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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
pPLAIIγ
genes, in which altered or partially deleted forms lead to haploid induction, were selected, and direct editing of Pak-choi
CENH3
and
pPLAIIγ
genes (
BcCENH3
and
BcpPLAIIγ
) was conducted using an
Agrobacterium
-mediated CRISPR/Cas9 system. First,
BcCENH3
and
BcpPLAIIγ
genes were characterized by analyzing the spatial expression patterns and subcellular localization. The
CENH3
expression levels in carpels and
pPLAIIγ
in various parts of Pak-choi flowers were higher than those of other parts. BcCENH3 and BcpPLAIIγ 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
BcpPLAIIγ
sgRNAs confirmed four independent
BcCENH3
-targeted transgenic lines with 2.1%, 1.8%, 1.8%, and 1.7% INDEL frequencies, and three independent
BcpPLAIIγ
-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
BcpPLAIIγ
as two haploid-inducer genes.