ACTA AGRONOMICA TOMUS 29. (A MTA AGRÁRTUDOMÁNYI KÖZLEMÉNYEI, 1980)

1980 / 1-2. szám - M. NAGY: Dormancy in fruits of Tilia platyphyllos Scop. IV. Changes in the endogenous gibberellin content during stratification

2 M. NAGY no importance in breaking the dormancy of Tilia seeds, since there are great differences between the gibberellins in their effect on germination (IKUMA— THIMANN 1963). In order to get a better knowledge of the role of endogenous gibberellins in developing the promotor level the forms of gibberellin occurring in the dormant seed and their changes during stratification were examined. Material and Method Material of examination. A population stock obtained from the Forestry of County Csongrád was used in the examinations. Stratification. After the pericarp had been removed the seeds were scarified with sul­phuric acid, then stratified in culture pots containing washed sand wetted to 80% of the full water capacity. Stratification was carried out in a refrigerator at 4—5°C. Treatment of seeds with CCC and ABA solutions. After scarification the seeds were kept in Petri-dishes between filter paper discs wetted with 500 ppm ABA solution or 1000 ppm CCC solution in a refrigerator (at 5°C) for 3 months, then placed at room temperature. Extraction and chromatographic separation of gibberellins. At the beginning of stratifica­tion, then after 3, 6, 9 and 12 weeks of stratification 2000 seeds were removed from the culture pots on each occasion. The sand was washed off the seeds, then the seeds were homogenized in a cooled homogenizator and extracted with 80% methanol in a refrigerator for 2 X24 hours. The combined methanol extract was separated into ethyl acetate soluble and butanol soluble acidic fractions by the method of HARADA —YOKOTA (1970). The ethyl acetate soluble acidic fraction which contained the free gibberellins was evaporated under reduced pressure and chromatographed on a silica gel G layer. The solvent was diisopropyl-ether : acetic acid (95 : 5) (REINHARD et al. 1964). The butanol soluble acidic fraction which contained the gibberellin conjugates was divided in two parts. One of them was first evaporated under reduced pressure and then chromatographed with chloroform : methanol : acetic acid : water (40 : 15 : 3 : 2) as solvent (HARADA—YOKOTA 1970), while the other part was hydrolysed with N H2SO, or /S-glucosidase at 60°C for 2 hours (YOKOTA et al. 1969). The gibberellins released during the hydrolysis were extracted with ethyl acetate, then the ethyl acetate extract was evaporated under reduced pressure and subjected to thin layer chromatography. The tissue homogenizate left behind after the methanol extraction, which contained the gibberellins bound to macromolecules, was suspended in phosphate buffer (pH 8.0) after the solvent residues had been evaporated. After centrifuging, the supernatant was treated with 10% TCA. After repeated centrifuging the precipitate was hydrolysed with 2 N NaOH at 40°C for 4 hours. After acidification with hydrochloric acid (pH 3.0) the gibberellins released were extracted with ethyl acetate, then evaporated and subjected to thin layer chromatography. Determination of biological activity Lettuce hypocotyl test. The chromatogram was divided into ten equal parts, and the silica gel was scraped into Petri dishes 7 cm in diameter. The powder was covered with a filter paper disc and wetted with distilled water. The biological activity was measured using the lettuce (variety: "Aranysárga kőfej") hypocotyl test, according to the FRANKLAND — WAREING (1960) method. As a control a developed and tested silica gel layer without plant extract was used. Barley endosperm test. The powder scraped off the layer was eluated with ethyl acetate or n-butanol. The eluate was evaporated to dryness under reduced pressure, the residue was taken up in 2 ml acetate buffer containing 20 /<М CaCl2 (pH 4.8) and tested with barley endo­sperm by the method of JONES — WARNER (1967) under sterile conditions. After 24 hours of incubation 1% starch, prepared with 1 ml acetate buffer containing 20 /iM CaCl2 (pH 4.8), was added to 1 ml of the incubation solution. It was incubated at 40°C for 10 minutes, then 1 ml was removed and a 0.0003 N KI-I2 solution, prepared with 10 ml 0.03 N hydrochloric acid, was added to it. The optical density of the mixture was measured at 580 nm. The amount of a-amylase was calculated with the formula given by JONES —WARNER (1967). As a control, a developed and tested silica gel layer containing no plant extracts was used. Acta Agronomicc Academiae Scientiarum Hungaricae 29, 1980

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