Abstract: The somatic hybridization of asymmetric fusion between protoplasts isolated from callus of Russian variegated alfalfa and ‘Gannong No. 4' alfalfa was obtained through electrofusion method. The effects of different inactivation pretreatments, electric field conditions and protoplast densities on alfalfa protoplast fusion were studied. Test results showed that the optimum inactivation pretreatments were that Russian variegated alfalfa protoplast was treated with ultraviolet radiation by 5 min and 'Gannong No.4’ alfalfa protoplast was treated with 6 mmol·L^-1 iodoacetamide. The optimum electrofusion parameters were AC electric field intensity for 15~20 V·cm^-1, frequency for 2000~2500 kHz, DC electric field intensity for 200~250 V·cm^-1, pulse width for 40 μs and pulse 3 times. The optimum protoplast density is 3×10⁵~5×10⁵ mL^-1. The highest protoplast binary fusion frequency reached 13.8% with above conditions.

Key words: Alfalfa, Protoplast, Cell fusion, Electrofusion, Optimum condition

摘要： 为改良培育苜蓿(Medicago)新品种,拟建立苜蓿原生质体融合体系。通过电融合方法,使俄罗斯杂花苜蓿(M. varia)原生质体和甘农4号紫花苜蓿(M. sativa ‘Gannong No. 4')原生质体进行非对称融合,获得了杂交细胞,研究不同失活处理条件、电场条件、原生质体密度对苜蓿原生质体融合的影响。结果表明:经过紫外灯辐射5 min的俄罗斯杂花苜蓿原生质体和6 mmol·L^-1 IOA处理的甘农4号紫花苜蓿原生质体,密度调至3×10⁵~5×10⁵ 个·mL^-1,以交流电场强度15~20 V·cm^-1、交流频率2000~2500 kHz,直流脉冲场强200~250 V·cm^-1、脉冲宽幅40 μs、脉冲个数为3的条件为最适电融合条件,此时一对一有效融合率最高可达13.8%。

关键词: 苜蓿, 原生质体, 细胞融合, 电融合, 适宜条件