提高二倍体马铃薯原始栽培种原生质体分离与分裂频率的研究

Studies on improving isolation and division frequency of diploid original cultivar potato protoplast

为获得较高的原生质体分离和分裂频率,试验以马铃薯二倍体原始栽培种‘47-33’‘5-19’试管苗叶片为材料,进行了原生质体游离和培养的研究.结果表明:培养21d的两品系试管苗叶片均在含有2.0%纤维素酶+0.5%果胶酶+0.25%离析酶,渗透压为0.35mol/L的酶解液中解离效果最好,原生质体产量分别为2.31×106个/gFW和2.52×106个/gFW;在28℃酶解温度条件下缓慢摇动14h或1h静置+13h缓慢摇动的,可促进叶片原生质体的大量释放.此外,1.0mg/L NAA+0.5mg/L 2,4-D+0.4mg/L BAP外源激素组合有利于叶片原生质体的分裂,‘47-33’‘5-19’原生质体一次分裂频率分别达到8.85%和12.56%.在0.35mol/L渗透压的液体培养基中,‘47-33’叶片原生质体分裂更早,分裂频率达13.85%.研究获得了稳定的原生质体分离体系以及较好的原生质体培养条件,为马铃薯二倍体原始栽培种的体细胞融合奠定了基础.

The potato leaves from 2 kinds of diploid original cultivar ‘47-33’and ‘5-19’were used to study protoplast isolation and division.The optimum combination of enzymes to separate protoplast from 21 d cultured leaves was 2.0% cellulase＋0.5% pectinase＋0.25% macerozyme.The optimum osmotic pressure was 0.35 mol/L.Under the condition of the above,the protoplast yields of ‘47-33’and ‘5-19’ were 2.52×106/gFW and 2.31 ×106/gFW respectively.The release of leaf protoplasts was significantly promoted by shaking samples for 13 h after 1 h stand or by shaking samples continuously for 14 h,both at 28 ℃ condition.In addition,exogenous hormone combination of 1.0 mg/L NAA＋0.5 mg/L 2,4-D＋0.4 mg/L BAP obviously enhanced the division of protoplast.The first division frequencies of protoplast from‘47-33’and ‘5-19’were up to 12.56% and 8.85% respectively.Furthermore,the division of protoplast of‘47-33’was earlier in the liquid medium with 0.35 mol/L osmotic pressure than solid or solid and liquid medium with same osmotic pressure.The division frequency reached to 13.85%.With this study,we ob-tained a stable protoplast isolation system and a reliable protoplast cultivation condition,which would be very useful to somatic cell fusion of potato original cultivar.