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Li Xi-wen An Li-jia Huang Bai-qu Hao Shui (Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024)  
The structural and functional changes of nucleoli and the synthesis of RNA and DNA during the early stages of mesophyll protoplast culture in Astragalus melilotoi-des var. tenuis were studied using conventional electron microscopy, radioactive precursor labelling and computerized image processing. The results demonstrated that the volume of the nucleoli began to increase after 4 h of protoplast culture. As calulated by the image processor, an increase of about 4 to 5 fold in nucleolar volume was reached after 3 to 5 days of culture. In the meantime, the ratio between the granular (g) and dense fibrillar components (DFC) in nucleoli increased continuously and at day 5, the G/DFC ratio increased by 8 fold. In addition, the number of fibrillar centers (FC) also increased. The radioactive labelling experiment showed that 3H-uridine incorporation occurred as soon as 4 h of culture and peaked at 24 h when the radioactive incorporation rate was nearly 10 fold as high as that in the newly isolated protoplasts. On the other hand, 3H-thymidine incorporation increased 48 h after in culture, and peaked at day 4. Moreover, the intranuclear inclusion (IN) became visible in nuclei of some protoplasts during 24 h of culture within 24 h. Under the EM, IN appeared as a bundle of fibres over 4μm in length composed of numerous fibres 7-10 nm in thickness. The diameter of these fibres was equivalent to that of the nuclear matrix filaments. On the basis of the structural and compositional changes and their timing during the in vitro culture of mesophyll protoplasts in A. melilotoides var. tenuis, the early development of cultured protoplasts is divided into three stages and the mechanisms of cell wall regeneration and cell de-differentiation during the early development of the mesophyll protoplasts cultured were discussed.
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