Effect of sucrose on adventitious root regeneration in apple

We have examined the effect of sucrose on adventitious root formation in apple microcuttings and in 1-mm stem slices cut from apple microcuttings. The sucrose concentration influenced the number of adventitious roots, but at a broad range of sucrose concentrations (1–9%) the effect was small. In addition, there was an interaction between sucrose and auxin: increasing the sucrose concentration shifted the dose–response curve of auxin to the right. When slices were cultured on medium without sucrose for the initial period (0–48 h), rooting was reduced whereas 48-h culture without application of sucrose had hardly any effect or even a slight promotive effect in a later period (48–120 h). The results show that during adventitious root formation, applied sucrose is used as a source of energy and building blocks but they are also in accordance with a possible regulatory role of sucrose.     

Toxicity of ethanol during proliferation and adventitious root formation in apple microcuttings in vitro

We have examined the toxicity of ethanol in tissue culture of the apple rootstock ‘Jork 9’. During proliferation through axillary branching, 0.2% (v/v) ethanol slightly stimulated proliferation whereas significant inhibition occurred at concentrations of 0.4 % or higher. In adventitious root formation, significant inhibition occurred at concentrations of 0.1 % or higher. The effect of ethanol was stage-dependent: during the induction period (i.e. from 24 to 72 h after the start of the rooting treatment), there was little or no inhibition. During autoclaving, ethanol evaporated to ca. 50 %. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of 1,2-benzisoxazole-3-acetic acid on adventitious shoot regeneration and in vitro rooting in apple

 The effect of 1,2-benzisoxazole-3-acetic acid (BOA), compared to 1-naphthaleneacetic acid (NAA), on adventitious shoot formation in leaf portions and compared to indolebutyric acid (IBA), on in vitro rooting in the apple (Malus domestica Borkh) cultivars McIntosh and Gala, and one rootstock, Jork 9, was investigated. BOA at 43.0 μm or 2.7 μm at NAA in combination with 17.8 μm benzyladenine (BA), induced the highest number of explants to produce adventitious shoots in Jork 9. In Gala, the combination of 21.5 μm BOA with 1.0 μm thidiazuron (TDZ) or with 22.0 μm BA induced the highest regeneration percentages, 58 and 54%, respectively, giving more satisfactory results than NAA (where only 42% of leaf explants exhibited shoot formation). In McIntosh, the highest percentage of regeneration was obtained with 1.3 μm NAA and 22.0 μm BA, while 51% was the highest response obtained with the BOA treatment. The combination of BOA with TDZ completely inhibited regeneration activity in leaf portions of this cultivar. The shoots of all the genotypes obtained with the most morphogenetic NAA or BOA treatments were excised, multiplied and successfully rooted and hardened. The results demonstrate that the synthetic auxin BOA is active in inducing shoot regeneration from leaf explants of apple and that the activity of BOA in plant regeneration is genotype dependent. When BOA was used to induce rooting in apple microcuttings, lower rooting percentages were obtained than with IBA, showing that the effect of BOA in inducing root formation is very low and that it cannot be used routinely to replace IBA in the in vitro rooting of microcuttings. Received: 18 June 1998 / Revision received: 4 January 1999 / Accepted: 29 January 1999     

Endogenous indole-3-acetic acid and abscisic acid in apple microcuttings in relation to adventitious root formation

The effects of applying indole-3-butyric acid (IBA) for periods up to 48 h were examined in difficult-to-root microcuttings (from newly-established cultures) and in easy-to-root microcuttings (from long-term subcultures) of Jonathan apple (Malus X domestica Borkh). In easy-to-root material, 20% of the microcuttings produced roots in the absence of IBA, while 6 h exposure to 10 M IBA gave 100% rooting of microcuttings. In contrast, root formation in difficult-to-root material was IBA-dependent. Maximum rooting of these microcuttings (50%) required 24 h exposure to 10 M IBA.Variation in the endogenous levels of free indole-3-acetic acid (IAA) during the course of root induction was similar in microcuttings of both types but there were marked differences in endogenous abscisic acid (ABA) levels. In easy-to-root microcuttings ABA remained at a constant low level, but in difficult-to-root material ABA exhibited marked fluctuations and was present at higher concentrations than in easy-to-root microcuttings.     

Rapid auxin-induced nitric oxide accumulation and subsequent tyrosine nitration of proteins during adventitious root formation in sunflower hypocotyls

Using NO specific probe (MNIP-Cu), rapid nitric oxide (NO) accumulation as a response to auxin (IAA) treatment has been observed in the protoplasts from the hypocotyls of sunflower seedlings (Helianthus annuus L.). Incubation of protoplasts in presence of NPA (auxin efflux blocker) and PTIO (NO scavenger) leads to significant reduction in NO accumulation, indicating that NO signals represent an early signaling event during auxin-induced response. A surge in NO production has also been demonstrated in whole hypocotyl explants showing adventitious root (AR) development. Evidence of tyrosine nitration of cytosolic proteins as a consequence of NO accumulation has been provided by western blot analysis and immunolocalization in the sections of AR producing hypocotyl segments. Most abundant anti-nitrotyrosine labeling is evident in proteins ranging from 25–80 kDa. Tyrosine nitration of a particular protein (25 kDa) is completely absent in presence of NPA (which suppresses AR formation). Similar lack of tyrosine nitration of this protein is also evident in other conditions which do not allow AR differentiation. Immunofluorescent localization experiments have revealed that non-inductive treatments (such as PTIO) for AR develpoment from hypocotyl segments coincide with symplastic and apoplastic localization of tyrosine nitrated proteins in the xylem elements, in contrast with negligible (and mainly apoplastic) nitration of proteins in the interfascicular cells and phloem elements. Application of NPA does not affect tyrosine nitration of proteins even in the presence of an external source of NO (SNP). Tyrosine nitrated proteins are abundant around the nuclei in the actively dividing cells of the root primordium. Thus, NO-modulated rapid response to IAA treatment through differential distribution of tyrosine nitrated proteins is evident as an inherent aspect of the AR development.     

Chloramphenicol effects on adventitious root production by radish hypocotyls

Abstract

The excision of the root accelerates greatly the formation of adventitious roots in the hypocotyl of etiolated radish seedlings, but if the seedlings develop in CAP 1×10^?4M, no adventitious root are induced after cutting. IAA either alone or associated with CAP, significantly increases the number of primordia in normal hypocotyls if given at the moment of cutting, while it has not stimulatory effect on the hypocotyls of seedlings grown in CAP. IAA has significant effect on both elongation and tickening of hypocotyl segments prepared from seedlings grown in CAP, and this could indicate a specific action of the inhibitor either on a particular process or on particular cells.

The endodermis and the pericycle, which are the two cell layers implicated in the formation of the adventitious roots, could be the mediators of this particular effect of CAP in rooting.     

Histological study of initiation and development in vitro of adventitious roots in minicuttings of apple rootstocks of M 26 and EMLA 9

Apple rootstocks M 26 and EMLA 9 'COST' shoots propagated in vitro were used for the histological study of initiation and development of adventitious roots after a brief induction pretreatment. The results show that there are differences in mode and timing of initiation and development of adventitious roots between the two varieties. In M 26, adventitious roots were directly initiated from the derivatives of the cambium, some of which were immediately transformed into meristemoids in situ 36 h after pretreatment. Subsequently, meristemoids differentiated into root primordia. Development of adventitious roots were completed when they emerged at the surface of stem bases 10 days after pretreatment. In EMLA 9, before the meristemoids formed, internal cell files were formed by continuous divisions of cambial cells. The cells were regularly arranged in files external to the cambium. On the fourth day after pretreatment, some cells in the outermost layers of these files became meristematic, started to divide and turned into meristemoids, which differentiated into root primordia. The cells of the files between the root primordium and the cambium were transformed into vascular tissues which connected the vascular systems of the adventitious roots and stems.     

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 light and riboflavin on indolebutyric acid-induced root formation on apple in vitro

Maximum root formation on apple ( Malus ) shoots cultured in vitro occurred after an incubation in the dark on medium containing 3.2 or 10 μ M indolebutyric acid (IBA) plus riboflavin. Omission of riboflavin or culture in light resulted in a significant decrease in the number of roots formed. About 95% of the absorbed IBA was inactivated by conjugation, ca 4% was extracted as the free IBA acid (IBAH) and only 1% as IAAH. It was investigated whether the decrease in root formation caused by exposure to light or omission of riboflavin during culture was parallelled by a shift in the concentrations of the physiologically active auxin compounds (IBAH and IAAH) in the stem base, i.e. the location where the roots emerge. At least 90% of the absorbed ³H-IBA was located in the stem base. Omission of riboflavin, either in the dark or in the light, had no effect on the IBAH and IAAH concentrations, whereas root formation decreased significantly. Incubation in the light on medium containing 10 μ M IBA with or without riboflavin and culture in the dark on medium containing 3.2μ M IBA plus riboflavin resulted in similar IBAH and IAAH concentrations. However, the number of roots was significantly lower after culture in the light. Therefore, we conclude that the synergistic effect of riboflavin and the antagonistic effect of light on IBA-induced root formation are not solely based on changes in the concentrations of the active auxin components resulting from IBA uptake.     

Effect of medium-pH and MES on adventitious root formation from stem disks of apple

We have examined the effect of medium-pH on rooting using 1-mm slices cut from stems of apple microshoots. Before autoclaving, the pH of the rooting medium was set at various pH values between 4.5 and 8.0. During autoclaving, the pH drifted in particular in the alkaline region. Additional changes occurred during culture and the range set at 4.5–8.0 had shifted to 5.2–6.0 after autoclaving and 3 weeks of culture. When 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) had been added as buffering agent, the pH was stable when set at 5.0–6.5. Highest rooting was achieved at pH ~5.3 with and without MES (pH measured after autoclaving). This maximum did not correlate with highest auxin uptake. MES inhibited adventitious root formation during the initial phase of root formation when the meristemoids are being formed (ca. 30% reduction at 10 mM) but was promotive during outgrowth of the meristemoids to roots (30% increase at 10 mM). Inhibition and promotion by MES were not related to its buffering action as they were observed at all pHs.     

不定根发生分子调控机制的研究进展

不定根发生问题,既是植物无性繁殖和工厂化育苗实践的核心问题,又是植物发育和形态建成等方面的重要理论问题。由于不定根发生过程的复杂性,到目前为止对其调控机制的了解还十分有限。大量研究证实,不定根发生与植物生长素类物质密切相关,因此现有的研究不仅围绕生长素及其信号传导途径展开,而且还涉及到基因表观遗传学调控水平。目前已经鉴定出一些与不定根发生相关的生长素信号传导因子,如NO、cGMP、microRNAs等。同时,还克隆到一些与不定根发生相关的基因,如OsPIN1、OsCKI1、NPK1、ARL1等。此外,发现DNA甲基化可以抑制DNA与蛋白(MeCP2) 的结合,从而抑制基因转录;microRNA可以使基因沉默来调控不定根的发生状况。本文围绕不定根发生的激素调控、不定根发生的基因调控、不定根发生的生长素信号传导机制、表观遗传调控等几个方面综述了近年来的研究进展。     

Review the formation of adventitious roots: New concepts, new possibilities

Summary Considerable progress has recently been made in understanding adventitious root formation using physiological studies. It is recognized that rooting is a process consisting of distinct phases, each with its own requirements. In this review, the successive phases in the rooting process are described and the possible roles of wounding-related compounds, auxin, ethylene and phenolic compounds during these specific phases are discussed. Recent results are assisting the development of advanced rooting treatments. Molecular studies on rooting are underway and will be essential in revealing the mechanisms underlying adventitious root formation.     

Endogenous gibberellin- and cytokinin-like substances in cultured shoot tissues of apple,Malus pumila cv. Jonathan,in relation to adventitious root formation

The frequencies of adventitious root formation in vitro of isolated shoots from bud cultures of apple (Malus pumila cv. Jonathan) after 1, 7 and 31 subcultures (weeks 5, 29 and 109 after the initial culture) were 5, 78 and 95% respectively. Endogenous gibberellin-like substances (GA) were extracted, chromatographed on SiO₂ partition columns, and assayed on dwarf rice (Oryza sativa cv. Tan-ginbozu). The levels of GA in shoots from the 1st, 7th and 31st subcultures were 40, 19 and 14 ng GA₃ eq./g dry weight of tissue, respectively, a trend which suggests an inverse relationship between endogenous GA level and rooting ability. This is consistent with the fact that applied GA₃ inhibits rooting in apple and many other species. The major peak of GA activity eluted coincidentally with GA₁/GA₃/GA₁₉. Endogenous cytokinin-like substances (CK) were chromatographed on paper and assayed with soybean hypocotyl sections. In contrast to the decrease in GA activity, CK activity increased 1.5–2.7 fold in the later subcultures (cytokinin activity per shoot, however, declined).     

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.     

温度对马尾松组培单芽不定根发生的影响

马尾松( Pinus massoniana)组织培养生根困难,该研究在严格控制光温条件的光照培养中,以马尾松无性系组培继代芽为材料,分析了在不同温度处理下马尾松组培单芽生根率、生根时间、根系条数和移栽成活率等生根能力指标以及生根解剖构造的变化。所有数据均采用SPSS 19.0统计分析软件,进行单因素分析(ANOVA)的显著性检验及最小显著性差异法(LSD)的多重比较。结果表明：在25℃最适温度培养下,马尾松组培生根效果较佳,移栽成活率最高,可达98.1％。低温处理下,生根时间显著延长,根系条数减少,生根率偏低;高温处理下,根茎愈伤组织明显,移栽成活率显著降低。从生根解剖构造来看,马尾松不定根主要由维管形成层细胞分化而成。与25℃适温处理相比较,在20℃较低温处理下,细胞分裂活动缓慢,不定根原始体诱发较少;而在30℃较高温处理下,细胞活动旺盛,但髓射线与维管组织细胞染色颜色加深、排列紧密,将诱发的不定根原基细胞团区隔成带状或片状。该研究结果探讨了培养环境温度对马尾松组培单芽生根能力的影响,为马尾松组培苗生产提供了参考。     

In vitro adventitious root induction in Antirrhinum majus L.

A broadly applicable method for the successful induction of root systems in a number of cultivars of A. majus has been determined. This involves a double filter-paper bridge with a liquid medium for root induction and allows the transfer of culture-grown plantlets to a glasshouse environment with minimal disturbance to the plant as a whole. 100% survival of transferred plantlets has been achieved with the inclusion of a few simple precautions upon shoot transfer and during the initial stages of plant establishment in vivo.     

Chemical induction of adventitious root formation in Taxus baccata cuttings

The effect of some auxins (IBA and NAA), phenolic compounds (phloroglucinol, gentisic acid and coumarin), a combination of auxins and phenolics, and a systemic fungicide (Bavistin) have been examined for stimulatory effects on adventitious root formation in stem cuttings (current season's growth) of Taxus baccata L. In general lower concentration (0.25 mM) of both IBA and NAA was more effective in inducing rooting of cuttings taken from both male and female trees. The combined treatment of IBA+NAA (0.25 mM each) showed some success in cuttings from male trees only (55%, compared to 15% rooting in cuttings from female trees). Generally, the callus formation was quite high (70%) in all auxin treatments (alone or in combination). Among the phenolics, 40% rooting success was achieved with phloroglucinol only, while coumarin and gentisic acid were ineffective. The combined treatment of auxins and phenolics also failed to promote rooting. On the other hand, Bavistin was extremely effective for callusing (90%) as well as rooting (80%). The effectiveness of various compounds tested for rooting of young stem cuttings declined in the order: 0.25 mM IBA>0.05% Bavistin>0.25 mM NAA>1.25 mM IBA>15 mM phloroglucinol>IBA+NAA (0.25 mM each). In addition to the auxins, IBA and NAA that are widely used for commercial propagation, the auxin-like properties of the fungicide Bavistin could be exploited for adventitious rooting in T. baccata, and in other plant species.     

Changes in peroxidase activity,auxin level and ethylene production during root formation by poplar shoots raised in vitro

Shoots of poplar (Populus tremula × P. tremuloïdes) were multiplied in vitro and rooted on a rooting medium in the presence of NAA. No rooting occurred in the absence of exogenous auxin. A peak of soluble peroxidase activity, which corresponded to a decrease in the free IAA level in the shoots, preceded rooting These events were considered as corresponding to the initiative phase of rooting. They are preceded by a peak in free IAA activity which might initiate the inductive phase of the rooting process. A burst of ethylene production was measured in both rooting and non-rooting shoots, but the ethylene peak from rooting shoots appeared earlier and was higher. The use of ACC indicated that the exogenous auxin might have enhanced ACC-synthetase activity.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - NAA naphthaleneacetic acid - IAA indole-3-acetic acid - 2-iP 2-isopentenyladenine - IAAsp indole-3-acetylaspartic acid - IBA indole-3-butyric acid - GC gas-chromatography