Cyto-swapping in maize by haploid induction with a cenh3 mutant.

Maize mutants of the centromeric histone H3 (CENP-A/CENH3) gene can form haploids that inherit only chromosomes of the pollinating parent but the cytoplasm from the female parent. We developed CENH3 haploid inducers carrying a dominant anthocyanin colour marker for efficient haploid identification and harbouring cytoplasmic male sterile cytoplasm, a type of cytoplasm that results in male sterility useful for efficient hybrid seed production. The resulting cytoplasmic male sterility cyto-swapping method provides a faster and cheaper way to convert commercial lines to cytoplasmic male sterile compared to conventional trait introgression.

Generation of paternal haploids in wheat by genome editing of the centromeric histone CENH3.

New breeding technologies accelerate germplasm improvement and reduce the cost of goods in seed production1-3. Many such technologies could use in vivo paternal haploid induction (HI), which occurs when double fertilization precedes maternal (egg cell) genome loss. Engineering of the essential CENTROMERIC HISTONE (CENH3) gene induces paternal HI in Arabidopsis4-6. Despite conservation of CENH3 function across crops, CENH3-based HI has not been successful outside of the Arabidopsis model system7. Here we report a commercially operable paternal HI line in wheat with a ~7% HI rate, identified by screening genome-edited TaCENH3α-heteroallelic combinations. Unlike in Arabidopsis, edited alleles exhibited reduced transmission in female gametophytes, and heterozygous genotypes triggered higher HI rates than homozygous combinations. These developments might pave the way for the deployment of CENH3 HI technology in diverse crops.

Plant DNA Repair Pathways and Their Applications in Genome Engineering.

Remarkable progress in the development of technologies for sequence-specific modification of primary DNA sequences has enabled the precise engineering of crops with novel characteristics. These programmable sequence-specific modifiers include site-directed nucleases (SDNs) and base editors (BEs). Currently, these genome editing machineries can be targeted to specific chromosomal locations to induce sequence changes. However, the sequence mutation outcomes are often greatly influenced by the type of DNA damage being generated, the status of host DNA repair machinery, and the presence and structure of DNA repair donor molecule. The outcome of sequence modification from repair of DNA double-strand breaks (DSBs) is often uncontrollable, resulting in unpredictable sequence insertions or deletions of various sizes. For base editing, the precision of intended edits is much higher, but the efficiency can vary greatly depending on the type of BE used or the activity of the endogenous DNA repair systems. This article will briefly review the possible DNA repair pathways present in the plant cells commonly used for generating edited variants for genome engineering applications. We will discuss the potential use of DNA repair mechanisms for developing and improving methodologies to enhance genome engineering efficiency and to direct DNA repair processes toward the desired outcomes.