摘要
【目的】探索双亚5号亚麻与罗布麻原生质体制备、电融合和培养的适宜条件,为进一步深入进行亚麻与罗布麻体细胞杂交育种研究奠定基础。【方法】以已建立的双亚5号亚麻和罗布麻胚性细胞悬浮系为材料,制备原生质体并进行电融合,培养。【结果】制备亚麻和罗布麻原生质体的最佳渗透剂分别是5.6%蔗糖和0.7mol/L甘露醇;游离亚麻和罗布麻原生质体的最适酶组合都是0.5%CellulaseOnozukaR-10+1.0%CellulaseOnozukaRS+0.2%PectolyaseY-23;最佳酶解时间分别是4和3h;亚麻细胞最佳继代时间是转瓶后的第3d,在此条件下原生质体存活率均为92.0%,产量分别达到1.24×106和22.4×106个/mL;2种原生质体电融合的最佳参数依次为:AC8v/cm,持续25s—DC1500v/cm,脉冲宽度为60μs,脉冲次数1次—交流电时间为9s,重复次数2次。在此条件下融合率达到20.5%。【结论】亚麻与罗布麻原生质体在一定的条件下可实现体细胞杂交。
[Objective]The suitable conditions of dissociate protoplast of Linum usitatissimum L.Shuangya No.5 and Apocynum venetum Linn.,electrofusion and protoplast culture were explored,to lay a foundation for further study of somatic hybridization breeding in the two species.[Method]The two kinds of protoplasts derived from established embryogenic cell suspension lines of Shuangya No.5 and A.venetum were fused with electrofusion and cultured.[Result]The proper penetrant on dissociate protoplast of flax and A.venetum were 5.6% sucrose and 0.7 mol/L mannitol,respectively.The proper combination of enzymes all were 0.5% Cellulase Onozuka R-10＋1.0% Cellulase Onozuka RS＋0.2% Pectolyase Y-23,the aptimal enzymolysis time were 4 h and 3 h,respectively.The proper subculture time of suspensionembryogenic line of flax was the third day.The survival rate of the two kinds of protoplasts all reached 92.0% and the protoplast yield of flax and A.venetum reached 1.24×106/mL and 22.4×106 /mL under optimum condition,respectively.The optimum electrofusion parameters of two kinds of protoplasts were AC 8 v/cm for 25 s—DC 1 500 v/cm for 60 μs in pulse length,one time—AC 8 v/cm for 9 s,twice repeated,under whose conditions the fusion rate reached 20.5%.[Conclusion]Flax and A.venetum could achieve somatic hybridization through protoplast fusion under definite conditions.
出处
《新疆农业科学》
CAS
CSCD
北大核心
2010年第7期1336-1342,F0002,共8页
Xinjiang Agricultural Sciences
基金
国家自然科学基金(30660095)
新疆维吾尔自治区教育厅重点科研项目(XJEDU2006108)
关键词
亚麻
罗布麻
胚性细胞系
原生质体
电融合
再生愈伤组织
flax
Apocynum venetum Linn
embryogenic cell line
protoplast
electrofusion
regenerative callus
