title: Efficient production of Aschersonia placenta protoplasts for transformation using optimization algorithms.
abstract: The insect pathogenic fungus Aschersonia placenta is a highly effective pathogen of whiteflies and scale insects. However, few genetic tools are currently available for studying 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 3 levels of 3 variables that was subsequently tested to verify its ability to predict protoplast production (R(2) = 0.9465). The optimized conditions resulted in the highest yield of protoplasts ((4.41 ± 0.02) × 10(7) 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 mol/L NaCl-Tris buffer. The latter was used as an osmotic stabilizer. The yield of protoplasts was approximately 10-fold higher than that of the nonoptimized conditions. Generated protoplasts were transformed with vector PbarGPE containing the bar gene as the selection marker. Transformation efficiency was 300 colonies/(μg DNA·10(7) 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.
authors.affiliation: a Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.|b Fujian-Taiwan Joint Centre for Ecological Control of Crop Pests, Fuzhou, Fujian 350002, China.|c State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Xiuyan
authors.initials: X
authors.lastname: Wei
authors.affiliation: a Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.|b Fujian-Taiwan Joint Centre for Ecological Control of Crop Pests, Fuzhou, Fujian 350002, China.|c State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Xinyue
authors.initials: X
authors.lastname: Song
authors.affiliation: a Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.|b Fujian-Taiwan Joint Centre for Ecological Control of Crop Pests, Fuzhou, Fujian 350002, China.|c State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Dong
authors.initials: D
authors.lastname: Dong
authors.affiliation: d Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611, USA.
authors.forename: Nemat O
authors.initials: NO
authors.lastname: Keyhani
authors.affiliation: a Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.|b Fujian-Taiwan Joint Centre for Ecological Control of Crop Pests, Fuzhou, Fujian 350002, China.
authors.forename: Lindan
authors.initials: L
authors.lastname: Yao
authors.affiliation: a Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.|b Fujian-Taiwan Joint Centre for Ecological Control of Crop Pests, Fuzhou, Fujian 350002, China.
authors.forename: Xiangyun
authors.initials: X
authors.lastname: Zang
authors.affiliation: a Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.|b Fujian-Taiwan Joint Centre for Ecological Control of Crop Pests, Fuzhou, Fujian 350002, China.
authors.forename: Lili
authors.initials: L
authors.lastname: Dong
authors.affiliation: a Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.|b Fujian-Taiwan Joint Centre for Ecological Control of Crop Pests, Fuzhou, Fujian 350002, China.
authors.forename: Zijian
authors.initials: Z
authors.lastname: Gu
authors.affiliation: a Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.|b Fujian-Taiwan Joint Centre for Ecological Control of Crop Pests, Fuzhou, Fujian 350002, China.
authors.forename: Delai
authors.initials: D
authors.lastname: Fu
authors.affiliation: c State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Xingzhong
authors.initials: X
authors.lastname: Liu
authors.affiliation: a Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.|b Fujian-Taiwan Joint Centre for Ecological Control of Crop Pests, Fuzhou, Fujian 350002, China.|c State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Junzhi
authors.initials: J
authors.lastname: Qiu
authors.affiliation: a Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.|b Fujian-Taiwan Joint Centre for Ecological Control of Crop Pests, Fuzhou, Fujian 350002, China.
authors.forename: Xiong
authors.initials: X
authors.lastname: Guan
mesh_terms: D000465:Algorithms; D005821:Genetic Techniques; D006999:Hypocreales / Q000235:genetics*; D009995:Osmosis; D011523:Protoplasts; D014170:Transformation, Genetic
keywords: Aschersonia placenta; condition de production; méthodologie des surfaces de réponses; production condition; protoplast yield; rendement de protoplastes; response surface method