Abstract: The aim of this study was to optimize the in vitro germination conditions of Medicago ruthenica pollen. A single-factor experimental design was employed preliminarily to determine the optimal concentration ranges of sucrose， boric acid， and calcium chloride. Subsequently， a three-factor， two-level experimental design was conducted to further optimize the culture conditions， and the pollen germination rates and pollen tube lengths under different treatments were statistically compared. The results demonstrated that medium composition， temperature， and sampling time had highly significant effects on pollen germination. The optimal medium composition was determined to be sucrose （200 g·L^-1）， boric acid （40 mg·L^-1）， and calcium chloride （30 mg·L^-1）. Under these conditions， pollen germination rate reached 75.98% and pollen tube length was 224.73 μm. The optimal culture time was 6 h， the optimum sampling time was 12：00-13：00. Pollen culture was more favorable under dark， the optimal culture temperature was 25℃ and the optimal storage temperature was 4℃. The findings of this study provide a theoretical foundation and technical reference for pollen biology research and breeding practices of M. ruthenica.

Key words: Medicago ruthenica, In vitro germination, Liquid medium, Culture conditions

摘要： 本研究旨在优化扁蓿豆（Medicagoruthenica）花粉离体萌发的培养条件。通过单因素试验初步筛选出蔗糖、硼酸和氯化钙的最适浓度范围，并利用三因素两水平试验进一步优化培养条件，比较不同处理下花粉萌发率和花粉管生长长度。结果表明，培养基组分、温度及采样时间等因素对花粉萌发有极显著影响。最优培养基组分为蔗糖（200 g·L^-1）、硼酸（40 mg·L^-1）和氯化钙（30 mg·L^-1），在此条件下，花粉萌发率达75.98%，花粉管长度为224.73 μm。最佳培养时间为6 h，最适采样时间为中午12：00—13：00，且在黑暗条件下花粉萌发效果更好，最佳培养条件为25℃，最佳贮藏温度为4℃。本研究结果为扁蓿豆花粉生物学研究及育种实践提供了理论依据。

关键词: 扁蓿豆, 离体萌发, 液体培养基, 培养条件