| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2000: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1999: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
The analysis methods for an intact single parenchyma cell of the fruit was examined in this research. The method developed is firstly due to collect vacuolar sap from a single cell of the fruit, and then to analyze at picolitre levels for studying sugar accumulation in a single parenchyma cell of the fruit.
1. The maicromanipulator system were used for collecting vacuolar sap. The glass micropipette was inserted into a targeted cell, and vacuolar sap was withdrawn from the cell by lowering the pressure. Then, the collected vacuolar sap was expelled into 5 μL of an oil drop and stored at -20℃ for sugar analysis. The content of glucose, fructose, sucrose, or sorbitol were determined by the intensity of NADPH, which was produced with enzymatic reaction of 15 pl vacuolar sap, using a fluorescence microscope equipped with a photometer produced. The invertase activities were also determined by the same method.
2. The method for determining the osmotic pressure and potassium content of the vacuolar sap from a single cell were tested. Osmotic pressure was determined by measuring freezing point depression of the sap using a picolitre osmometer, and potassium content was determined by X-ray microanalysis using a scanning electron microscopy (SEM) with energy dispersive spectrometers.
3. Using above methods mentioned, analysis of sugar accumulation was conducted to the fruit of persimmon, grape, peach, Japanese pear, and apple on a single parenchyma cell basis. It was revealed that sugar accumulation pattern of a single cell is coincident with that of bulk tissue of the fruit in every kind of fruit, and that increase in osmotic pressure is due to accumulation of the sugars in the cells. Furthermore, individual cells showed different sugar compositions in some fruit, but it had the same compositions in other fruit.
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