Simulated Microgravity On Plant Cell Physiology And Cell Electric Fusion

Cell electrofusion was one of the practical and space developmental biotechnology. It was reported that the somatic cell hybrids were obtained by cell electrofusion technology after 1970s. However, the cell electrofusion technology was not developed and applied on the industrial and agricultural production due to the low cell fusion rate. It was well known that the influence factors which were the gravitation sedimentation and the hot convection were not occured in the space, the rate of fusion and livability for the hybrid cells were increased. The space carrying experiment was expensive and was limited by means of flight time and instrument weight that was why the reason it was not easy to repeat the same experiment. In general, the biological research of simulated weightlessness was performed with simulated rotator under the gravitational condition. At persent, the main research object was concentrated on animal cells, algae cells, the whole plants and seeds under the simulated weightlessness condition and only a few research work about the plant cells were reported. In this paper, the plant protoplasts were rotatived by means of RCCS equipment under the simulated weightlessness condition, the research works which were about the plant protoplast state, the metabolic reaction at the physiological and biochemistrical level and the change of cell fusion rate were investigated. It provided a theory foundation for the plant cell electrofusion mechanism.This paper mostly had three parts:The callus induction and plantlet regeneration were researched on the different plant hormone combination media with Datura innoxia stem as explants. The results showed that the embryonic callus were induced on the modified MS medium containing vitamin B1 0.5 mg/L, 2,4-D 2 mg/L without glycine, pH5.5. The callus were subcultured on the MS medium with 0.5 mg/L 2,4-D .The regeneration plants were obtained on the MS differentiation medium adding sucrose 20 g, agar 8 g, hydrolyze casein 0.1 g and 6-BA 0.5 mg/L. The root growth was performed on the 1/2 MS medium supplementing 0.2 mg/L IBA.The protoplasts were isolated from tobacco leaves (Nicotiana tabacum cv. Wisconsin 38), the cell of tobacco suspension system BY-2 and Datura stem callus. The protoplast livability and the coloration effects of cell nucleus were compared with different dyes. The result showed that considerable quantity protoplasts were collected from tobacco suspension system BY-2. They had high livability and strong tolerance characterization and were suitable for simulation experiments under the microgravity condition. The protoplast livability was observed by means of FDA method, which was higher definition than others. The protoplast livability was determined through MTT staining method quantitatively. The coloration effect of DAPI was better than theother dyes for the different kinds of cell nucleus. The protoplasts could be counted and observed with DAPI method clearly.The change of physiological character and electrofusion rate were researched with protoplasts from tobacco suspension system BY-2 under the simulated microgravity condition. The results showed that 15rpm rotation was suitable for simulation microgravity experiments as material of protoplasts from tobacco suspension system BY-2. The most of tobacco protoplasts in shape didn't change after rotation, the cytoplasm and vacuole in part of protoplasts were separated. The protoplast livability was decreased compared with control. The regenerative cell wall was observed under the simulated microgravity condition, which was no difference compared with the control. The content of NADH in tobacco protoplasts was higher than the control in early stage of rotation. The content of NADH was decreased gradually along with the extension time and was lower than the control at last. It suggested that the necessary energy for the tobacco protoplasts was decreased under the simulated microgravity condition. In addition, the microspike skeleton (F-actin) in tabacco protoplasts was displaymerized in the early stage of simulated rotation and the content of F-actin was lower than the control. The content of F-actin in tabacco protoplasts with simulated rotation treatment was increased gradually and was the same content as the control. The content of F-actin in tabacco protoplasts was higher than the control after rotating 24 hours. It was necessary to study the reason further. The cell electrofusion research was performed for the tabacco protoplasts which was treated under the simulated microgravity condition at last. The results showed that there was no significant change between the cell electrofusion rate and electrofusion time. The fused protoplast livability was decreased, which was not the expectant result about the space electrofusion.