The use of starch and skim milk in the regeneration of Bacillus amylotiquefaciens and Bacillus subtilis protoplasts

Abstract

The addition of starch or skim milk to bovine serum albumin‐containing regeneration medium enhances the regeneration of both Bacillus amyloliquefaciens and B. subtilis protoplasts. With B. amyloliquefaciens starch could replace bovine serum albumin; with B. subtilis either starch or skim milk could be used instead of bovine serum albumin. The maximum regeneration frequency achieved was 10% with B. amyloliquefaciens and 290% with B. subtilis.

Protoplast Transformation of Recalcitrant Alkaliphilic Bacillus sp. with Methylated Plasmid DNA and a Developed Hard Agar Regeneration Medium

“…For reversion of the protoplast to the bacillary form, several modifications of SA1.2 medium had been examined, including addition of autoclaved Bacillus sp. N16-5 cells [31] , substitution of bovine serum albumin (BSA) by maize starch or skim milk [32] , replacement of sodium succinate by sucrose [33] or mannitol [34] , addition of 2.5% gelatin [34] , removal of BSA, and lowering the incubation temperature from 37°C to 25°C. However, neither L colonies nor bacillary colonies arose when sucrose or mannitol was used as the osmotic stabilizer and BSA was substituted by skim milk.…”