Efficient production of Aschersonia placenta protoplasts for transformation using optimization algorithms

Aschersonia placenta is a highly effective pathogen of whiteflies and scale insects. Few genetic tools, however, are currently available for this organism. Here we report on the conditions for the production of transformable A. placenta protoplasts using an optimized protocol based on the response surface method (RSM). Critical parameters for protoplast production were modelled by using a Box-Behnken design (BBD) involving three levels of three variables, and subsequently experimentally verified in its predictability of protoplast production (R2 = 0.9465). The optimized conditions resulted in the highest yield of protoplasts (4.41 ± 0.02 ×107 cells/ml of culture, mean ± SE) when fungal cells were treated with 26.1 mg/ml of lywallzyme for 4 h of digestion, and subsequently allowed to recover for 64.6 h in 0.7 M NaCl-Tris buffer. The latter used as an osmotic stabilizer. The yield of protoplasts was approximately 10-fold higher than that of the non-optimized conditions. Generated protoplasts were transformed with vector PbarGPE containing bar gene as the selection marker. Transformation efficiency was 300 colonies/g DNA/107 protoplasts, and integration of the vector DNA was confirmed by PCR. The results show that rational design strategies (RSM and BBD methods) are useful to increase the production of fungal protoplasts for a variety of downstream applications.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author