Li Jiawen, Xu Zhizhuo, Zeng Tuo, Zhou Li, Li Jinjin, Hu Hao, Luo Jing, Wang Caiyun
Key Laboratory for Biology of Horticultural Plants, Ministry of Education, College of Horticulture & Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, China.
School of Life Sciences, Guizhou Normal University, Guiyang 550025, China.
Plants (Basel). 2022 Jun 15;11(12):1575. doi: 10.3390/plants11121575.
Pyrethrum () is one of the most important industrial crops for the extraction of pyrethrins, which are natural insecticidal compounds. Progress in pyrethrum molecular breeding with the objective of increasing pyrethrin content has been slow for lack of a suitable gene transfer system. Regeneration recalcitrance is a crucial barrier to establishing a genetic transformation system in pyrethrum. Therefore, in this study, an Agrobacterium-mediated transformation system in pyrethrum was developed using shoot apical meristems from germinated seedlings. Factors affecting transformation efficiency were optimized. Optimal conditions included explants at the "no true leaf" stage with a half apical meristem, an cell density of OD = 0.5, two days of cocultivation, and the incorporation of 1.5 mg L 6-BA and 30 mg L kanamycin into the selection medium. Under the optimized conditions, two expression cassettes ( and ) were successfully transformed into pyrethrum. Polymerase chain reaction (PCR), Southern blotting, reverse-transcription quantitative PCR (RT-qPCR), and histochemical staining confirmed the identity of transgenic plants. PCR and RT-qPCR analyses confirmed the identity of transgenic plants. The transformation efficiency was 0.83% (5 transgenic lines/600 infected explants). The relative concentration of pyrethrins in transformants (OX T0-1: 1.50% or OX T0-2: 1.24%) was higher than that in nontransformed plants (WT: 0.76%). Thus, the genetic transformation system overcame the low regeneration efficiency and integrated a foreign gene into the pyrethrum genome. The new system is a suitable and effective tool for creating high-yielding cultivars of pyrethrum.
除虫菊（）是提取除虫菊酯的最重要的工业作物之一，除虫菊酯是天然杀虫化合物。由于缺乏合适的基因转移系统，以提高除虫菊酯含量为目标的除虫菊分子育种进展缓慢。再生难是在除虫菊中建立遗传转化系统的关键障碍。因此，在本研究中，利用发芽幼苗的茎尖分生组织开发了一种农杆菌介导的除虫菊转化系统。优化了影响转化效率的因素。最佳条件包括处于“无真叶”阶段的外植体，带有半个茎尖分生组织，细胞密度为OD = 0.5，共培养两天，以及在选择培养基中加入1.5 mg/L 6 - BA和30 mg/L卡那霉素。在优化条件下，两个表达盒（和）成功转化到除虫菊中。聚合酶链反应（PCR）、Southern杂交、逆转录定量PCR（RT - qPCR）和组织化学染色证实了转基因植株的身份。PCR和RT - qPCR分析证实了转基因植株的身份。转化效率为0.83%（5个转基因株系/600个感染外植体）。转基因植株（OX T0 - 1：1.50%或OX T0 - 2：1.24%）中除虫菊酯的相对浓度高于未转化植株（WT：0.76%）。因此，该遗传转化系统克服了低再生效率问题，并将外源基因整合到除虫菊基因组中。新系统是创建除虫菊高产栽培品种的合适且有效的工具。
