FIELD: agriculture.
SUBSTANCE: invention relates to the field of agricultural biotechnology, in particular, to methods for in vitro cultivation of somatic cells and genomic editing of higher plants. The invention constitutes a new method for isolating wheat protoplasts for genome editing. Said method includes effective isolation of wheat protoplasts from 7 to 10-day shoots. The effectiveness of isolation is achieved due to the low-temperature treatment of donor plants and vacuum infiltration of the cooled enzyme solution. In the process of crushing the shoots and prior to processing with enzymes, leaf tissues are sustained on an osmotic medium in order to reduce the cell damage during mechanical impact on tissue cells. Transfection of protoplasts can be executed by a wide range of genetic constructs. The system for transfection and imaging of protoplasts can be used with various marker selection systems, including selection with reporter genes.
EFFECT: invention can be used for comparative estimation of the effectiveness of wheat genome editing by various genetic engineering constructs by means of transfection of protoplasts with an effectiveness of up to 40.5%.
1 cl, 1 dwg, 1 tbl
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