Mosquito fungus Pythium Guiyang Protoplast Preparation and Regeneration of.doc

 PAGE \* MERGEFORMAT 12 Mosquito fungus Pythium Guiyang Protoplast Preparation and Regeneration of [Abstract] Objective: To establish Guiyang Pythium Protoplast preparation and regeneration system for the bacteria’s genetic engineering and cell engineering is to provide appropriate fungal condition. Methods: The impact of protoplast formation of various factors, including the bacteria age, the type and proportion of digestive enzymes, digestion time, and osmotic pressure stabilizer composition and concentration of Experimental Comparison. Results: 1% cellulase and 1% lywallzyme 1:1 combination of the joint off-wall, 0.6 mol / L of mannitol as osmotic stabilizer, enzyme using KPYG2 culture medium 52 ~ 54 h of the Guiyang-rot fungus hyphae, digestion 5 h obtained the largest number of protoplasts, protoplast concentration to reach 6.48 * 106 / ml; in 0.6 mol / L mannitol as osmotic stabilizer KPYG2 medium, the protoplast regeneration rate of 0.043 %. Conclusion: The experiments summarized in protoplast ways to get mutations in the gene transfer or cell protoplasts induced by the concentration required. [Keywords:] fungi; Guiyang Pythium; Protoplast; biological mosquito control; osmotic pressure [Abstract] Objective: To provide a suitable fungal material for protoplast-mediated genetic transformation and mutation induction of Pythium guiyangense Su. Methods: Conditions for the fungal protoplast preparation and regeneration including mycelium incubation time, various compounding of enzymes and osmotic stabilizers were examined. Results: Under the conditions as following: mixture of 1% lywallzyme and 1? llulose (1:1) as digestive enzyme solution; 0.6mol / L D-Mannitol as osmotic stabilizer, and 5h, of digestive time the obtained protoplast amount reached 6.48 * 106 each milliliter. A regeneration rate of 0.043% was obtained. Conclusion: A satisfied concentration of P. guiyangense protoplasts can be achieved with the preparation system developed in this research.