甘蓝型油菜小孢子培养子叶形胚发育成苗研究

Study on seedling development of cotyledon embryo in microspore culture of Brassica napus L.

甘蓝型油菜小孢子细胞诱导胚状体以子叶胚和非子叶胚如球形胚、心型胚和类似于胚的结构形态存在。子叶胚在成苗培养基上能够快速生长成植株,但是非子叶胚难以一次成苗,容易褐化死亡,因此减少非子叶状胚、培育高质量子叶胚是提高油菜小孢子再生成植株效率的关键步骤。为了提高甘蓝型油菜小孢子培养过程中诱导子叶胚的数目,对3个甘蓝型油菜品系分离得到的小孢子细胞在25℃下暗培养2周,之后转移到固液双层培养基上培养。通过对摇床的振荡周期、培养温度、固液双层培养基中活性炭的浓度以及6-BA不同梯度的处理,分析诱导子叶胚和非子叶胚发生的数目。结果显示,3个品系小孢子细胞在固液双层培养基上诱导得到的子叶胚数目显著高于液体培养基上诱导发生的数目。小孢子细胞在固液双层培养基上振荡培养1周所得子叶胚数目显著增加。降低小孢子培养温度至23℃,出胚总数和对照差异不显著,但子叶胚的数目显著增加。在固液双层培养基中的下层固体培养基中加入0.1%活性炭和0.25 mg/L 6-BA能够有效提高子叶胚的数目。此外,相比前人只用液体培养基培养的方法,本试验建立了两步培养方法(先液体培养后固-液双层培养)能有效获得子叶胚,从而快速生成植株,提高了甘蓝型油菜小孢子培养技术创制育种资源的效率。

The embryos induced by microspore cells in Brassica napus L.were cotyledon type and non-cotyledon type such as globular embryo,heart-shaped embryo and unregular embryos structure.Cotyledon embryos can grow into plantlets quickly on seedling medi⁃um,but non-cotyledon embryos are easy to browning and die.Therefore,reducing non-cotyledon embryos and obtaining cotyledon embryos is the key point to improve the efficiency of microspore regeneration.In this research,we isolated microspore cells from three varieties and transferred them to different media and culture methods.In order to optimize the experiment,the microspores isolated from three varieties were cultured in the dark at 25℃for 2 weeks.They were transferred from the solid-liquid double-layer culture me⁃dium and treated with different agitation period,temperature,concentrations of activated carbon and 6-BA.The results showed that the number of cotyledon embryos grown on the solid-liquid two-layer medium was significantly increased than that in the liquid medi⁃um.The number of cotyledon embryos obtained from the combination of solid-liquid double-layer medium and agitation for 1 week was evidently increased and the number of non-cotyledon embryos was significantly reduced.When microspores were cultured at 23℃,there was no significant difference between the total number of embryos and the control,but the number of cotyledon embryos increased significantly.The result on the effect of various concentrations of activated charcoal and 6-BA on sublayer medium indicated the combination of 0.1%and 0.25 mg/L 6-BA was very promising,as it outperformed the control treatment in the yield of normal look⁃ing embryos.In this study,we developed a two-step(liquid culture and solid-liquid double-layer culture)microspore culture proto⁃col.It can effectively obtain high-quality cotyledon embryos with ability to regenerate seedlings,which may greatly accelerate the ap⁃plication of microspore-derived DH plants as breeding tool.