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以油菜品种“蜀杂6号”的薹茎段髓部为材料分离原生质体,产量一般在每g鲜重2×10~6个左右。在每L附加1.5mgBA、0.4mgNAA、1.5mg2,4-D、200mg水解酪蛋白、250mg木糖、0.3M蔗糖和0.1M葡萄糖的Nitsch培养基中,对原生质体进行液体浅层静置培养。2～7天内原生质体出现第一次分裂,分裂频率最高可达49.2％。培养15天后逐渐形成肉眼可见的小愈伤组织。21天时将培养基改换成每L附加2mgBA、0.2mg 2,4-D和3％蔗糖的MS扩增培养基,45天后可形成5mm左右的愈伤组织。大于2mm的愈伤组织可在每L含有1.0mgBA/3.0mgZT和0.1mgNAA的MS琼脂培养基上分化出芽,分化频率可达36.7％。在1/2MS无激素培养基中,1cm左右的分化芽有85.9％生根,形成完整的植株。
The protoplast was isolated from the stem of sedge stem segment of hybrid variety “Shuza No.6” with the yield about 2 × 10 ~ 6 per g fresh weight. Protoplasts were subjected to shallow supernatant culture in Nitsch medium supplemented with 1.5 mg BA, 0.4 mg NAA, 1.5 mg 2,4-D, 200 mg hydrolyzed casein, 250 mg xylose, 0.3 M sucrose and 0.1 M glucose per L. Protoplasts showed the first division within 2-7 days, with a frequency of up to 49.2%. After 15 days of cultivation, small macroscopically visible callus gradually formed. At 21 days, the medium was changed to MS expansion medium supplemented with 2 mg BA, 0.2 mg 2,4-D and 3% sucrose per L, and about 5 mm callus was formed after 45 days. Callus greater than 2 mm could differentiate on MS agar medium containing 1.0 mg BA / 3.0 mg ZT and 0.1 mg NAA per L, with a frequency of differentiation of up to 36.7%. In 1 / 2MS hormone-free medium, about 1 cm differentiation buds have 85.9% rooting, forming a complete plant.