Endogenous Indole-3-acetic Acid Research Articles

The Effects of Exogenous Auxins on Endogenous Indole-3-Acetic Acid Metabolism (The Implications for Carrot Somatic Embryogenesis).

The effect of auxin application on auxin metabolism was investigated in excised hypocotyl cultures of carrot (Daucus carota). Concentrations of both free and conjugated indole-3-acetic acid (IAA), [2H4]IAA, 2,4-dichlorophenoxyacetic acid, and naphthaleneacetic acid (NAA) were measured by mass spectroscopy using stable-isotope-labeled internal standards. [13C1]NAA was synthesized for this purpose, thus extending the range of auxins that can be assayed by stable-isotope techniques. 2,4-Dichlorophenoxyacetic acid promoted callus proliferation of the excised hypocotyls, accumulated as the free form in large quantities, and had minor effects on endogenous IAA concentrations. NAA promoted callus proliferation and the resulting callus became organogenic, producing both roots and shoots. NAA was found mostly in the conjugated form and had minor effects on endogenous IAA concentrations. [2H4]IAA had no visible effect on the growth pattern of cultured hypocotyls, possibly because it was rapidly metabolized to form inactive conjugates or possibly because it mediated a decrease in endogenous IAA concentrations by an apparent feedback mechanism. The presence of exogenous auxins did not affect tryptophan labeling of either the endogenous tryptophan or IAA pools. This suggested that exogenous auxins did not alter the IAA biosynthetic pathway, but that synthetic auxins did appear to be necessary to induce callus proliferation, which was essential for excised hypocotyls to gain the competence to form somatic embryos.

The response to auxin of rapeseed (Brassica napus L.) roots displaying reduced gravitropism due to transformation by Agrobacterium rhizogenes.

It has recently been documented that, compared to untransformed controls, the roots of oilseed rape (Brassica napus L. CV CrGC5) seedlings transformed by Agrobacterium rhizogenes A4 show a reduced gravitropic reaction (Legue et al. 1994, Physiol Plant 91: 559-566). After stimulation at 90 degrees C or 135 degrees, the transformed root tips curve. but never reach a vertical orientation. In the present study, we investigated the causes of reduced gravitropic bending observed in stimulated transformed root tips. First, we localized the gravitropic curvature in normal and in transformed roots after 1.5 h of stimulation. The cells involved in root curvature (target cells) corresponded at the cellular level to the apical part of the zone of increasing cell length. In transformed roots grown in the vertical position, these cells showed a reduction in cell length compared to controls. Because auxin is considered to be the gravitropic mediator, the response of normal and transformed roots to exogenous auxin was studied. Indole-3-acetic acid (IAA) was applied along the first 3 mm using resin beads loaded with the hormone. In comparison to normal roots, transformed roots showed reduced bending toward the bead at all points of bead application. Moreover, the cells which responded to IAA corresponded to the target cells involved in the gravitropic reaction. The level of endogenous IAA was lower in transformed roots. Thus, it was concluded that the modified behavior of transformed roots during gravitropic stimulation could be due to differences either in IAA levels or in reactivity of the target cells to the message from the cap.

Endogenous Hormone Contents in Standard and Dwarf Persimmon (Diospyros kaki Linn. cv.)

`Xiao Fang Shi' is a rare, dwarf cultivar of persimmon (Diospyros kaki Linn cv.) recently found north of Shanghai, China. The tree starts to bear fruit at 2 years of age, while standard trees start fruiting at 5 or 6 years of age. Dwarf and standard cultivars have about equal spring shoot growth, but the dwarf cultivar has little fall growth. To determine the mechanisms of dwarfness and early fruiting, enzyme-linked immunosorbant assay (ELISA) was used to analyze the endogenous indoleacetic acid (IAA), gibberellic acid (GA1+3) and abscicic acid (ABA) contents in leaves and shoot tips of dwarf (`Xiao Fang Shi') and standard (`Da Fang Shi' and `Zhu Sha Hong') persimmon. The measurement was done during the entire growing season. The results showed that IAA, GA1+3, and ABA contents were influenced by cultivars, ages of trees, and phenophases. The dwarf cultivar `Xiao Fang Shi' has lower IAA and GA1+3 but higher ABA contents than the two standard cultivars during the growing season. These correlations are especially evident when the fruit is ripening. The correlation coefficiency between contents of IAA and GA1+3 and tree height was 0.9704, while that between ABA content and tree height is –0.9697. The low IAA and GA, and high ABA contents may be responsible for little shoot growth of the dwarf cultivar in the fall.

Indole-3-acetic Acid (IAA) Content and Axillary Bud Development in Relation to Russet Spotting in Harvested Iceberg Lettuce

This study was undertaken to determine if endogenous IAA content and axillary bud development correlate with phenylalanine ammonia-lyase (PAL) induction and russet spotting (RS) susceptibility among RS susceptible and resistant cultivars of Iceberg lettuce (Lactuca sativa L.). Final levels of ethylene-induced PAL activity and RS development were highly correlated among cultivars, field conditions, and harvest dates. Harvested Iceberg lettuce midribs contained relatively low amounts of free IAA (maximum of 5.2 ng·g-1 fresh weight). There was poor correlation between free IAA content in lettuce leaf midribs and final RS development among all cultivars, growing conditions, and harvest dates. Axillary bud development, as measured by the number of visible buds per head, bud weight, or bud length, were not significantly correlated with final RS development or midrib IAA content. Cultivars with higher initial free IAA content lost much of their IAA after 8 days storage at 5C in air ± ethylene.

Paclobutrazol affects the extension growth and the levels of endogenous IAA of almond seedlings

The effect of paclobutrazol on plant height and the concentration of free indole-3-acetic acid (IAA) in almond seedlings grown in 0.5 L pots with moist perlite and subjected to 15°C and photoperiod of 16 hours was studied.

Indole-3-acetic acid in pea cotyledons induces α-amylase in Situ

Summary The development of α-amylase activity in detached cotyledons of Pisum sativum was accelerated dramatically during incubation with the synthetic auxins, 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlo-rophenoxyacetic acid at the concentration of 1μmol·L−1 and 10μmol·L−1, and the natural auxin, indole-3-acetic acid (IAA) at the high concentration of 1 mmol·L−1 but IAA at physiological concentrations of 1 μmol·L−1 and 10μmol·L−1 had only a small effect (Hirasawa, 1989). We examined whether IAA can induce α-amylase at the physiological concentrations. Almost all indole-3-[5(n)-3H]-acetic acid supplied to detached cotyledons was converted to other metabolites during incubation after treatment, even at the concentration as high as 1 mmol·L−1. Indole-3-[3H]-acetic acid that had been applied to the buds of pea seedlings was metabolized to a lesser extent in attached cotyledons than in detached cotyledons to which indole-3-[3H]-acetic acid had been applied. The inflow of tritium and the development of α-amylase activity in the attached cotyledons were simultaneously inhibited after application of 2,3,5-triiodobenzoic acid to stems under the buds of seedlings during germination. These results suggest the possibility that endogenous IAA from buds induces α-amylase in cotyledons during germination.

Changes in ABA, IAA and GAs Contents in Reproductive Organs of Satsuma Mandarin.

The endogenous levels of abscisic acid (ABA), indole-3-acetic acid (IAA) and gibberellins (GAs) in reproductive organs of satsuma mandarin (Citrus unshiu Marc. cv. Ozaki) were determined by gas chromatograph (GC)-electron capture detector, GC-mass spectrometry-selective ion monitoring and dwarf rice bioassay. Their probable roles are discussed.1. As anthesis approached, IAA concentration in styles and stamens did not change, while ABA concentration of styles increased six-fold. Until 39 days after flowering (DAF), the fresh weigth of fruitlets increased dramatically, while that of 'sepals' which included floral disks, sepals and receptacles increased a little.2. Fruitlets attained their maximum concentration of ABA 4 DAF and then declined rapidly. Its level remained much lower than that of 'sepals' from 7 DAF. IAA concentrations of fruitlets reached a maximum 7 DAF, and thereafter decrease; that in the 'sepals' increased from anthesis to 31 DAF surpassing that of fruitlets about 15 DAF.3. GAs concentrations of fruitlets continued to increase from 7 to 31 DAF, and decreased sharply 39 DAF.4. Because there was a sequence of peaks of endogenous ABA, IAA and GAs levels in fruitlets, it seemed that these hormones may play a sequential and synergistic role in the retention and growth of fruitlets.

Auxin-polyamine interaction in the control of the rooting inductive phase of poplar shoots in vitro

Changes in endogenous free IAA and its conjugate IAAaspartate as well as in endogenous polyamines were analysed in in vitro poplar shoots during their inductive phase, under the effect of the rooting auxin NAA, in combination or not with polyamines or inhibitors of polyamine biosynthesis. In vitro raised poplar shoots rooted 100% when treated by NAA (0.3 mg/l) for 7 h, the previously determined duration of the inductive phase. Spermidine and aminoguanidine (AG, an inhibitor of diamine oxidase) alone were unable to promote rooting under these conditions, and counteracted the NAA rooting effect. Putrescine and cyclohexylamine (CHA, an inhibitor of spermidine synthase) on the contrary did not oppose the NAA effect and promoted up to 40% rooting when applied alone. The levels of free indoleacetic acid (IAA) and of its aspartate conjugate IAAsp elevated up to peaks situated at the 7th and 8th h, respectively in the basal parts of the NAA-treated shoots. Putrescine, when added toegether with NAA, did not affect the typical IAA and IAAsp increases (except a displacement of the peaks to the 8th and 10th h, respectively); when applied alone, it provoked elevation of their levels to limited extents. Quite similar results were recorded with CHA. Spermidine and AG counteracted the NAA induced elevations of IAA and IAAsp. The level of endogenous putrescine typically increased up to a peak at the 6th h in the basal part of the NAA-treated shoots only, was slightly affected by exogenous putrescine and CHA application but significantly reduced by spermidine and AG supply. The NAA inducing rooting treatment did not affect the variation of the levels of endogenous spermidine and spermine but putrescine treatment (also spermidine to a lesser extent) led to increases of their levels. The relationships between auxin and putrescine temporary accumulation are discussed as well as the involvement of the different polyamines in the rooting inductive process.

A Microscale Technique for Gas Chromatography-Mass Spectrometry Measurements of Picogram Amounts of Indole-3-Acetic Acid in Plant Tissues.

A microscale technique has been developed for routine quantifications of picogram amounts of indole-3-acetic acid (IAA) in plant tissues by combined gas chromatography-mass spectrometry. Low- and high-resolution selected-ion-monitoring and selected-reaction-monitoring mass spectrometry techniques were compared for selectivity and precision. The best selectivity was obtained with selected-reaction-monitoring analysis, and 1-mg samples containing 500 fg of IAA could be analyzed accurately with this method. This technique was used to investigate the IAA distribution pattern along the longitudinal axis of tobacco (Nicotiana tabacum [L.]) leaves. In young, developing leaves an increase of endogenous IAA from the leaf tip to the base of the leaf was observed, whereas the level of IAA was uniform along this axis in mature leaves.

Effects of the Indole-3-Acetic Acid (IAA) Transport Inhibitors N-1-Naphthylphthalamic Acid and Morphactin on Endogenous IAA Dynamics in Relation to Compression Wood Formation in 1-Year-Old Pinus sylvestris (L.) Shoots.

Both N-1-naphthylphthalamic acid (NPA) and methyl-2-chloro-9-hydroxyfluorene-9-carboxylic acid (CF) inhibit the polar transport of indole-3-acetic acid (IAA) and, therefore, are attractive tools for investigating IAA's role in the regulation of plant growth. Ringing an intact conifer shoot with lanolin containing NPA or CF induces the formation of compression wood above the ring. This induction has been attributed to a postulated accumulation of IAA above the application site of the IAA transport inhibitor, but the validity of this postulation has never been confirmed. Using gas chromatography-selected ion monitoring-mass spectroscopy with [13C6]IAA as an internal standard, we measured the levels of endogenous free and conjugated IAA in 1-year-old Pinus sylvestris (L.) shoots ringed with NPA or CF. The level of free IAA was dramatically decreased below the ring, indicating that the polar transport of endogenous IAA was inhibited by the treatment. However, the free IAA level above the ring, where compression wood was formed, was also slightly lower than in control shoots. The lack of IAA accumulation above the site of the IAA transport inhibitor could not be explained by an increase in IAA conjugation. Furthermore, the turnover of [2-14C]IAA, measured using high-performance liquid chromatography with on-line radioactivity monitoring, was the same in NPA-treated and control shoots. The decrease in IAA level above a NPA or CF ring is attributed to these substances being transported acropetally and interfering with polar IAA transport along the shoot. It is concluded that compression wood formation above a NPA or CF ring is not associated with an overall increase in cambial region IAA level or increased IAA turnover. Instead, we suggest that acropetally transported NPA and CF induce compression wood formation by interacting with the NPA receptor in differentiating tracheids, thereby locally increasing IAA in these cells.

Assimilation of exogenous 2′-14C-indole-3-acetic acid and 3′-14C-tryptophan exposed to the roots of three wheat varieties

This study was conducted to determine if plants can assimilate indole-3-acetic acid (IAA) from rooting media and if exogenous L-tryptophan (L-TRP) can be assimilated and converted by plants into auxins. The addition of 2′-14C-IAA (3.7 kBq plant-1) to wheat (Triticum aestivum L.) seedlings of three varieties grown in nutrient solution resulted in the uptake (avg.=7.6%) of labelled IAA. Most of the label IAA was recovered in the shoot (avg.=7.2%) with little accumulation in the root (avg.=0.43%). A portion of the assimilated IAA-label in the plant was identified by co-chromatography and UV spectral confirmation as IAA-glycine and IAA-aspartic acid conjugates. Little of the assimilated IAA label was found as free IAA in the wheat plants. These same assimilation patterns were observed when 2′-14C-IAA was added to wheat plants grown in sterile and nonsterile soil. In contrast, the wheat varieties assimilated considerably less (avg.=1.3%) of the added microbial IAA precursor, 3′-14C-L-TRP (3.7 kBq plant-1) and thus much lower amounts of IAA conjugates were detected. Glasshouse soil experiments revealed that 2 out of 3 wheat varieties had increased growth rates and increased yields when L-TRP (10-5 and 10-7M) was added to the root zone. It is surmised that this positive response is a result of microbial auxin production within the rhizosphere upon the addition of the precursor, L-TRP. The amino acid composition of the root exudates plays a critical role in microbial production of auxins in the rhizosphere. This study showed that wheat roots can assimilate IAA from their rooting media, which will supplement the endogenous IAA levels in the shoot tissue and may positively influence plant growth and subsequent yield.

IAA Distribution within Tomato Flower and Fruit

‘Zuisyu’ tomato (Lycopersicon esculentum Mill.) fruit growth is believed to depend on auxin content, because exogenously applied auxin promotes tomato fruit growth in vivo (Asahira et al., 1968). Iwahori (1967) and Abdel-Rahman (1977) found a rapid increase of auxin in whole tomato fruit after anthesis, and they stressed the importance of auxin in fruit set and development. However, to our knowledge, the IAA distribution within tomato flowers and fruit has not been fully described. Tomato plants were grown in a greenhouse located at Hiroshima Prefectural Agricultural Expt. Station, Japan. Fruit set was induced by natural pollination [experiments on flower parts sampled 10 days after flowering (DAF )] or by natural pollination combined with 4chlorophenoxy acetic acid (4-CPA) treatment when four buds had opened in a truss (experiments on fruit parts). Fruit with mean weights of ≈3.4, 24, 62, 110, and 140 g were sampled. The heaviest fruit were mature green and almost full size. After harvest, fruit were immediately separated into locules (with seeds), pericarps, and calyxes. From the remaining tissues (axes and partitions), only the white and central parts of the axes were excised because fixing the boundary between axes and partitions was impractical. Separated tissues were weighed and then immersed in 80% ethanol. The samples were stored in a freezer at –80C until analyzed. Indole-3-acetic acid (IAA) was extracted according to Kojima et al.’s (1993) method and determined with a high-performance liquid chromatograph equipped with a fluorometric detector (Japan Spectrosopic Co., Hachioji, Japan), according to Akiyama et al.’s (1983) method. Endogenous IAA content was calculated from the peak area of IAA :

Interaction of Ethylene and Auxin in the Regulation of Hook Growth II. The Role for Ethylene in Different Growing Regions of the Hypocotyl Hook of Phaseolus vulgaris

In different growing regions of the hypocotyl hook of Phaseolus vulgaris synthesis and levels of endogenous ethylene are asymmetrically distributed. Significant difference exists between waterstress- and IAA-induced ethylene formation from isolated hook segments, which fits to the former results (Schwark and Schierle. J. Plant Physiol. 140, 562–570, 1992) that also endogenous auxin levels are asymmetrically distributed in the hook. 1-N-Naphthylphthalamic acid (NPA), a specific inhibitor of polar transport of indole-acetic-acid (IAA) equalizes the asymmetric distribution of aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene. This result indicates, that transport of endogenous IAA is causally linked with the asymmetric synthesis of ethylene in the hook.

The differential effect of N6-benzyl-adenine and N6-(?2-isopentenyl)-adenine on in vitro propagation of Paeonia suffruticosa Andr. is correlated with different hormone contents

N(6)-benzyl-adenine (BA) enhanced phyllogenesis and axillary bud development of Paeonia suffruticosa during in vitro culture allowing good propagation while N(6)-(Δ(2)isopentenyl)adenine (iP) did not. During the first five days of culture, the mitotic activity of BA-treated explants was higher than in the iP-treated ones. High BA levels were detected in the BA-treated explants, and this was correlated with the absence of or the low indole-3-acetic acid (IAA) content. The low iP levels measured in iP-treated explants were correlated with high endogenous IAA content; the new cytokinin / auxin ratio could explain the lack of axillary buds and the development of only one leaf. Abscisic acid (ABA) was detected neither in the controls nor in the cytokinin-treated explants during the first week. However, intensive restoration of ABA accumulation was observed in controls from the third week onwards. Both BA and iP-treated explants accumulated less ABA than the controls but this hormone appeared later in the BA-treated explants than in the iP-treated ones.

Magnitude and Kinetics of Stem Elongation Induced by Exogenous Indole-3-Acetic Acid in Intact Light-Grown Pea Seedlings.

Exogenously applied indole-3-acetic acid (IAA) strongly promoted stem elongation over the long term in intact light-grown seedlings of both dwarf (cv Progress No. 9) and tall (cv Alaska) peas (Pisum sativum L.), with the relative promotion being far greater in dwarf plants. In dwarf seedlings, solutions of IAA (between 10-4 and 10-3 M), when continuously applied to the uppermost two internodes via a cotton wick, increased whole-stem growth by at least 6-fold over the first 24 h. The magnitude of growth promotion correlated with the applied IAA concentration from 10-6 to 10-3 M, particularly over the first 6 h of application. IAA applied only to the apical bud or the uppermost internode of the seedling stimulated a biphasic growth response in the uppermost internode and the immediately lower internode, with the response in the latter being greatly delayed. This demonstrates that exogenous IAA effectively promotes growth as it is transported through intact stems. IAA withdrawal and reapplication at various times enabled the separation of the initial growth response (IGR) and prolonged growth response (PGR) induced by auxin. The IGR was inducible by at least 1 order of magnitude lower IAA concentrations than the PGR, suggesting that the process underlying the IGR is more sensitive to auxin induction. In contrast to the magnitude of the IAA effect in dwarf seedlings, applied IAA only doubled the growth in tall seedlings. These results suggest that endogenous IAA is more growth limiting in dwarf plants than in tall plants, and that auxin promotes stem elongation in the intact plant probably by the same mechanism of action as in isolated stem segments. However, since dwarf plants to which IAA was applied failed to reach the growth rate of tall plants, auxin cannot be the only limiting factor for stem growth in peas.

Growth patterns and endogenous indole‐3‐acetic acid concentrations in current‐year coppice shoots and seedlings of two Betula species

Apical dominance, internode elongation, radial growth and xylem cell size in coppice and apical shoots of Betula pubescens B. Pendula were determined and related to endogenous indole‐3‐acetic acid (IAA) levels, measured by gas chromatography‐selected ion monitoring in the apical bud and at three positions along the stem. The effects of defoliation and debudding on morphological and anatomical characters and endogenous IAA levels were also investigated. The coppice shoots displayed superior stem elongation and increased branching during the initial phase of growth, after which their growth pattern was similar to that of the seedlings; however, their radial growth was greater throughout the experiment. Both plant types produced smaller‐sized xylem cells at the top of the shoot than at the bottom with coppice shoots tending to form larger tracheids and smaller vessels than the seedlings. There was no consistent difference in IAA concentration between the coppice shoots and the seedlings. Defoliation and debudding reduced the IAA level in the stem within 36 h and it was still low after 25 days. Although the extent of the IAA decrease was similar in both coppice shoots and seedlings, the treatments affected the morphological and anatomical characters differently in the two plant types. The results suggest that the observed differences between seedlings and coppice shoots were not mediated through a drastic change in IAA level.

Relations between exogenous growth regulators and endogenous indole-3-acetic acid and abscisic acid in the expression of somatic embryogenesis in Hevea brasiliensis (Müll. Arg.)

Endogenous IAA (indole-3-acetic acid) and ABA contents were measured during the somatic embryogenesis process from Hevea brasiliensis (Müll. Arg.) callus. The decrease in 3,4-D (3,4-dichlorophenoxyacetic acid) and BAP (6-benzylaminopurine) supply in the culture medium and the addition of ABA both stimulated embryogenic induction. But only exogenous ABA sustained embryogenic expression of the callus. The two operations resulted in a decrease in endogenous ABA and an increase in IAA in callus. Low endogenous ABA levels (2–4 nmol/g dry wt.) appear to be necessary for the acquisition and conservation of a embryogenic state. Non-embryogenic calli accumulated high levels of ABA (25–40 pmol/g dry wt.). This accumulation confirm the hypothesis that non-embryogenic Hevea calli were water stressed.

Endogenous levels of IAA in relation to parthenocarpy in cucumber ( Cucumis sativus L.)

The role of endogenous indole-3-acetic acid (IAA) on parthenocarpic fruit set in cucumber was investigated. Parthenocarpic fruit set was closely related to the accumulation of IAA in the ovary. Application of N-1-napthylphthylamic acid (naptalam) and 2,3,5-triiodobenzoic acid (TIBA), auxin transport inhibitors, to the ovary or peduncle significantly increased the IAA content in the ovary. Exogenously applied auxins, naphthaleneacetic acid (NAA) and 4-chlorophenoxyacetic acid (4-CPA), gibberellic acid (GA 3) and cytokinins, N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) and 6-benzylaminopurine (BA), also increased it. It is suggested that endogenous IAA is the main hormonal factor for parthenocarpic fruit set and exogenously applied hormones are considered to promote IAA accumulation in the ovary.

Auxin dependent growth of rhizoids of Chara globularis

Auxin greatly influences plant cell elongation, particularly in the organs of shoots but also in roots. Earlier reports are limited to organ and/or cell growth connected with a mosaic type of cell elongation. The present paper describes auxin sensitivity of polarly growing rhizoid cells of Chara globularis Thuill. where auxin‐dependent growth could be observed in two different ways: (1) Auxin had no effect when applied to intact Chara explants with developed thizoids, but decapitated explants reacted to auxin with optimal growth at 1 μM indole‐3‐acetic‐acid (IAA). (2) N‐I‐Naphthylphthalamic acid (NPA) at 10 and 100 μM caused a strong inhibition in rhizoid growth of intact Chara explants. Auxin applied at the same time abolished this inhibition but, due to lack of plant material, endogenous IAA content could not be measured. Chara explants pre‐fed with 1‐[14C] IAA from a 3.5 μM solution for 8 h, then washed and transferred for 11 h to auxin free solution containing 0, 10 or 100 μM NPA, showed an effect of NPA upon IAA accumulation. Therefore, NPA may inhibit auxin secretion in Chara, 100. Our data are in agreement with earlier results on auxin regulated cell elongation and H−‐secretion, and show that auxin secretion may also be an essential step in endogenous regulation of polar growth in Chara rhizoids.

Auxin levels at different stages of carrot somatic embryogenesis

The role of auxin in somatic embryogenesis was evaluated by characterizing the changes in the concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), and their conjugates in callus suspension cells and developing embryos of Daucus carota. Both embryogenic and non-embryogenic lines exhibited similar growth rates and levels of IAA and 2,4-D on 2,4-D-supplemented medium. Total endogenous IAA in both lines exposed to 2,4-D reached high levels greater than 600 ng g −1 fresh weight which suggests that IAA levels in carrot callus are not regulated via auxin feedback mechanisms. After being transferred to 2,4-D-free medium, the embryogenic line exhibited a rapid decline in both free and conjugated 2,4-D metabolites within seven days, while IAA levels remained relatively steady for seven days in the preglobular stage after which the levels declined steadily in all subsequent stages of embryo development. Individual analyses of different embryo fractions collected from asynchronous cultures confirmed that each stage in embryo development had lower IAA levels than the preceding stage. The non-embryogenic line maintained similar 2,4-D levels but higher IAA levels than the embryogenic line throughout the experiment. The present results suggest that high IAA levels may be necessary but are not sufficient for the initial events in plant embryogenesis, whereas low IAA levels are associated with the later stages of embryo development.