The invention provides a preparation and regeneration method of
pythium oligandrum protoplasts. The reparation and regeneration method comprises the following steps: a, preparing thallospore hybrids; b, preparing protoplasts; c, purifying the protoplasts; and d, regenerating the protoplasts. According to the method, a complex
enzyme system containing lysing
enzyme, cellulsase and lyticase is adopted for carrying out enzymolysis on the mycelia of
pythium oligandrum, then the
pythium oligandrum protoplasts are obtained, and the protoplasts are regenerated through liquid shaking culture adopting a regeneration culture medium taking
mannitol as an
osmotic pressure stabilizer. The number of the
pythium oligandrum protoplasts prepared by adopting the method is large, the yield is high, and the activity is good; through the
liquid culture regeneration path, relatively high regeneration rate can be obtained, and the regeneration rate can achievce 1.25%. The method is easy and convenient to operate and good in stability, the obtained
pythium oligandrum protoplasts can completely satisfy the research and application such as late
gene genetic transformation and
cell fusion. The invention further provides composite enzymatic
hydrolysate for preparing the
pythium oligandrum protoplasts, the composite enzymatic
hydrolysate is a key factor in the preparation process of the pythium oligandrum protoplasts, and the enzymatic
hydrolysate is suitable for being further developed and applied.