Culture And Chemical Fusion Of Plant Protoplasts In A Microfluidic Device

Since the seventies of 20th century, the protoplast have been extensively studied and applied by scholars, as plant protoplasts can be used much more easily without cell wall. The studies of protoplast mainly include protoplast culture and fusion. Cell regeneration process can be observed and secondary metabolites can be obtained through protoplast culture; while the protoplast fusion can transfer genetic material and get the virus infection to gain the high-quality hybrid breeding and improvement of genetic traits. Plant protoplasts have been used in many areas of life science, its importance is self-evident. Microfluidic device is a new technology which has far-reaching implications in the history of science and technology in the nineties of 20th century, and foreword area of basis and application in kinds of interdisciplinary science.Many works has been done about cells in microfluidic chips, mostly focused on animal cells, bacteria, insect, seldom for plant cells. Based on the importance of plant protoplasts and the advantage of microfuidic chip, we designed a integrated microfluidic device, in which tobacco protoplasts and cotyledon protoplasts of Brassica campestris ssp. chinensis Makino were cultured, and chemical (PEG) fusion was finished for the first time. The main results are as follows:(1) The necessary microfluidic device has been devised and made.(2) Cultivating seedling of Brassica campestris ssp. chinensis Makino, protoplasts that were got by enzyme digestion and purification had been detected by FDA for vitality. Protoplasts were cultivated in both the traditional dish and the microfluidic device, the results of which were compared.(3) Under the laboratory conditions, the sterile tobacco leaves were treated by enzyme and purification to obtain the clean tobacco mesophyll protoplasts which had vitality of 95% by activity assay. At the same temperature and lighting conditions, tobacco protoplasts cultivated in the microfluidic device had the same process as in the dish by comparing traditional culture and culture in the microfluidic device, but the start division time of tobacco protoplasts within the microfluidic device is faster than traditional culture about 1 day. Application of three kinds of liquid medium (NT6, NT1 and MS medium) on the tobacco mesophyll protoplasts culture, the NT1 was the most suitable medium for mesophyll protoplast culture of tobacco through compared the frequency of the cell divison and the formation of the derived cell clusters.(4) Application of PEG chemical fusion method, Cotyledon protoplasts of Brassica campestris ssp. chinensis Makino Chinese cabbage and tobacco mesophyll protoplasts were fused by PEG. tobacco mesophyll protoplasts were fused in the microfluidic device and the dish, comparison of fusion rate and the two sources of integration rate showed that the fusion of two sources of tobacco mesophyll protoplast more favorable occurred in the microfluidic device.