%0 Journal Article
%T Efficient gene targeting in soybean using Ochrobactrum haywardense-mediated delivery of a marker-free donor template.
%A Kumar S
%A Liu ZB
%A Sanyour-Doyel N
%A Lenderts B
%A Worden A
%A Anand A
%A Cho HJ
%A Bolar J
%A Harris C
%A Huang L
%A Xing A
%A Richardson A
%J Plant Physiol
%V 189
%N 2
%D 06 2022 1
%M 35191500
%F 8.005
%R 10.1093/plphys/kiac075
%X Gene targeting (GT) for precise gene insertion or swap into pre-defined genomic location has been a bottleneck for expedited soybean precision breeding. We report a robust selectable marker-free GT system in soybean, one of the most economically important crops. An efficient Oh H1-8 (Ochrobactrum haywardense H1-8)-mediated embryonic axis transformation method was used for the delivery of CRISPR-Cas9 components and donor template to regenerate T0 plants 6-8 weeks after transformation. This approach generated up to 3.4% targeted insertion of the donor sequence into the target locus in T0 plants, with ∼ 90% mutation rate observed at the genomic target site. The GT was demonstrated in two genomic sites using two different donor DNA templates without the need for a selectable marker within the template. High-resolution Southern-by-Sequencing analysis identified T1 plants with precise targeted insertion and without unintended plasmid DNA. Unlike previous low-frequency GT reports in soybean that involved particle bombardment-mediated delivery and extensive selection, the method described here is fast, efficient, reproducible, does not require a selectable marker within the donor DNA, and generates nonchimeric plants with heritable GT.