Auxin Pulse Research Articles

Morphoanotomical aspects of auxin herbicides-induced somatic embryogenesis in Euterpe edulis Martius, a symbol and threatened species of the Atlantic Forest

The palm Euterpe edulis presents primary meristematic growth without sprouting at the base, which prevents conventional vegetative propagation. Here, we assessed the effect of the herbicides 2,4-D and picloram on somatic embryogenesis as an alternative means for propagating and in vitro conservation of pro-embryogenic cultures of E. edulis, as their seeds are recalcitrant. Embryos could be effectively induced with 2,4-D (159.42 μmol L−1) and PIC (300 μmol L−1), totaling 44.33 and 76.33 embryos, respectively. Moreover, proembryos, embryogenic clusters, and somatic embryos were clearly visible. Embryogenic and meristematic cells presented marked staining of the cytoplasm, spherical shape of the cells and nucleus, and rapidly dividing nucleoli. Maturation of explants induced with 2,4-D and PIC was improved by adding 2iP (12.3 μmol L−1), NAA (0.6 μmol L−1), and AC (0.3 g L−1). During germination, BAP (1 μmol L−1) and GA3 (0.5 μmol L−1) promoted primary root protrusion and emergence of the shoot. In summary, PIC (300 μmol L−1) is suggested for embryogenic induction of E. edulis calli, and medium containing 2iP, NAA, and AC is suggested for maturation. Finally, medium containing BAP and GA3 without an auxin pulse favors the conversion of E. edulis somatic embryos into plants.

AUXIN PULSE IN THE INDUCTION OF SOMATIC EMBRYOS OF Eucalyptus

ABSTRACT The objective of this study was to evaluate the effect of auxin pulse intervals on the induction of somatic embryos of Eucalyptus grandis x E. urophylla and to describe the embryogenic behavior of callus under the effect of auxinic stress. Cotyledons were inoculated in culture medium containing 207.07 µM picloram, a treatment considered as auxin pulse. Explants that were in the auxin pulse treatment were transferred to semisolid or liquid medium containing 20.71 µM picloram after one, two, four or eight days of auxin pulse. In a second experiment, explants that were on auxin pulse treatment were transferred to semi-solid medium containing 20.71 µM picloram after one, two or three days of auxin pulse. Auxiliary picloram pulse treatments (207.02 µM) can be used as an initial source of stress for the acquisition of embryogenic competence. The oxidation of cotyledonary explants may be considered as an indication of the formation of embryogenic calli. The presence of pectins in peripheral regions of somatic pro-embryos can be considered as a marker of somatic embryogenesis in cotyledonary explants of Eucalyptus grandis x E. urophylla.

Improved in vitro rooting of Prunus avium microshoots using a dark treatment and an auxin pulse

The influence of a four-day dark treatment and a 48h IBA pulse on in vitro root formation of Prunus avium microshoots was examined. Both treatments led to significantly higher rooting percentages in the majority of the six genotypes. While the IBA pulse led to a disproportional growth of roots compared to shoots, the plantlets under the dark treatment during root induction were healthy and were not visually distinct from the control plants. Further data collected during the commercial production of 17 genotypes verified the improved root induction using the dark treatment and identified the gelling agent Gelcarin (a carrageenan) as an additional factor with positive effects on rooting. In addition, the importance of in vitro rooting for the successful transfer to greenhouse conditions was confirmed. Rooted plantlets (>3 roots) had a significantly higher survival percentage and showed improved growth during acclimatization compared to unrooted microshoots.

The Shape of an Auxin Pulse, and What It Tells Us about the Transport Mechanism.

Auxin underlies many processes in plant development and physiology, and this makes it of prime importance to understand its movements through plant tissues. In stems and coleoptiles, classic experiments showed that the peak region of a pulse of radio-labelled auxin moves at a roughly constant velocity down a stem or coleoptile segment. As the pulse moves it becomes broader, at a roughly constant rate. It is shown here that this ‘spreading rate’ is larger than can be accounted for by a single channel model, but can be explained by coupling of channels with differing polar transport rates. An extreme case is where strongly polar channels are coupled to completely apolar channels, in which case auxin in the apolar part is ‘dragged along’ by the polar part in a somewhat diffuse distribution. The behaviour of this model is explored, together with others that can account for the experimentally observed spreading rates. It is also shown that saturation of carriers involved in lateral transport can explain the characteristic shape of pulses that result from uptake of large amounts of auxin.

Refinement of Cost Reduction Technology in Sugarcane Micro propagation

In the present study, an efficient ex-vitro rooting technique was developed for sugarcane commercial cultivar CoS 96268 based on the assessment of several factors. Axillary bud cultures of nodal segments were used in this experiment. The parameters optimized for ex vitro rooting includes the effects of auxin concentration, propagule size, shoot size, duration of auxin pulse treatment, sucrose concentration in multiplication medium, ex-vitro rooting medium were studied. The results showed that the auxin concentration @ 20 mg/l gave maximum rooting percentage. Among 0h, 6h,12h and 24h treatment duration with auxin, 24h treatment duration developed better roots. 90 to 95% rooting was observed in three shoot propagule with 5 to 6 cm length of in vitro micro shoots. It has been observed that sucrose concentration in multiplication medium also influenced the ex vitro rooting. Sucrose at 45 g/l gave good results. Sand was best rooting medium among the peat, peat + soil and vermiculite for the development of ex vitro roots in green house conditions. Ex vitro rooted plants were transplanted in field and 9098% survival percent was found same as in vitro propagated plants of sugarcane.

Thidiazuron enhances axillary and adventitious shoot proliferation in juvenile explants of Mediterranean provenances of maritime pine Pinus pinaster

The effectiveness of thidiazuron (TDZ) in promoting in vitro organogenesis in cultured embryo axes and cotyledons from two Spanish Mediterranean provenances of Pinus pinaster Aiton is reported. Application of 44.4 μM TDZ as a 6-d pulse on DCR medium and subsequent transference of maritime pine explants to the same medium without growth regulators, but supplemented with activated charcoal, produced between 17 and 19 axillary shoots per embryo axis with no differences between the two provenances. The same sequential treatment allowed adventitious shoot differentiation on 75% of the cotyledons, with a mean of 12.2 shoots regenerated per explant. Axillary and adventitious shoots were rooted ex vitro after an auxin pulse of indole-3-butyric acid and 1-naphthaleneacetic acid. The micropropagation protocols presented here should enable vegetative multiplication of selected families, and elite trees from these Spanish Mediterranean provenances of maritime pine.

Auxin pulse treatment holds the potential to enhance efficiency and practicability of somatic embryogenesis in potato

The objective of the current study was to simplify existing somatic embryogenesis systems in potato (Solanum tuberosum L.) cv. Desiree. The project targeted the agar-based induction phase of the potato somatic embryogenesis process as the key area for improvement. Experiments were established to ascertain the effect of a 2,4-D (2,4 dichlorophenoxyacetic acid) pulse, applied to the primary internodal section explant source and its subsequent effect on embryo induction. Parameters tested were the duration of the auxin pulse in a range from 0 to 300 min, and the concentrations of 2,4-D applied, in a range from 0 to 5,120 microM. The mean number of somatic embryos formed per explant was recorded after 4 and 8 weeks culture. Our findings indicated that the somatic embryogenesis in potato internodal segments could be evoked by an auxin (2,4-D) pulse treatment over a wide concentration and duration range. The results further suggested that a simple 20 microM 2,4-D pulse treatment could replace a lengthy 2 week induction phase in potato somatic embryogenesis and thus improve the system's practicability for wider uptake.

A liquid 2,4-D pulse increased shoot and root regeneration from leaf explants of adult Prunus rootstocks

Regeneration of adult plant material is one of the main limitations for successful Prunus rootstock transformation. Results herein show that a liquid pulse (90 min) of 2,4-dichlorophenoxyacetic acid (2,4-D) (1.7 μM), applied to leaf explants, greatly improved shoot regeneration in Marianna 2624 ( Prunus cerasifera × munsoniana) and Myrobalan 605 AD ( P. cerasifera); and induced roots in Adafuel ( Prunus × amigdalo-persica) when placed in regeneration medium. Whole leaves and basal leaf explants of Marianna 2624 regenerated shoots in a higher proportion of explants after the pulse (up to 58.9% in whole leaves) than medium or tip leaf segments, whereas the leaf tip was the explant that showed less regeneration. In the regeneration medium, in the presence of BAP, NAA was more effective than 2,4-D. The application of an auxin pulse is a simple method that could enhance adult plant regeneration in commercial rootstocks.

Immuno-cytochemical localization of indole-3-acetic acid during induction of somatic embryogenesis in cultured sunflower embryos

Immature zygotic embryos of sunflower (Helianthus annuus L.) produce somatic embryos when cultured on medium supplemented with a cytokinin as the sole source of exogenous growth regulators. The timing of the induction phase and subsequent morphogenic events have been well characterized in previous work. We address here the question of the role of endogenous indole-3-acetic acid (IAA), since auxins are known to have a crucial role in the induction of somatic embryogenesis in many other culture and regeneration systems. The fact that in the sunflower system no exogenous auxin is required for the induction of somatic embryos makes this system very suitable for the study of the internal dynamics of IAA. We used an immuno-cytochemical approach to visualize IAA distribution within the explants before, during and after the induction phase. IAA accumulated transiently throughout cultured embryos during the induction phase. The detected signal was not uniform but certain tissues, such as the root cap and the root meristem, accumulated IAA in a more pronounced manner. IAA accumulation was not restricted to the reactive zone but the kinetics of endogenous variations strikingly mimic the pulse of IAA that is usually provoked by exogenous IAA application. The direct evidence presented here indicates that an endogenous auxin pulse is indeed among the first signals leading to the induction of somatic embryogenesis.

Micropropagation of Juvenile Eucalyptus regnans (Mountain Ash)

Node-derived shoot cultures of Eucalyptus regnans were established from in vitro grown seedlings on Murashige and Skoog basal medium supplemented with 0.5 mg L-1 (2 μm) zeatin and 0.05 mg L-1 (0.3 μm) napthaleneacetic acid. A double sterilisation method was essential to obtain clean material from seed. Microcuttings from established cultures were used to develop an efficient method for in vitro rooting. Rooting was best after a 7 day pulse on 20 mg L-1 (98 μm) indolebutyric acid. Hoagland's or Woody Plant Medium supported better rooting than MS basal medium and rooting was significantly enhanced by subculture to activated charcoal after the auxin pulse. Carbohydrate (sucrose or glucose) was essential for rooting while high light intensity was inhibitory. Optimal light conditions were a 12 h day (17 W m-2). In all, 90% of plantlets established in the nursery survived the winter.

Micropropagation of eastern white pine (Pinusstrobus L.)

Cotyledons, epicotyls, and hypocotyls from embryos and "seedlings" of Pinusstrobus formed shoots following exposure to cytokinin on a Schenk and Hildebrandt medium. Benzyladenine and 2-isopentenyladenine were both effective at inducing shoot formation but benzyladenine was 5–10 times more potent. There was no synergistic interaction between these two cytokinins. Benzyladenine concentrations of 0.5–1.0 mg L−1 gave consistently good results with cotyledons. A 2-week cytokinin exposure produced uniformly good results and longer exposures caused callusing and vitrification. Pretreating seeds with hydrogen peroxide for 3 days stimulated caulogenesis. Addition of phenolics to medium containing benzyladenine also stimulated shoot formation and di-substituted phenolics were more effective than p-hydroxybenzoic acid. A short liquid pulse with high concentrations of benzyladenine induced caulogenesis but was not as effective as the application of cytokinin through agar-solidified culture medium. Cotyledons excised from embryos in cultured megagametophytes or from cultured embryos were more caulogenic than cotyledons from imbibed seeds. Best caulogenesis occurred after 1 week of direct embryo culture or culture of the embryo inside the megagametophyte. The caulogenic capacity of cotyledons from cultured embryos declined after 1 week but they retained competence to form shoots for 1 month. Epicotyls and hypocotyl sections from 1-week-old cultured embryos were also caulogenic but roots were not. Epicotyls responded best at benzyladenine levels less than 1 mg L−1, but hypocotyls required higher concentrations. Caulogenesis from hypocotyls was polar in that most shoots formed at the upper end of the apical hypocotyl half. The basal hypocotyl half was less caulogenic and produced callus. Roots only produced callus. Charcoal inhibited shoot induction when it was included with benzyladenine. Charcoal also inhibited rhizogenesis when included in the final rooting medium. Shoot elongation was promoted by charcoal when it was given during the second subculture on basal medium for 1 month. Shoot elongation occurred on full-strength basal medium. Reduced concentrations of minerals and organics failed to enhance shoot elongation. Approximately 80% of microshoots rooted invitro on quarter-strength basal medium without any hormone application. An auxin pulse treatment had little effect on rooting. Application of rooting powders containing indolebutyric acid increased the number of roots formed but had little effect on rooting frequency.

De novo shoot organogenesis of Pinus eldarica Medw. in vitro

Seedling-derived explants of the Afghan pine, Pinus eldarica, were cultured in a triplicate experiment to produce callus that was serially subcultured for up to three years. Callus was removed at various times and induced to regenerate shoots by de novo organogenesis. The shoot regeneration process involved the identification of four discrete developmental steps, each requiring a separate cultural manipulation. In one case a regenerated shoot was induced to root following an auxin pulse treatment. Induction and limited development of buds in callus derived from mature-tree explants was also achieved. This is the first reproducible system for shoot regeneration from long-term callus cultures of a conifer.

Cell wall pH and auxin transport velocity.

According to the chemiosmotic polar diffusion hypothesis, auxin pulse velocity and basal secretion should increase with decreasing cell wall pH. Experiments were designed to test this prediction. Avena coleoptile sections were preincubated in either fusicoccin (FC), cycloheximide, pH 4.0, or pH 8.0 buffer and subsequently their polar transport capacities were determined. Relative to controls, FC enhanced auxin (IAA) uptake while CHI and pH 8.0 buffer reduced IAA uptake. Nevertheless, FC reduced IAA pulse velocity while cycloheximide increased velocity. Additional experiments showed that delivery of auxin to receivers is enhanced by increased receiver pH. This phenomenon was overcome by a pretreatment of the tissue with IAA. Our data suggest that while acidic wall pH values facilitate cellular IAA uptake, they do not enhance pulse velocity or basal secretion. These findings are inconsistent with the chemiosmotic hypothesis for auxin transport.

Xylogénèse chez les dicotylédones arborescentes. III. Transport de l'auxine et activité cambiale dans les jeunes tiges de Hêtre

The pattern of [3H]-labelled auxin transport has been studied in the beech (Fagus sylvatica L.) (a diffuse-porous tree), in young shoots where the slow basipetal cambial reactivation depends on the buds. At 20 °C, most [3H]-labelled IAA molecules proceed downwards in active shoots as a wave, whose average speed is 13 to 15 cm a day; the velocity of the front being at least 18 to 19 cm a day. The speed of the [3H]-labelled IAA wave depends on the temperature; it is about 7 cm a day at 9 °C, which is the average temperature in the forest during the cambial reactivation period. In natural conditions, the auxin pulse velocity is comparable to that of the cambial reactivation. The ability of tissues to transport auxin at 20 °C depends on the season. During winter rest, the pattern of [3H]-labelled IAA movement is diffusive and very slow. During the 2 months preceding cambial reactivation, the tissues become gradually capable of active auxin transport, the speed and the intensity of which increase at the same time. Microautoradiographs show that the cambium and its differentiating derivatives are the site of auxin transport and also of its utilization: the hormone occupies a preferential site for xylogenesis regulation.

Distribution of labelled auxin and derivatives in stem tissues of intact and decapitated broad‐bean plants in relation to apical dominance

Abstract[3H]‐auxin (0.13 to 0.18 nmol) was applied to the apical bud of broadbean plants (Vicia faba L. cv. Aguadulce). After 24 h, the exportation from the donor organ was ended. After 48 h, i.e. 10–15 h after the passage of the [3H]‐auxin pulse into the root system, the distribution and the nature of labelled molecules located in the basal part of the stem and in the axillary buds were investigated. Chromatographic analyses concerned both intact plants and plants decapitated 12 h, 24 h or 42 h after the [3H]‐auxin application. In intact plants, there was no significant amount of [3H]‐auxin in the axillary buds, whose radioactivity was very low compared to the stem tissues. The labelled molecules with the Rf of auxin represented 50% or more of the whole radioactivity of the stem tissues. The distribution of [3H]‐auxin was not uniform along the stem. In particular, the cotyledonary node zone, bearing the most inhibited buds, which is known to be an important centre of label retention, contained the highest amounts of labelled auxin both in intact and decapitated plants. The decapitation was quickly followed by a decrease of the [3H]‐auxin amount in the stem base more than 15 cm away from the wound, particularly in the scale leaf nodes, whose axillary buds were mainly the ones to grow after relief from apical dominance. The induction of this early decrease was clearly distinct in plants decapitated when auxin exportation from the donor organ was ended.

Differences in Velocity of Auxin Movement Obtained by Intercept and Peak Arrival Methods

AbstractVelocity of IAA movement was determined by noting the time of arrival of an ether‐soluble auxin wave, at a fixed distance, after presentation of an auxin pulse to bean (Phaseolus vulgaris L. cv. Pinto) hypocotyl segments. The effects of IAA adsorption on wave symmetry were reduced by monitoring the arrival of ether‐soluble auxin molecules. Velocity of auxin movement, as estimated by wave arrival, was slower than velocity estimated by the intercept method in controls and in both iodoacetate (IOAA) and 2,3,5‐triiodobenzoic acid (TIBA) treated tissue. Velocity of wave migration was reduced by both treatments but intercept velocity was reduced only by TIBA treatment. Velocity of IAA migration was faster in segments (independent of method of measurement) than from segments into agar by a factor of 4 to 8. The rate limiting step of auxin migration in the traditional agar‐plant sandwich is the partitioning of IAA between the tissue and agar. It was suggested that arrival curves for pulsed auxin migration are analogous to elution profiles of chromatographic columns and that at least two populations of mobile molecules with different velocities exist. It was also suggested that the two velocities represent migration of auxin on two different pathways: the faster velocity representing auxin movement of water films which coast highly crosslinked polymers in the segment and the slower component representing a population which moves primarily within the matrices of crosslinked polymers. Velocity of both populations may be a function of tissue hydration and charge interactions of mobile molecules and matrix polymers.

Time Course of Auxin Stimulations of Growth

Measurements of the time course of growth responses of corn coleoptile sections to pulses of auxin (10(-5)m indoleacetic acid) establish that the growth rate changes in a regular pattern around the auxin pulse: a latent phase of 12 to 15 minutes is followed by an acceleration of growth rate lasting 15 to 20 minutes, after which a fairly steady rate is maintained. When the auxin source is withdrawn, there is an after-effect of about 15 minutes followed by a decay of growth rate, which reaches 50% decay after a further 15 to 40 minutes. The decay phase appears to be a function of the transport of auxin out of the sections. The 50% decay of growth for single cells is estimated at 30 minutes from the time of withdrawal of an exogenous supply of auxin. The regulation of growth by auxin is rapidly imposed or dissipated as auxin enters and exits, respectively, suggesting a facile association and disassociation of auxin with a growth-limiting site in the cell. It is proposed that the growth-stimulated state is dissipated at once when the transportable auxin has passed out of the cell.

Ethylene Modification of an Auxin Pulse in Cotton Stem Sections

The effect of ethylene on the basipetal movement of indole-3-acetic acid-1-(14)C through cotton stem sections (Gossypium hirsutum, L. var. Stoneville 213) was studied apart from processes involved in the uptake and exit of auxin by the section. Stem sections 60 mm in length were pretreated with ethylene or placed in room air (control) and pulse labeled for 20 min with IAA-1-(14)C. In both the ethylene treated and control sections, the IAA-1-(14)C taken up moved basipetally as a peak of radioactivity. Generally, the applied pulse moved down the stem sections at an average velocity of approximately 5.8 mm per hr. In some experiments, however, ethylene slightly reduced the velocity of auxin transport. Although the peak of radioactivity became broader and more dispersed during its migration through the section, it was still distinguishable after 7 hr of transport.As the ethylene pretreatment periods were increased from 1.5 to 3.0 hr there was a progressively greater loss of activity from the pulse of applied IAA-1-(14)C during its basipetal movement. On the average, 4% more activity was lost from the applied pulse at 1.5 hr, 15% more at 2.0 hr and 26% more at 3.0 hr when compared to control stem sections.The data establish that ethylene inhibits auxin transport in vivo, and it is proposed that the effect is due possibly to increased rates of auxin immobilization.