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Shoot elongation in many coniferous species is predetermined during bud formation the year before the shoot extends. This implies that formation of the primordial shoot within the bud is the primary event in annual shoot growth. Hormonal factors regulating bud formation are consequently of utmost importance. We followed the levels of the endogenous cytokinins zeatin riboside (ZR) and isopentenyladenosine (iPA) in terminal buds, whorl buds and lower lateral buds of the uppermost current-year whorl shoots of 15- to 20-year-old trees of Norway spruce [Picea abies (L.) Karst.] from June to September. Cytokinins were isolated with affinity chromatography columns, purified by high performance liquid chromatography, and quantified by ELISA. The level of ZR was low in June but increased gradually in all buds until September. Throughout the measurement period, the ZR level was highest in terminal buds and lowest in the scattered lateral, buds, with the whorl buds intermediate. The level of iPA peaked in July and decreased later without any consistent differences among the three classes of buds. The development of different kinds of buds was followed by scanning electron microscopy. We found that bud growth was greatest during August and September. The final size of primordial shoots within the buds varied considerably and the weight of the terminal bud was three times that of the whorl buds and more than five times that of the other lateral buds. We conclude that the increase in ZR level during the period of active bud development is indicative of the importance of cytokinin for this process. Furthermore, the positive correlation between the level of ZR and bud growth during the period of predetermination of next year's branch growth suggests that this hormone indirectly controls the form of single branches in the spruce tree.

In conifers such as Norway spruce, the extent of shoot growth is predetermined by the size and number of embryonal organs of the buds laid down the previous year. As it is known that cytokinins have a key role in bud development a possible hypothesis is that the level of cytokinin in the buds during their formation determines their size and complexity. As a first step to test this hypothesis we compared cytokinin levels in buds of different size of annual shoots from 15- to 20-year-old trees of Picea abies (L.) Karst. Apical buds from the leaders, and from branches in lower parts of the trees, were collected in April, July and August. The difference in size of the buds and the shoots growing from them was considerable in these three positions. Extracts were purified by immunoaffinity columns, and the retained compounds were separated by high-performance liquid chromatography (HPLC). Quantification was made by enzyme-linked immunosorbent assay (ELISA) ; and the accuracy of this method was checked by measurements with liquid chromatography-mass spectrometry (LC-MS) and UV absorption. Zeatin riboside (ZR) was the most abundant cytokinin, but isopentenyladenosine (iPA) was also present in all samples. The large apical bud of the leader contained much higher cytokinin concentrations than the considerably smaller buds from lower positions, and during the period of secondary growth in July, similar relationships were found for annual stem tissue from different positions. The possible role of ZR as a controlling factor in bud development and apical control is discussed. Our conclusion is that the level of zeatin-type cytokinins appears to play an important role in the establishment of differences in bud size and, thereby, the architecture of the tree crown.

Cuttings of pea (Pisum sativum L. cv Marma) were treated with 1-aminocyclopropane-l-carboxylic acid (ACC). This treatment caused increased ethylene production and reduction of root formation. The effect of 0.1 mM ACC on the level of endogenous indole-3-acetic acid (IAA) in the rooting zone and in the shoot apex was analyzed by gas chromatography-single ion monitoring mass spectrometry or by high pressure liquid chromatography with fluorimetric detection (HPLC). Concentrations of indole-3-acetylaspartic acid (IAAsp) in the stem bases were also determined using HPLC. The ACC treatment had little effect on the IAA level in the base measured after 24 h, but caused a considerable decrease during the 3 following days. IAAsp increased in the base on days 1, 2 and 3 and then declined. The build up of IAAsp in the base was not affected by ACC during the first two days of the treatment, but later this conjugate decreased more rapidly than in controls. No effect of the ACC treatment was found on the level of IAA in the apex. IAA (1 µM) applied to the cuttings during 24 h reduced the number of roots formed. The possibility that IAA-induced ethylene is involved in this response was investigated. Our results support earlier evidence that the inhibitory effect of ethylene on rooting in pea cuttings is due to decreased IAA levels in the rooting zone. The inhibitory effect of applied IAA is obtained if the internal IAA level is maintained high during the first 24 h, whereas stimulation of rooting occurs if the internal IAA level remains high during an extended period of time. Our results do not support the suggestion that ethylene mediates the inhibitory effect of applied IAA.

Etiolated pea seedlings (Pisum sativum L. cv. Weibull's Marma) were used to investigate the effects of exogenous cytokinins on root growth. Benzylaminopurine (BAP) added to the growth solution inhibited the elongation and formation of lateral roots and stimulated swelling of the root tips. Similar effects were obtained with zeatin. The effects were obtained over a wide concentration range down to 0.01 μM. Growth responses appeared only after treatment for several hours, and the duration of treatment had an important influence on the degree of the effects. BAP caused a moderate increase in ethylene production as measured in excised 10-mm-long root tips. Lowering ethylene production by treatment with cobalt ions counteracted both the inhibition and swelling caused by BAP. Treatment with silver ions also reversed the effect to some extent, indicating that ethylene is involved in the response of the roots to BAP. To further study the involvement of the increased ethylene production in the elongation and swelling response, the effects were compared with those obtained after application of 1-aminocyclopropane-1-carboxylic acid (ACC) in relation to the ethylene produced from this compound. This comparison showed that the increase in ethylene production caused by BAP was too low to explain the response of the roots. However, ACC treatment caused a considerable lowering of the content of indole-3-acetic acid (IAA) in the root tips, whereas BAP did not; instead, BAP increased the amount of IAA per root tip. It is concluded that cytokinins influence growth processes in roots via several mechanisms. A synergistic interaction between endogenous IAA, maintained at a high level by the cytokinin treatment, and the increased ethylene levels appears to explain most of the cytokinin effects during the first day of treatment.

The influence of nitrate availability on growth of seminal roots, and root cytokinin levels, was studied in barley (Hordeum vulgare L. cv Golf). Nitrate was continuously supplied to initially N-starved seedlings at relative addition rates (RA) of 0.03 to 0.21 per day (standard cultures) or at RA 0.09 per day in split root cultures with the nitrate additions distributed in ratios of 100:0 or 80:20 to the two subroots. Data were collected both during a phase of acclimation (first 10 days of N additions) and in the acclimated stage (>10 days after onset of N additions). Limitation of whole-plant growth was observed at RA

Levels of endogenous indole-3-acetic acid (IAA) and indole-3-acetylaspartic acid (IAAsp) were monitored in various parts of leafy cuttings of pea (Pisum sativum L. cv. Marma) during the course of adventitious root formation. IAA and IAAsp were identified by combined gas chromatography—mass spectrometry, and the quantitations were performed by means of high performance liquid chromatography with spectrofluorometric detection. IAA levels in the root forming tissue of the stem base, the upper part of the stem base (where no roots were formed), and the shoot apex remained constant during the period studied and were similar to levels occurring in the intact seedling. A reduction of the IAA level in the root regenerating zone, achieved by removing the shoot apex, resulted in almost complete inhibition of root formation. The IAAsp level in the shoot apex also remained constant, whereas in the stem base it increased 6-fold during the first 3 days. These results show that root initiation may occur without increased IAA levels in the root regenerating zone. It is concluded that the steady-state concentration is maintained by basipetal IAA transport from the shoot apex and by conjugation of excessive IAA with aspartic acid, thereby preventing accumulation of IAA in the tissue.

The influence of exogenous indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) on the internal levels of these auxins was studied during the first 4 days of adventitious root formation in cuttings of Pisum sativum L. The quantitations were done by high performance liquid chromatography with spectrofluorometric detection. IBA, identified by combined gas chromatography-mass spectrometry (GC-MS), was found to naturally occur in this plant material. The root inducing ability of exogenous IBA was superior to that of IAA. The IAA level in the tissue increased considerably on the first day after application of IAA, but rapidly decreased again, returning to a level twice the control by day 3. The predominant metabolic route was conjugation with aspartic acid, as reflected by the increase in the level of indole-3-acetylaspartic acid. The IBA treatment resulted in increases in the levels of IBA, IAA, and indole-3-acetylaspartic acid. The IAA content rapidly returned to control levels, whereas the IBA level remained high throughout the experimental period. High amounts of indole-3-butyrylaspartic acid were found in the tissue after feeding with IBA. The identity of the conjugate was confirmed by (1)H-nuclear magnetic resonance and GC-MS. IBA was much more stable in solution than IAA. No IAA was detected after 48 hours, whereas 70% IBA was still recovered after this time. The relatively higher root inducing ability of IBA is ascribed to the fact that its level remained elevated longer than that of IAA, even though IBA was metabolized in the tissue. Adventitious root formation is discussed on the basis of these findings.

Ethylene regulation of cell division in the vascular cambium and cell wall formation was studied in hypocotyls of Norway spruce (Picea abies [L.] Karst.) seedlings. Cuttings from 6-week-old seedlings were placed in water culture to which compounds affecting the synthesis and action of ethylene were added. After a 3-week treatment period, growth, ethylene production, morphology and cell wall composition of the hypocotyls were determined. Addition of high concentrations of the potent ethylene releasing agent 2-chloroethylphosphonic acid (ethrel), which increased ethylene emission by more than twice compared to control plants, inhibited the expansion of xylem cells while stimulating the incorporation of cell wall material, especially cellulose. Addition of small amounts of ethrel, which slightly stimulated ethylene emission, led to increases in the size of xylem cells, the amount of phloem tissue and the number of intercellular spaces in the cortex, and thus to increased hypocotyl diameter. However, no significant change in cell wall composition was detected. When ethylene production was decreased by adding Co2+ to the nutrient solution, differentiation of new xylem was disturbed, but the rate of cell division was not affected. Although the incorporation of cell wall material was inhibited, the proportions of lignin and cellulose in the wall appeared to remain unchanged. Silver ions stimulated the expansion of both xylem and cortex cells, but had no significant effect on cell wall formation. We conclude that ethylene has a role in regulating the incorporation of wall carbohydrates.

Cuttings from seedlings of Picea abies L. (Karst.) grown under high light (HL). rooted slowly and incompletely, whereas rooting in cuttings from seedlings grown under low light (LL) was rapid and almost complete. Immunoaffinity chromatograpriy (IAC) with antibodies against isopentenyl adenosine ([9-RIiP) and trans-zeatin riboside (I9R]Z) was used to isolate cytokinin-like compounds from extracts of the cuttings. These compounds were separated by HPLC and quantified with a UV-detector. [9R]iP-type and [9R]Z-type compounds were also quantified using an enzyme immunoassay (ELISA). Levels of both [9R]iP-type and [9R]Z-type cytokinins were considerably higher in seedlings grown under HL than in those grown under LL. An unidentified compound (X), occurring in high amounts in the hypocotyls of HL seedlings and isolated by IAC and HPLC, inhibited rooting in cuttings from LL seedlings. Compound X was metabolised during the rooting period. Although X was retained on the imnimmoaffinity column, it did not bind to the antibodies to the degree necessary to allow quantification by ELISA. Treatment with acid phosphatase (EC 3.1.3.2) and β-glucosidase (EC 3.2.1.21) converted X into other unknown compounds. Some of these cross-ieacted with [9R)iP antibodies in ELISA.

Cuttings were taken from 4-week-old seedlings of Norway spruce (Picea abies L. Karst.) raised at two different irradiation levels. Rooting experiments showed that root formation was increased by the ethylene formed by adding 1-aminocyclopropane-1-carboxylic acid ACC or Ethrel, especially in the slowly rooting cuttings grown under high light (HL). Cobaltousion. an ethylene synthesis inhibitor, delayed rooting, especially in the easily rooted cuttings grown under low light (LL). Compounds isolated from the cuttings using immunoaffinity chromatography, on a column with antibodies against cytokinins, and separated by HPLC decreased in amount during the first week of the rooting period. An increase in ethylene production accelerated this process, especially in cuttings grown under HL, whereas cobaltous ion delayed it. We suggest that ethylene stimulates rooting by enhancing the degradation of cytokinins.

Light inhibits root elongation, increases ethylene production and enhances the inhibitory action of auxins on root elongation of pea (Pisum sativum L. cv. Weibulls Marma) seedlings. To investigate the role of ethylene in the interaction between light and auxin, the level of ethylene production in darkness was increased to the level produced in light by supplying 1-aminocyclopropane-1-carboxylic acid (ACC) or benzylaminopurine (BAP). Ethylene production was measured in excised root tips after treatment of intact seedlings for 24 h, while root growth was measured after 48 h. Auxin, at a concentration causing a partial inhibition of root elongation, did not increase ethylene production significantly. A 4-fold increase in ethylene production, caused either by light, 0.1 μM ACC or 0.1 μM BAP, inhibited root elongation by 40–50%. The auxins 2,4-dichlorophenoxyacetic acid and indolebutyric acid applied at 0.1 μM inhibited root elongation by 15–25% in darkness but by 50–60% in light. Supply of ACC or BAP in darkness enhanced the inhibitory effects of auxins to about the same extent as in light. The inhibition caused by the auxins as well as by the BAP was associated with swelling of the root tips. ACC and BAP treatment synergistically increased the swelling caused by auxins. We conclude that auxin and ethylene, when applied or produced in partially inhibitory concentrations, act synergistically to inhibit root elongation and increase root diameter. The effect of light on the response of the roots to auxins is mediated by a light-induced increase in ethylene production.

Levels of isopentenyladenosine and zeatin riboside were monitored in buds and needles of Scots pine (Pinus sylvestris L.) seedlings grown under controlled climatic conditions and in field-grown trees. Extracts were purified by immunoaffinity chromatography and high-performance liquid chromatography. Cytokinin levels were quantified with an enzyme-linked immunosorbent assay. The cytokinin content was low in buds and needles of dormant seedlings but increased during dormancy release, reaching peak values in buds just before resumption of shoot growth. Samples collected in the field also showed a marked increase in the levels of cytokinins just prior to bud burst. Further, the biological activity of applied cytokinins in activating the dormant buds of Scots pine is shown. The results indicate a probable role of cytokinins in seedlings during dormancy release.

The relationship between endogenous cytokinin content and relative growth rate (RGR) was studied in cultures of Lemna gibba L. G3 supplied with daily doses of mineral nutrients that were increased exponentially over time. At the optimal level of nutrient supply the RGR was 30–35% day-1. The RGR was regulated by adjusting the rate of nitrogen supply, or it was restricted by addition of 0.5 μM abscisic acid (ABA). Another approach used to investigate the specific roles of nitrogen (N) and phosphorus (P), was to transfer optimally growing plants to media without N or P but otherwise complete. The plants were harvested at regular intervals for determination of the RGR and levels of cytokinins of the isopentenyladenosine (iPA) and zeatinriboside (ZR) types with an enzyme-linked immunosorbent assay (ELISA). Levels of both iPA- and ZR-type cytokinins decreased when nitrogen was applied to cultures in growth limiting amounts. The cytokinin levels decreased more rapidly than the RGR when either N or P was lacking in the medium, suggesting an early influence of nutrient availability on cytokinin levels which in turn may induce adaptive response by the plant. RGR retardation induced by ABA did not affect cytokinin levels during the first 4 days of the treatment, and the later effects were small. The experiments gave no indication that ABA is involved in the adaptation response of Lemna plants to nutritional stress.

Cuttings of Norway spruce seedlings were placed in liquid media containing various concentrations of Ca 2+ . The effects of Ca 2+ concentrations on the deposition of total cell wall material as well as on the deposition of cellulose, lignin and non cellulosic polysaccharides in the hypocotyls were investigated

The formation of adventitious roots in pea (Pisum sativum L) cuttings was studied in relation to the endogenous concentration levels of auxin and cytokinin. Now we are engaged in work to identify genes involved in the rooting process and regulated by these two hormones.

Ethylene concentrations were determined in gas samples extracted from sealed holes made in the sapwood and heartwood of stems of 70-100-year-old Scots pine (Pinus sylvestris L). Gas could be collected from the heartwood holes by lowering the pressure by means of a large syringe. However, attempts to extract gas from air spaces in the sapwood with the same technique failed, presumably because of lack of an interconnected system of gas-filled canals. High ethylene concentrations, usually in excess of 1 ppm, accumulated in the sapwood holes within one day after sealing. Ethylene concentrations in the sapwood rose to 3-7 ppm during the growing season, and decreased to 0.1-0.3 ppm during the winter. In response to extreme drought, sapwood ethylene concentration increased to 30 ppm, followed by a rapid decrease after the onset of rain. Ethylene concentrations in gas samples from the heartwood were consistently lower than 1 ppm. The lowest values, about 0.1 ppm, were found during the autumn and early winter, whereas values around 0.5 ppm were typical from February to August.

Eliasson, L. and Bollmark, M. 1988. Ethylene as a possible mediator of light-induced inhibition of root growth. - Physiol. Plant. 72: 605–609. Pea seedlings (Pisum sativum L. cv. Weibull's Marma) were used to investigate the possible role of ethylene in light-induced inhibition of root elongation. Illumination of the roots with white light inhibited root elongation by 40–50% and increased ethylene production by the roots about 4-fold. Our main approach was to use exogenous 1-aminocyclopropane-1-carboxylic acid (ACC), supplied in the growth solution, to monitor ethylene production of the roots independent of light treatment. Ethylene production of excised root tips increased with increasing ACC concentrations. The rate of ethylene production in dark-grown roots treated with 0.1 μM ACC was similar to that caused by illumination. Low ACC concentrations (0.01–0.1 μM) decreased the rate of root elongation, especially in seedlings grown in the dark, and 0.1 μM ACC inhibited elongation to about the same extent as light. In light the roots curved and grew partly plagiogravitropically. This effect was also simulated by the 0.1 μM ACC treatment. At 1 μM and higher concentrations, ACC inhibited root growth almost completely and caused conspicuous curvatures of the root tips both in light and darkness. Inhibitors of ethylene synthesis and action partially counteracted the inhibition of root elongation caused by light. These observations suggest that the increase in ethylene production caused by light is at least partly responsible for the decreased growth of light-exposed roots.

Irradiation of the base internode of stem cuttings of Pisum sativum L. with white light during the rooting period resulted in the formation of fewer roots. IBA applied to the rooting solution for 4 days counteracted this inhibition even at low concentrations. IBA stimulated rooting, also, in cuttings grown under low irradiance where availability of photosynthetic products is probably the limiting factor for rooting. IAA, however, did not reverse the inhibition of rooting, resulting from unfavourable light conditions, at concentrations lower than 10−5M, but 10−4M IAA had a similar action to IBA. Under light conditions giving good root formation IAA over a concentration range from 10−7M to 10−5M decreased the number of roots formed. It is hypothesized that light prevents the action of endogenous auxin, or low exogenous concentrations of IAA, in the root-forming process while the synthetic auxin IBA is fully active in irradiated tissues.

Indol-3yl-acetic acid (IAA) present in the rooting solution for 1–4 days appreciably decreased the number of roots subsequently formed in pea stem cuttings. Removal of the lowest 10 mm of the cutting base after IAA treatment abolished the inhibitory effect of IAA almost completely. The number of roots formed from the remaining part of the base internode after excision of the basal centimeter of the internode 4 days after the cuttings were taken was similar to that of the control. It is concluded that the first root primordia, which develop near the cut surface, have an inhibitory influence on development of further roots. The inhibitory effect of IAA may be explained as a strengthening of this dominance phenomenon. Addition of charcoal to the rooting solution delayed the appearance of roots but increased the number of roots developed during a standard rooting period. Adsorption of stimulatory and inhibitory compounds at the surface of the cutting base is believed to be the reason for these results as no evidence of accumulation of inhibitory compounds in the solution was obtained. Charcoal reversed the inhibitory effect of IAA if added after the IAA treatment. This reversal was almost complete if the IAA treatment lasted for one day only but decreased drastically if the IAA treatment was extended over 4 days.

The time course of growth of Lemna gibba L. in low concentrations of abscisic acid (ABA) and of uptake of 14C-labelled ABA into the plants was followed for up to 15 days. Fresh weight increase was arrested when the plants were transferred to ABA solutions. This inhibition was greatest during the first days after which a marked recovery took place. Dry matter production was only slightly affected by ABA. On a culture basis, the uptake of ABA was almost linear with time during the experimental period. The rate of uptake on a fresh weight basis was highest during the first two days but decreased rapidly from the third day. Most of the applied 14C remained in the culture solution throughout the experiments. Plants transferred to a solution without ABA retained most of their accumulated 14C. Possible explanations for the decrease in rate of ABA uptake and restoration of growth are discussed.

The rooting of softwood cuttings of Alnus incana (L.) Moench in nutrient solution was studied under controlled conditions. Cuttings consisting of one internode with the leaf and axillary bud attached rooted easily and more rapidly than shoot tip cuttings. Light was necessary for rooting but good rooting was obtained in photon flux densities of both 40 and 190 μmol m-2s-1. Root number and root length was reduced when light reached the base of the cuttings. Treatment with indolebutyric acid (10-6–10-4M) increased the number of roots but 10-4M delayed rooting and decreased the root length. Debudded internode cuttings rooted as well as intact cuttings, and detached leaves also contained sufficient substances for rooting.

Summary Buds pre-treated with 50 μM benzyladenine (BA) and shoots of four willow clones ( Salix spp. ) cultured in vitro on hormone-free medium and on medium with 1 μM BA were analysed for cytokinins using an enzyme linked immunosorbent assay with polyclonal antibodies against isopentenyladenosine (iPA) and zeatin riboside (ZR). The clone which was most difficult to root yielded the highest ZR titers. A clone with poor growth and a tendency to become necrotic had very high iPA titers, possibly indicating uptake of BA to a toxic level, since BA cross-reacts with antibodies against iPA. Other genotypic differences relating to axillary shoot production, shoot elongation growth and vitrification could not be correlated to cytokinin contents.

Benzylaminopurine (BAP) or zeatin continuously supplied through the rooting solution to cuttings of pea (Pisum sativum L. cv. Weibull's Marma), inhibited root formation down to a concentration of 3.10−9M. The inhibitory effect of BAP in the concentration range 10−8–10−7M was readily reversible if the cuttings were transferred to solutions without cytokinin after treatment for 1–4 days. A slight increase in the number of roots formed was obtained after treatment with low cytokinin concentrations for 1–2 days. Evidence from microscopic studies of primordia formation indicates that BAP inhibits differentiation of primordia at an early stage in their development. Growth of already formed primordia, or root elongation, was considerably less sensitive to the inhibitory effect of BAP. The results indirectly support the hypothesis that endogenous cytokinins prevent root formation in stems of intact plants and may be of importance for the regulation of rooting in cuttings.

The possibility was investigated that the inhibition of rooting in pea (Pisum sativum L. cv. Weibull's Marma) cuttings caused by low indol-3yl-acetic acid (IAA) concentrations is due to ethylene produced as a result of IAA treatment. Treatment with 10 uμ IAA reduced the number of roots to about 50% of the control and increased ethylene production in the stem bases by about 20 times the control value during the two first days of treatment. Ethylene-releasing compounds (ethephon and 1-amino-cyclopropane-1-carboxylic acid, ACC), in concentrations giving a similar ethylene release, inhibited rooting to the same extent or more strongly than IAA. These results indicate that IAA-induced ethylene is at least responsible for the negative component in IAA action on root formation in pea cuttings. A higher IAA concentration (100 μ) and indol-3yl-butyric acid efficiently counteracted the negative effect of ethylene on root formation.

Shoots of apple (rootstock A2, Malus sylvestris (L.) Mill var. domestica Borkh.) were grown in vitro on agar media containing 4.4 μM benzylaminopurine (BAP) during the whole of a 36-day culture period or during the first 6 days of the period. Development of lateral shoots and an appreciable increase in fresh weight were obtained only when BAP was present during the whole period. Treatment for 6 days only was not sufficient for shoot proliferation although the transferred shoot continued its growth on fresh medium without BAP. Uptake of the cytokinin was studied by adding [8–14C]-benzylaminopurine to the medium and then following the distribution of the isotope in the shoots during the 36-day culture period. There was a steady, almost linear, uptake of 14C during the whole period. Most of the activity accumulated in the lower part of the shoot. It is concluded that BAP, due to slow translocation, is active mainly in the basal parts of the shoot where it stimulates formation and growth of buds. Its action is dependent on continuous supply of new BAP from the medium, indicating metabolism of BAP in the tissue.

Light-grown stem cuttingss of Pisum sativum L. cv. Weibull's Marma were rooted in a nutrient solution. The presence of 10 μM indolylacetic acid (IAA) in the solution for 24 h or longer periods decreased the number of roots subsequently formed to about 50% of control, provided IAA was present in the solution during any of the 4 first 24 h periods. Treatment for 6 h or shorter periods caused no or small response. IAA did not appreciably change the time needed for root formation, the time course of root appearance or the pattern of root distribution along the basal internode. IAA at 100 μM usually increased the number of roots although variable results were obtained with this IAA concentration. The number of roots was strongly increased by treatment with indolylbutyric acid (IBA) or 2,4-dichlorophenoxyacetic acid (2,4-D). None of these or other synthetic auxins decreased the number of roots in suboptimal concentrations. Experiments with 10 μM IBA showed that stimulation of rooting was obtained only if the auxin was present in the rooting solution for several days. Simultaneous treatment with IAA decreased the stimulating effect of IBA to some extent, whereas no such response was obtained if IAA was combined with 2,4-D. IAA applied in lanolin to the stem of intact cuttings decreased the number of roots formed. Decapitation and debudding of the cuttings decreased the number of roots formed. If at least 2 leaves were left this decrease was efficiently counteracted by an optimal IAA dose applied to the upper part of the stem. A five times higher dose was less effective, indicating a negative effect on rooting also by IAA applied to the shoots.

The effect of light on rooting of leafy cuttings of aspen (Populus tremula × tremuloides) and a willow hybrid (Salix caprea × viminalis) was investigated under controlled conditions in water culture. Two levels of irradiance were used, 40 and 8 W m−2. The lower level gave the best rooting of aspen cuttings, both when applied to the stock plants before the cuttings were taken and when given to the cuttings during the rooting period. Irradiation of the cutting base during the rooting period inhibited rooting almost completely in aspen and decreased the number of roots formed in the Salix hybrid. Net photosynthesis in the cuttings of Salix decreased considerably after excision and increased again after formation of roots. Indirect evidence indicated that photosynthesis was even more affected in aspen cuttings. The possible roles of carbohydrates and inhibitors in the light effects are discussed.

ON THE REASON FOR THE DIFFERENT PHOTOSYNTHETIC RATES OF SEEDLINGS OF PINUS-SILVESTRIS AND BETULA-VERRUCOSA

Excised stem sections from growing plants of Populus tremula L. and Pisum sativum L. including lateral buds were treated with indole-3-acetic acid in a phosphate buffer solution. In control sections the level of the abscisic acid-like inhibitor decreased strongly during 24 h as did the level of the endogenous auxin. Exogenous indoleacetic acid counteracted the decrease in the inhibitor level to a considerable extent. Implications of this auxin effect in relation to apical dominance are discussed.

Indole-3-acetic acid (IAA) contents in plant extracts were determined spectrofluorometrically after conversion of the compound into indolo-α-pyrone. The method is sensitive and accurate and showed good reproducibility. The values obtained by this method corresponded well with those obtained with the Avena coleoptile straight-growth assay. Impurities in extracts often cause various difficulties. Colored compounds influence the readings and may even make indole-3-acetic acid determination impossible by strong quenching of the fluorescence light. Insoluble compounds can be formed in the reaction and cause increased opalescence and light scattering which interferes with the fluorescence measurements. The necessities for the method to be reliable are that the insoluble compounds are removed or brought into solution and that the exciting light is sufficiently monochromatic. Indolo-α-pyrone is fairly stable in the dark but is rapidly broken down by UV radiation. This decay may be used as a check to see if the measured light represents fluorescence of indolo-α-pyrone.

Cuttings obtained from seedlings of Pisum sativum L. were rooted in water solution. Shoot growth continued after excision and shoot length increased considerably before roots emerged. Increase in dry weight was strongly dependent on light supply. Continued growth was dependent on supply of mineral nutrients to the rooting solution. Mineral nutrients had no or slight influence on the number of roots formed on cuttings from stock plants grown in fertilized soil, but the growth in length of the roots was dependent on the presence of calcium in the solution. Root formation was dependent on photosynthetic products formed after excision. No roots were formed on cuttings kept in the dark. The number of roots increased with increasing irradiance given to the leafy part of the cutting. At a low level of irradiance sucrose supply through the rooting medium increased the number of roots. Light given to the basal part of the cuttings had a strongly inhibitory effect on the number of roots formed. It is concluded that the carbohydrate level easily becomes a limiting factor for root formation in growing pea cuttings. Availability of mineral nutrients influences in the first place the growth of the shoots.

Summary Pea (Pisum sativum, cv. Weibulls Marma) stem cuttings were rooted in nutrient solution. The development of root primordia at the base of the cuttings was followed microscopically. The levels of zeatin-type and isopentenyladenosine-type cytokinins were determined in bleeding sap and in extracts of the lowermost centimeter of the stem base with an enzyme-linked immunosorbent assay (ELISA). Extracts were purified with the help of immunoaffinity chromatography. The bleeding sap as well as the tissue contained cytokinins of both types. In the stem tissue the level of the cytokinins decreased during the day after the cuttings were made. They remained at low levels for 3-4 days during the early phases of primordial development and increased later when the primordia began elongating. The results support the hypothesis that endogenous cytokinins are involved in the regulation of adventitious root formation.

The seasonal variation in capacity to form suckers and in auxin level in bark and wood was determined in root segments of aspen (Populus tremula L.). Auxin occurred in the roots from May to October but not in November. The highest auxin level was found during the period of shoot growth. During this period the capacity of root segments to form suckers was low. Auxin level decreased in isolated root segments during the first 24 hours after excision and was low during the period of sucker induction. The relation of endogenous auxin level to control of sucker formation is discussed. The experiments do not exclude the possibility that the auxin effect is mediated through inhibitors.

The content of acid ether-soluble auxins and inhibitors in vegetative tissues of aspen (Populus tremula L.) was determined with the Avena coleoptile straightgrowth assay. A growth promotor, tentativety identified as indol-3-yl-acetic acid, occurred in highest concentration in the growing stem parts. It was also detected in various parts of the roots. Large amounts of inhibitors (inhihitor β) were found in growing stem tissue and in upper mature stem parts. Considerably lower content of inhibitors was found in leaves, in the basal stem part and in the roots. Nonwoody growing roots and the wood of older root segments, especially, contained very smalt quantities of inhibitors. The elution volume of a part of the inhibitory activity on a Sephadex column was similar to that of abscisic acid.

Translocation and metabolism of 4-amino-3,5,6-trichloropicolinic acid (picloram) and 2,4-dichlorophenoxyacetic acid (2,4-D) in small plants of aspen (Populus tremula L.) were studied. In most experiments 14C-carboxyl-labelled herbicides were used. Considerable quantities of both herbicides were retained in the treated leaf. Translocation was mainly upwards into the growing shoot tip. Only minute quantities were found in the roots. Injection of the herbicides through a cut stem surface increased the translocation into the roots very little. One of the reasons for the limited downward translocation is considered to be a ready transfer of the herbicides between the phloem and the xylem. Both herbicides were incorporated into complexes from which the active herbicides could be released. However, this complex formation can only partly account for the retention of the herbicides in the treated leaves. The differences in metabolism found between 2,4-D and picloram cannot explain the considerable difference in toxicity between the compounds.

Uptake, translocation and metabolism of 14C-labelled 4-amino-3,5,6-trichloropicolinic acid (picloram) and 2,4-dichlorophenoxyacetic acid (2,4-D) in seedlings of wheat (Triticum aestivum L.) were studied. The uptake of the herbicides through the upper surface of the first leaf was slow but was almost complete after nine days. Picloram was absorbed faster than 2,4-D. Picloram was also translocated into the stem and the untreated leaves to a greater extent than 2,4-D. Only small fractions of the activity were recovered from the roots and from the nutrient solution. Picloram and 2,4-D formed water-soluble conjugates in the tissues. These conjugates were very labile and hydrolyzed under release of the unchanged herbicides. The isotope from 2,4-D was also incorporated in an insoluble fraction, containing cell walls and proteins. Also from this fraction biologically active 2,4-D could be released by hydrolysis. The formation of the complexes was partly prevented by cycloheximide. It is suggested that herbicide detoxification through complex formation is of importance for the relatively low sensitivity of wheat to auxin herbicides.

Experiments with small plants of Populus tremula L. growing in solution culture indicate that polarly transported auxin is an important factor in the control of axillary bud growth. If the auxin supply from the growing apex is eliminated, the number of buds released is influenced by factors translocated in the transpiration stream from the roots. Suckers may be induced to develop from aspen roots, the age of which is six weeks or more. Removal of the growing apex and the axillary buds or stoppage of shoot growth by short day treatment were effective in inducing abundant suckering in small aspen plants. Some mature leaves had to be maintained, indicating the dependence of sucker formation on carbohydrate supply. These treatments are known to decrease auxin production in the shoots. Extraction and biological assay showed a decrease in the content of auxin in the roots as a consequence of removal of growing shoot parts. The results indicate that suckering in roots of intact aspen plants is prevented by auxin transported into the roots from growing shoot parts.

White light increases the inhibition of growth in length of pea seedling roots caused by 2,4-D. This effect is most pronounced in the case of lateral root growth. The effect is obtained also if the shoots above the cotyledons are removed and is probably due to the direct influence of light on the roots. While light also weakly enhances the inhibition of pea root growth caused by 1-naphthylacetic acid, light does not increase the sensitivity of wheat roots to 2,4-D or naphthylacetic acid.

Young seedlings of Picea abies Karst, grown in nutrient solution were treated with the growth retardants Amo-1618, B-995, and CCC. These were added to the nutrient medium. B-995 and CCC retarded root and shoot growth in the concentrations 100, 10, and 1 mg/l. Growth was almost entirely inhibited by 300 mg/l, obviously due to toxicity. The effects of Amo-1618 were similar but more varying. GA counteracted the effects of all the retardants on shoot growth, but not on root growth.

Hoot elongation of rooted cuttings of aspen, Populus tremula L., was followed on moist filter paper. The time course of root growth during treatments which decrease or interrupt the supply of carbohydrates to the roots was studied. At light intensities below 4000 lux the rate of root elongation was found to berelated to the light intensity over the leaves. In the dark, root growth decreased within 24 hours and the roots stopped growing completely during the second or third day. Excision of the leaves or steam-killing of the tissue in a stem section below the leaves was followed by root growth stoppage within 24 hours. These results imply that the rate of root growth under certain conditions is determined by the supply of carbohydrates from the leaves.