Chadegani M, Brink J J, Shehata A, Ahmadjian V
Department of Biology, Clark University, Worcester, MA 01610-1477.
Mycopathologia. 1989 Jul;107(1):33-50. doi: 10.1007/BF00437588.
Factors affecting high yields, regeneration frequencies, and viability of protoplasts from clonal cultures of Microsporum gypseum were investigated. Maximum yields of protoplasts were obtained after 6 hrs digestion of 2-4 days old mycelium with Novozyme 234 using CaCl2 (0.4 M) as an osmotic stabilizer and glycine + HCl (pH 4.5) as the buffer system. Mercaptoethanol + dithiothreitol (0.01 M) proved to be the best pretreatment of mycelium prior to digestion with enzyme. A regeneration frequency of 94.4% was obtained using the top agar method with complete medium (pH 6.5) containing 0.5% agar and 0.4 M CaCl2 as an osmoticum. Colonies from regenerated protoplasts on medium containing CaCl2 were pigmented and completely powdery with high sporulation. Protoplast viability was studied in osmotic stabilizer supplemented with glucose or glutamine. After 24 hrs, glucose (2%) and glutamine (2%) enhanced protoplast viability by 22% and 23%, respectively. Protein synthesis, as measured by 3H-lysine uptake, matched the viability profile determined by fluorescence microscopy.
研究了影响石膏样小孢子菌克隆培养物原生质体高产率、再生频率和活力的因素。使用氯化钙（0.4M）作为渗透稳定剂和甘氨酸+盐酸（pH 4.5）作为缓冲系统，用诺维信234对2 - 4日龄的菌丝体消化6小时后，可获得最大原生质体产量。巯基乙醇+二硫苏糖醇（0.01M）被证明是在用酶消化之前对菌丝体进行预处理的最佳方法。使用顶部琼脂法，在含有0.5%琼脂和0.4M氯化钙作为渗透压调节剂的完全培养基（pH 6.5）上，获得了94.4%的再生频率。在含有氯化钙的培养基上，再生原生质体形成的菌落有色素沉着，完全呈粉末状，产孢量大。在补充有葡萄糖或谷氨酰胺的渗透稳定剂中研究了原生质体活力。24小时后，葡萄糖（2%）和谷氨酰胺（2%）分别使原生质体活力提高了22%和23%。通过3H - 赖氨酸摄取量测定的蛋白质合成与通过荧光显微镜确定的活力情况相符。
