The invention discloses a
protoplast isolation and genetic transformation method of
Brassica napus without restriction of
genotype and a regeneration
system thereof, and provides a method for transforming exogenous
nucleic acid molecules by using
Brassica napus protoplasts. The method comprises the following steps of: 1) preparing protoplasts by enzymolysis of
Brassica napus leaves; 2) transferring the exogenous
nucleic acid molecules into the protoplasts to obtain the protoplasts with the transferred exogenous
nucleic acid molecules; and dividing and culturing the protoplasts with the transferred exogenous nucleic acid molecules in a C: B culture medium to obtain
cell clusters formed by division of the protoplasts. According to the invention, a sterile
seedling of the Brassica napus is used as a
raw material, the mesophyll tissue of the Brassica napus is digested by
cellulase R-10 and macerozyme R-10, the protoplasts are isolated by using a
density gradient sedimentation method to obtain the high-purity protoplasts. The
target gene is introduced into the
genome of the Brassica napus protoplasts through the mediation of
polyethylene glycol, and a large number of Brassica napus transgenic plants are cultured through shallow
liquid culture and subsequent differentiation and regeneration.