growth rate but in which theequilibrium level of 2,4-D was 2?3 ? 10^–6 M (state Iculture), an enhanced degree of cell aggregation, enhanced meancell volume, and the presence of cells giving a generalizedlignin reaction with extracellular lignin-positive material.The state IV culture showed a proportion (10–15 per cent)of cells having ultrastructural features not observed in thestate I culture. Some of the cells, located at the surface ofthe cellular aggregates, were small, rounded, highly cytoplasmic,and rich in rough endoplasmic reticulum. Further within theaggregates there occurred some cells showing abnormal or incompletecytokinesis and having irregularly thickened walls. Locatedcentrally in the aggregates were cells showing massive accumulationsof electron-dense material and with cell walls showing bandsof thickening alternating with thinner wall regions traversedby plasmodesmata. The latter cells are interpreted as cellsshowing intense polyphenol metabolism and imperfect xylogenicdifferentiation.