甘肃野生草地早熟禾原生质体分离与培养研究

建立高效的原生质体再生体系是通过原生质体融合技术培育草地早熟禾(Poa pratensis)新品种的重要前提。以甘肃陇西野生草地早熟禾(LX)和定西野生草地早熟禾(DX)胚性愈伤组织为材料,探索其原生质体游离和培养条件。结果表明,LX和DX原生质体分离的最佳酶液组合为1.5%纤维素酶R-10+0.5%果胶酶Y-23+1.0%离析酶R-10+0.3%崩溃酶;酶解时间为16 h;LX原生质体最适宜的甘露醇浓度为0.6 mol·L-1,而DX原生质体的最适甘露醇浓度为0.5 mol·L-1。LX原生质体的产量最高可达6.59×106个·g-1,DX可达5.95×106个·g-1。DX原生质体培养的最适密度为3.0×105个·m L-1,最适2,4-D浓度为1.0 mg·L-1,此时,DX原生质体再生细胞的分裂频率可达9.56%,植板率为4.62%。

Protoplast isolation and culture of wild Kentucky Bluegrass in Gansu

In the present study, the conditions of protoplast isolation and culture were explored using embryogenic callus from wild Kentucky Bluegrass variety Longxi (LX) and Dingxi (DX). The results showed that the optimal conditions for protoplast isolation was enzyme composition including 1.5% Cellulase R 10+0.5% Pectolase Y 23+1.0% Macerozyme R 10+0.3% Driselase with 16 h digestion. The optimal mannitol concentration was 0.6 mol·L-1 for LX protoplast and 0.5 mol·L-1 for DX protoplast. The highest yield of protoplasts was 6.59×106 number·g-1 for LX and 5.95×106 number·g-1 for DX. After purification, DX protoplasts were cultured in KM8P liquid medium. With 3.0×105 number·mL-1 plating density and 1.0 mg·L-1 2,4 D, DX protoplasts had highest cell division frequency of 9.56% and plating efficiency of 4.62%.