Bioassay seedling

The extraction and purification, leading to the crystallization of abscisin II, were guided by an abscission bioassay (1). Seedlings of... 

Various bioassay methods have been used to detect the "natural" release of allelopathic agents. Sane authors preferred, after partial purification, to assay the extracts by petri dish methods for gemination, growth of roots or shoots and other symptoms of seedlings. The bioassays also included tests in soil or sand and also in nutrient solution (Table 3). 

In our efforts to detect and isolate the allelcpathic agents from tall fescue and several other grass species, we extracted the detached plant material with water and/or organic solvents. Either solvent extraction method yielded extracts that were inhibitory to the seed germination and seedling growth in our bioassay systems. 

Abdul-Wahab and Rice (9) found that boiling water extracts of johnsongrass leaves inhibited germination and seedling growth of several bioassay species at concentrations 80 times that used in this present study. 

Bioassay on Solid Medium. A-9, a medium previously shown to be favorable for antibiotic production by actinomycetes in shake flasks (36), was modified for bioassays on solid medium. We halved the concentration of components in A-9 and adjusted the pH to 6.9-7.1 with KOH to reduce the possibility of osmotic or toxic effects of medium components themselves on seed germination and seedling growth. The medium was amended with 15 g agar per liter and poured into 10 x 10 x 1.5 cm square plastic petri dishes, about 60 ml per plate. 

Bioassay of Extracts. Extracts tested for the presence of cyclohexi-mide were also bioassayed for phytotoxicity. The extracts were redis-sOlved in acetone, and 0.2 mg in 2 pi was applied to 6-cm-dia disks of filter paper. The extract was distributed on the paper with 0.2 ml of methanol. The disks were dried with warm air, placed in 1.5 x 6 cm petri dishes, and moistened with 1.5 ml distilled water. Ten cress seeds were placed on the paper, and after incubation for 3 d at 28 C radicle length of the seedlings was measured. 

Figure 3. Histograms showing growth response of cress, barnyard grass, and cucumber to 347 microbial isolates bioassayed on solid medium. Growth response 1) seedling growth minimal or germination nil, 2) pronounced inhibition, 3) slight inhibition, 4) growth comparable to controls, 5) stimulation over controls.

Procedure From bioassays, control and treated seedlings (seed and roots at 48 and 72 h of treatment) are stained for 10-15 min with 25 pM DCFDA in distilled water. Then fluorescence intensity of the dye is observed using the confocal microscope BIORAD 1024 (488nm dichroic and 510-560 nm emission). DCFDA fluorescence increases as the dye is oxidized by ROS to dichlorofluorescein (DCF). 

Compounds 58 and 59 showed phytotoxic effects when tested against seedlings of A. hypochondriacus using a Petri dish bioassay. Compounds 58 [IC50 = 6.57 xM] and 59 [IC50 = 3.86 xM] inhibited radicle growth of this species with a similar potency to 2,2—dichlorophenoxyacetic acid [2,4-D IC50 = 18 xM], which was used as a positive control. 

I often observed that under greenhouse conditions there was considerable variation in the effect a particular concentration of a phenolic inhibitor had on the growth of seedlings. Since procedures used in these bioassays were quite uniform, it was logical that environmental factors were influencing the results. 

Seedlings of two species of plants were used for bioassay to detect inhibition the dwarf-1 mutant of maize, Zea mays L., and the Little Marvel cultivar of peas,... 

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