Cheng Y, Bélanger R R
Centre de Recherche en Horticulture, Département de Phytologie, Université Laval, Quebec, Canada.
FEMS Microbiol Lett. 2000 Sep 15;190(2):287-91. doi: 10.1111/j.1574-6968.2000.tb09300.x.
This paper describes a specific protocol for yielding and regenerating protoplasts from spores of the recently described powdery mildew biocontrol agent Pseudozyma flocculosa. With this protocol, a large quantity of protoplasts was obtained from beta-mercaptoethanol-pretreated spores in 3-day-old cultures of P. flocculosa grown in YMPD. Enzymatic digestion was optimal with 0.5% Novozym 234 and 5% Glucanex prepared in 0.6 M KCl in 0.1 M citrate buffer. All liberated protoplasts fluoresced in the presence of fluorescein diacetate indicating that viability was nearly 100%. The regeneration rate was equally outstanding reaching 75% when 0.8 M sucrose was used as osmotic stabilizer in the regeneration medium. This protocol will find useful applications in genetic studies of this poorly characterized and understood biocontrol agent.
本文描述了一种从最近描述的白粉病生物防治剂絮凝假丝酵母（Pseudozyma flocculosa）的孢子中制备和再生原生质体的特定方案。通过该方案，在YMPD培养基中培养3天的絮凝假丝酵母培养物中，从经β-巯基乙醇预处理的孢子中获得了大量原生质体。在0.1 M柠檬酸盐缓冲液中用0.6 M KCl配制的0.5%诺维信234和5%葡聚糖酶的酶解效果最佳。所有释放的原生质体在荧光素二乙酸存在下发出荧光，表明活力接近100%。当在再生培养基中使用0.8 M蔗糖作为渗透稳定剂时，再生率同样出色，达到75%。该方案将在这种特征和了解较少的生物防治剂的遗传研究中找到有用的应用。
