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.

Doubled haploid (DH) technology is an essential component in producing inbred lines for a competitive maize (Zea mays L.) breeding program. The R1-navajo (R1-nj) gene provides phenotypic marker that insures only variable reliability for seed selection of haploid embryos. Therefore, in the present study we outline a complex protocol for early stage genome size determination that integrates the phenotypic screening with the flow cytometry of nuclei from root tips and with the use of DNA isolated from seedlings for molecular marker-based genotyping. In a representative experiment with three genotypes, only 59% of the color marker pre-selected seeds were confirmed to be haploid by cytometric analysis of nuclei isolated from root tips. As a novel tool we have identified the UMC1152 SSR marker being polymorphic between the haploid inducer line (K405) and the K4390 hybrid as parents to screen seedlings pre-selected with the R1-navajo marker. Using this molecular marker, alleles characteristic for the inducer K405 line could not be detected in 83% of seedlings previously selected as haploid candidate. Seedlings identified as haploids were exposed to 0.06% colchicine solution for rediploidization. This procedure resulted in doubled haploids with 3% frequency relative to the initial population as it was quantified by the number of mature maize plants with fertile tassel. The described complex approach can support safer identification of haploids at early seedling stage in a hybrid population derived from crossing with a haploid inducer line.