The Microscopic Analysis Of Spontaneous Fluorescence In The Tea Leaves Suffering From The Fluorescent Green Spot Disease

The Fluorescent Green Spot Disease was a physiological disease of tea mature leaves occurred all over the state in China. The representative symptom of this disease was green spots with fluorescence and irregular in shape.In order to investigate the rule of the disease occurrence and the spectra of yellow-greenish fluorescent compounds, the fluorescence microscopy, microscopic fluorescence spectral imaging, laser scanning confocal microscopy and flow cytometry were used in this research. This experiment covered the condition of microscopic observation, emission spectral characters of yellow-greenish spontaneous fluorescence, and its localization in the diseased tea leaves, the distribution of the free Ca2+ and intracellular pH. The results showed that:(1) The diseased leaves could emit several kinds of spontaneous fluorescence by irradiating different lights, and the optimal microscopic condition for observing the yellow-greenish fluorescence was that the diseased leaves were excited by blue light and images recorded in multicolor and green color mode, in which there were three fluorescence peaks at 515 nm, 535 nm and 585 nm within the light range of yellow and green.(2) The yellow-greenish fluorescence was firstly emitted in the sheath cell of vascular bundle and then in the barrier cells, sponge cells and the epidermis cells. In these diseased cells, The chloroplasts disaggregated, the intension of their red fluorescence weakened and the yellow-greenish fluorescence was bright. And many diseased cells congregated closely,so the bright fluorescence spots can be observed. The fluorescence located in the vacuole of the diseased cells.(3) The average intracellular pH of the normal leaves was 5.35 and the diseased leaves was 6.89 and the intracellular pH of the diseased leaves increased as the disease aggravated. The enhancement of intracellular pH was one cause of intensification of yellow-greenish fluorescence. (4) The concentration of intracellular free Ca2+ of the normal leaves was higher than the diseased leaves. There was area which has high concentration Ca2+. In the diseased leaves, the distribution of free Ca2+ polarized seriously in primary diseased cells and there was no free Ca2+ in the entirely diseased cells. It was better loading the probe under low temperature than at room temperature.