蝴蝶兰F3'5'H基因转化OT杂种百合Robina的研究

摘要

为了定向育种获得蓝色百合,该研究以百合Robina为蓝色基因最佳受体,以其花丝诱导产生的胚性愈伤组织和再生小植株小鳞片作为转化材料,利用农杆菌介导法,将蝴蝶兰F3'5'H基因导入百合Robina中.结果表明:以小鳞片为转化材料,预培养3d,OD600为0.8,侵染10 min,共培养3d,加入100 μmol/L AS稳定转化率最高为12.78％;而以胚性愈伤为转化材料,预培养2d,OD60.为0.8,侵染10 min,共培养3d,加入100 μmol/L AS稳定转化率最高为12.22％.2种转化材料的最适潮霉素筛选浓度均为20 mg/L.对抗性植株分别进行PCR和反转录PCR检测,获得9个阳性株系,Southern印记分析进一步确定了6株转基因百合中携带蓝色基因F3'5'H,为后续进一步获得蓝色百合奠定了基础.%To obtain bluish Lilium spp.in direct breeding,we screened the suitable variety-OT lily Robina for genetic transformation.Here,both embryonic callus induced from filament and regenerated bulb scales of OT lily Robina were used as the transformation material.Agrobacterium-mediated transformation of Phalaenopsis F3'5'H was studied.The results showed:with the pre-culture 3 d,OD600 =0.8,infection time 10 min,co-cultured for 3 d,100 μμmol/L acetosyringone conditions,the stable transformation rate of regenerated bulb scales could reach to the highest 12.78％;however,embryogenic callus pre-culture 2 d,OD600 =0.8,infection time 10 min,co-cultured 3 d,with 100 μμmol/L acetosyringone conditions,which the stable transformation rate was the highest 12.22％.In all the conditions,the best hygromycin-rcsistant screening concentrations always was 20 mg/L.PCR and reverse transcription PCR assay showed 9 putative transgenic plants were obtained.Southern hybridization analysisfurther proved 6 plants that the transgenic lilium flowers carry blue gene F3'5'H.The results provided technical support and material basis for the continuing development of novel bluish Lilium flowers.