大豆花穗置低温(7°—8℃)冷处理5、8、10、12天,取花药置培养皿中,用盖玻片压出花粉粒,去掉残渣,加入液体培养基。基本培养基是改良的B_5,参照高国楠原生质体培养基附加糖、椰乳、水解酪蛋白等有机物,0.05—0.2mg/l 2.4—D,0 1～0.5mg/l Zeatin,0.5—1mg/l NAA。20天后见花粉分裂,30天见细胞团,40—50天形成肉眼可见的愈伤组织。分裂的花粉最后达接种时培养皿中花粉的60—70％以上。活体观察花粉分裂大致有多细胞型和游离核型。将直径2mm大小的愈伤组织转至固体培养基继代增殖而获得大量愈伤组织。分化培养获得根,未见芽的分化。 Soybean spikes at low temperature (7 ° -8 ℃) cold treatment 5,8,10,12 days, take anther petri dish, with a cover glass pressure out of pollen grains, remove the residue, add liquid medium. The basic medium is modified B_5, reference to the high protoplast culture medium attached sugar, coconut milk, hydrolyzed casein and other organic matter, 0.05-0.2mg / l 2.4-D, 0 1 ~ 0.5mg / l Zeatin, 0.5-1mg / l NAA. See pollen division 20 days later, see cell mass 30 days, 40-50 days to form macroscopic callus. Split pollen last up to 60-70% of the pollen in the dish when inoculated. In vivo observation pollen split roughly multicellular and free karyotype. Callus tissues of 2 mm in diameter were transferred to solid medium and subcultured to obtain a large amount of callus. Differentiation culture obtained roots, no bud differentiation.