A rapid, sensitive and accurate determination of indolyl-3-acetic acid

Knegt, Erik; Bruinsma, J.

doi:10.1016/0031-9422(73)80672-7

Cited by 112 publications

(52 citation statements)

References 2 publications

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“…The seedlings (3-5 g fresh weight) were ground to a fine powder in a prechilled mortar and pestle. The powdered material was extracted, the extract was purified, and IAA was assayed by procedures that were slight modifications of those described by Knegt and Bruinsma (16). The ground tissue was extracted three times with 20 ml of cold methanol in a glass Buchner funnel with a fritted glass disc.…”

Section: Methodsmentioning

confidence: 99%

Endogenous Levels of Auxin and Tumorigenesis in a Nicotiana Amphiploid

Ames¹

1974

Plant Physiol.

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Young seedlings of the tumor-prone amphiploid Nicotiana suaveolens X N. langsdorjjii were grown aseptically on nutrient medium in a controlled environment chamber. At regular intervals after the seeds had been sown, the incidence of tumor formation was scored and plants were harvested. Indole-3-acetic acid was extracted, purified, and assayed spectrofluorometrically. A close correlation in time between onset of tumor formation and decline in endogenous level of indole-3-acetic acid was demonstrated.It has been suggested that the trigger for tumor induction in certain Nicotiana hybrids is a reduction in the endogenous level of auxin (5). There is considerable evidence in support of this hypothesis, and it is beginning to look as though the mechanism of tumor induction in this system is similar to that by which axillary buds are released from the dominance of the apical meristem. First, it has been demonstrated that correlative inhibition of axillary buds diminishes as a plant gets older (18). Tumors appear most frequently on mature Nicotiana hybrids during and after the flowering period when growth of the shoot apex is reduced (15). Second, growth of inhibited axillary buds can be stimulated by decapitation of the apical bud, and this loss of apical dominance can be prevented by applying IAA to the cut surface of decapitated plants (27). Decapitation of primary and secondary shoots accelerates tumor formation in Nicotiana, and the appearance of internodal tumors can be suppressed with exogenous IAA (5). Third, triiodobenzoic acid has been shown to break apical dominance in a variety of plants (6,13,20,25,28) While the available evidence supports the suggestion that auxin is involved in the trigger for tumor induction in genetically tumor-conditioned Nicotiana hybrids, a reduction in auxin at potential centers of growth prior to tumor initiation has not been reported. The aim of the present investigation was to study the rate of tumor formation and fluctuations in the endogenous level of IAA with time in young tumor-prone Nicotiana seedlings. MATERIALS AND METHODSSeeds of the amphiploid Nicotiana suaveolens X N. lanigsdorffii were surface-sterilized in a Clorox-Kromet solution for 12 min, and then sown in 125-ml Erlenmeyer flasks on 30 ml of a modified Hildebrandt-Riker nutrient medium (pH 5.3-5.4) which was solidified with 1.0% (w/v) agar (14). Seedlings were grown in a controlled environment chamber at 22 ± 1 C and 60 to 65% relative humidity under 18 hr illumination (1900 -4-150 ft-c). Beginning 11 days after the seeds had been sown, the plants were examined at regular intervals in order to score the incidence of tumor formation as evidenced by visible outgrowth of undifferentiated tissue.In order to determine the endogenous level of IAA, plants were harvested, the roots were removed, and the tops were immediately frozen in liquid nitrogen. The seedlings (3-5 g fresh weight) were ground to a fine powder in a prechilled mortar and pestle. The powdered material was extracted, the extract was purified, ...

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Section: Methodsmentioning

confidence: 99%

Endogenous Levels of Auxin and Tumorigenesis in a Nicotiana Amphiploid

Ames¹

1974

Plant Physiol.

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“…The extract was purified by solvent partitioning (17) followed by column chromatography on polyvinylpyrrolidone (Polyclar AT GAF) (13 7) with increasing concentrations of methanol as follows-5% min-' for 5 min, 2% min-' for 10 min, 1% min-' for 5 min, 2% min-' for 15 min-gave a retention time for authentic methyl indol-3yl-acetate of 20 min. The appropriate fraction was collected from the column and used for GC-MS.…”

Section: Methodsmentioning

confidence: 99%

“…The methanolic extracts were filtered and the filtrates were combined and concentrated to small volume under reduced pressure at 30°C. The concentrated extract was purified by solvent partitioning in the manner described by Knegt and Bruinsma (17). The purified extract contained pigments which caused considerable quenching of fluorescence, hence, a further purification step was used before the spectrophotofluorimetric assay.…”

Section: Methodsmentioning

confidence: 99%

“…The ether extract was concentrated to small volume under reduced pressure at 30°C, and the IAA content was determined by the spectrophotofluorimetric method of Knegt and Bruinsma (17) modified by the use of methanol to stop the reaction. Losses during extraction were accounted for by isotope dilution.…”

Section: Methodsmentioning

confidence: 99%

“…GC-MS has been used for identification of indole compounds in vegetative plant material (3,4) and a spectrophotofluorimetric method has been developed which makes possible routine estimation of the amounts of free indol-3yl-acetic acid in plant material (17). These techniques have been used in the present investigation.…”

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Auxin Requirements of Sycamore Cells in Suspension Culture

Moloney¹,

Hall²,

Robinson³

et al. 1983

Plant Physiol.

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Sycamore (Acerpseudopkwanus L.) cell suspension cultures (strain OS) require 2,4-dichlorophenoxyacetic acid (2,4-D) The classical assumption (32) that there was a precise correlation between auxin content and growth of plant parts has been vigorously challenged (e.g. 2,20). Critics of the classical view have made important points about the deficiencies of experimentation and interpretation by early workers but have, perhaps, given too little attention to the importance of choice of both the plant system and the auxin assay system. Plant cell suspension cultures provide more uniform systems in which the correlative influences present in the intact plant are removed and in which differential patterns of distribution of nutrients and hormones between the cells are essentially eliminated. It is anticipated that the specific factors regulating the onset and termination of cell division and enlargement will be identified by their use (5).The English sycamore (Acerpseudoplatanus L.) cell suspension cultures (strain OS) used in this investigation were maintained in a culture medium (30) which contained 2,4-D at a concentration of 4.5 JiM. Street et al. (29) noted that sycamore cells transferred to standard culture medium from which 2,4-D had been omitted began to lyse so that cultures actually showed a reduction in cell number during the standard 3-week passage. King (16) found that when cells from batch cultures were washed and transferred to 2,4-D-free culture medium there was a rapid decline in the rate of cell division, and in a subsequent passage in 2,4-D-free medium no increase in cell number occurred. The data of Simpkins et al. (27) produced by callus tissues ofPanax ginseng and Nicotiana tabacum (24), and possible correlations between production of IAA and control of cell division in tobacco cells grown on 2,4-D-containing medium were discussed by Nishinari and Yamaki (22, 23). However, the role of 2,4-D was not considered by these workers. In the present investigation, consideration is given to the relationships between exogenous and endogenous auxins in control of growth of sycamore cells maintained in batch culture.For critical studies involving hormones, it is essential that the active molecule(s) be identified and precisely estimated (3). GC-MS has been used for identification of indole compounds in vegetative plant material (3, 4) and a spectrophotofluorimetric method has been developed which makes possible routine estimation of the amounts of free indol-3yl-acetic acid in plant material (17). These techniques have been used in the present investigation. MATERIALS AND METHODSCulture of Sycamore Cells. The origin of Acer pseudoplatanus cell suspension cultures and general techniques of culture have been described previously (14). The basic synthetic culture medium was that developed by Stuart and Street (3) except that kinetin was omitted. Cells were subcultured (as 70-ml cultures) at 23-d intervals. Inoculum densities were 2.5 x 105 cells/ml, and final (stationary phase) cell densities were 2....

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Direct determination of indole‐3‐acetic acid in plant tissues by electroanalytical techniques using a carbon paste modified with OV‐17 electrode

et al. 1994

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The electrochemical behavior of indole-3-acetic acid (LAA) with a silicone OV-17 modified carbon paste electrode was explored for the determination of this compound in plant tissues. Under optimum conditions, the procedure gave a linear concentration range up to 0.6 pg mL-l. with a limit of detection of O.Oj3 pg mL-', containing the preconcentration solution 0.08 hl HAc/Ac-buffer (pH 4 ) and 0.4 M phosphate buffer (pH 12) as a supporting electrolyte in the measurement cell. The accumulation time was 10 minutes~and the sweep potentials were carried out with a scan rate of 30 mV s-' with a pulse amplitude (AE) of 100 my. This method is available for the determination of IAA in plant tissues.KEY WORDS: OV-17 carbon paste modified electrode, Differential pulse voltammetn-, Indole-3-acetic acid determination.

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A rapid, sensitive and accurate determination of indolyl-3-acetic acid

Cited by 112 publications

References 2 publications

Endogenous Levels of Auxin and Tumorigenesis in a Nicotiana Amphiploid

Endogenous Levels of Auxin and Tumorigenesis in a Nicotiana Amphiploid

Auxin Requirements of Sycamore Cells in Suspension Culture

Direct determination of indole‐3‐acetic acid in plant tissues by electroanalytical techniques using a carbon paste modified with OV‐17 electrode

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