Shoot RNA, cambrial activity and indolebutyric acid effectivity in seasonal rooting of juvenile and mature Ficus pumila cuttings

The seasonal influence on adventitious root formation was studied in woody leaf bud cuttings of Ficus pumila L., creeping fig. Juvenile cuttings rooted easily, whereas only mature cuttings treated with indolebutyric acid (IBA) exceeded 30% rooting. Greater rooting occurred in IBA-treated juvenile and mature cuttings than controls, regardless of the month each experiment was initiated. Seasonal changes influenced rooting in all treatments except IBA treated juvenile cuttings where percentage rooting was not affected. Higher vascular cambial activity and shoot RNA levels occurred in juvenile and mature forms during peak rooting periods. Highest RNA was recorded with juvenile materials during maximum rooting periods, while lowest RNA was observed in mature shoots during low rooting intervals.     

Seasonal Changes in Auxin Effect on Rooting of Douglas-Fir Stem Cuttings as Related to Bud Activity

The relation of seasonal bud activity to the periodicity of rooting in Douglas-fir, Pseudotsuga menziesii (Mirb.) Franco, stem cuttings was studied in combination with auxin and cold storage treatments. Cuttings were collected in all months except April and May, for 3 years. Rooting was least in September and October when bud dormancy was most pronounced, greatest in December and January if exogenous auxin was applied, or in February and March if no auxin was used. The buds contributed significantly to rooting from January to April, and were responsible for differences in rooting of terminal and lateral cuttings during this period. Auxin did not enhance rooting in September and October, but at other times it replaced or supplemented the role of vegetative buds in promoting rooting. Auxin also removed the differences in rooting between lateral and terminal cuttings. Cold treatment in October and November removed bud dormancy and enhanced rooting. After November the need for auxin or cold treatment diminished and rooting without either treatment reached a maximum in February and March. Auxin did not change the seasonal pattern of rooting but broadened and enhanced the rooting response in favor of earliness. These results are discussed in relation to the effect of bud activity on auxin response and root initiation. The hypotheses are proposed that cambial dormancy or auxin deficiency is not the limiting factor during bud dormancy, and that cold treatments have the effect of bringing inhibitors and promoters into proper balance for optimum rooting response.     

Seasonal Changes in Auxin Effects on Rooting of Stem Cuttings of Populus nigra and its Relationship with Mobilization of Starch

Stem cuttings of Populus nigra were treated with 10 and 100 mg/1 each of IAA., IBA, 2,4-D and NAA at one month intervals and observations were recorded for the morphophysiological status of the branches, their starch content and their rooting response. — The first phase characterized by delayed, short and scarce roots and the high starch content of cuttings coincided with the onset of winter dormancy in November lasting through February. It was followed by a phase of vigorous rooting and low starch content of cuttings coinciding with the renovation of growth activity in February lasting through October, except in April and May when rooting was more or less completely nullified. — The poor rooting in winter was caused by low activity of hydrolyzing enzymes not mobilizing starch into soluble sugars; and profuse rooting during active growth period by high activity of hydrolyzing enzymes caused by endogenous auxin, resulting in mobilization of reserved food materials necessary for the initiation and development of roots. The low rooting in April and May is ascribed to the fact that bulk of the mobilized food was used up in the growth of sprouted branches leaving very little for rooting when these cuttings were planted. — The seasonal changes in the effectiveness of exogenously applied auxins also appear to be related with the level of endogenous auxin. In June endogenous auxin was high due to high meristematic activity, the exogenously applied auxins raising it to supra-optimal levels that were inhibitory. On the other hand, in October exogenously applied auxins enhanced rooting by raising it to an optimal level as the production of endogenous auxin had been decreasing gradually due to lowering temperatures. — The results demonstrate that auxin effect on differential rooting with season in this plant is determined by the physio-morphological status of the branches that govern the production of endogenous auxin and is mediated primarily through its effect on mobilization of reserve food materials caused by enhanced activity of hydrolytic enzymes.     

N,N′-bis-(2,3-Methylenedioxyphenyl)urea and N,N′-bis-(3,4-methylenedioxyphenyl)urea enhance adventitious rooting in Pinus radiata and affect expression of genes induced during adventitious rooting in the presence of exogenous auxin

We have analyzed the effect of N,N′-bis-(2,3-methylenedioxyphenyl)urea (2,3-MDPU) and N,N′-bis-(3,4-methylenedioxyphenyl)urea (3,4-MDPU), two symmetrically substituted diphenylurea derivatives with no auxin or cytokinin-like activity, on the rooting capacity of Pinus radiata stem cuttings. Results indicate that both diphenylurea derivatives enhance adventitious rooting in the presence of exogenous auxin (indole-3-butyric acid, IBA), even at low auxin concentration, in rooting-competent cuttings, but have no effect on the adventitious rooting of low or null competent-to-root cuttings. Histological analyses show that, in the simultaneous presence of MDPUs and low concentration of exogenous auxin, adventitious root formation is induced in the cell types that retain intrinsic competence to form adventitious roots in response to auxin. The time course of cellular events leading to root formation and the time of root emergence are closely similar to that observed in cuttings treated only with higher auxin concentration. In addition, the mRNA level of a P. radiata SCARECROW-LIKE gene, which is significantly induced in the presence of the optimal concentration (10 μM) of exogenous auxin needed for cuttings to root, is increased in the presence of MDPUs and low concentration of exogenous auxin (1 μM). The expression of a P. radiata SHORT-ROOT gene in rooting-competent cuttings during adventitious rooting is also affected by the presence of MDPUs when combined with auxin. As MDPUs do not affect the expression of either gene in the absence of exogenous auxin, but only in its presence, we suggest that MDPUs could interact, directly or indirectly, with the auxin-signalling pathways in rooting-competent cuttings during adventitious rooting.     

Isolation and characterization of a cDNA clone encoding an auxin influx carrier in carnation cuttings. Expression in different organs and cultivars and its relationship with cold storage

Polar auxin transport (PAT) is necessary for the formation of adventitious roots in the base of leafy stem cuttings, as has been demonstrated in several studies in which the application of PAT inhibitors strongly inhibited the rooting of cuttings. However, unlike in the case of lateral roots, there is almost no information on the molecular mechanism that controls PAT in the formation of adventitious roots. A novel cDNA encoding an auxin influx carrier has been isolated and characterized from carnation (Dianthus caryophyllus) cuttings. The full length of DcAUX1 was obtained and the deduced aminoacid sequence revealed a high degree of identity with the corresponding auxin carrier proteins from several species. The expression of this gene depended on the organ, the carnation cultivar and the length of time cuttings had been stored in a cold chamber. As a rule, expression was higher in stem than in leaves, in the basal than in the first internode and in mature than in young leaves irrespective of the cultivar and the duration of the storage. This pattern of expression agrees with the results of a previous study showing that auxin from mature leaves was essential for rooting, while exogenous auxin applied to mature leaves was polarly transported in the stem and accumulated in the basal internode (the rooting zone). Variations in the expression observed during storage (depending of the cultivar) might be related to the variation in PAT and rooting reported in previous studies.     

Auxin Stimulation of Cambial Activity in Pinus silvestris II. Dependence upon Basipetal Transport

Natural auxin content has been determined in the cambial region of large Pinus silvestris L. trees at various dates during the year. The tissue was collected from the stem of intact or ring-barked trees and from stumps remaining after the trees were cut down at breast height in early summer or late autumn. No seasonal decrease of concentration of the extractable auxin in the cambial region could be detected. Decapitation or ring-barking produced severe reduction in auxin content and arrested cambial division. In the next season the auxin level and the cambial activity remained completely depressed. It is concluded that without tissue continuity in the region external to xylem and without basipetal supply of substances, no mechanism operated by roots or remaining stem tissue near the tree base can ensure a high level of auxin in the cambial region or activate and maintain the cambial division. The activity of extracted pine auxin was found not to be identical with the stimulatory potential of authentic IAA determined by standard bioassays. The possibility of interaction with other extracted substances is discussed.     

Participation of auxin and abscisic acid in the regulation of seasonal variations in cambial activity and xylogenesis

Summary The current notion that hormonal level and cell response are clearly correlated has often been challenged recently. During the period of cambial activity, auxin content seems to control the intensity of mitosis and some features of the resulting wood, but not the duration of the active period itself. During cambial rest, the indole-3-acetic acid (IAA) level often remains high in the cambium, but the cell sensitivity to auxin is low. The decrease of auxin transport in autumn is sometimes interpreted as a major qualitative change affecting the pattern of transport, and sometimes as a secondary change occurring later than rest onset. The causes of the seasonal variation of cambial response remain unknown. A hypothesis is proposed that accounts for the structural-functional changes occurring in cambial cells during the onset of dormancy. Abscisic acid (ABA) may reduce wood production and xylem cell enlargement in late summer. An important amount of ABA may be present in the cambial zone in autumn after drought stress and in spring in the young growing shoot. Changes in ABA level do not appear to be clearly correlated with the different steps of cambial rest and activity. Beyond the role of ABA as a stress mediator, its participation in the annual regulation of cambial activity remains unclear. Its distribution in the most alkaline compartments may account for the particularities of its seasonal activity. The involvement of IAA and ABA in cambial growth is discussed within the scope of a possible annual alternation of two different metabolisms in the cambial cell.Abbreviations ABA abscisic acid - DPA dihydrophaseic acid - GA gibberellic acid - GC-MS gas chromatography-mass spectrometry - IAA indole-3-acetic acid - PA phaseic acid - RNA ribonucleic acid - SICM single ion current monitoring - SIM selected ion monitoring     

The Effects of Ascorbate on Root Regeneration in Seedling Cuttings of Tomato

The changes in ascorbate (ASC) and dehydroascorbate (DHA) levels and the activities of ascorbate metabolising enzymes were examined during adventitious root formation in cuttings of tomato (Lycopersicon esculentum Mill. cv. Paw) seedlings. The effects of ASC, DHA and the immediate ascorbate precursor – galactono-γ-lactone (GalL) supplemented to the culture medium on the rooting response, ascorbate content and the activities of the ASC-metabolising enzymes were also investigated. The cuttings treated with abovementioned compounds formed more roots then control plants. However, in contrast to the number of regenerated organs, the elongation of newly formed roots was markedly inhibited. Treatment with auxin (IAA) resulted in a similar phenotype. The inhibitor of auxin polar transport-TIBA (2,3,5-triiodobenzoic acid) effectively blocked rooting. The inhibitory effect of TIBA was reversed by auxin and ASC treatments, while DHA and GalL were ineffective. Both auxin and ASC stimulated cell divisions in an area of pericycle layer of TIBA-treated rooting zones, that enabled cuttings to form roots in the presence of the inhibitor of auxin polar transport. It has been found that the first stages of rooting, preceding the emergence of roots, are accompanied by an increase in endogenous content of ASC with a peak in the 3rd day of rooting. Subsequent stages, when elongation of newly formed roots occurs, are characterised by low level of ASC. The activities of the ascorbate peroxidase (APX), ascorbate oxidase (AOX), ascorbate free radical reductase (AFRR) and dehydroascorbate reductase (DHR) increased in the first 3 days of root formation. The initial period of rooting was also accompanied by the increase of the hydrogen peroxide content and the activities of catalase (CAT) and guaiacol peroxidase (GPX) in the rooting zones. IAA, ASC, DHA as well as Gal stimulated the APX activity, however the rise of the enzyme's activity induced by ASC, DHA and Gal was reversed by TIBA, which was found to inhibit APX. Only exogenous IAA was able to maintain the high level of APX activity in the TIBA-treated cuttings. AOX was strongly affected by ASC and GalL – treatments, its activity increased in the cuttings grown on the media containing ASC in the absence as well as in the presence of TIBA. On the other hand, GalL-dependent stimulation of its activity was suppressed if TIBA was present in a rooting medium.     

Hormonal control of cambial activity and vessel differentiation in Quercus robur

Cambial activity and vessel differentiation of the Quercus robur stem were investigated in relation to concentration of growth regulators and sucrose, seasonal changes in the sensitivity of cambial cells, and axial polarity of the stem. Basipetal efflux of natural auxin was measured in the oak stem cambial region. IAA, GA₃, kinetin and sucrose affected cambial activity and/or initiation of vessel differentiation differently, depending upon concentration. Depending upon the season, kinetin increased or reduced the stimulation of cambial activity caused by IAA and GA₃, but it did not affect the differentiation of vessels. Supply of sucrose in higher concentrations reduced the number of differentiated vessels but did not decrease the stimulation of cambial divisions.Unlike stimulation of cambial activity by GA₃, auxin stimulation of cambial divisions and differentiation of vessels were highly dependent upon stem polarity, 2,3,5-triiodobenzoic acid (TIBA) inhibited formation of vessels, but not cambial activity. The oscillations in basipetal efflux of natural auxin from the cambial stem region of successive 6 mm long sections substantiate the hypothesis that the histogenesis of xylem tissue in ring-porous species is under control of the vectoriat field that is associated with oscillatory phenomena in polar auxin transport.     

Auxins or Sugars: What Makes the Difference in the Adventitious Rooting of Stored Carnation Cuttings?

Cold storage of cuttings is frequently applied in the vegetative propagation of ornamental plants. Dianthus caryophyllus was used to study the limiting influences of auxin and sugars on adventitious root formation (ARF) in cuttings stored at 5°C. Carbohydrate levels during storage were modulated by exposing cuttings to low light or darkness. The resulting cuttings were treated (or not) with auxin and planted, and then ARF was evaluated. Carbohydrate levels in the cuttings were monitored and the influence of light treatment on indole-3-acetic acid (IAA) and zeatin (Z) in the basal stem was investigated. Dark storage for up to 4 weeks increased the percentage of early rooted cuttings and the final number and length of adventitious roots, despite decreased sugar levels in the stem base. Light during cold storage greatly enhanced sugar levels, particularly in the stem base where the Z/IAA ratio was higher and ARF was lower than observed in the corresponding dark-stored cuttings. Sugar levels in nonstored and dark-stored cuttings increased during the rooting period, and auxin application enhanced the accumulation of sugars in the stem base of nonstored cuttings. Auxin stimulated ARF most strongly in nonstored, less so in light-stored, and only marginally in dark-stored cuttings. A model of auxin-sugar interactions in ARF in carnation is proposed: cold storage brings forward root induction and sink establishment, both of which are promoted by the accumulation of auxin but not of sugars, whereas high levels of sugars and probably also of cytokinins act as inhibitors. Subsequent root differentiation and growth depend on current photosynthesis.     

Stimulation of adventitious root formation in non-woody stem cuttings by uridine

Uridine strongly stimulated adventitious root formation in stem cuttings of sunflower (Helianthus annuus L.), mung bean (Vigna radiata L.) and common bean (Phaseolus vulgaris L.). A dose response curve of uridine induced rooting showed that the optimum concentration of uridine was 0.1 µM. At all concentrations employed, uridine had no significant effect on root elongation. The rooting response of stem cuttings to the optimal concentration of indole-3-butyric acid (10 µM) in combination with 0.1 µM uridine did not significantly differ from their response to either of these compounds when applied alone. However, the rooting response of the cuttings to sub-optimal IBA (0.01 µM) was significantly stimulated by uridine. These findings suggested that uridine may have stimulated rooting by increasing the sensitivity of the rooting tissue to auxin.     

Adventitious rooting: examining the role of auxin in an easy- and a difficult-to-root plant

Therooting responses of cuttings of difficult-to-root lilac (Syringavulgaris) and easy-to-root forsythia(Forsythia×intermedia)were compared. The rooting ability of lilac cuttings declined over the growingseason (May–June). There was also a decline in the initial concentrationof free IAA at the base of the cuttings, but there was not a tight relationshipbetween basal IAA concentration and rooting ability. Polar auxin transportability was measured in lilac and forsythia during the period of maximum growthby [³H]IAA application to stem internodal tissue. Transport abilitydeclined in lilac over this time period, particularly in terms of transportintensity and percentage of [³H]IAA transported. In contrast thechanges in polar auxin transport ability in forsythia were less marked. Thisdifference between species was maintained in winter hardwood cuttings, withforsythia tissue showing greater polar auxin transport ability than lilac. Theimportance of polar auxin transport for adventitious rooting was demonstratedinboth lilac and forsythia softwood cuttings by use of the polar transportinhibitor 2,3,5-triiodobenzoic acid (TIBA). Overall the results indicate thatdifferences in polar auxin transport ability between lilac and forsythiacontribute to differences in rooting ability.     

Auxin Stimulation of Cambial Activity in Pinus silvestris I. The Differential Cambial Response

Isolated stem segments of Pinus silvestris L. produce new xylem in sterile culture for 5 weeks if sucrose and IAA are present in the medium. The response of cambium varies in the course of the season and along the tree stem. The cambium is more sensitive in spring and in the stem portion closer to tree apex than later in the season and closer to the stem base. Spring initiation of cambial activity in adult pine trees under natural conditions could not be correlated with any consistent concentration gradient of natural auxin extracted from the cambial region. Thus, the relation between concentration of auxin and the activity of cambium is complex and involves changes of cambial responsivity. Interaction with gibberellic acid or kinetin and changing concentration of sucrose were studied during the season, but none of these substances alone appeared to be responsible for the observed variation in cambial response to auxin.     

Metabolic changes during rooting in stem cuttings of five mangrove species

Vegetative propagation through rooting in stem cuttings in five tree mangroves namely Bruguiera parviflora, Cynometra iripa, Excoecaria agallocha, Heritiera fomes, and Thespesia populnea using IAA, IBA and NAA was reported. Spectacular increase in the root number was noted in the cuttings of H. fomes and C. iripa treated together with IBA (5000 ppm) and NAA (2500 ppm). The highest number of roots was obtained with IBA (2500 ppm) and NAA (500 ppm) in E. agallocha. B. parviflora and T. populnea responded better to IAA and IBA treatment. The species specific variation in the rooting response to exogenous application of auxins was reflected in the metabolic changes during initiation and development of roots in cuttings. Biochemical analysis showed increase of reducing sugar in the above-girdled tissues at initiation as well as subsequent development of roots which was further enhanced by the use of auxins. Decreases in the total sugar, total carbohydrate and polyphenols and increase in total nitrogen were recorded in the girdled tissues and the high C/N ratio at the initial stage helped in initiation of roots in all the species. Interaction of IBA and NAA promoted starch hydrolysis better than IAA and IBA during root development and subsequently reduced the C/N ratio and increased the protein-nitrogen activity during root development which suggest the auxin influenced mobilization of nitrogen to the rooting zone.Abbreviations IAA Indole-3-acetic acid - IBA Indole-butyric acid - NAA A-naphthalene acetic acid     

Juvenility and endogenous rooting substances inCastanea sativa Mill

The rooting response to exogenous auxin of cuttings in a juvenile phase of growth from plants ofCastanea sativa Mill. was determined and simultaneously the rooting potential of the water extracts was evaluated in presence of IAA by a bean rooting test. The level of the extractable rooting promoters was high in the cuttings which exhibited the highest percentage of rooting. An inhibition of the effect of IAA on rooting was detected in the cuttings which showed the lowest rooting response, the histogram differing not much from that of the adult plant. The results indicate that in chestnut the juvenile condition, easy rooting, is associated with high levels of endogenous rooting promoters.     

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.     

Distribution of indole-3-acetic acid in Petunia hybrida shoot tip cuttings and relationship between auxin transport, carbohydrate metabolism and adventitious root formation

To determine the contribution of polar auxin transport (PAT) to auxin accumulation and to adventitious root (AR) formation in the stem base of Petunia hybrida shoot tip cuttings, the level of indole-3-acetic acid (IAA) was monitored in non-treated cuttings and cuttings treated with the auxin transport blocker naphthylphthalamic acid (NPA) and was complemented with precise anatomical studies. The temporal course of carbohydrates, amino acids and activities of controlling enzymes was also investigated. Analysis of initial spatial IAA distribution in the cuttings revealed that approximately 40 and 10 % of the total IAA pool was present in the leaves and the stem base as rooting zone, respectively. A negative correlation existed between leaf size and IAA concentration. After excision of cuttings, IAA showed an early increase in the stem base with two peaks at 2 and 24 h post excision and, thereafter, a decline to low levels. This was mirrored by the expression pattern of the auxin-responsive GH3 gene. NPA treatment completely suppressed the 24-h peak of IAA and severely inhibited root formation. It also reduced activities of cell wall and vacuolar invertases in the early phase of AR formation and inhibited the rise of activities of glucose-6-phosphate dehydrogenase and phosphofructokinase during later stages. We propose a model in which spontaneous AR formation in Petunia cuttings is dependent on PAT and on the resulting 24-h peak of IAA in the rooting zone, where it induces early cellular events and also stimulates sink establishment. Subsequent root development stimulates glycolysis and the pentose phosphate pathway.     

Effect of abscisic acid on endogenous IAA, auxin protector levels and peroxidase activity during adventitious root initiation in Vigna radiata cuttings

Endogenous levels of free and conjugated IAA, auxin protectors (Prs) and peroxidase (PER) activity and their relation to adventitious root initiation (ARI) were investigated at the potential sites of adventitious rooting in relation to exogenous application of 250 μM ABA during the first 120 h after treatment. Cuttings from 7-day-old mung bean [Vigna radiata (L.) Wilcz.] seedlings were treated with 125, 250, and 500 μM ABA for 24 h. ABA significantly stimulated ARI but extremely inhibited epicotyl growth as compared to control. Free and conjugated IAA were measured by reversed-phase high performance liquid chromatography while Prs and PER activities were measured spectrophotometrically. The present results also indicate that endogenous free IAA levels peaked later in ABA-treated cuttings than that in control, suggesting that ABA extended the length of the induction phase of rooting process in treated cuttings and that might explain the significant delay of the appearance of roots at the treated cuttings. Higher level of IAA conjugates was found in ABA-treated cuttings than that in untreated ones. Pr level also peaked later in ABA-treated cuttings than that in control, indicating that ABA extended the period of Pr activity. An initial temporary decrease of PER activity was found in associating with high levels of free IAA and Prs during most of the primary events, while the opposite occurred during the secondary events of adventitious rooting process in both treated and untreated cuttings. Thus, ABA may stimulate ARI in mung bean Vigna radiata cuttings by regulating the concentration and /or activities of endogenous IAA, Prs, and PER activity in favor of inducing a large number of adventitious roots at their potential sites of adventitious rooting.     

The role of auxin stored in scots pine trunk during spring activation of cambial activity

In a 9-year-old pine girdled during the winter cambial activity was observed below the girdle in the next spring. This indicates that cambial activity was initiated without auxin produced in the spring by buds. The auxin produced in apical shoots successively flows down the stem, where as a result of periodic restriction in transport it remains over the winter till the next year. This auxin of apical origin but locally stored over the winter in the stem is responsible for the activation of cambium before the new flow of auxin produced in the apical meristems arrives. Calculations based on seasonal changes in auxin levels can explain both, earlier spring activation of cambium in the crown and the temporary cambial divisions below the girdle, without assumption of direct auxin synthesis in the lateral meristems.