微流控芯片内植物原生质体的培养及其化学融合
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摘要
植物原生质体因少了细胞壁更便于各种实验研究,自20世纪七十年代以来,已被学者们广泛的研究和应用。植物原生质体方面的研究主要包括原生质体的培养与融合两大方面,通过原生质体培养可以观察细胞再生过程、获得细胞次生代谢产物等,而原生质体融合可以进行外缘遗传物质的导入、病毒的转染等,从而选育优质杂种、改良遗传性状等。植物原生质体已被应用于生命科学的许多领域,其重要性是不言而喻的。微流控芯片技术是20世纪九十年代发展起来的一项新兴技术,被誉为在20世纪科技史上有深远影响的事件之一,成为诸多交叉领域中基础与应用的重要发展前沿。
微流控芯片上对细胞的研究有很多,但多集中在动物细胞、细菌、昆虫方面,对于植物细胞的相关研究很少,基于植物原生质体的重要性与微流控芯片的技术优势,本研究设计了一个微流控芯片,在这个芯片内实现了烟草叶肉原生质体与小白菜无菌苗子叶原生质体的培养,并首次采用PEG化学融合的方法,实现了原生质体芯片内的融合。
主要结果如下:
(1)设计并制备实验所需的微流控芯片。
(2)培育出实验所需的小白菜无菌苗,经酶解纯化获得小白菜子叶原生质体,并用二乙酸荧光素检测活性。在相同实验条件下,分别在传统培养皿和微流控芯片内对小白菜原生质体进行培养,并对两种方法进行比较。
(3)在实验室条件下种植得到烟草叶片,将无菌处理过的烟草叶片酶解纯化及活力检测,得到干净且活力为95%的烟草叶肉原生质体。在相同温度及光照条件下,比较传统培养与微流控芯片培养的异同,得出微流控芯片内烟草原生质体培养与传统培养皿内培养经历相同的过程,但是为微流控芯片内烟草原生质体在启动分裂的时间上比传统培养要快1 d左右。应用三种液体培养基(NT6、NT1和MS培养基)对烟草叶肉原生质体进行培养,通过比较烟草原生质体一次分离频率及形成细胞团频率,得出最适合烟草原生质体培养的培养基为NT1。
(4)应用PEG化学融合法,对小白菜无菌苗子叶原生质体及烟草叶肉原生质体进行化学融合,同时应用微流控芯片与传统方法对烟草叶肉原生质体进行融合,通过对融合率及二源融合率比较发现,微流控芯片内更有利与烟草叶肉原生质体二源融合的发生。
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.
微流控芯片上对细胞的研究有很多,但多集中在动物细胞、细菌、昆虫方面,对于植物细胞的相关研究很少,基于植物原生质体的重要性与微流控芯片的技术优势,本研究设计了一个微流控芯片,在这个芯片内实现了烟草叶肉原生质体与小白菜无菌苗子叶原生质体的培养,并首次采用PEG化学融合的方法,实现了原生质体芯片内的融合。
主要结果如下:
(1)设计并制备实验所需的微流控芯片。
(2)培育出实验所需的小白菜无菌苗,经酶解纯化获得小白菜子叶原生质体,并用二乙酸荧光素检测活性。在相同实验条件下,分别在传统培养皿和微流控芯片内对小白菜原生质体进行培养,并对两种方法进行比较。
(3)在实验室条件下种植得到烟草叶片,将无菌处理过的烟草叶片酶解纯化及活力检测,得到干净且活力为95%的烟草叶肉原生质体。在相同温度及光照条件下,比较传统培养与微流控芯片培养的异同,得出微流控芯片内烟草原生质体培养与传统培养皿内培养经历相同的过程,但是为微流控芯片内烟草原生质体在启动分裂的时间上比传统培养要快1 d左右。应用三种液体培养基(NT6、NT1和MS培养基)对烟草叶肉原生质体进行培养,通过比较烟草原生质体一次分离频率及形成细胞团频率,得出最适合烟草原生质体培养的培养基为NT1。
(4)应用PEG化学融合法,对小白菜无菌苗子叶原生质体及烟草叶肉原生质体进行化学融合,同时应用微流控芯片与传统方法对烟草叶肉原生质体进行融合,通过对融合率及二源融合率比较发现,微流控芯片内更有利与烟草叶肉原生质体二源融合的发生。
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.
引文
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