甘蓝型油菜DH系培养技术优化

[目的]研究消毒处理方式、培养基成分和胚状体培养方法对油菜游离小孢子培养的影响。[方法]以B5培养基为基础培养基,添加不同浓度的蔗糖、琼脂及不同激素组合进行试验,对油菜DH系的组培技术进行优化。[结果]含2%Cl-的NaClO消毒15 min与0.1%HgCl2消毒10 min培养效果较好,都能达到良好的消毒和产胚效果;在游离小孢子提取的过程中,B5液体培养基中蔗糖浓度为2%时能达到较好的产胚效果;小孢子培养首先在32℃暗培养5~7 d,然后25℃暗培养12~15 d,最后25℃振荡暗培养3~7 d胚状体就可完全成熟;1/2 MS培养基(附加1.2%琼脂+0.02%NAA+2.0 mg/L6-BA+3.4 mg/LAgNO3+2%蔗糖)有利于胚的分化和苗的形成,其褐化程度小,玻璃化程度低。[结论]该研究结果有助于甘蓝型油菜DH系的规模化生产及高效转化体系的建立。

Optimization of Culture Techniques for DH Line in Brassica napus L.

[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 2% 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 3-7 d,when the embryoid would become full ripeness;1/2 MS 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.