Studies on Protoplast Isolation of Pleurotus cornucopiae

노랑느타리버섯의 원형질체(原形質體) 분리(分離)에 관한 연구(硏究)

The optimal conditions for high yields of mycelial protoplasts from P. cornucopiae were established. The concentraion of enzyme system containing Novozym 234, ${\beta}-D-glucanase$ and ${\beta}-glucuronidase$ was $5mg\;ml^{-1}$ each. The osmotic stabilizer most effective for protoplast isolation was O.6 M sucrose. The optimal reaction time of mycelium with the lytic mixture was 90 min in a shaking condition at 120 strokes $min^{-1}$. When the myelium of P. cornucopiae was cultured for 4 days on mushroom complete medium at $28^{\circ}C$, the formation of protoplast was effective. When the pH of the digestion mixture with O.6 M sucrose as stabilizer varied between pH 4.0 and 7.0, the production of protoplasts was effective in phosphate buffer (pH 6.2) and Na-maleate buffer (pH 5.0). Generally, phosphate buffer was more effective for protoplast isolation than Na-maleate buffer, but 0.6 M sucrose osmotic stabilizer without adjusting pH was most effective. Using these conditions, protoplasts from P. cornucopiae were obtained at a ratio $1{\times}10^7\;ml^{-1}$.

버섯의 품종개발(品種開發)과 유전연구(遺傳硏究)를 위해서 느타리 균(菌)의 일종(一種)인 P.cornucopiae 노랑 느타리의 원형질체(原形質體) 분리(分離)에 적합한 제조건(諸條件)을 조사하였다. 원형질체(原形質體) 분리(分離)의 최적(最適) 조건(條件)으로 Novozym 234농도(濃度)는 $5\;mg{\cdot}ml^{-1}$이었고 Novozym $234+{\beta}-D-glucanase\;+{\beta}-glucuronidase\;mixture$에서 원형질체수(原形質體數)는 $10.55{\times}10^6\;ml^{-1}$으로 가장 높았으며, 삼투압(渗透壓) 조절제(調節劑)와 농도(濃度)는 0.6 M sucrose에서 효과적(效果的)이었고 혼합효소액(混合酵素液)과의 반응(反應) 90분일때 원형질체(原形質體) 분리량(分離量)은 $2.2{\times}10^7\;ml^{-1}$으로 높았다. 완충용액(緩衝溶液)과 pH는 0.6 M sucrose+Na-maleate buffer (pH 5.0), 0.6 M sucrose+phosphate buffer(pH 6.2)에서 원형질체(原形質體) 분리량(分離量)이 좋았으며 대체로 phosphate buffer가 효과적인 것으로 나타났고 pH를 조절하지 않은 0.6M sucrose 삼투압 조절제(調節劑)가 원형질체(原形質體) 분리(分離)에 가장 알맞았다. 그리고 배양(培養) 일수(日數) 및 균사체량(菌絲體量)을 보면 cellophane MCM에서 4일 배양(培養)한 균사체(菌絲體) 6colonies와 혼합효소(混合酵素) 4 ml과 혼합하여 $28^{\circ}C$에서 90 분간 흔들었을 때 원형질체(原形質體)가 가장 많이 분리되었다.