Method for separating, purifying and instantaneously and efficiently converting root, stem and leaf protoplasts of taxodium distichum

Abstract

The invention discloses a method for separating, purifying and instantaneously and efficiently converting root, stem and leaf protoplasts of taxodium distichum. The method comprises the following steps: 1) a taxodium distichum seedling with 1-3 rounds of grown leaves is selected and cultured for 24 hours in a dark place, cleaning is conducted once with 75% alcohol firstly, then cleaning is conducted with deionized water for three times, and then the leaves, stem segments and roots of the taxodium distichum seedling are separated by a blade; (2) the roots, stems and the leaves of the taxodium distichum are subjected to enzymolysis through cellulase and pectinase to obtain the protoplasts, and the obtained protoplasts are separated, purified and pretreated; and (3) the protoplasts of different tissue are taken as receptors, a transient expression vector containing an exogenous gene is transferred into the protoplasts through a PEG-mediated transformation method, and through culturing, the exogenous gene is expressed in the protoplasts. The method is easy to operate and easy to implement, a large number of the complete and pure root, stem and leaf protoplasts of the taxodium distichumcan be obtained, the conversion efficiency reaches up to 50% or above, and the technical basis is provided for transient expression detection of a taxodium distichum gene, protoplast fusion and the like.

Description

technical field

[0001] The invention belongs to the field of cell biology and biotechnology, in particular to a method for separating, purifying and instantaneously transforming protoplasts of bald cypress roots, stems and leaves. Background technique

[0002] Plant cells form protoplasts after removal of the cell wall, which includes the cell membrane, cytoplasm, and nucleus. In short, plant protoplasts are "naked cells" surrounded by plasma membranes. With the continuous development of science and technology, protoplasts have gradually become an effective tool for researchers to study the biological functions of genes and proteins and analyze plant signal transduction pathways. Protoplast isolation is the first step in protoplast cultivation, which directly affects the success of protoplast cultivation. At present, the separation of protoplasts by enzymatic hydrolysis is recognized as the most convenient and effective method. Cellulase is used to digest the cell wall, p...

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