A rooting inhibitor present in Norway spruce seedlings grown at high irradiance - a putative cytokinin

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.     

Effect of changing the endogenous concentration of auxins and cytokinins and the production of ethylene in pea stem cuttings on adventitious root formation

Decapitation or treatment with naphthylphthalamic acid (NPA) ndash; an inhibitor of IAA transport ndash; or a synthetic cytokinin N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) caused a decrease in rooting of pea cuttings. However the mode of action of the above treatments was different. Decapitation resulted in a decrease of indole-3-acetic acid (IAA) and a simultaneous increase in cytokinin content at the base of the cuttings. NPA decreased IAA even more, but did not influence cytokinins. CPPU alone or in combination with amino-ethoxy-vinylglycine (AVG) ndash; an ethylene biosynthesis inhibitor ndash; increased IAA at the rooting zone, but CPPU was transported from apex to the base of the cuttings where it inhibited rooting. NAA, applied alone after decapitation, stimulated rooting, probably partly by being an auxin, and partly by inhibiting the accumulation of cytokinins at the base of the cuttings. Treatment with AVG neither influenced rooting nor auxin or cytokinin content. Therefore, ethylene production does not seem to be one of the main factors involved in the reduced rooting after the various treatments.     

The Role of Ethylene in the Inhibition of Rooting under Low Oxygen Tensions

A 60-fold increase in ethylene content was observed in stem cuttings of chrysanthemum (Chrysanthemum × morifolium Ramat.) held in aero-hydroponics under anoxic conditions during the 8 to 12 days necessary for adventitious root formation. Ethylene, 1-aminocyclopropane-1-carboxylic acid, and 10-(malonylamino) cyclopropane-1-carboxylic acid contents were highest in the immersed portion of the cuttings, but there was substantial ethylene produced by the anoxic, misted portions of the cutting above the liquid. Application of ethylene (10 microliters per liter) to chrysanthemum cuttings stimulated root development in cuttings held in high dissolved oxygen concentrations (8.0 milligrams per liter). Since the application of ethylene did not inhibit rooting in cuttings held at low dissolved oxygen concentrations (2.0 milligrams per liter), the inhibition of rooting under low oxygen concentrations is not mediated by the observed increase in endogenous ethylene content.     

Root Formation in Ethylene-Insensitive Plants

Experiments with ethylene-insensitive tomato (Lycopersicon esculentum) and petunia (Petunia x hybrida) plants were conducted to determine if normal or adventitious root formation is affected by ethylene insensitivity. Ethylene-insensitive Never ripe (NR) tomato plants produced more below-ground root mass but fewer above-ground adventitious roots than wild-type Pearson plants. Applied auxin (indole-3-butyric acid) increased adventitious root formation on vegetative stem cuttings of wild-type plants but had little or no effect on rooting of NR plants. Reduced adventitious root formation was also observed in ethylene-insensitive transgenic petunia plants. Applied 1-aminocyclopropane-1-carboxylic acid increased adventitious root formation on vegetative stem cuttings from NR and wild-type plants, but NR cuttings produced fewer adventitious roots than wild-type cuttings. These data suggest that the promotive effect of auxin on adventitious rooting is influenced by ethylene responsiveness. Seedling root growth of tomato in response to mechanical impedance was also influenced by ethylene sensitivity. Ninety-six percent of wild-type seedlings germinated and grown on sand for 7 d grew normal roots into the medium, whereas 47% of NR seedlings displayed elongated tap-roots, shortened hypocotyls, and did not penetrate the medium. These data indicate that ethylene has a critical role in various responses of roots to environmental stimuli.     

生长素、乙烯和一氧化氮对拟南芥下胚轴插条形成不定根的调节

研究生长素、乙烯和一氧化氮（NO）对拟南芥下胚轴插条形成不定根的调节,以及生长素和乙烯信号转导成员在IAA促进不定根形成中的作用的结果表明：拟南芥切条以IAA和硝普钠（N0供体）单独处理7d后的不定根形成均受到促进,其中以50μmol·L^-1 IAAμmol·L^-1 SNP的促进作用为最强,乙烯的促进作用不明显;生长素运输和信号转导以及乙烯信号转导相关突变体对IAA促进生根作用的敏感性比野生型有所下降,特别是IAA14功能获得型的突变体。IAA和NO在促进不定根形成中有协同效应。     

Effects of pre-severance light quality on the vegetative propagation ofEucalyptus grandis W. Hill ex Maiden

Two-leaf, two-node cuttings were taken fromEucalyptus grandis stockplants grown under different light qualities (red to far-red ratios of 0.4, 0.7, 1.3, 3.5 and 6.5) at a constant photon flux density (200 μmol m^-2 s^-1). Two experiments tested effects of pre-severance light quality on cutting morphology, post-severance gas exchange, carbohydrate status and rooting of cuttings. The best rooting percentage was achieved by cuttings with longer stems and greater stem volume from stockplants grown at lower red to far-red (R∶FR) ratios. Generally, rooting success was associated with low pre-severance starch and water-soluble sugar concentrations, and a greater total water-soluble carbohydrate (TWSC) content per cutting. Rooting was associated with well maintained stem starch and an increase in stem TWSC during the propagation period. Gas exchange of cuttings was measured between 28 and 33 days after severance. Rooting percentages at 35 days after severance were positively and linearly related to net photosynthetic rate and stomatal conductance. In unrooted cuttings there was a net release of CO₂ which increased significantly with an increase in pre-severance R∶FR ratio. These results demonstrate that stockplant environment may significantly modify the morphology and physiology of subsequent cuttings, and that cutting morphology, and stored and current photosynthates have a significant influence on rooting. ITE is a component of the Edinburgh Centre for Tropical Forestry     

The role of light and polar auxin transport in root regeneration from hypocotyls of tomato seedling cuttings

A relationship between light conditions, auxin transport and adventitious root formation by hypocotyls of tomato seedling cuttings was demonstrated. Effective rooting of tomato seedling cuttings was observed under continuous white light (WL) irradiation. However, root formation was reduced in darkness or under red (RL) or blue light (BL). At least 3/4-day-long irradiation treatment with (WL) was necessary to increase the number of roots formed in comparison with control cuttings grown in darkness. Light was most effective if applied during the first half of the 13-day-long rooting period. The role of photoreceptor-dependent light perception in the light-regulation of rooting was tested using tomato photomorphogenic mutants: aurea (au) and high pigment (hp). When exposed to WL both mutants generated fewer roots then their isogenic wild type (WT). In darkness or under BL and RL less roots were formed on all plants and no difference was observed between mutants and WT plants. TIBA (2,3,5-triiodobenzoic acid) inhibited rooting in a dose-dependent manner both in darkness and under WL. However, although rooting was suppressed by 0.75 M TIBA in the dark, 8 M TIBA was necessary to block root formation in continuous WL. Inhibition of rooting by TIBA was most efficient when applied at the initial period of rooting, a 1-day-long treatment with TIBA being sufficient to suppress rooting if given during the first 2 days of culture. Later treatment had much less effect on the root formation.     

The effect of etiolation and shading of stock plants on rhizogenesis in stem cuttings of <Emphasis Type="Italic">Cotinus coggygria</Emphasis>

In order to improve vegetative propagation of a difficult to root Cotinus coggygria the stock plants were subjected to: etiolation, shading and spraying with IBA, combined with the application of two commercially available rooting powders. The IBA treatment was more suitable for rooting of C. coggygria cuttings than the NAA application and it enhanced rhizogenesis regardless of the form of auxin application (foliar application to a stock plant or a rooting powder used directly on cuttings) and the amount of light provided to stock plants. Etiolation did not improve rhizogenesis in stem cuttings, however, reduction of light intensity by 50% and 96% of the ambient prior to harvest of cuttings affected rooting positively. Positive effects of shading can be ascribed to changes in shoot anatomy, i.e. a weaker sclerenchyma development. Synergistic effect of shading and foliar auxin application can result from the increase in leaf blade area and/or thinner lower epiderm. Enhanced rooting in cuttings from shoots grown out under reduced light intensity was accompanied by decrease in the contents of total soluble sugars, soluble proteins and free ABA and by increase in total chlorophyll, free amino acids, polyphenolic acids and free IAA contents.     

Conditions for rooting of leafy cuttings of Alnus incana

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.     

Effects of auxin and irradiance on the rooting of cuttings of Pinus sylvestris

Abstract Seedlings of Pinus sylvestris L. were grown under controlled conditions (temperature 20°C, photoperiod 17 h) at two irradiances, 8 or 40 W m^-2. Hypocotyl cuttings were excised and rooted at different irradiances in tap water solutions of indolebutyric acid (IBA). The fastest rooting and highest rooting percentage were obtained with cuttings from stock plants grown at 8 W m^-2 and treated with 10^-5M IBA for 21 days. The concentration of 10^-4M IBA inhibited root formation. In comparable treatments rooting was always better in cuttings from stock plants grown at 8 W m^-2 than in cuttings from stock plants grown at 40 W m^-2. The irradiance during the rooting period had only a minor influence on rooting. When cuttings from plants irradiated with 40 W m^-2 were treated with 10^-5M IBA for 21 days the rooting percentage almost reached the same level as in untreated cuttings from stock plants given 8 W m^-2. In cuttings treated with IBA during the whole rooting period, rooting was depressed in comparison to untreated cuttings. Aeration of the 10^-4M IBA solution increased the rooting percentage, but aeration had no effect on untreated cuttings and on cuttings treated with lower IBA concentrations.     

Seasonal changes in adventitious root formation in hypocotyl cuttings of Pinus sylvestris: Influence of Photoperiod during stock plant growth and of indolebutyric acid treatment of cuttings

Seedlings of Pinus sylvestris were grown for 6 weeks under natural light conditions in a temperature controlled environment room. Cuttings from these plants were rooted in tap water or in indolebutyric acid (IBA) solutions for 60 days at an irradiance of 16 W m^-2. Experiments were performed at 3-week intervals during two growth seasons. — Seasonal changes in root formation were found in control cuttings as well as in IBA treated cuttings. The number of roots and the percentage of cuttings that rooted were high during early spring and autumn. During the summer period hardly any roots were formed. Stimulation of root formation by IBA occurred manily during spring and autumn when cuttings already possessed the ability to form roots. — The influence of photoperiod during stock plant growth was also investigated. Shorter photoperiod resulted in an increase in the number of roots and rooting percentage. The period during summer where rooting was inhibited under natural light conditions was considerably shortened when stock plants were grown at a photoperiod of only 4 h. The results demonstrate the importance of the growing conditions for stock plants for subsequent root formation. The results are discussed with special reference to the role of irradiance.     

Ethylene and auxin-ethylene interaction in adventitious root formation in mung bean (Vigna radiata) cuttings

The role of ethylene in adventitious root formation and its involvement in auxin-induced rooting were investigated in cuttings ofVigna radiata (L.). Treatment with 30 M indole-3-acetic acid (IAA) for 24 h slightly inhibited rooting, whereas the same concentration of indole-3-butyric acid (IBA) significantly stimulated it. Ethylene derived from 1-aminocyclopropane-1-carboxylic acid (ACC) increased the number of adventitious roots but inhibited their emergence and elongation. Endogenous levels of ethylene, ACC, and malonyl-ACC (MACC) were initially higher in cuttings treated with IAA. This trend was quickly reversed, and cuttings, particularly hypocotyls, treated with IBA produced higher levels of ethylene and had more ACC and MACC during most of the rooting process. Aminoethoxyvinylglycine significantly inhibited rooting, but its inhibitory effect could not be reversed by ACC. The data suggest that the stimulating effect of IBA on rooting is closely associated with its induction of ACC and ethylene biosynthesis.     

The effect of shading of stock plants on rhizogenesis in stem cuttings of Berberis thunbergii ‘Red Rocket’

In order to improve vegetative propagation of Berberis thunbergii ‘Red Rocket’, stock plants were subjected to shading and IBA treatment, as well as to the application of two commercially available rooting powders. Spraying stock plants with IBA 24 hours prior to cutting harvest gave the effect comparable to the use of rooting powders. Reduction of light intensity by 50% and 96% of ambient prior to harvest of cuttings improved rooting. Positive effect of shading results in changes in shoot anatomy, i.e. a weaker sclerenchyma development. Enhanced rooting in cuttings from shoots grown under reduced light intensity was accompanied by a decrease in the concentration of soluble proteins and by an increase in total chlorophyll content while the concentration of total soluble sugars remained unaffected.     

Effects of combinational treatment with ethephon and indole-3-butyric acid on adventitious rooting of <Emphasis Type="Italic">Pinus thunbergii</Emphasis> cuttings

Adventitious rooting is essential for cutting propagation of pine wilt-resistant Pinus thunbergii. To examine a variety of adventitious rooting potentials among donor plants, cuttings were taken from 31 seedlings within a half-sib family. Rooting abilities of cuttings from each seedling ranged from 0 to 100%. When 11 ortets and 11 ramets (clonally propagated from each ortet) were used as donor plants, there was a positive correlation between rooting abilities of cuttings from ortets and ramets, suggesting that adventitious rooting is dependent on genetic factors in the donor plants. To promote adventitious rooting of cuttings by hormonal treatment, we examined the effect of soaking time in Oxyberon (19.7 mM indole-3-butyric acid (IBA) solution) on rooting. Ten minutes was the best soaking time for rooted cuttings to produce more adventitious roots without impairing normal growth. When cuttings were soaked in Ethrel diluent (69.2 μM ethephon) for 24 h before soaking in Oxyberon for 10 min, a significantly higher rooting ability was observed than those soaked in Oxyberon alone. Ethrel on its own barely affected rooting ability. The positive effect of the combinational treatment was confirmed in a similar experiment using authentic ethephon and IBA instead of Ethrel and Oxyberon. When cuttings were soaked in a mixture of ethephon and silver thiosulfate (STS), an ethylene action inhibitor, before IBA-soaking, the effect was partially diminished compared with combinational treatment without STS. These findings suggest that ethylene action caused by ethephon treatment promotes IBA-mediated adventitious rooting of P. thunbergii cuttings.     

Nitrogen- and storage-affected carbohydrate partitioning in high-light-adapted Pelargonium cuttings in relation to survival and adventitious root formation under low light

BACKGROUND AND AIMS: The aim of this study was to determine the role of nitrogen- and storage-affected carbohydrate availability in rooting of pelargonium cuttings, focusing on the environmental conditions of stock plant cultivation at low latitudes, transport of cuttings, and rooting under the low light that prevails during the winter rooting period in Central European greenhouses. METHODS: Carbohydrate partitioning in high-light-adapted cuttings of the cultivar 'Isabell' was studied in relation to survival and adventitious root formation under low light. Effects of a graduated supply of mineral nitrogen to stock plants and of cutting storage were examined. KEY RESULTS: Nitrogen deficiency raised starch levels in excised cuttings, whereas the concentrations of glucose and total sugars in leaves and the basal stem were positively correlated with internal total nitrogen (Nt). Storage reduced starch to trace levels in all leaves, but sugar levels were only reduced in tissues of non-nitrogen deficient cuttings. Sugars accumulated in the leaf lamina of stored cuttings during the rooting period, whereas carbohydrates were simultaneously exhausted in all other cutting parts including the petioles, thereby promoting leaf senescence. The positive correlation between initial Nt and root number disappeared after storage. Irrespectively of storage, higher pre-rooting leaf glucose promoted subsequent sugar accumulation in the basal stem and final root number. The positive relationships between initial sugar levels in the stems with cutting survival and in leaves with root formation under low light were confirmed in a sample survey with 21 cultivars provided from different sources at low latitudes. CONCLUSIONS: The results indicate that adventitious rooting of pelargonium cuttings can be limited by the initial amount of nitrogen reserves. However, this relationship reveals only small plasticity and is superimposed by a predominant effect of carbohydrate availability that depends on the initial leaf sugar levels, when high-light adaptation and low current light conditions impair net carbon assimilation.     

Physical conditions of propagation media and their influence on the rooting of cuttings

Summary The formation and subsequent growth of roots by cuttings of poinsettia, hydrangea, rose and azalea in various propagation media, Jiffy-7, Jiffy-9 and Grodan under different conditions of aeration was investigated. The interrelationships of the effects of air content of the media, temperature and light intensity on the rooting of poinsettia cuttings was also studied.With low air contents (0 cm moisture tension) in the propagation media the formation and growth of roots was strongly inhibited. The rooting performance of rose appeared to be less affected by the poor aeration. Increasing air content improved rooting but best results were obtained at moisture tensions of 4 to 8 cm. Rooting seems to be better correlated with oxygen diffusion rate (ODR) than with air content.For poinsettia cuttings the optimum temperature for rooting was 24 to 28°C. At low temperatures rooting was delayed while at higher temperatures it was almost completely inhibited. Callus formation increased with temperature but decreased with increasing moisture tension. Conditions which induced large callus formation inhibited root formation.High light intensity during rooting reduced overall rooting performance and the inhibition was most pronounced in conjunction with high moisture tensions.Report No. 255.     

Reduced irradiance and applied auxin influence carbohydrate relations in Pinus banksiana cuttings during propagation

Untreated and indole-3-butyrie acid-treated (IBA) cuttings from 90-day-old Pinus banksiana Lamb, stock plants were propagated under normal greenhouse irradiance (max. 900 $$mol m^-2 s^-1) and shade (max. 120 $$mol m^-2 s^-1) to determine effects on adventitious rooting and on reducing sugar and starch concentrations in needles and basal stems. In one experiment, cuttings were assessed at days 15 and 25 of propagation for basal 1-cm stem fresh weight, proportion rooted, number of roots and longest root length. In a second experiment with cuttings, basal 1-cm stem fresh weight and concentrations of reducing sugar and starch in needles and basal stems were measured each day for the first 10 days of propagation. Carbohydrate measurements were also made for seedling stock plants as controls for the second experiment. Carbohydrate data for cuttings were primarily evaluated based on net (cutting minus seedling) concentrations, to correct for changes in cuttings not related to adventitious rooting. Increase of basal stem fresh weight and rooting of cuttings, based on all measured variables, occurred in the order: light + IBA > light > shade + IBA > shade. The best rooting required the greater irradiance. Compared to results from cuttings in the light, shading resulted in lesser accumulations of reducing sugars and starch in needles and basal stems. Reducing sugar: starch concentration ratios were significantly greater in shade- vs light-propagated cuttings, IBA treatment did not offset the effects of shade on rooting or on reducing sugar and starch concentrations or ratios. Overall, the results suggested that decreased reducing sugar and starch concentrations and/or their increased ratios are associated with shade-induced poor rooting of P. banksiana cuttings.     

Etiolation of stock plants affects adventitious root formation and hormone content of pea stem cuttings

Stock pea plants (Pisum sativum L.) were etiolated fully or partially at the third internode that acted as the cutting base. The etiolation started the fifth day after sowing and lasted till cutting preparation. Cuttings derived from partially etiolated plants rooted more than non-etiolated ones while fully etiolated ones rooted more only after treatment with 1% sucrose solution for 4 days. Endogenous IAA in the base of etiolated cuttings was higher during the first 24 h after cutting preparation than in the control. Z/ZR did not show significant differences while iAde/iAdo was higher in the control. Ethylene was increased 24 h after cutting preparation and the increase was greater from partially etiolated cuttings. The results showed that besides IAA and cytokinins, which played a role in the rooting of cuttings, sucrose influenced rooting in the case of fully etiolated stock plants.     

Effect of Ethephon, Indole Butyric Acid, and Treatment Solution pH on Rooting and on Ethylene Levels within Mung Bean Cuttings

Light-grown mung bean (Phaseolus aureus Roxb.) cuttings were treated with buffered and nonbuffered solutions of Ethephon, indole butyric acid (IBA), and the combination of both. Ethephon treatment resulted in increased tissue ethylene levels with increasing solution pH, but had no effect on rooting. IBA treatment had no effect on tissue ethylene levels, but strongly promoted rooting. Combinations of Ethephon and IBA had no effect on rooting of mung bean cuttings beyond that obtained by IBA alone.     

Micropropagation of Wild Service Tree (Sorbus torminalis [L.] Crantz): The Regulative Role of Different Aromatic Cytokinins During Organogenesis

The influences of three different aromatic cytokinin derivatives [6-benzylaminopurine, meta-topolin, and 6-(3-methoxybenzylamino)purine-9-ß-D-ribofuranoside (MeOBAPR)] on in vitro multiplication and rhizogenesis of the wild service tree (Sorbus torminalis [L.] Crantz) were compared. The highest micropropagation rate (24 new shoots per explant after 3 months of cultivation) was achieved on media containing BAP. On the other hand, the best rooting microcuttings were those multiplied on a medium containing MeoBAPR. To compare these results with the levels of endogenous cytokinins in multiplied explants, a newly developed UPLC-ESI(+)-MS/MS method was used to determine levels of 50 cytokinin metabolites in explants cultivated 12 weeks on media supplemented by BAP and of the two other aromatic cytokinin analogs used. Several significant differences among the levels of endogenous cytokinins, extracted from the explants, were found. The concentration of BAP9G, an important metabolite suspected to be responsible for inhibition of rooting and acclimatization problems of newly formed plantlets, was found to be the highest in microcuttings grown on media supplemented with BAP. This agrees well with the results of our rooting experiments; the lowest percentages of rooted plantlets 6 weeks after transferring shoots on rooting medium were present on explants multiplied on BAP. In contrast, BAP was still the most effective for the induction of bud formation on primary explants. Levels of the most active endogenous isoprenoid cytokinins, tZ, tZR, and iPR, as well as O-glucosides were also suppressed in explants grown on BAP compared with those of explants treated with other cytokinin derivatives. This may be the result of a very high BAP uptake into the explants grown on this cytokinin. On the other hand, endogenous concentrations of cis-zeatin derivatives as well as dihydrozeatin derivatives were not affected. Differences in the production of another plant hormone, ethylene, that plays an important role in controlling organogenesis in tissue culture, were also observed among S. torminalis plantlets grown in vitro on media containing different cytokinins tested. The highest ethylene levels were detected in the vessels containing media supplemented with mT. They were two to four times higher compared with the production by the S. torminalis explants cultivated on other media used. Finally, the levels of free IAA were also determined in the explants. S. torminalis plantlets grown on media containing BAP contained the lowest level of auxin, which is again in good agreement with their loss of rooting capacity. The results found in this study about optimal plant hormone concentrations may be used to improve in vitro rooting efficiency of the wild service tree and possibly also of other plant species.