灵芝原生质体分离与再生研究

STUDY ON THE SEPARATION AND REGENERATION OF GANODERMA PROTOPLAST

首次详细、系统地探讨了灵芝原生质体分离与再生的条件，结果表明：用0．6mol／L甘露醇配制成含2％溶壁酶和0．5％崩溃酶的复合酶，在30℃，pH为6.0时酶解3小时，可得到最高的原生质体得率。但考虑到原生质体的再生，以酶解2．5小时为最适。酶解2．5小时所得原生质体经精制，用纤维二糖培养基（以0.6mol／L甘露醇为渗透压稳定剂）进行双层平板（上层平板含0．2％琼脂）培养法再生，原生质体的再生率最高。本研究为以后进行灵芝原生质体的融合以及灵芝的转基因研究打下了基础。

The conditions of separation and regeneration of Ganoderma tsugae protoplast were studied in detail and systematically for the first time. Results show that the highest production rate of protoplast can be obtained by using compound enzyme composed of 0.6mol / L mannitol, 2% lywallzyme and 0.5% driselase and digesting for 3 hours at the condition of 30℃ and pH value 6.0. However, taking account of the regeneration of protoplast, it is most suitable to digest for 2.5 hours.The protoplast produced by 2.5-hour enzymolysis was purified and regenerated through double layers plate method in cellobiose medium with 0.6mol / L mannitol used for stabilizing osmotic pressure. The highest regeneration rate was obtained. This study has laid the foundation of protoplast fusion and transgenic research of Ganoderma in the future.