铁皮石斛叶肉原生质体的分离与培养研究

Study on isolation and culture of mesophyll protoplasts of Dendrobium officinale Kimura et Migo

对铁皮石斛无菌试管苗叶片原生质体的游离及培养条件进行了研究.结果表明原生质体分离的适宜酶液配比是0.5%Pectinase和1.0%CellulaseOnozukaR-10,渗透压调节剂的浓度为0.5mol/L,以蔗糖作为渗透压调节剂优于甘露醇和葡萄糖,质壁分离处理后的叶片原生质体产量提高了16.6%,存活率提高了14.2%.培养在1/10MS附加2mg/LNAA+0.5mg/L6-BA和多种有机成分培养基上的铁皮石斛无菌苗叶片原生质体,3～5d后开始第一次分裂,30d后长成胚性细胞团.弱光照(1501x)和稍低的温度((21±0.5)℃)适于原生质体的培养.

The isolation and culture of protoplasts of plantlet leaves of Dendrobium officinale Kimura et Migo on asepsis tube were discussed. The appropriate condition in isolation is basic solution containing 0.5% pectinase and (1.0%) cellulase onozuka R-10. Osmotic medium concentration is 0.5 mol/L. Sugar is superior to mannitol and glucose. Moreover,if the young leaf pieces were plasmolyzed before incubated in enzyme solution will increased the yield and activity of protoplast up to 16.6% and 14.2%, respectively. The first division of protoplast occurred within 3 to 5 days in modified MS liquid media ( 1/10 strength macroelement) supplemented with 2 mg/L NAA+0.5 mg/L 6-BA and several organic compounds. The embryogenic cell cluster could be observed 30 days after culture. Relatively low light intensity (150 1x) and low temperature (21±0.5℃)are preferable in culture.