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考察了细胞壁降解酶的种类、酶解时间、培养基种类和涂布方式对顶头孢霉原生质体制备和再生的影响,结果提示顶头孢霉菌丝形态对原生质的制备和再生也有重要影响。顶头孢霉生长过程可分为3个时期:细长时期、中度膨大时期和高度膨大时期。本试验利用显微图像分析法确定了各时期菌丝的形态参数,描述了培养过程中的菌丝形态变化规律,以及不同菌丝形态对原生质体释放和再生率的影响。最终优化条件为:选择中度膨大初期的菌丝体,使用1%溶壁酶,30℃酶解2 h后采用双层平板法接种于原生质体再生培养基I(HCM)中。每1 ml原生质体悬浮液中原生质体的释放数可达2.51×10~8个,再生率大于20%。
The effects of cell wall degrading enzyme type, enzymolysis time, medium type and coating method on the preparation and regeneration of Cephalosporium acremonium were investigated. The results indicated that the morphology of Cephalosporium acremonium also had an important effect on the preparation and regeneration of protoplasts. Cephalosporium growth process can be divided into three periods: slender period, moderate swelling period and highly swollen period. In this experiment, the morphological parameters of mycelium were determined by microscopic image analysis. The changes of mycelium morphology and the effects of different mycelial morphology on protoplast release and regeneration rate were described. The final optimization conditions were as follows: The mycelium was selected at the initial stage of moderate expansion, and then it was inoculated into protoplast regeneration medium I (HCM) by double layer plating method using 1% lywallzyme at 30 ℃ for 2 h. The number of protoplasts released per ml of protoplast suspension was up to 2.51 × 10 ~ 8 and the regeneration rate was more than 20%.