Yamada Hajime, Kato Norio, Ichikawa Masako, Mannen Keiko, Kiba Takatoshi, Osakabe Yuriko, Sakakibara Hitoshi, Matsui Minami, Okamoto Takashi
Agri-Bio Research Center, KANEKA CORPORATION, Higashibara 700, Iwata, Shizuoka, 438-0802 Japan.
Plant Innovation Center, Japan Tobacco, Inc., Higashibara 700, Iwata, Shizuoka, 438-0802 Japan.
Plant Cell Physiol. 2024 May 30;65(5):729-736. doi: 10.1093/pcp/pcae010.
Genome-editing tools such as the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system have become essential tools for increasing the efficiency and accuracy of plant breeding. Using such genome-editing tools on maize, one of the most important cereal crops of the world, will greatly benefit the agriculture and the mankind. Conventional genome-editing methods typically used for maize involve insertion of a Cas9-guide RNA expression cassette and a selectable marker in the genome DNA; however, using such methods, it is essential to eliminate the inserted DNA cassettes to avoid legislative concerns on gene-modified organisms. Another major hurdle for establishing an efficient and broadly applicable DNA-free genome-editing system for maize is presented by recalcitrant genotypes/cultivars, since cell/tissue culture and its subsequent regeneration into plantlets are crucial for producing transgenic and/or genome-edited maize. In this study, to establish a DNA-free genome-editing system for recalcitrant maize genotypes/cultivars, Cas9-gRNA ribonucleoproteins were directly delivered into zygotes isolated from the pollinated flowers of the maize-B73 cultivar. The zygotes successfully developed and were regenerated into genome-edited plantlets by co-culture with phytosulfokine, a peptide phytohormone. The method developed herein made it possible to obtain DNA- and selectable-marker-free genome-edited recalcitrant maize genotypes/cultivars with high efficiency. This method can advance the molecular breeding of maize and other important cereals, regardless of their recalcitrant characteristics.
诸如成簇规律间隔短回文重复序列/Cas9（CRISPR/Cas9）系统等基因组编辑工具已成为提高植物育种效率和准确性的重要工具。在世界上最重要的谷类作物之一玉米上使用此类基因组编辑工具，将极大地造福农业和人类。传统的用于玉米的基因组编辑方法通常涉及在基因组DNA中插入一个Cas9导向RNA表达盒和一个选择标记；然而，使用此类方法时，必须消除插入的DNA盒，以避免对基因改造生物的立法担忧。难以转化的基因型/品种给建立一个高效且广泛适用的玉米无DNA基因组编辑系统带来了另一个主要障碍，因为细胞/组织培养及其随后再生为幼苗对于生产转基因和/或基因组编辑玉米至关重要。在本研究中，为了建立针对难以转化的玉米基因型/品种的无DNA基因组编辑系统，将Cas9-gRNA核糖核蛋白直接导入从玉米-B73品种授粉花中分离出的合子中。通过与一种肽植物激素植物磺肽素共培养，这些合子成功发育并再生为基因组编辑幼苗。本文开发的方法使得能够高效获得无DNA和无选择标记的基因组编辑的难以转化的玉米基因型/品种。该方法可以推动玉米和其他重要谷类作物的分子育种，而不论其是否具有难以转化的特性。
