Germination bioassay

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. 

Table VI. Okra germination bioassay of root exudates...

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 for Toxicity of Inhibitory Compound(s). Since the identity of the inhibitory compound was determined to be ferulic acid (4-hydroxy-3-methoxycinnamic acid), ferulic acid obtained from Sigma Chemical Company was used in germination bioassays. 

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. 

The germination bioassay consisted of germinating the seeds of a number of crop and weed species (Table I) for 72 h in the dark at... 

The fourth isolated and identified compound from Palmer amaranth is chondri 11 asterol (5a-stigmasta-7,22-dien-30-ol), a sterol closely related structurally to the major plant sterols, stigmasterol and sitosterol. This compound, isolated as the free sterol, is not soluble in water or 0.1% DMS0, and germination bioassays required pretreatment of the test seed with a 0.1 mM solution of the sterol in DCM. 

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. 

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.

Bioassay of solid medium Bioassay of liquid medium Microbial Growth rating Germination Radicle length isolate Cucu Bygr Cress Cucu Bygr Cress Bygr Cress ... 

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. 

Spore germination and protonemal growth and morphogenesis are therefore, useful systems to test in vitro, with good reproducibility, potential allelochemicals both through direct co-existence test in vitro or using the described bioassays to monitor/guide isolation, purification, characterization of chemical structure of bioactive compounds. 

Principle Phytotoxic effects of the aqueous leachate of an allelopathic plant can be tested in vitro bioassays. Test or target plants are placed in contact with 0.5 % aqueous leachate from the allelopathic plant. Germination and radicle growth can be monitored during time-course experiments (i.e. after 24,48 and 72 h of treatment), but in this chapter we will include only the results obtained after 72 h of treatment. 

Bioassays are performed under sterile conditions in a laminar flow hood. Tomato seeds are previously washed and disinfected with 1% sodium hypochlorite. Seeds are germinated in the Petri dishes containing the S. deppei aqueous leachate. For control, seeds are germinated in 1% agar. Twelve seeds are placed on each Petri dish and kept in the dark at 27°C in a growth chamber. For enzyme activities, 40-50 Petri dishes are used per treatment. Primary roots (radicles) are excised after 72 h, frozen in liquid nitrogen and kept at -70 °C until use. For root growth response, experiments... 

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