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.     

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     

Changing cell aggregations and lignification in tobacco suspension cultures

The formation and dissociation of cell aggregates in tobaccocell suspensions were analyzed during 2,4-dichlorophenoxyaceticacid (2,4-D) (10^–6 M) stock culture and during 6-benzyladenine(BA) (10^–6 M) culture, for their effects on lignification. Aggregates were fractionated according to size by sieving them.Each fraction was cultured in both media and changes in thedistribution of cells in the cluster fractions were followedduring culture, after which the formation and dissociation ratesof cells in aggregates were estimated. There was a significant difference in the formation and dissociationof cell aggregates between the 2,4-D culture and the BA culture.In the 2,4-D culture, aggregates dissociated to smaller onesor to single cells in the early growth stage, and then grewinto large aggregates. Moreover, most cells had a uniform capacityfor aggregate formation. In the BA culture, aggregates grewrapidly to a large size in the early growth stage. Lignification and tracheary element formation in the BA culturewere especially stimulated in large aggregates derived fromthe large aggregates of the 2,4-D culture. Results of successivecultures of a particular cell aggregate fraction indicated thatmost cells had the potential to lignify or to differentiatetracheary elements. Thus, the switch-on of lignification ortracheid formation in the BA culture was determined only bycells in aggregate. (Received October 22, 1977; )     

Effect of Auxin and Gibberellin on Conidial Germination in Neurospora crassa II. "Conidial Density Effect" and Auxin

Conidial germination in liquid shake culture of Neurospora crassawas affected by the number of conidia in the medium (conidialdensity effect) with the optimum density being around 2?10⁶conidia/ml. The conidial density effect at 2?10⁴ and 2?10⁵ conidia/mlwas eliminated by the addition of auxin, IAA or 2,4-D with theoptimum concentrations of IAA and 2,4-D being around 10^–6M. IAA and 2,4-D had no effect on the effect at 2?10⁷ conidia/ml.An active substance(s) for conidial germination which was relatedto the conidial density effect was detected in the cell-freefiltrate of the germination medium, and was found to be non-dialyzableand thermolabile. At the early stage of germination, the concentrationof active substance(s) in the medium increased in proportionto the conidial density and reached a supraoptimum amount forgermination at 2?10⁷ conidia/ml. IAA (10^–6M) enhancedthe concentration. Endogenous auxin concentrations in filtratesof the germination media containing 2?10⁵, 2?10⁶ and 2?10⁷ conidia/mlwere 5.8?10^–12, 4.6?10^–11 and 1.7?10^–10M IAAequivalent, respectively. The conclusion reached was that auxinmay control conidial germination with mediation by the activesubstance(s). (Received June 8, 1982; Accepted September 27, 1982)     

Effects of Culture Conditions on Cell Division and Composition of Regenerated Cell Walls in Vinca rosea Protoplasts

Protoplasts prepared from suspension-cultured Vinca rosea cellswere grown in a liquid medium, and the effects of osmolarityand growth regulators on cell division and on the compositionof regenerated cell walls were investigated. The concentrationof mannitol optimal for cell division was 0.3–0.4 M. Thepresence of 2,4-D was essential for cell division, and 6-benzylaminopurine(BAP) enhanced cell division at concentrations of 0.03–0.1ppm. However, the composition of regenerated cell walls wasabnormal under suspension culture; the predominant sugar wasglucose, indicating that the regenerated cell walls consistedmostly of glucans, and that the other cell-wall components werereleased into the medium. Mannitol, 2,4-D, and BAP at variousconcentrations did not significantly affect the sugar compositionof the regenerated cell walls. Compared with liquid culture,cell division was stimulated when protoplasts were culturedon agar, and their regenerated cell walls had a compositionsimilar to that of the original culture. The importance of thephysical environment for the deposition of polysaccharide componentsin cell walls and the interrelationship between cell divisionand the regeneration of cell walls by protoplasts are discussed. ¹ Present address: Division of Environmental Biology, NationalInstitute for Environmental Studies, Yatabe, Tsukuba, Ibaraki305, Japan. (Received June 10, 1981; Accepted December 12, 1981)     

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.     

Wheat Callus Culture: the Initiation, Growth and Organogenesis of Callus Derived from Various Explant Sources

Callus was obtained from mature excised embryos of wheat, fromnodal and internodal stem segments and from rachis segmentsusing the medium of Murashige and Skoog(1962)(M medium), containing1-0mg l^–1 2,4-D, and from immature embryos using the mediumof Green and Phillips (1975) containing 2 mg l^–1 2,4-D.Callus yield from mature embryos depended upon the cultivarused. No callus could be obtained from leaf segments. Callusderived from mature embryos and nodal stem segments was successfullymaintained by serial sub-culture on the M medium containing2,4-D for up to 3 years although its growth rate declined toa lower level as culture proceeded. Such cultures consistently produced roots when transferred toa medium containing a low level of 2,4-D or no 2,4-D. The presenceof the auxin was essential for continued proliferation of thecallus tissue. Shoot initiation was infrequent, did not occurafter the first few sub-cultures and could not be enhanced byvarious auxin and cytokinin additions to the medium. Callusderived from immature embryos did not have an enhanced potentialfor shoot initiation. Triticum aestivum, wheat, callus culture, organogenesis     

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; )     

The Growth, Anatomy and Morphogenetic Potential of Callus and Cell Suspension Cultures of Hevea brasiliensis

Callus cultures have been initiated from stem explants of youngplants of Hevea brasiliensis and maintained over long periodsat 30 ?C by serial subculture in Murashige and Skoog mediumcontaining 2 mg 1^–1 2,4-D and 0.5 mg 1^–1 kinetin.Newly-initiated cultures spontaneously initiated roots but,on serial subculture, this property was lost and the culturesbecame heterogeneous (consisting of proliferating light segmentsand darker compact non-growing segments). Serially propagatedcultures continued to differentiate a few scattered latex vesselscontaining particulate material similar to that in the rootlaticifers. This callus (O callus) did not yield a growing cellsuspension when transferred to agitated liquid medium. However,the large cell aggregates which could be recovered after twopassages in liquid medium, when again grown on solid mediumyielded a highly friable light-coloured fast-growing homogeneouscallus (R callus) which retained its distinctive character onsubculture. This callus when transferred back to agitated liquidmedium yielded a fine rapidly growing cell suspension culturewhich could be serially propagated at 30 ?C in the same mediumas that used for callus culture. Both the O and R cultures were2,4-D dependent, but differed in their responses to 2,4-D. Bothretained their diploid character when serially propagated. Serially-propagatedsuspensions came to contain a proportion of polyploid cells.When the suspensions were maintained for several months withoutsubculture the larger cell aggregates which developed gave riseto embryo-like structures. Attempts to promote the further developmentof these embryo-like structures into plantlets were unsuccessful.     

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)     

In vitro Propagation of Red Cabbage (Brassica oleracea L. var. capitata)

By manipulation of various growth regulators and physical conditions,plants have been regenerated from excised roots, stem segments,cotyledons, leaves, and callus cultures of red cabbage (Brassicaoleracea var. capitata) grown under in vitro conditions. Shootbuds were induced on isolated root segments (1 cm long) culturedon Murashige and Skoog's medium and the frequency of bud formationwas greatly enhanced by the addition of kinetin (0.5 part 10^–6).Callus obtained from the seeds, cotyledons, and hypocotyl segmentscultured on a medium fortified with 2,4-D (1 part 10^–6),kinetin (0.1 part 10^–6), and coconut milk (10%, v/v) hasbeen repeatedly subcultured. The callus is slow growing, andon transference to a kinetin (2 parts 10^–6) and IAA (2parts 10^–6) medium underwent morphogenesis to give riseto plants. The significance of the propagation of red cabbageby in vitro culture is pointed out.     

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     

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)     

The Effect of 2,4-Dichlorophenoxyacetic Acid and Related Compounds on the Fine Structure of the Primary Leaves of Phaseolus vulgaris

The epidermal cuticle of the abaxial surface of the primaryleaves of Phaseolus vulgaris used for these experiments is notmore than 0.15 µm thick except on the walls of basal cellsof epidermal hairs. The cuticle apparently has no internal structure;neither channels or canals can be seen passing through it. An examination of the fine structure of leaf tissue 24 h afterthe application of 1.6 x 10^–3 M solutions of 2-chlorophenoxyaceticacid (2-CPA), 4-chlorophenoxyacetic acid (4-CPA), 2,6-dichlorophenoxyaceticacid (2,6-D), and 2,4-dichlorophenoxyacetic acid (2,4-D) hasshown that 2,4-D alone, and then only in the light, induceschanges in the morphology and internal structure of the chloroplast.As early as 4 h after application, there is an apparent breakdownin the structure of the membrane systems of the cells of theepidermis, palisade, and mesophyll. After 8 h the chloroplastsare distorted and contain electron-dense granules, and the cellsappear to be plasmolysed. Subsequently these changes becomemore pronounced. It is possible that the specific disturbancesbrought about by 2,4-D reflect its phytotoxic properties.     

Induction of DNA synthesis and callus formation from tuber tissue of Jerusalem artichoke by 2,4-dichlorophenoxyacetic acid

Cellus induction was observed from Jerusalem artichoke tubertissue on a synthetic medium containing 2,4-D at 10^–6,10^–5 (optimum conc.) and 10^–4 M. The first DNA synthesis(thymidine incorporation) was observed only at 2,4-D concentrationsof 10^–5 to 10^–4M. In 10^–5 M 2,4-D treatedtissue, DNA synthesis increased after a 20 hr lag and reacheda maximum at 36 hr, after which it decreased. Actinomycin Dand 8-aza-guanine; inhibitors of RNA synthesis, inhibited DNAsynthesis completely. 2,4-D caused the characteristic changesin RNA and protein syntheses. In comparison with the control,RNA and protein syntheses were first repressed then inducedbefore the peak of DNA synthesis. Treatment with cycloheximide(10^–4M) for one hour before inoculation inhibited proteinsynthesis completely for 12 hr; consequently DNA synthesis wasalso delayed. The results suggest that RNA and protein synthesesneeded for callus induction are regulated by 2,4-D in the firstDNA synthesis. (Received July 19, 1973; )     

Modification in Cell Shape Unrelated to Cellulose Microfibril Orientation in Growing Thallus Cells of Chaetomorpha moniligera

Cellulose microfibril orientation patterns in thallus cellsof Chaetomorpha moniligera were studied, and the relationshipbetween the microfibril and the peripheral microtubule arrangementsduring cell-shape modification by colchicine was examined. Inthe cuttings from growing thalli, linearly arranged cylindricalcells developed into cask-shaped cells during 4–6 daysof culture at 27?C. In the cylindrical cells, microfibrils formingthe innermost portion of the wall were arranged alternatelyin longitudinal and transverse directions, but peripheral microtubuleswere always arranged only in a longitudinal direction. Thesefeatures were also noted in the cask-shaped cells. Colchicineat 10^–3M and 3?10^–3M accelerated both cell expansionand wall thickening with matrix deposition, but the directionsin which both microfibrils and microtubules were arranged werethe same as those of the cylindrical cells. These results indicatethat (1) the microfibril and microtubule arrangements of Chaetomorphaare not necessarily correlated, (2) changes in cell shape ofChaetomorpha are not necessarily accompanied by changes in thearrangement of cell-wall microfibrils, and (3) colchicine playsa role in the loosening and thickening of cell walls by enhancingmatrix deposition. (Received June 2, 1986; Accepted February 13, 1987)     

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     

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)     

Frond and flower production in Lemna gibba G 3 in presence of respiratory inhibitors

Effects of respiratory inhibitors on frond and flower productionin light culture of a long-day duckweed, Lemna gibba G 3, wereinvestigated. The inhibitors examined could be divided into3 groups based on their specific actions: (A) 2,4-Dinitrophenol(10^–6M), arsenate (10^–4M), malonate (10^–2M),o-phenanthroline (10^–6M), ,'-dipyridyl (10^–5M) andazide (10^–6M) inhibited flower production by suppressingthe rate of flower production without affecting the inductionperiod. Frond production, however, was promoted by these reagents.Effective time of application came one day after the end ofthe induction period. (B) Iodoacetate (10^–6M) and fluoride(10^–4M) inhibited both flower production and, less significantly,frond production. Reduced rate of flower production was responsiblefor the inhibition of flowering. Effective time of applicationpreceded by one day that of A group inhibitors. (C) Salicylaldoxime(10^–6M), diethyldithiocarbamate (10^–6M) and 8-hydroxyquinoline(10^–7M) enhanced flower production by reducing the lengthof the induction period, and simultaneously slightly inhibitedfrond production. Effective time of application was the latterhalf of the induction period. The implications of these findingsare discussed with special reference to the component processesinvolved in photoperiodic induction of flowering in duckweed. (Received March 27, 1969; )