Tsuruta Momi, Maruyama Tsuyoshi E, Ueno Saneyoshi, Hasegawa Yoichi, Moriguchi Yoshinari
Department of Forest Molecular Genetics and Biotechnology, Forestry and Forest Products Research Institute, Tsukuba, Japan.
Graduate School of Science and Technology, Niigata University, Niigata, Japan.
Front Plant Sci. 2021 Oct 12;12:748110. doi: 10.3389/fpls.2021.748110. eCollection 2021.
Pollen allergy caused by sugi (Japanese cedar, ) is a serious problem in Japan. One of the measures against pollinosis is the use of male-sterile plants (MSPs; pollen-free plants). In this context, the development of a novel technique for the efficient production of sugi MSPs, which combines marker-assisted selection (MAS) with somatic embryogenesis (SE), was recently reported by our research group. To improve the efficiency of MSP production, in this paper we report improved MAS for male-sterile individuals from embryogenic cells, cotyledonary embryos, and somatic plants of sugi using a newly developed marker in the form of the causative mutation of itself, selecting individuals with and male-sterile mutations. We also describe simplified methods for extracting DNA from different plant materials and for MAS using LAMP diagnostics. Finally, we show that MAS can be efficiently performed using the one-step indel genotyping (ING) marker developed in this study and using InstaGene for DNA extraction. The combination of SE and 100% accurate marker selection during the embryogenic cell stage enables the mass production of male-sterile sugi seedlings.
由柳杉（日本柳杉）引起的花粉过敏在日本是一个严重问题。防治花粉症的措施之一是使用雄性不育植株（MSP；无花粉植株）。在此背景下，我们研究小组最近报道了一种将标记辅助选择（MAS）与体细胞胚胎发生（SE）相结合的高效生产柳杉MSP的新技术。为提高MSP的生产效率，本文报道了利用柳杉自身致病突变形式的新开发标记，对柳杉胚性细胞、子叶胚和体细胞植株中的雄性不育个体进行改良的MAS，筛选具有特定雄性不育突变的个体。我们还描述了从不同植物材料中提取DNA以及使用环介导等温扩增（LAMP）诊断进行MAS的简化方法。最后，我们表明使用本研究开发的一步插入缺失基因分型（ING）标记并使用InstaGene进行DNA提取能够高效地进行MAS。在胚性细胞阶段将SE与100%准确的标记选择相结合能够大量生产雄性不育柳杉幼苗。
