Chinese cabbage, a major crop in Korea, shows self-incompatibility (SI). SI is controlled by the type 2A serine/threonine protein phosphatases (PP2As). The PP2A gene is controlled by regulatory subunits that comprise a 36 kDa catalyst C subunit, a 65 kDa regulatory A subunit, and a variety of regulatory B subunits (50–70 kDa). Among them, the PP2A 55 kDa B regulatory subunit (PR55/B) gene located in the A05 chromosome has 13 exons spanning 2.9 kb, and two homologous genes, Bra018924 and Bra014296, were found to be present on the A06 and A08 chromosome, respectively. In this study, we performed a functional analysis of the PR55/B gene using clustered regularly interspaced short palindromic repeats/CRISPR-associated system 9 (CRISPR/Cas9)-mediated gene mutagenesis. CRISPR/Cas9 technology can be used to easily introduce mutations in the target gene. Tentative gene-edited lines were generated by the Agrobacterium-mediated transfer and were selected by PCR and Southern hybridization analysis. Furthermore, pods were confirmed to be formed in flower pollination (FP) as well as bud pollination (BP) in some gene-edited lines. Seed fertility of gene-edited lines indicated that the PR55/B gene plays a key role in SI. Finally, self-compatible T-DNA-free T₂ gene-edited plants and edited sequences of target genes were secured. The self-compatible Chinese cabbage developed in this study is expected to contribute to Chinese cabbage breeding.

Due to the sudden change in temperature in spring, Chinese cabbage, a leafy vegetable cultivated for consumption, loses its commercial value due to the onset of bolting—the phenomenon of switching from vegetative to reproductive growth. In this study, we applied clustered regularly interspaced short palindromic repeats/(CRISPR)-associated system 9 (CRISPR/Cas9) technology to analyze AGAMOUS-like genes. We performed functional analysis of AGL19 and AGL24 genes related to bolting and flowering using CRISPR/Cas9-mediated Chinese cabbage transformation. Single-guide RNA (sgRNA) sequences were created with a low off-targeting probability to construct gene-editing vectors. Agrobacterium-mediated transformation was conducted, and tentative E₀ AGL-edited lines were analyzed using molecular biotechnological methods. Two AGL19-edited lines with nucleotide sequence mutations in the target sequence of the AGL19 genes and four AGL24-edited lines with nucleotide sequence mutations in the target sequence of the AGL24 genes showed particularly late bolting compared to the inbred line ‘CT001.’ Generational progression using bud pollination obtained T-DNA-free E₁ AGL-edited lines, which also showed late bolting. The loss of function of the AGL protein was caused by the occurrence of an indel mutation in the AGL19 and AGL24 genes, which results in an early stop codon. Furthermore, frameshift mutations led to structural changes and the introduction of an early stop codon in the AGL19 and AGL24 proteins. Our results indicate that CRISPR/Cas9-mediated editing of AGAMOUS-like genes results in a late-bolting phenotype and that CRISPR/Cas9 is a useful technology for analyzing gene function in Chinese cabbage (Brassica rapa ssp. pekinensis).

Chinese cabbage has unintended bolting in early spring due to sudden climate change. In this study, late-bolting Chinese cabbage lines were developed via mutagenesis of the BrLEAFY (BrLFY) gene, a transcription factor that determines floral identity, using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system. Double-strand break of the target region via gene editing based on nonhomologous end joining (NHEJ) was applied to acquire useful traits in plants. Based on the ‘CT001’ pseudomolecule, a single guide RNA (sgRNA) was designed and the gene-editing vector was constructed. Agrobacterium-mediated transformation was used to generate a Chinese cabbage line in which the sequence of the BrLFY paralogs was edited. In particular, single base inserted mutations occurred in the BrLFY paralogs of the LFY-7 and LFY-13 lines, and one copy of T-DNA was inserted into the intergenic region. The selected LFY-edited lines displayed continuous vegetative growth and late bolting compared to the control inbred line, ‘CT001’. Further, some LFY-edited lines showing late bolting were advanced to the next generation. The T-DNA-free E₁LFY-edited lines bolted later than the inbred line, ‘CT001’. Overall, CRISPR/Cas9-mediated mutagenesis of the BrLFY gene was found to delay the bolting time. Accordingly, CRISPR/Cas9 is considered an available method for the molecular breeding of crops.

Transgenic plants are usually produced through tissue culture, which is an essential step in Agrobacterium-mediated plant transformation. However, genomic variations, termed somaclonal variations, have been detected in transgenic plants cultured in vitro. The occurrence of these variations should be as low as possible to secure the stability of transgenic crops. Determining the cause and mechanism of somaclonal variations in tissue culture-derived plants will help reduce the rate of variation and promote the stable expression of genes in transgenic plants. In order to determine the genetic variability in transgenic Chinese cabbage plants, we performed whole-genome resequencing and compared the sequencing data with the ‘CT001’ reference genome. The variation candidates that were expected to consistently occur in the transgenic lines were selected and validated. The single nucleotide polymorphism (SNP) and insertion and deletion (InDel) candidates were identified using the resequencing data and validated by reverse transcription (RT)-PCR analysis. The deduced amino acid sequences were used to determine whether the variations caused changes in the resulting polypeptide, and the annotations of the mutated genes were analyzed to predict the possible effects of the SNPs on gene function. In conclusion, we selected and validated the genetic variations identified in transgenic Chinese cabbage plants. Their genomes were expected to be affected by the process of Agrobacterium-mediated transformation. The findings of our study will provide a genetic basis for transgenic plant research.

Transposable elements (TEs) are DNA fragments that can be replicated or transposed within a genome. TEs make up a high proportion of the plant genome and contribute to genetic diversity and evolution, affecting genome structure or gene activity. Miniature inverted-repeat transposable elements (MITEs) are short, non-autonomous class II DNA transposable elements. MITEs have specific sequences, target site duplications (TSDs), and terminal inverted repeats(TIRs), which are characteristics of the classification of MITE families. In this study, a Stowaway-like MITE, PTE-2, was activated in transgenic Chinese cabbage lines. PTE-2 was revealed by in silico analysis as the putative activated element in transgenic Chinese cabbage lines. To verify the in silico analysis data, MITE insertion polymorphism (MIP) PCR was conducted and PTE-2 was confirmed to be activated in transgenic Chinese cabbage lines. The activation tendency of the copy elements of PTE-2 at different loci was also analyzed and only one more element was activated in the transgenic Chinese cabbage lines. Analyzing the sequence of MIP PCR products, the TSD sequence and TIR motif of PTE-2 were identified and matched to the characteristics of the Stowaway-like MITE family. In addition, the flanking region of PTE-2 was modified when PTE-2 was activated.

Plant tissue culture is an in vitro technique used to manipulate cells, tissues, or organs, and plays an important role in genetic transformation. However, plants cultured in vitro often exhibit unintended genetic and epigenetic variations. Since it is important to secure the stability of endogenous and exogenous gene expressions in transgenic plants, it is preferable to avoid the occurrence of such variations. In this study, we focused on epigenetic variations, exclusively on methylation level changes of DNA, in transgenic Chinese cabbage (Brassica rapa ssp. pekinensis) plants. To detect these methylation level changes of DNA, bisulfite sequencing was performed and the obtained sequences were compared with the ‘CT001’ reference genome. Differentially methylated regions (DMRs) of DNA between the non-transgenic and transgenic lines were detected by bisulfite sequencing, and ten DMRs located in exonic regions were identified. The regions with methylation variations that were inherited and consistently maintained in the next generation lines were selected and validated. We also analyzed the relationship between methylation status and expression levels of transformant-conserved DMR (TCD) genes by quantitative reverse transcription-PCR. These results suggested that the changes in methylation levels of these DMRs might have been related to the plant transformation process, affecting subsequent gene expression. Our findings can be used in fundamental research on methylation variations in transgenic plants and suggest that these variations affect the expression of the associated genes.