The VERNALIZATION1 (VRN1) gene is a crucial transcriptional repressor involved in triggering the transition to flowering in response to prolonged cold. To develop Chinese cabbage (Brassica rapa L. ssp. pekinensis) plants with delayed flowering time, we designed a multiplex CRISPR/Cas9 platform that allows the co-expression of four sgRNAs targeting different regions of the endogenous BrVRN1 gene delivered via a single binary vector built using the Golden Gate cloning system. DNA sequencing analysis revealed site-directed mutations at two target sites: gRNA1 and gRNA2. T₁ mutant plants with a 1-bp insertion in BrVRN1 exhibited late flowering after the vernalization. Additionally, we identified ‘transgene-free’ BrVRN1 mutant plants without any transgenic elements from the GE₁ (gene-editing 1) and GE₂ generations. All GE₂ mutant plants contained successful edits in two out of three BrVRN1 orthologs and displayed delayed flowering time. In GE₂ mutant plants, the floral repressor gene FLC1 was expressed during vernalization; but the floral integrator gene FT was not expressed after vernalization. Taken together, our data indicate that the BrVRN1 genes act as negative regulators of FLC1 expression during vernalization in Chinese cabbage, raising the possibility that the ‘transgene-free’ mutants of BrVRN1 developed in this study may serve as useful genetic resources for crop improvement with respect to flowering time regulation.

Maize seeds harvested in the field have higher vitality than those harvested in greenhouses but have higher contamination rates in terms of fungal or bacterial infection. It is important to disinfect maize seeds before sowing because seeds are a source of infection and damage crop production. In this study, we aimed to provide an efficient seed sterilization method to manage fungal or bacterial infections of field-harvested maize seeds. The optimized sterilization protocol was set up according to the disinfectant liquid immersion time, inverting RPM (rotations per minute), number of seeds, and black layer removal. We put 20 grains of maize seeds in 100% commercial bleach disinfectant containing 4–5% NaClO and performed 20 min of inversion at 45 RPM. After standing without inverting for the next 25 min in the sterile hood, inversion at 45 RPM for another 40 min was performed. By using this protocol, microorganisms occurred at a low rate with an average of 11.7%. Moreover, it was shown that microorganisms occurred at the lowest rate (average of 0.29% of seeds) when the black layer was removed. In addition, this sterilization method did not affect the growth and development of maize plants. These results revealed that black layer removal from maize seeds is a highly efficient, easy, and inexpensive sterilization method and can be used for seeds of various maize lines.

Drought stress is a serious abiotic factor limiting the quality and yield of maize (Zea mays). To produce maize plants with enhanced drought tolerance, we generated transgenic maize plants overexpressing OsPYL/RCAR7, encoding an abscisic acid receptor. We crossed the selected lines with maize variety B73 and obtained F1 hybrid seeds. Initial screening suggested that the transgenic lines were more drought tolerant than wild-type plants. Analysis using the DroughtSpotter platform indicated that expressing OsPYL/RCAR7 enhanced drought resistance in transgenic maize seedlings by reducing water loss. In addition, the stomatal conductance of the leaf surface was 30% lower in OsPYL/RCAR7-overexpressing plants than in wild-type ones. After drought treatment, OsPYL/RCAR7-overexpressing maize showed a much higher survival rate than the wild type, suggesting that expressing OsPYL/RCAR7 reduced the negative effects of drought exposure on stomatal conductance and enhanced water use efficiency. Furthermore, the expression levels of drought-tolerance–related abscisic acid–signaling genes ABP2 and RAB16A were higher in the transgenic plants than in the wild type. Taken together, our data indicate that the seedlings of transgenic maize expressing the gene OsPYL/RCAR7 showed increased tolerance to drought stress, raising the possibility that stress-related genes from monocotyledonous crops could be used as genetic resources to improve the agricultural traits of maize.