Establishment of an Agrobacterium-mediated genetic transformation and CRISPR/Cas9-mediated mutagenesis of haploid inducer genes in Pak-choi plants (Brassica rapa ssp. chinensis)

Pak-choi (Brassica rapa ssp. chinensis) is a popular vegetative crop in southern China, East Asia, and Southeast Asia. Owing to the threat of climate change, rapid breeding strategies for vegetable cultivars that are tolerant to abiotic and biotic stresses are required. Thus, the rapid fixation of useful agronomic traits using doubled haploid technology is urgent. The haploid-inducer gene is key to doubled haploidization. Two known CENH3 and pPLAII gamma genes, in which altered or partially deleted forms lead to haploid induction, were selected, and direct editing of Pak-choi CENH3 and pPLAII gamma genes (BcCENH3 and BcpPLAII gamma) was conducted using an Agrobacterium-mediated CRISPR/Cas9 system. First, BcCENH3 and BcpPLAII gamma genes were characterized by analyzing the spatial expression patterns and subcellular localization. The CENH3 expression levels in carpels and pPLAII gamma in various parts of Pak-choi flowers were higher than those of other parts. BcCENH3 and BcpPLAII gamma proteins targeted in the nucleus and plasma membrane, respectively. Whole plants were successfully regenerated from the shoot apical meristem (SAM) regions of Pak-choi seedlings using the optimized procedure and culture conditions. The regeneration results of SAM explants after Agrobacterium-mediated transformation of constructs expressing CRISPR/Cas9 and BcCENH3 or BcpPLAII gamma sgRNAs confirmed four independent BcCENH3-targeted transgenic lines with 2.1%, 1.8%, 1.8%, and 1.7% INDEL frequencies, and three independent BcpPLAII gamma-targeted transgenic lines with 24.5%, 33.7%, and 33.0% INDEL frequencies. Thus, our results suggested the possibility of developing transgenic Pak-choi lines by applying the CRISPR/Cas9 genome editing technology to BcCENH3 and BcpPLAII gamma as two haploid-inducer genes.