To improve the amylose content (AC) and resistant starch content (RSC) of maize kernel starch, we employed the CRISPR/Cas9 system to create mutants of starch branching enzyme I (SBEI) and starch branching enzyme IIb (SBEIIb). A frameshift mutation in SBEI (E1, a nucleotide insertion in exon 6) led to plants with higher RSC (1.07%), lower hundred-kernel weight (HKW, 24.71 ± 0.14 g), and lower plant height (PH, 218.50 ± 9.42 cm) compared to the wild type (WT). Like the WT, E1 kernel starch had irregular, polygonal shapes with sharp edges. A frameshift mutation in SBEIIb (E2, a four-nucleotide deletion in exon 8) led to higher AC (53.48%) and higher RSC (26.93%) than that for the WT. E2 kernel starch was significantly different from the WT regarding granule morphology, chain length distribution pattern, X-ray diffraction pattern, and thermal characteristics; the starch granules were more irregular in shape and comprised typical B-type crystals. Mutating SBEI and SBEIIb (E12) had a synergistic effect on RSC, HKW, PH, starch properties, and starch biosynthesis-associated gene expression. SBEIIa, SS1, SSIIa, SSIIIa, and SSIIIb were upregulated in E12 endosperm compared to WT endosperm. This study lays the foundation for rapidly improving the starch properties of elite maize lines.

Waxy maize is a specialty maize that produces mainly amylopectin starch with special food or industrial values. 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-DTM^wx. 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-DTM^wx. 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.