Phytohormone contents in Corylus avellana and their relationship to age and other developmental processes

Endogenous levels of indole-3-acetic acid, abscisic acid, and cytokinins (Z-type: dihydrozeatin, dihydrozeatin riboside, zeatin, and zeatin riboside; iP-type: N ⁶-isopentenyl adenine and N ⁶-isopentenyl adenosine), were determined in leaves of hazelnut (Corylus avellana L.) (adult material from spring, autumn and forced outgrowth, and juvenile material). Our results showed high levels of indole-3-acetic acid, abscisic acid and total cytokinins in spring samples and low levels of the same hormones in autumn and forced outgrowth materials. The ratios of iP-type/Z-type cytokinins were low in autumn and spring leaves, while they were high in the juvenile and forced outgrowth samples. Both juvenile and forced-outgrowth hazel tissues also showed a high morphogenetic potential, suggesting that the ratio of iP-type/Z-type cytokinins may be a good index of in vitro potential of hazelnut materials.     

Endogenous hormonal content and somatic embryogenic capacity of Corylus avellana L. cotyledons

Endogenous indole-3-acetic acid (IAA), abscisic acid (ABA) and cytokinins [zeatin (Z) zeatin riboside, dihydrozeatin, dihydrozeatin riboside, N⁶-isopentenyl adenine (iP) and N⁶-isopentenyladenine riboside] were evaluated in hazelnut (Corylus avellana L.) cotyledons of different developmental stage and genetic source for their somatic embryogenic capacity. There was an inverse correlation between the embryogenic potential of cotyledons and the degree of maturity of zygotic embryos, the first characteristic being associated with iP-type cytokinins and the second with Z-type cytokinins. Although the differences in total cytokinin, ABA and IAA contents between the cotyledons were small, the IAA/ABA and, mainly, the iP-type/Z-type cytokinin ratios were found to be two good indexes of the embryogenic competence of explants, suggesting that the endogenous hormonal balance is a very important factor defining the in vitro potential of hazelnut cotyledons. Received: 6 January 1997 / Revision received: 3 March 1997 / Accepted 1 April 1997     

Endogenous plant growth regulators in carnation tissue cultures under different conditions of ventilation

Endogenous indole-3-acetic acid (IAA), abscisic acid (ABA) and cytokinins (zeatin, zeatin riboside, dihydrozeatin, (diH)Z, dihydrozeatin riboside, (diH)[9R]Z, N⁶-isopentenyl adenine and N⁶-isopentenil adenine riboside) levels were evaluated in normal (N) and hyperhydric (H) microplants of Dianthus caryophyllus cultured under different aeration conditions in hormone-free liquid medium. The morphological differences between N and H explants grown under ventilated conditions were correlated with differences in their endogenous hormonal levels: after 15 and 30 days of culture, H explants showed lower IAA and ABA contents than N explants, as well as higher cytokinin levels, mainly of (diH)Z and (diH)[9R]Z. This was associated with less tissue differentiation and with an inability of H microplants to survive under ex vitro conditions. However, these relationships could not be observed between H and N explants grown under non-ventilated conditions probably due to the difficulty in discerning the plant status (N or H) and therefore, an underestimation of H microplants. This assumption is supported by the low ability for acclimatization to ex vitro of N plants grown without ventilation.     

In vitro propagation of Cassia angustifolia through leaflet and cotyledon derived calli

High efficiency shoot regeneration was achieved through leaflet and cotyledon derived calli in Cassia angustifolia - an important medicinal plant. Dark brown compact callus was induced at the cut ends of the explants on Murashige and Skoog's (MS) medium augmented with 1 μM N⁶-benzyladenine (BA) + 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Such callus pieces on transfer to cytokinins (BA or kinetin) supplemented medium differentiated shoots within 10 – 15 d. Of the two cytokinins, 5 μM BA was optimum for eliciting morphogenic response in 83.33 and 70.83 % cultures with an average of 4.16 ± 0.47 and 3.70 ± 0.56 shoots in cotyledon and leaflet derived calli, respectively. The addition of 0.5 μM α-naphthaleneacetic acid (NAA) to MS + 5 μM BA further elevated the maximum average number of shoots to 12.08 ± 1.04 and 5.37 ± 0.52 for cotyledon and leaflet calli, respectively. The excised shoots were transferred to a rooting medium containing either IAA (indole-3-acetic acid), IBA (indole-3-butyric acid) or NAA. Nearly 95 % shoots developed an average of 5.4 ± 0.41 roots on half strength MS medium supplemented with 10 μM IBA.     

Mixture screening and mixture-amount designs to determine plant growth regulator effects on shoot regeneration from grapefruit (<Emphasis Type="Italic">Citrus paradisi</Emphasis> macf.) epicotyls

The objective of this study was to improve shoot regeneration from grapefruit. Because many commercially grown citrus types are apomictic, important in vitro applications such as Agrobacterium-mediated transformation commonly use epicotyl explants from in vitro seedlings; thus, adequate adventitious shoot production is an important prerequisite for efficient use of these applications. Eight plant growth regulators were studied—six cytokinins (6-benzylaminopurine, kinetin, zeatin trans-isomer, 6-[γ,γ-dimethylallylamino] purine, zeatin riboside trans-isomer and meta-topolin) and two auxins (α-naphthalene acetic acid and indole-3-acetic acid). An iterative design strategy was followed that included mixture and mixture-amount experimental designs suitable for resolving proportional and concentration effects; in vitro effects of cytokinins and auxins are affected by both proportion and concentration. One-centimeter-long explants were excised from the epicotyl of etiolated, in vitro-grown seedlings. Explants were placed onto experimental formulations and cultured in growth cabinets at 27°C over 6 wk, which included 2 wk in the dark followed by 4 wk in the light. The results indicated that (1) 6-benzylaminopurine or zeatin riboside were the most effective cytokinins for inducing shoot regeneration in citrus; (2) zeatin riboside singly or in combination with indole-3-acetic acid resulted in the highest quality, the greatest number of explants with buds/shoots, and the greatest shoot number; and (3) 6-benzylaminopurine and indole-3-acetic acid improved shoot regeneration vs. 6-benzylaminopurine at a considerably lesser cost than zeatin riboside and indole-3-acetic acid.     

Endogenous hormone levels during direct somatic embryogenesis in Medicago falcata

Endogenous indole-3-acetic acid, abscisic acid and cytokinins (zeatin, zeatin riboside, N-isopentenyladenine and N-isopentenyladenosine) were evaluated in initial explants (leaves) of in vitro propagated plants of alfalfa ( Medicago falcata L.) lines varying in embryogenic capacity and during the somatic embryogenesis process. Fast embryo-genic induction was correlated with high IAA and low ABA levels in the initial explants. No significant differences were observed in the cytokinin contents. Our results suggest that a certain hormone balance is necessary to allow the expression of the embryogenic potential. The consistent stages of the direct somatic embryogenesis are also characterized by changes in hormonal levels.     

Further studies on the effects of different auxins and cytokinins on flower formation in thin cell layers of Nicotiana tabacum: The effects of zeatin riboside, dihydrozeatin and dihydrozeatin riboside

Organogenesis in thin cell layers of Nicotiana tabacum L. was studied in relation to the effects of natural and synthetic auxins in combination with various cytokinins. All cytokinins tested, benzyladenine (BA), kinetin, zeatin (Z), zeatin riboside (ZR), N⁶-Δ²-isopentenyl) adenine (IPA), dihydrozeatin [(diH)Z] and dihydrozeatin riboside [(diH)ZR], seem to be active in flower bud formation. In addition to the initiation of flower buds, vegetative buds or roots were also formed on the explants in the presence of BA, Z or IPA as exogenous cytokinins. Only dihydrozeatin and its riboside stimulated the initation of flower buds alone (as is known for kinetin), especially if supplemented with indole-3-acetic acid (IAA) as exogenous auxin. A high number of explants with flower buds was also found with high cytokinin/2,4-D ratios. In these conditions the presence of (diH)Z yielded the higest number of flower buds per explant.     

Fluctuation in levels of endogenous plant hormones in ovules of normal and mutant cotton during flowering and their relation to fiber development

The contents of indole-3-acetic acid (IAA), gibberellins (GAs), abscisic acid (ABA), and cytokinins were determined in ovules of normal cotton (Tm-1) and a kind of fiber differentiation mutant (Xin) before and after flowering by enzyme-linked immunosorbent assays. It was found that 24 h before flowering, a peak of IAA content was observed in ovules of Tm-1, whereas in ovules of Xin, a low level of IAA was determined. From –1 day (1 day before flowering) to +3 days (3 days after flowering), GA₁₊₃ levels in ovules of Xin were 40–70% lower than those of Tm-1; GA₄₊₇ levels were very low, and there was no visible difference in GA₄₊₇ content between normal and mutant cotton. The ABA content in ovule of Tm-1 decreased by 70% 3 days after flowering, whereas that of Xin only decreased by 20%. The levels of cytokinins in ovules of Tm-1 decreased after flowering, and those of Xin kept up a steady increase.Abbreviations IAA indole-3-acetic acid - GA gibberellin - ABA abscisic acid - ELISA enzyme-linked immunosorbent assay - FW fresh weight - PBS phosphate-buffered saline - iPA isopentenyladenosine - ZR zeatin riboside - DHZR dihydrozeatin riboside - CTK cytokinin     

Effects of water stress on cellular ultrastructure and on concentrations of endogenous abscisic acid and indole-3-acetic acid in Fatsia japonica leaves

Ultrastructural alterations in mesophyll cells as well as variations in bulk leaf endogenous ABA and IAA concentrations were studied in water-stressed field-grown plants of Fatsia japonica. Under water deficit cellular membranes were modified and an increase in vesicles was observed. The main damage to the chloroplasts included thylakoid swelling and disruption of the chloroplast envelope. Concomitant variations in abscisic acid and indole-3-acetic acid were observed. Despite the expected increased in endogenous ABA concentration in relation to water stress, after the highest concentration of ABA, observed at predawn in severely stressed plants (29-1), there was a sharp decline from 2768 pmol g fw^–1 to 145 pmol g fw^–1; thus in severely stressed plants ABA levels were not related to changes in bulk leaf ABA contents. Water stress did not influence the concentrations of indole-3-acetic acid, although the increase in the endogenous abscisic acid concentration could be related with the ultrastructural changes.Abbreviations ABA abscisic acid - IAA indole-3-acetic acid - leaf water potential     

The content of endogenous hormones and sugars in the process of early somatic embryogenesis in the tree fern <Emphasis Type="Italic">Cyathea delgadii</Emphasis> Sternb.

Somatic embryogenesis (SE) of Cyathea delgadii presents a model system for investigating the mechanisms associated with the acquisition of embryogenic competence by single epidermal cells of stipe explants cultured on plant growth regulator-free medium. The present work reveals relationship between endogenous hormone and sugar content in the process of early SE in C. delgadii. By comparing two types of initial explants, i.e. incapable (non-etiolated) and capable (etiolated) of SE, it was established that in etiolated explants, the glucose, fructose, sucrose, and abscisic acid (ABA) contents diminished, but indole-3-acetic acid (IAA) and cytokinins (CKs; i.e. cis/trans zeatin, cis/trans-zeatin riboside, kinetin, kinetin riboside, isopentenyladenosine) contents increased. The ratios between phytohormones revealed that a high concentration of ABA is the main factor inhibiting SE induction. Because of explant excision, a dramatic reduction in concentration of all phytohormones studied was observed, but hormonal balance and sugar content remained almost unchanged. During the 14-day-long culture, the ABA/CKs and ABA/IAA ratios remained constant, whereas the greatest differences were detected for the IAA/CKs and Z-type/iPA cytokinin ratios. Excluding day 6 of culture, cytokinins were found to be the predominant phytohormones over IAA. An almost 12-fold increase in soluble sucrose concentration at day 6 of culture might be the switch to the SE expression phase. Frequent cell divisions leading to somatic embryo formation are clearly associated with increase in trans-zeatin riboside content.     

Phytohormone production and colonization of canola (Brassica napus L.) roots by Pseudomonas fluorescens 6-8 under gnotobiotic conditions

Pseudomonas fluorescens 6-8, a rhizosphere isolate previously shown to enhance root elongation of canola ( Brassica napus L.), was characterized for its ability to produce indole-3-acetic acid and cytokinins in pure culture and in the rhizosphere of canola under gnotobiotic conditions in comparison with the cytokinin-producing strain P. fluorescens G20-18 and its mutant CNT2. Strain 6-8 produced isopentenyl adenosine, zeatin riboside, and dihydroxyzeatin riboside at levels similar to those of G20-18, but only very low concentrations of indole-3-acetic acid. In a gnotobiotic assay canola inoculated with 6-8 and G20-18 had higher concentrations of isopentenyl adenosine and zeatin riboside in the rhizosphere and greater root length than the noninoculated control. The ability of strain 6-8 to colonize canola roots was assessed following transformation with the green fluorescent protein and inoculation onto canola seed in a gnotobiotic assay. Higher populations of strain 6-8 were observed on the proximal region of the root closest to the seed than on the mid and distal portions 9?days after seed inoculation. The ability of P. fluorescens 6-8 to produce cytokinins, colonize the roots of canola seedlings, and enhance root elongation may contribute to its ability to survive in the rhizosphere and may benefit seedling growth.     

Effect of phytohormones on fiber initiation of cotton ovule

In order to study the effect of phytohormones on cotton fiber initiation, contents of four endogenous phytohormones and activities of four related enzymes in ovules (in vivo) of a fuzzless–lintless mutant (fl) and its wild-type (FL) line were measured from 4 days before anthesis (day −4) to 4 days after anthesis (day 4). The results showed that contents of indole-3-acetic acid, gibberellic acid (GA), and zeatin riboside in fl ovules were lower than those in FL ovules. Therefore, indole-3-acetic acid, GA, and zeatin riboside were thought to be the promoters of fiber initiation. Although abscisic acid (ABA) content in fl ovule was slightly higher than that in FL ovule on day 0, which might imply that ABA inhibited fiber initiation. Fiber initiation could also be influenced by enzyme through regulating synthesis and degradation of related phytohormones since fl ovules were significantly higher in activities of indole-3-acetic acid oxidase, cytokinin oxidase and peroxidase, but lower in activity of tryptophan synthetase than those in FL ovules. To test the above hypothesis, exogenous plant growth regulators were also applied for the culture of ovules from fl and FL in vitro. When no regulators were added, no fiber was induced on fl ovule, but a few fibers were induced in FL ovule. Higher total fiber units (TFU) were observed when indole-3-acetic acid and gibberellic acid (GA₃) were applied either separately or in combination to media. TFU did not increased with zeatin riboside alone, but the highest TFU was achieved when zeatin riboside was applied together with indole-3 acetic acid and GA₃, which implied that fiber initiation could be promoted by them as additive.     

Enhanced oxidative stress in the ethylene-insensitive (ein3-1) mutant of Arabidopsis thaliana exposed to salt stress

To better understand the role of ethylene signaling in plant stress tolerance, salt-induced changes in gene expression levels of ethylene biosynthesis, perception and signaling genes were measured in Arabidopsis thaliana plants exposed to 15 days of salinity. Among the genes analyzed, EIN3 showed the highest expression level increase under salt stress, suggesting a key role for this ethylene-signaling component in response to salt stress. Therefore, we analyzed the salt stress response over 15 days (by adding 100 mM NaCl to the nutrient solution) in the ein3-1 mutant compared to the wild-type (Col-0) in terms of growth, oxidative stress markers (lipid peroxidation, foliar pigments and low-molecular-weight antioxidants) and levels of growth- and stress-related phytohormones (including cytokinins, auxins, gibberellins, abscisic acid, jasmonic acid and salicylic acid). The ein3-1 mutant grew similarly to wild-type plants both under control and salt stress conditions, which was associated with a differential time course evolution in the levels of the cytokinins zeatin and zeatin riboside, and the auxin indole-3-acetic acid between the ein3-1 mutant and the wild-type. Despite showing no signs of physiological deterioration under salt stress (in terms of rosette biomass, leaf water and pigment contents, and PSII efficiency) the ein3-1 mutant showed enhanced lipid peroxidation under salt stress, as indicated by 2.4-fold increase in both malondialdehyde and jasmonic acid contents compared to the wild-type. We conclude that, at moderate doses of salinity, partial insensitivity to ethylene might be compensated by changes in endogenous levels of other phytohormones and lipid peroxidation-derived signals in the ein3-1 mutant exposed to salt stress, but at the same time, this mutant shows higher oxidative stress under salinity than the wild-type.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Capsicum chinense</Emphasis> Jacq.

An efficient micropropagation protocol was established for Capsicum chinense Jacq. cv. Umorok, a pungent chilli cultivar. Shoot-tip explants were cultured on Murashige and Skoog (MS) medium containing cytokinins (22.2–88.8 μM 6-benzylaminopurine, BAP, 23.2–93.0 μM kinetin, Kin, or 22.8–91.2 μM zeatin, Z) alone or in combination with 5.7 μM indole-3-acetic acid (IAA). Maximum number of shoots were induced on medium containing 91.2 μM Z or 31.1 μM BAP with 4.7 μM Kin. The separated shoots rooted and elongated on medium containing 2.5 or 4.9 μM indole-3-butyric acid (IBA). Axillary shoots were induced from in vitro raised plantlets by decapitating them. The axillary shoot-tip explants were used for further multiple shoot buds induction. A maximum of about 150 plantlets were obtained from a single seedling. Hardened and acclimatized plantlets were successfully established in the soil.     

A comparative study of different cytokinins on the formation of Rhododendron forrestii Balf. f. ex diels. axillary shoots in vitro

The effect of different concentrations and activities of cytokinins on the morphogenesis of regenerated Rhododendron forrestii Balf. f. ex Diels. shoots taken from nodal segments were tested. We evaluated zeatin, zeatin riboside, izopentyladenine, izopentyladenine riboside, kinetin, kinetin riboside, benzylaminopurine, benzylaminopurine riboside. The experimental results were evaluated by mathematical methods and regression analysis describing the effect of isoprenic and aromatic type of cytokinins. On the basis of this modelling, maximum axillary shoot production was attained with medium supplemented with 2.0 mg·l⁻¹ izopentyladenine riboside, 2.0 mg·l⁻¹ benzylaminopurine and 20 g·l⁻¹ sucrose. Minimal axillary shoots were produced with kinetin and kinetin riboside.     

Morphogenetic potential of foliar explants in Duboisia myoporoides R.Br. (Solanaceae)

The effects of serial combinations of either indole-3-acetic acid, indole-3-butyric acid or -naphthaleneacetic acid (0.5–10.0 mg/l) with either kinetin, 6-benzyl-amino-purine, zeatin or 6-methylaminopurine (0.5–5.0 mg/l) have been investigated to assess the morphogenetic potential of foliar explants of Duboisia myoporoides. Shoot buds developed either directly or via a callus interphase. Combinations involving indole-3-acetic acid with any of the cytokinins were more effective in inducing shoot bud formation compared to those containing indole-3-butyric acid or -napthalenacetic acid as an auxin. Among cytokinins, zeatin, kinetin and 6-benzylamino-purine were equally effective for shoot formation. However, optimum response with zeatin could be achieved at low concentrations (0.5–2.0 mg/l), while kinetin and 6-benzylamino-purine exhibited comparable efficacy at higher levels (3.0–5.0 mg/l). 6-Methylaminopurine proved least effective in all concentrations and combinations tested. Rooting of the differentiated shoots was readily achieved with -naphthaleneacetic acid alone (0.5 mg/l) after changing the physical form of the medium from gel to static liquid. Regenerated plantlets were transferred to pots and grown to maturity in the field with a high rate of survival (80–90%).Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MAP 6-methylaminopurine - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic acid - PVP polyvinyl pyrrolidone     

An improved bioassay for cytokinins using cucumber cotyledons

The cucumber cotyledon greening bioassay is frequently used for detecting cytokinins. Beneficial modifications of the original technique included using 5-day-old cucumber (Cucumus sativus L., cv. National Pickling) cotyledons treated with combinations of 40 millimolar KCl and various concentrations of cytokinins. A dark incubation period of 20 hours was followed by an exposure to light for 3.5 hours. Under these conditions, extremely low (0.0001 milligram per liter) concentrations of N⁶-benzyladenine, zeatin, kinetin, or zeatin riboside can be detected. Of the four cytokinins tested, kinetin appeared to be the least active. With these improvements, the assay is 10 times more sensitive than is the previously described cucumber cotyledon greening bioassay for cytokinins.     

Grain filling pattern and cytokinin content in the grains and roots of rice plants

Grain filling patterns and their relationships withzeatin (Z), zeatin riboside (ZR), indole-3-acetic acid(IAA) and gibberellin (GA) contents in the grains androots during grain development were examined in sixrice (Oryza sativa L.) genotypes grown in thefield and in water culture. Three grain fillingpatterns based on the filling rate of superior andinferior spikelets were observed, i.e., fastsynchronous: all spikelets started filling early andfast at the early filling stage; slowsynchronous: all spikelets filled slowly at the earlyfilling stage and reached the maximum filling ratelate; and asynchronous: superior spikeletsstarted filling and reached the maximum filling ratemuch earlier than the inferior ones. The order ofgrain filling percentage in the three types of grainfilling patterns was: fast synchronous >asynchronous > slow synchronous. Changes in Z + ZRcontents in the superior and inferior spikelets wereassociated with the grain filling patterns. Grainfilling percentage was significantly correlated withZ + ZR contents in the grains and roots at the earlyand middle grain filling stages. IAA and GA(GA¹ + GA³ + GA⁴)contents in the grains and roots were notsignificantly correlated with grain fillingpercentage. The results suggest that cytokinins in thegrains and roots during the early phase of graindevelopment play an important role in regulating grainfilling pattern and consequently influence grainfilling percentage.     

Plantlet regeneration from primary callus cultures of Lilium brownii F.E.Br. ex Miellez var. giganteum G. Y. Li & Z. H. Chen,a rare bulbous germplasm

Lilium brownii F.E.Br. ex Miellez var. giganteum G. Y. Li & Z. H. Chen, an endangered valuable genetic resource, was used to establish and optimize a callus propagation system and to investigate the effects of internal and external phytohormones for the purpose of germplasm conservation. Of the combinations and concentrations of auxins and cytokinins examined, Murashige and Skoog (MS) medium supplemented with 8 g L⁻¹ agar, 30 g L⁻¹ sucrose, 0.45 μM 2,4-dichlorophenoxyacetic acid, 2.69 to 5.37 μM α-naphthaleneacetic acid, and 0.44 μM 6-benzyladenine, 0.45 μM thidiazuron, and 0.28 μM zeatin riboside generated the best results, effectively promoting callus proliferation. Four callus types could be discriminated, of which type A (yellowish, granular) and type B (yellow, medium-granular) were dry, friable, and grew well. Periodic acid-Schiff staining revealed small and regular cells, with numerous starch granules surrounding each nucleus. In culture, callus clumps produced an average of 14.33 shoots under “MS + 7-d-dark–light” treatment with 100% regeneration frequency. Bulblets formed within 60 d after shoot transfer to bulblet formation medium. Type A and B callus was likely to be embryogenic, according to morphology, cytology, and high shoot regenerating capacity. Examination of endogenous phytohormone levels showed that the abscisic acid to indole-3-acetic acid (ABA/IAA) ratio gradually increased with increasing diameter of callus clumps treated with all exogenous phytohormones, except zeatin riboside, leading to the hypothesis that callus induction competence was closely associated with endogenous ABA/IAA ratio. This first report should assist further genetic studies of this rare Lilium and other bulbous plants.

     

Organogenesis in leafy spurge (Euphorbia esula L.)

Summary All parts of leafy spurge seedlings can be regenerated when isolated and placed onto B5 medium. One-centimeter isolated hypocotyl segments were tested successfully for their usefulness as a bioassay system by comparing the response of auxins, herbicides, and cytokinins. Indole-3-acetic acid (IAA) was the most effective auxin to stimulate root formation. IAA was effective whether the hypocotyl segments remained on the same medium up to 60 days, or the segments were transferred to basal media after 2 or 5 days (pulse treatment). Pulse treatments with the other auxins resulted in stimulation of root formation; continuous or 5-day pulses of higher concentrations of indole-3-butyric acid,&#x03B1;-naphthaleneacetic acid and especially 2,4-dichlorophenoxyacetic acid and picloram formed excessive callus instead of roots. Picloram did not stimulate root formation, whether the treatment was continuous or pulse-treated. No roots formed with continuous picloram at 0.1 mg/liter or greater, but transfer to basal media did result in root and shoot formation at about 50% of the number formed on the controls. Lesser picloram concentrations had no effect. Shoots formed readily on untreated (control) segments, but continuous treatment with all three cytokinins, kinetin, zeatin, and zeatin riboside, increased the numbers of shoots about equally. Root formation was inhibited by the cytokinins at the higher concentrations (0.1 to 0.2 mg/liter). With the exception of a 5-day pulse of 0.04 mg/liter IAA, the auxins did not stimulate shoot formation, but generally inhibited shoot formation, even in pulse-treated cultures.