Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070, People's Republic of China.
Plant Cell Rep. 2010 Apr;29(4):371-81. doi: 10.1007/s00299-010-0828-6. Epub 2010 Feb 24.
A protocol for chloroplast transformation of an elite rapeseed cultivar (Brassica napus L.) was developed based on optimized conditions for callus induction and regeneration from cotyledonary tissues. Comparison of six different media with three elite cultivars showed that B5 medium plus 3 mg/l AgNO(3) supplemented with 0.6 mg/l 2,4-dichlorophenoxyacetic acid and 0.2 mg/l 6-furfurylaminopurine was optimal for callus formation and maintenance without differentiation, while the medium suitable for regeneration was B5 medium supplemented with 1 mg/l 6-benzylaminopurine, 1 mg/l 6-furfurylaminopurine and 0.5 mg/l alpha-naphthaleneacetic acid. A rapeseed-specific chloroplast transformation vector was constructed with the trnI and trnA sequences amplified from the rapeseed chloroplast genome using two primers designed according to Arabidopsis homologs. The aadA gene was used as a selection marker regulated by the ribosome-binding site from the bacteriophage T7 gene 10L, the tobacco 16S rRNA promoter and the psbA terminator. After bombardment, cotyledonary segments were cultured for callus formation on media containing 10 mg/l spectinomycin and regeneration was carried out on medium with 20 mg/l spectinomycin. Heteroplasmic plastid transformants were isolated. An overall efficiency for the chloroplast transformation was one transplastomic plant per four bombarded plates. Southern blot analyses demonstrated proper integration of the target sequence into the rapeseed chloroplast genome via homologous recombination. The expression of the aadA gene was confirmed by Northern blot analysis. Analysis of T1 transplastomic plants revealed that the transgenes integrated into the chloroplast were inheritable with a ratio of about 8%. These results suggest that rapeseed may be a suitable crop for chloroplast transformation with cotyledons as explants under appropriate conditions.
基于优化的子叶组织愈伤组织诱导和再生条件，开发了一种用于油菜（Brassica napus L.）优良品种叶绿体转化的方案。将六种不同的培养基与三个优良品种进行比较表明，B5 培养基+3 mg/L AgNO3，补充 0.6 mg/L 2,4-二氯苯氧乙酸和 0.2 mg/L 6-糠基氨基嘌呤，对于无分化的愈伤组织形成和维持最为理想，而适合再生的培养基是 B5 培养基，补充 1 mg/L 6-苄氨基嘌呤、1 mg/L 6-糠基氨基嘌呤和 0.5 mg/L α-萘乙酸。利用根据拟南芥同源物设计的两个引物，从油菜叶绿体基因组扩增 trnI 和 trnA 序列，构建了油菜特异性叶绿体转化载体。aadA 基因作为选择标记，由噬菌体 T7 基因 10L 的核糖体结合位点、烟草 16S rRNA 启动子和 psbA 终止子调控。轰击后，子叶段在含有 10 mg/L 壮观霉素的培养基上培养形成愈伤组织，在含有 20 mg/L 壮观霉素的培养基上进行再生。分离异质质体转化体。叶绿体转化的总体效率为每四个轰击板一个转基因植株。Southern blot 分析表明，通过同源重组，靶序列正确整合到油菜叶绿体基因组中。通过 Northern blot 分析证实了 aadA 基因的表达。对 T1 转基因植株的分析表明，整合到叶绿体中的转基因是可遗传的，比例约为 8%。这些结果表明，油菜可能是一种适合的作物，在适当的条件下，以子叶作为外植体进行叶绿体转化。
