Protoplasts Isolation from Cell Culture of Soybean (Glycine max L.)

Abstract

For the protoplasts isolation from callus, only friable callus which was cultured on B5 medium under the presence of 2,4-D and BA could be used as a source. The optimum conditions for protoplasts isolation from callus were demonstrated by using 4% Cellulase, 0.5% Macerozyme in and 13% mannitol at pH 5.6 and at log phase stage/ Appropriate incubation time was found at 2 hour in the dark on the rotary shaker. Three percents Cellulase and 0.5% Macerozyme in 10% mannitol at pH 5.2 were found to be the proper combination for the isolation of protoplasts from intact cell growing in liquid culture. BA and 2,4-D were proved to be essential components for the cell lines used in protoplast isolation. Same as callus the appropriate stage was log phase. Cells regeneration from protoplasts started to divide in four days after cultivated in K8P liquid medium without hormone.