The objective of this study was to overcome the limitations of wx mutant allele acquisition and breeding efficiency by conversion of parental lines from normal to waxy maize. The intended mutation activity was achieved by in vivo CRISPR/Cas9 machinery involving desired-target mutation of the Wx locus in the ZC01 background, abbreviated as ZC01-DTMwx. Triple selection was applied to segregants to obtain high genome background recovery with transgene-free wx mutations. The targeted mutation was identified, yielding six types of mutations among progeny crossed with ZC01-DTMwx. The amylopectin contents of the endosperm starch in mutant lines and hybrids averaged 94.9%, while those of the wild-type controls were significantly (P < 0.01) lower, with an average of 76.9%. Double selection in transgene-free lines was applied using the Bar strip test and Cas9 PCR screening. The genome background recovery ratios of the lines were determined using genome-wide SNP data. That of lines used as male parents was as high as 98.19% and that of lines used as female parents was as high as 86.78%. Conversion hybrids and both parental lines showed agronomic performance similar to that of their wild-type counterparts. This study provides a practical example of the efficient extension of CRISPR/Cas9 targeted mutation to industrial hybrids for transformation of a recalcitrant species.
Figure 2: Generation of targeted ZmWX mutations using in vivo DTM activity among BC2F2 progeny of two parental lines, 35Mu and 35Fu. Nucleotides in red and underlined lettering indicate the protospacer-motif (PAM); Nucleotides in bold blue lettering indicate the spacer region; Nucleotides or dashes in bold red lettering indicate created mutations.