Fariña J I, Molina O E, Figueroa L I C
Planta Piloto de Procesos Industriales Microbiológicos (PROIMI-Biotecnología), Universidad Nacional de Tucumán, Argentina.
J Appl Microbiol. 2004;96(2):254-62. doi: 10.1046/j.1365-2672.2003.02145.x.
Different cultural conditions for forming and reverting protoplasts were systematically studied to establish a rapid and efficient protocol for Sclerotium rolfsii ATCC 201126.
Osmotic stabilizer, lytic enzymes and mycelial age were the main factors influencing protoplast yields. An optimized protocol involving 1-h hydrolysis of 45-h-old mycelium with Trichoderma harzianum enzymes in a 1 : 1 (w/w) biomass : enzyme ratio and 0.6 mol l-1 MgSO4 as osmotic stabilizer was designed to produce approx. 2 x 109 protoplasts per gram biomass dry weight, with 99% viability. Differences on the lytic activity between batches of commercial enzymes were clearly evidenced. Protoplast release was highly efficient showing no remaining cell wall material as witnessed by fluorescent brightener 28. Up to 26% of purified protoplasts developed into the typical filamentous form after 50 h of incubation on 0.6 mol l-1 sucrose agar media.
The methodology herein proposed allowed a rapid, inexpensive and efficient protoplast production. Optimum yields were higher or in the order of that elsewhere reported for other S. rolfsii strains and the required lytic time was significantly shorter. Purified protoplasts successfully reverted to the filamentous morphology.
The present research reports the former protocol for the isolation and reversion of protoplasts in S. rolfsii ATCC 201126 providing key factors to ensure optimum results. In addition, the described procedure constitutes a starting point for downstream genetic manipulation.
系统研究形成和再生核盘菌（Sclerotium rolfsii）ATCC 201126原生质体的不同培养条件，以建立一种快速有效的方法。
渗透稳定剂、裂解酶和菌丝体年龄是影响原生质体产量的主要因素。设计了一种优化方法，用哈茨木霉（Trichoderma harzianum）酶在生物量与酶质量比为1:1（w/w）的条件下对45小时龄的菌丝体进行1小时水解，并以0.6 mol l-1 MgSO4作为渗透稳定剂，每克干重生物量可产生约2×109个原生质体，活力为99%。不同批次商业酶的裂解活性差异明显。荧光增白剂28显示原生质体释放高效，无残留细胞壁物质。在0.6 mol l-1蔗糖琼脂培养基上培养50小时后，高达26%的纯化原生质体发育成典型的丝状形态。
本文提出的方法可快速、廉价且高效地生产原生质体。最佳产量高于或与其他核盘菌菌株的报道产量相当，所需裂解时间明显更短。纯化的原生质体成功再生为丝状形态。
本研究报告了核盘菌ATCC 201126原生质体分离和再生的前一个方法，提供了确保最佳结果的关键因素。此外，所描述的程序是下游基因操作的起点。
