Genome-editing tools from Oligonucleotide-Directed Mutagenesis (ODM) to CRISPR system use synthetic oligonucleotides for targeted exchange of nucleotides. Presently, majority of genome-editing protocols are dependent on the in vitro cell or tissue culture systems with somaclonal variation, and limitations in plant regeneration. Therefore, here, we report an alternative in planta cellular test system for optimization of the ODM, based on the injection of oligonucleotide solution into the apical meristematic region of haploid maize seedlings. Using 5′-fluorescein-labeled oligonucleotides, we detected accumulation of synthetic DNA molecules in cells of the shoot apical meristem and of the vascular bundles of leaf primordia. For silencing or knocking down of the phytoene desaturase gene in somatic cells, 41-mer long single-stranded oligonucleotides with TAG stop codon were injected into maize seedlings. We detected out-growing M1 plantlets that developed leaves with white stripes or pale-green color. Confocal microscopy of white stripes showed that in addition to the chlorophyll fluorescence-deficient tissue region, chlorophyll containing cells are present in white stripes. The Ion Torrent sequencing of DNA samples from the white stripes indicated 0.13–1.50% read frequency for the TAG stop codon in the phytoene desaturase gene. Appearance of chlorotic abnormalities supports the mutagenic nature of oligonucleotide molecules after injection into the shoot apical meristem region of maize seedling. The described protocol provides basis for early seedling stage characterization of functionality of a mutagenic oligonucleotide with different chemistry and testing efficiency of various treatment combinations at plant level.

从寡核苷酸定向诱变（ODM）到CRISPR系统的基因组编辑工具使用合成寡核苷酸进行核苷酸的靶向交换。目前，大多数基因组编辑方案都依赖于具有体细胞克隆变异的体外细胞或组织培养系统，以及植物再生的局限性。因此，在这里，我们在Planta中报告了一种替代方案基于将寡核苷酸溶液注入单倍体玉米幼苗的顶端分生组织区域中的用于优化ODM的细胞测试系统。使用5'-荧光素标记的寡核苷酸，我们检测到合成的DNA分子在茎尖分生组织和叶原基血管束的细胞中积累。为了沉默或敲除体细胞中的八氢番茄红素去饱和酶基因，将具有TAG终止密码子的41个mer长的单链寡核苷酸注入玉米幼苗。我们检测到生长出带有白色条纹或淡绿色叶子的M1小植株。共聚焦显微镜下的白色条纹显示，除了叶绿素荧光不足的组织区域之外，白色条纹中还包含含叶绿素的细胞。白色条纹上的DNA样品的离子激流测序表明，在番茄红素去饱和酶基因中TAG终止密码子的读取频率为0.13-1.50％。叶绿素异常的出现支持寡核苷酸分子注入玉米幼苗的茎尖分生组织区域后的诱变特性。所描述的方案为具有不同化学性质的诱变寡核苷酸的功能的幼苗早期表征提供了基础，并在植物水平上测试了各种处理组合的效率。