利用荧光染色和流式细胞技术辅助卵孢小奥德蘑原生质体制备与再生研究

本研究以卵孢小奥德蘑液体培养菌丝作为实验材料,利用单因子变量法探索研究了菌丝培养时间、酶浓度、酶解时间、酶解温度、稳渗剂类型对卵孢小奥德蘑原生质体制备的影响,并对原生质体再生培养基进行选择和优化。通过荧光染色,利用激光共聚焦显微镜和流式细胞仪对原生质体的制备过程、得率和活力进行研究。结果表明,将卵孢小奥德蘑菌丝在液体培养基中培养5d收集菌丝体,以甘露醇作为渗透压稳定剂,在溶壁酶浓度2%、30℃条件下酶解5h,获得的原生质体得率最高,达2.0×10 ⁷个/mL;通过流式细胞仪分析,约57.69%的原生质体细胞为活细胞;在RM培养基中再生效果最好,再生率为(0.103±0.025)%。研究结果可以为卵孢小奥德蘑育种与食用菌原生质体制备再生提供研究基础。

Protoplast preparation and regeneration of Oudemansiella raphanipes using fluorescent staining and flow cytometry

The single-factor experiment was used to analyze the effects of mycelium cultivation time, enzymolysis time, enzymolysis temperature, enzyme concentration, and type of osmotic stabilizer on the preparation of the protoplast in Oudemansiella raphanipes. The medium for protoplast regeneration was optimized. Through fluorescence staining, the process of protoplast preparation was observed by laser scanning confocal microscope, and the yield and viability of protoplasts were determined by flow cytometer. The results showed that the highest protoplast production was 2.0×10 ⁷cells/mL under the optimal conditions as follows: cultivation in liquid medium for 5 days, using mannitol as osmotic pressure stabilizer, and lywallzyme digestion for 5h at the enzyme concentration of 2% under 30°C. Flow cytometer analysis indicated that about 57.69% of the protoplast cells were alive. In the optimal solid regeneration medium (RM), the regeneration rate of protoplasts was (0.103±0.025)%.