Cadmium (Cd) in agricultural soils readily accumulates in crop grains and shoots, posing serious risks to food and feed safety, especially in maize (Zea mays L.). Natural resistance-associated macrophage proteins (NRAMPs) constitute a key family of metal transporters; however, their role in Cd uptake in maize remains unclear. In this study, the maize gene ZmNRAMPL5 mediated Cd uptake when expressed in yeast. ZmNRAMPL5 knockout mutants, which exhibited no differences from wild type under normal conditions but showed reduced Cd accumulation, alleviated toxicity, increased tolerance under Cd stress. Pot experiments under simulated light and moderate Cd-contaminated soils further showed that knockout lines maintained silage biomass and exhibited over 60% and 80% increases in grain yield per ear compared with wild type, respectively. Moreover, Cd concentrations in stems, leaves, and grains of knockout lines were significantly reduced and remained well below China’s national safety thresholds. Thus, ZmNRAMPL5 might be used in developing Cd-tolerant, low-Cd maize germplasm for grain and feed production in Cd-contaminated farmlands.

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.