Abstract: To find effective sterilization methods for rhizobia inside alfalfa seeds,the seeds of Alfalfa (Medicago sativa ‘Gannong No.9’) were used as materials in the experiment. Four sterilization methods including DF (iodophor immersion 6 min),DS (iodophor soaked 5 min to sterile water rinse,ST solution -0.9% sterile sodium chloride solution and 0.5% Tween 80 soaked 1 min),SH3 (3% sodium hypochlorite soaked 6 min) and SH10 (10% sodium hypochlorite immersion 6 min) were compared. The intact seeds,peeled seeds,seed coat,cotyledon,embryo,cotyledon + embryo,junction of cotyledon-embryo,and seedlings were taken as research materials,and control materials were soaked in sterile water for the corresponding time. The effects of the four sterilization methods on the sterilization of endophytic rhizobia in the tested materials,seed germination capacity,and seedling growth were analyzed. Result shows that:Endophytic rhizobia were mainly distributed in cotyledon + embryos (52.7 per seed) of alfalfa seeds,with a minor distribution in the seed coat (16 per seed). The cotyledon and embryo tissues were mainly distributed at the junction of cotyledon-embryo (38.33 per seed),and the rest were distributed in cotyledons (9.67 per seed) and embryos (4.17 per seed). The seeds could be completely sterilized by DS treatment,and the germination rate,germination potential,germination index,radicle length,radicle length,and seedling length of intact seeds under DS treatment were not significantly different from those of the control. In conclusion,the cotyledon embryo junction is the main existing site of rhizobia inside M. sativa seeds,and the DS treatment is the best sterilization method for rhizobia inside M. sativa seeds.

Key words: Medicago sativa, Seed, Endophytic rhizobium, Sterilization method

摘要： 为探寻苜蓿种子内生根瘤菌的有效灭菌方法，试验以‘甘农9号’紫花苜蓿(Medicago sativa ‘Gannong No.9’)种子为材料，设置DF(碘伏浸泡6min),DS(碘伏浸泡5min→无菌水冲洗→ST液-0.9%无菌氯化钠溶液和0.5%吐温80浸1min),SH3(3%次氯酸钠浸泡6min)和SH10(10%次氯酸钠浸泡6min)4种灭菌方法，分别以完整种子、去皮种子、种皮、子叶、胚、子叶+胚、子叶-胚连接处、幼苗为研究材料，以无菌水浸泡相应时间处理为对照，分析4种灭菌方法对供试材料内生根瘤菌的灭菌情况以及对种子萌发能力及幼苗生长的影响。结果表明：内生根瘤菌主要分布于紫花苜蓿种子的子叶+胚(52.7个·粒^-1)中，种皮有少量分布(16个·粒^-1)，而在子叶+胚组织中内生根瘤菌主要分布于子叶-胚连接处(38.33个·粒^-1)，其余分布数量依次为子叶(9.67个·粒^-1)、胚(4.17个·粒^-1)；DS处理可对种子完全灭菌，且DS处理下完整种子的发芽率、发芽势、发芽指数、胚芽长、胚根长和幼苗长与CK差异不显著。综上，子叶-胚连接处是紫花苜蓿种子内生根瘤菌的主要存在部位，DS处理是紫花苜蓿种子内生根瘤菌的最佳灭菌方法。

关键词: 紫花苜蓿, 种子, 内生根瘤菌, 灭菌方法