Factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum
Factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum
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DOI：
10.1590/s1413-70542010000100009
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发表时间：
2010-02-01
期刊：
影响因子：
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通讯作者：
Dias, Eustáquio Souza
中科院分区：
文献类型：
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作者：
Ishikawa, Francine Hiromi;Barcelos, Quélen de Lima;Dias, Eustáquio Souza
本文报道了影响林氏炭疽菌原生质体产生和再生的因素。原生质体分离的实用性与许多不同的应用相关，并主要用于涉及基因操作的程序中。研究了渗透压稳定剂、裂解酶、培养时间和菌丝年龄对原生质体产量的影响。原生质体产生的最适条件为：幼菌丝体在0.6moL L(-1)氯化钠渗透压稳定剂中培养48h，哈茨木霉裂解酶用量为30mgml(-1)，培养3h。在此条件下，原生质体产量可达10(6)个原生质体m l(-1)以上，可用于真菌转化实验。蔗糖浓度为1.2molL(-1)和1molL(-1)是最适合48h再生的渗透压稳定剂，其稳定率分别为16.35%和14.54%。本研究提供了一种生产原生质体的有效方法，并利用一株炭疽菌LV115菌株将其恢复为典型的菌丝形态。
The present work reports factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum. The usefulness of protoplast isolation is relevant for many different applications and has been principally used in procedures involving genetic manipulation. Osmotic stabilizers, lytic enzymes, incubation time and mycelial age were evaluated in terms of their effects on protoplast yield. The optimal condition for protoplast production included the incubation of young mycelia (48 h) in 0.6 mol l(-1) NaCl as the osmotic stabilizer, with 30 mg ml(-1) Lysing Enzymes from Trichoderma harzianum for 3 h of incubation. In these conditions protoplasts production was higher than 10(6) protoplatos ml(-1) in the digestion mixture, number suitable enough for experiments of transformation in fungi. Sucrose concentrations of 1.2 mol l(-1) and 1 mol l(-1) were the most suitable osmotic stabilizers for the regeneration after 48 h, with rates of 16.35% and 14.54%, respectively. This study produced an efficient method for protoplast production and reverted them into a typical mycelial morphology using a Colletotrichum lindemuthianum LV115 isolate.