The natural occurrence of cytokinins existing both in a free form and as a constituent of transfer RNA was examined in serial segments of young seedling roots of pea. Purified ethanol extracts of root apices were resolved into four factors capable of inducing soybean callus tissue proliferation. The most active factor was identified as zeatin or some closely related compound; it produced polyploid divisions and tracheary element differentiation when tested on cultured pea root segments. 2Abbreviations: IPA: 6-(3-methyl-2-butenylamino)-9-,3-D-ribofuranosylpurine or isopentenyladenosine; 2iP: 6-(3-methyl-2-butenylamino)purine or isopentenyladenine; ZR: 6-(4-hydroxy-3-methyl-2-cisbutenylamino)-9-,8-Dribofuranyosylpurine or zeatin ribonucleoside; Z: 6-(4-hydroxy-3-methyl-2-transbutenylamino)purine or zeatin. Extraction of Free Cytokinins. Immediately after harvesting, the different fractions of pea root segments were immersed separately in precooled absolute ethanol at -72 C. The frozen segments were collected by filtration on Whatman No. 1 filter paper and homogenized in an all-glass homogenizer in cold 80% (v/v) ethanol. The homogenized tissue was extracted for 3 hr in two portions of 80% ethanol and centrifuged at 10,000g for 15 min at 4 C. The original absolute ethanol fraction and the two 80% ethanol extracts were combined and the alcohol evaporated in vacuo. The water phase was adjusted to pH 8.0 and extracted with three equal volumes of 1-butanol which were then combined and taken to dryness in vacuo. The residue was redissolved in about 100 ml of water and adjusted to pH 2.0 with concentrated H2SO,. Fifteen milliliters of cold saturated AgNO, solution were added, and the mixture constantly was stirred for 12 hr at 4 C and centrifuged at 7000g for 15 min. The precipitate was washed with cold 2% AgNO, solution and stirred with 20 ml of 0.2 N HCl at 50 C for 30 min before being centrifuged at 7000g. This procedure was repeated twice. The combined acidic supernatants were adjusted to pH 2.5 with concentrated NaOH and percolated through a Dowex 50W-X8, H+ (200-400 mesh) column, 1.0 cm X 25.0 cm, which was then washed with 100 ml of water and eluted with 300 ml 3 N NH,OH. The eluate was evaporated in vacuo to about 50 ml and extracted at pH 8.0 with three equal volumes of 1-butanol which were combined and taken to dryness in vacuo. The resulting residue was dissolved in 80% ethanol and applied as a streak on Whatman No. 1 paper and developed with solvent system A, 1-butanol-acetic acid-water (12: 3:5 v/v). In addition, the following solvents were employed: B: 30 mM borate buffer at pH 8.4; C: ethyl methyl ketone saturated with water; D: l-butanol-1 N ammonium hydroxide (10:7 v/v); E: 1-butanol saturated with water. Each chromatogram was thoroughly air dried and cut into 10 equal RF strips and eluted with 80% ethanol. The eluates were reduced in volume and tested for cytokinin activity using the soybean callus bioassay.
A forty‐fold increase in tissue fresh weight has been achieved for Allium cepa stem tissue callus grown on a modified B5 medium with increased ammonium, phosphate and nitrogen levels; and decreased level of 2,4‐dichlorophenoxyacetic acid. Growth was examined on several standard media, using fresh weight, dry weight and cell number as parameters over an 8‐week growth period. Further, tissue grown upon this medium is more friable than on other media. The value of such friable callus for suspension culture initiation and for single cell cloning experiments is discussed.
Abstract. Experiments on the effects of stockpiling soil on an opencast coal mine in Derbyshire showed that there were significant changes in the microbial community. Numbers of aerobic bacteria in stored soils ranged from 0.5 to 12.8 ± 107 colony‐forming‐units (CFU)g‐1 with the smallest values being in the deepest parts of the oldest stores, whereas an adjacent undisturbed soil contained 6.6 ± 107 CFU g‐1. There was a greater effect on the numbers of fungal spores, which ranged from 0.1 to 6.7 ± 105 CFU g‐1 soil, all less than the 10 ± 105 CFU g‐1 recorded for the undisturbed control soil. The number of fungal spores in the deepest part of the older soil stores was only 1/100 of the number in the undisturbed soil. This was mirrored by the biomass values, as determined by adenosine triphosphate (ATP) assay. Values of ATP ranged from 0.38 to 13.13 nmol g‐1 as compared to 5.8 nmol g‐1 in the undisturbed soil. All three of these microbiological properties decreased in value with both age and depth of storage. Neither anaerobic nor spore‐forming bacterial numbers were greatly affected by storage. The pH values tended toward neutrality in the deeper parts of the soil stores, and there was less organic matter in the older stores.
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