Seed bioassay germination

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. 

Root elongation bloassay of root exudates. Five ml aliquots of the root exudates were pipetted onto three layers of Anchor1 germination paper In a 10 by 10 by 1.5 cm plastic petri dish. Twenty five radish or tomato seeds were placed in a 5x5 array in each petri dish. Radish seeds were incubated at 20C for 96 hours tomato seeds were incubated at 20C for 168 hours, before the root length was measured. Experimental design was a completely randomized design with three replications (dishes) per treatment per bioassay seed species. The bioassay was repeated each week for 23 weeks. 

Seed germination bioassay of root exudates. Bioassay results are presented as a 23 week mean for each germination count time (Table III, IV, V, VI). Means were separated by LSD after data normalization by the inverse sine transformation. 

Some Palmer amaranth samples prepared at SRRC were first extracted with hexane (HX), rather than PE. The organic solvent extracts were supplied for bioassay in the form of oils or solids remaining after solvent removal in vacuo. After each extraction step aliquots of the crude extracts from the roots, stems, or leaves (and thyrses) were evaluated for seed germination regulatory activity. 

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. 

Most of the work on correlation of molecular structure with bioactivity in witchweed seed germination has been produced by two groups (7-10). Johnson, et al. (7-9) prepared and evaluated a large number of strigol analogs and many approached the activity of strigol. In many studies by others, the results of the bioassays are presented, but the compounds from Johnson are described only by GR-number. GR-7 and -24, probably the more promising of these compounds, have been used in extensive field studies, and their structures are known. 

The accumulated assay results obtained with the 4-alkyl-2-methylpyrrolo[2,3-d]pyrimidines are of special interest. Can-pounds 25 and 26 exhibited significant cytokinin activity in all assays utilized. On the other hand, compound 27 was a potent antagonist in the tobacco bioassay but had little activity in the other two assay systems, while 29 acted as an anticytokinin in the tobacco bioassay and a cytokinin in the lettuce seed germination assay, but was without activity in the Amaranthus test system. 

Among the compounds that have been isolated from the brassins were a new group of glucosyl esters of fatty acids, the structures of which were established by spectral methods (254, 255) as well as by chemical and biochemical synthesis (256, 257, 258). Although these esters were much less active than brassinolide in the bean second internode bioassay, their presence may be essential for seed germination since the enzymatic synthesis of these esters was correlated to germination (257, 258). 

Some SL tested on Striga asiatica seed germination bioassays. 

Seed germination and seedling growth of radish in petri dishes were designed to evaluate the phytotoxicity of OMW fractions, thirteen polyphenols isolated from RO fraction and a mixture of these compounds. Radish seed (Raphanus sativus L. cv Saxa), collected during 1999, were purchased from Improta Co., Naples. To test the inhibitory effect of OMW fractions and RO, 20 seeds of radish were placed on two layers of filter paper (Whatman No. 1) in Petri dishes (90 mm diameter). The paper was wetted with 4 mL of buffered distilled water (BDW) with MES (2-N-[morpholino]ethanesulfonic acid) 10 mM, or test solution (undiluted fraction and a series of dilutions 1 2, one part of fraction to two parts of DW, 1 6 1 8 1 10 and 1 14). All pH values were adjusted to 6.0 before bioassay with MES. Experiments were made in triplicate. 

Another weak point of many allelopathic research studies reported in the literature is the lack of proper statistical analysis. Probably the two most critical errors are the lack of proper controls and insufficient replication. Appropriate controls need to be included even when a minimal amount of a solvent [e.g., dimethyl sulfoxide (DMSO), acetone, etc.] is used to solublilize a compound or for extraction of the test plant material. In this situation, it is also helpful to include positive controls (known compounds at similar concentrations) for comparison to the unknown or suspected allelochemicals. As discussed earlier, it is also useful to include various species so that a range of sensitivity to the test material can be observed. For example, a bioassay using seed germination might include lettuce seed, generally a sensitive species, and other species which might vary in response to the allelochemical(s) or extract. Such selection can demonstrate plant... 

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