[Objective] The aim of this study is to reveal the disinfectants and disinfection methods, medium components, and embryoid culture method on dissociative microspore culture. [Method] B5 as basic medium appended with different concentrations of sucrose, agar and different hormone combinations was used to optimize the culture technique for DH line in Brassica napus L. [Result] Both the 15 min disinfection of NaClO containing 5% Cl- and 10 min disinfection of 0.1% HgCl2 performed well in disinfection and subsequent embryo production; in the extraction process of dissociative microspores, B5 medium containing 2% sucrose could achieve a good embryo production effect; under dark condition microspores were firstly incubated at 32 ℃ 5-7 d, then at 25 ℃ 12-15 d, and finally transferred to 25 ℃ oscillator(60-65 r/min) for 3-7d, when the embryoid would become full ripeness; 1/2MS medium appended with 1.2% agar, 0.02% NAA, 2.0 mg/L 6-BA, 3.4 mg/L AgNO3 and 2% sucrose was helpful for embryoid differentiation and plantlet generation, presenting low degree of browning and slight vitrification. [Conclusion] The results may facilitate DH Line in rape production in large scale and high efficient transformation system.