草地早熟禾原生质体培养与融合

以草地早熟禾品种RugbyⅡ成熟种子诱导的松软胚性愈伤组织为材料,以酶解法分离出原生质体,进行了原生质体培养;利用PEG融合法对MidnightⅡ和Nuglade的原生质体进行了融合,获得了体细胞杂种愈伤组织。研究了不同酶液组成、酶解时间、愈伤组织继代时间和渗透压对草地早熟禾原生质体游离及生长的影响;预处理对亲本原生质体活力的影响和适宜的融合条件。结果表明:采用继代培养8～10d的胚性愈伤组织,在1.0%纤维素酶+1.0%离析酶+0.5%果胶酶+0.3%崩溃酶条件下,酶解14～17h可得到产量和活力较高的原生质体;原生质体在KM8P培养基中培养3d后出现第1次细胞分裂,2～3周后形成小细胞团,此时添换低渗培养基2～3次,有利于小细胞团持续分裂并形成愈伤组织;试验获得了草地早熟禾品种RugbyⅡ原生质体来源的愈伤组织;5mmol/ml碘乙酰胺处理Nuglade原生质体10min及40μg/ml罗丹明处理MidnightⅡ原生质体5min,能有效地使Nuglade和MidnightⅡ原生质体失活;两品种融合PEG(6000)适宜浓度35%,融合率为9.8%。

Protoplasts Culture and Fusion from Callus of Poa pratensis L.

Abstract：The friable calli induced from mature seeds of Rugby Ⅱ (Poa pratensis L.) were used for protoplast preparation, and the protoplasts were isolated through enzyme digestion and were cultured. Midnight Ⅱ protoplasts and Nuglade prtoplasts of Poa pratensis L. by ployethylene glycol (PEG) method was fused, and callus had been induced. The effects of different composition of enzyme, time of enzyme digestion, calli age and osmoticum on protoplast isolation and the effects of pretreatment and the optimal fusion condition on the activity of protoplasts were studied. The results showed that highter yields and activities of protoplasts were obtained from embryogenic calli 8 to 10d with 10% cellulase Onozuka R 10, 10% macerozymeR 10, 03% driselase, 05% pectolase Y 23 and 14~17h of enzyme digestion. The first divisions occurred in 3 d and small cell clusters could be seen after 2 to 3 weeks in the culture. At this moment, the addition of the protoplast culture medium with 01~02mol/L mannitol 2 or 3 times was needed for the continuous protoplasts division to form calli. The calli were obtained from Rugby Ⅱ. The protoplast of Nuglade treated  with 5mmol/ ml IOA 10min and the protoplast of Midnight Ⅱ treated with 40μg/ml R 6G 5min could be killed. The best fusion condition with PEG(6000) was 35% for the hybrid cell and the fusion rate was 98%.