Petri dishes germination

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-... 

Five ml aliquots of leachate or extract were pipetted onto 3 sheets of germination paper in a petri dish. Twenty five tomato or radish seeds were placed in each dish. Each treatment/seed combination was replicated five times. The assays were incubated at 20°C radish roots length was measured at 96 hrs and tomato at 168 hrs. 

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. 

Carolina in 1981. Each sample (1.4 ml of methanolic sample) was placed into a 3-cm petri dish and the solvent evaporated under a laminar flow hood at room temperature. Seventy seeds (0.035 g) were then placed into the petri dishes and 1.4 ml of sterilized (0.2 pm-filter) 15 mM Mes [2-(N-morpholino)ethanesulfonic acid Sigma Chemical Co.] buffer adjusted to pH 5.5 was added. The dishes were kept in the dark at 25°C2for 84 hr, exposed to 12-hr fluorescent light (250 p einsteins/m /sec), and then placed back in the dark for an additional 4 days (17). Percent germination, root and hypocotyl lengths were then determined. 

Table I. Germination of rape and birdsfoot trefoil seeds in petri dishes containing fescue extracts ...

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. 

Procedure Pollen develops on the nutrition medium, forming pollen tube (Fig. 3). The nutrient medium for pollen was 10% sucrose solution and tested compounds were also dissolved in 10% sucrose. The pollen develops till the formation of pollen tube that lasts from 2-3 h (fresh collected and one week stored pollen) to 24 h (stored > 1 week, but < 1.5-2 months). All experiments were done at room temperature 20-22 °C. The growth occurred in the solution studied (0.05 ml = 1 drop) on the slides (object glasses) put on wet paper in Petri dishes. Five ml of water was added to the bottom of every dish and 4-5 dishes with the slides were used per treatment. Using light microscope, we determined the microspores germination (%) 2-24 h after moistening. The number of developed pollen tubes was counted. 

Procedure Germination conditions were 25 1°C under continuous fluorescent light of 25 mE m 2 sec-1. Seed germination was monitored by observing the seeds directly in the Petri dishes with a stereomicroscope. They were considered germinated, when the radicle had protruded through the seed coat. Seeds sampled at different times after the beginning of imbibition were used for microscopy studies. 

Procedure Purslane seeds were collected from crop fields near Naples. Five hundred seeds were sown in 10 Petri dishes (0 =90 mm), containing 5 layers of Whatman filter paper impregnated with 7 ml of water (control) or 7 ml rue infusion/chromatographic fractions or isolated compounds as per treatment. Thereafter, daily 3 ml water was added to each Petri plate. Germination conditions were 30 1°C with a continuous light of 25 4,E/m2/... 

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... 

A defined number of seeds of each plant was germinated in Petri dishes kept in a Phytotron growth chamber at 21 1°C in the dark. Root tips ( 2 mm) were collected after 5 or 7 days of germination on dependence on the plant species, and subjected to Feulgen staining procedure before the preparation of permanent slides for the observation at an Olympus CX40 microscope. 

Germinate 20 mg fresh weight lots of Lilium longiflorum, cv. Ace pollen" in Petri dishes containing 10 mL medium supplemented with 0.1 p i[14C]-proline (pro) for 4 h at 27 2°C. 

To germinate lily pollen, spread 20 mg g fresh weight lots onto the surface of 10-mL sterile SYB (46,47) or Dickinson s (48) media in sterile Petri dishes. 

Over 50 different pyridazin-3-ones were evaluated for biological activity in a wheat (Triticum aestivum L.) test system described previously (1). Briefly, seeds were germinated in 9-cm petri dishes on three layers of filter paper. Pyridazinones were dissolved in acetone and the filter papers were impregnated with 1 ml of acetone solution. After the soluent evaporated, 10 ml of distilled water were added to form an inhibitor concentration of 100 yM. Seeds were planted directly on the moist papers and germinated for 4 days in a controlled environment chamber on a 16-hr photoperiod with 27+lC day temperature and 21+lC night temperature. Light intensity from both fluorescent and incandescent bulbs was 28 klux at dish level. Lipids were extracted and recovered from 1 g of lyophilized shoot tissue, separated into membrane and non-membrane lipids, and analyzed by gas chromatography as described (1). 

In 1963, Fletcher and Renney (.2) detected a plant growth inhibitor in knapweed and they suggested an allelopathic role for the substance(s) that could promote the spread of knapweed. Although these results are widely quoted, the inhibitor has never been identified nor has its existence been confirmed. Twenty years later, knapweed allelopathy was reexamined in our laboratory. A series of diffuse knapweed isolates were found to be inhibitory to ryegrass germination in petri dish assays... 

Extracts (12 mL each) were added to Petri dishes 10 cm in diameter containing 50 g of 30-mesh washed sand covered with filter paper circle (7-cm diameter, Whatman 1). Controls were moistened with doubly distilled water. Ten indicator seeds were placed on the filter paper in each dish with the embryo down and the hypocotyl pointed to the center of the Petri dish. Each indicator/extract combination had 3 replicates. The Petri dishes were kept in a dark growth chamber for approximately 72 hr at 25°C. The radicle length of each germinated seedling was measured at 72 hr. 

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. 

After the conditioning period, 500 pL of an aqueous solution of GR-24 (used as internal standard) or the test compounds were added to every Petri dish, sealed with parafilm and incubated for another 4 days in darkness at 20°C. Germination was observed under a microscope (30x) and the germinated seeds expressed as a percentage of the total seeds. Germination was considered when the radicle was at least 0.2 mm long. 

---