Complete chloroplast long-time isolated culture method

Abstract

The invention discloses a complete chloroplast long-time isolated culture method. Sterile seedling cotyledons of cucumber, tobacco and spinach are used as materials respectively; citric acid, NaCl, Tris and ethylene diamine tetraacetic acid (EDTA) are used as chloroplast extraction reagents of main components; complete chloroplast (figure 1) is separated by the operations of grinding, gauze filtration, differential centrifugation and the like of the plant materials; and MnCl2, MgSO4, NaCl, sorbitol and the like are used as culture reagents of the main components, and the isolated chloroplast is subjected to liquid culture. After being cultured in vitro for about 20 days, the chloroplast still keeps green and is microscopically normal (figure 2), and the membrane structure is complete (figure 3). In two weeks of isolated culture, the chloroplast is split and propagated to reach relatively stable number (figure 4). Proved by isolated transformation and exogenous gene detection of the chloroplast, the DNA in the chloroplast cultured for long time is not degraded (figure 5). The method can provide convenience and a new path for basic theories and application research of chloroplast function, genetic transformation, cell engineering and the like.

Description

【Technical field】: [0001] The invention belongs to the technical field of plant genetic engineering, and mainly relates to the technology and implementation of complete plant chloroplast isolation and in vitro cultivation, including preparation of reagents for chloroplast isolation and in vitro cultivation and related operating procedures. 【Background technique】: [0002] Chloroplast is an important organelle for plants to convert solar energy into chemical energy, and is the main source of energy intake for living organisms on the earth. Therefore, the structure, function, genome and gene expression regulation of chloroplasts have attracted much attention in life sciences. Relevant basic theories and applied research are closely related to human survival such as food, energy, and environment. Although the chloroplast in eukaryotic cells has an independent genome and double-layer biofilm coating, it is a semi-autonomous organelle. Its occurrence, development and function are...

AI Technical Summary

Problems solved by technology

At present, the technology of introducing exogenous genes into chloroplasts is still limited to the main gene gun method in the world, and the screening of chloroplast mutants and transformants is still relatively difficult, which has become a key bottleneck restricting the development of related research and application of chloroplast transformation. The operation of chloroplasts is an important breakthrough. If the isolated chloroplasts can remain intact for a long time, it will provide convenience and open up new ways for chloroplast genetic engineering operations.

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