Coleoptile test

The first plant bioassay [Avena sativa L (oat) coleoptile test] was employed by F.W. Went in the 1920 s to demonstrate the existence of and to quantitatively assess the first growth-modifying substance [indole-3-acetic acid (IAA)] isolated from plants.122 Plant bioassays have been extremely useful and intimately linked to the discovery and characterization of the major classes of plant hormones. In fact, many of the bioassays used now were developed for PGRs. Bioassays have been used to screen, evaluate phytotoxicity or plant growth promotion, study mode... 

Various assay methods have been used to detect the presence of inhibitory substances. These include some of the classical tests used by investigators of growth-promoting substances—i.e., the various Avena coleoptile assays which utilize intact, decapitated, or isolated cylinders and the split pea stem test. Effects on seed germination and seedling shoot or root growth and development have also been measured in addition to other visible expressions of inhibition. Details of many of these tests have been compiled by Mitchell et al. (99). Tests have been carried out in Petri dishes, with various solution culture techniques, and by sand and soil culture. Effects so measured may or may not be similar to those obtained under field situations— i.e., the establishment of inhibition under controlled conditions pro-... 

Protoanemonin, which has been isolated from Anemone pulsatilla and Ranunculus spp., was reported to inhibit root growth by slowing down metabolism and blocking mitosis 35). Erickson and Rosen 35) observed cytological effects in corn root tips at concentrations of 10M and lower. Cells undergoing division appeared to accumulate in the interphase or prophase stages. Metaphase, anaphase, and telophase stages were not observed. Cytoplasmic and vacuolar structures were disturbed and the presence of mitochondria could not be demonstrated in treated tissue. Thimann and Bonner 141) reported that protoanemonin was 10 to 30 times more inhibitory than coumarin in coleoptile and split pea stem tests, and that BAL prevented the inhibitory action. 

Rothwell and Wain (126) have isolated in crystalline form a growth inhibitor from Lupinus luteus (yellow lupine) pods which they have partially characterized. Analytical data suggested that the inhibitor possessed the characteristics of an unsaturated hydroxyketo acid. Inhibition in the wheat coleoptile cylinder test was obtained with concentrations of 0.25 to 1.0 p.p.m. 

Fig. 10. Diffusion of auxin into agar blocks in three hours from corn coleoptile tips. The numbers given are curvatures obtained from a standard auxin test, i,.e. a measure for the auxin diffused into the agar. (A) dark control bisecting does not significantly reduce the total amount of auxin. Unilateral bluelight (B) the total amount of flavin is not significantly reduced by irradiation (C) partial bisecting indicates strong lateral-basipetal auxin transport23). (D) Radio-actively labeled auxin, symmetrically applied to the coleoptile tip, proves the conclusion drawn from experiment (C)13 G...

Fig. 12. Curvature responses of decapitated avena coleoptiles as a function of auxin concentration. (A) curvature obtained in 90min, auxin applied asymmetrically (standard auxin test). (B) curvature produced in 60 min by symmetrical application of auxin upon succeeding transverse electrical stimulation. Bending occurs towards the positive pole, as sketched187 ...

Replication. Six Petri dishes each containing ten seeds were used for each control and for each treatment Controls accompanied all experiments. Results, parameters, and measurements. Counts of germinated vs. ungerminated seed, length and width of coleoptile, and length of central root and stem were recorded. Means per dish and per treatment (four to six dishes) were calculated and standard statistical tests were used in the analysis. 

In 1989 Cutler et al.1 reported the isolation of 3,7-dimethyl-8-hydroxy-6-methoxyisochro-man (1) from Penicillium corylophilum and demonstrated that it inhibited etiolated wheat coleoptiles at 10-3 and 1(T4M, as did the acetoxy (2) and methoxy (3) derivatives (Figure 5.1).2 The Parent compound had originally been isolated from moldy millet hay implicated in the death of cattle,3 but the metabolite had not been tested in plants. Because of the encouraging results obtained in the wheat coleoptile bioassay, isochromans 1,2, and 3 were assayed on greenhouse-grown bean, com, and tobacco plants. The methyl ether exhibited the greatest herbicidal activity in all the plants treated, while the parent and its acetoxy derivative were active only on corn. 

A novel fungal metabolite, hydroxyterphenyllin (242) from Aspergillus candidus found in unbleached flour was shown to be an active inhibiting ingredient when tested in wheat coleoptile bioassays at 10 3 - 10-5M. Terphenyllin (244, 245) was also identified from the same source but was less active (at 10 % ). ... 

Following concentrations were obtained by dilution. The bioassays were performed in 10 mL test tubes five coleoptiles were added to each test tube containing 2 mL of the test solution. Three replicates were made for each test solution, and the experiments were run in duplicate. Test tubes were placed in a roller tube apparatus and rotated at 0.25 rpm for 24 h at 22°C in the dark. All manipulations were done under a green safelight. The coleoptiles were measured by digitalization of their photographic images and the data were statistically analyzed. 

Aldehyde oxidase purified from maize coleoptiles is a multicomponent enzyme that contains a molybdenum cofactor, nonheme iron, and flavin adenine dinucleotide (FAD) as prosthetic groups.111 When substrate specificity of the aldehyde oxidase was tested, good activity was detected with IAAld, indole-3-aldehyde, and benzaldehyde among others. The addition of NADP and NADPH did not change the activity. In contrast, in maize endosperm, tryptophan-dependent IAA biosynthesis was dependent on an NADP/NADPH redox system, which may mean that the two tissues of maize are utilizing different pathways or different redox systems for IAA biosynthesis.112... 

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