Chemical agents such as trichostatin A (TSA) can assist in optimization of doubled haploidy for rapid improvements in wheat germplasm and addressing recalcitrance issues in cell culture responses. In wheat, plant regeneration through microspore culture is an integral part of doubled haploid (DH) production. However, low response to tissue culture and genotype specificity are two major constraints in the broad deployment of this breeding tool. Recently, the structure of chromatin was shown to be linked with cell transitions during tissue culture. Specifically, repression of genes that are required for cell morphogenesis, through acetylation of histones, may play an important role in this process. Reduction of histone acetylation by chemical inhibition may increase tissue culture efficiency. Here, the role of trichostatin A (TSA) in inducing microspore-derived embryos was investigated in wheat. The optimal dose of TSA was determined for wheat cultivars and subsequently validated in F hybrids. A significant increase in the efficiency of DH production was observed in both cultivated varieties and F hybrids. Thus, the inclusion of TSA in DH protocols for wheat breeding programs is advocated.

Fusarium head blight (FHB) is a devastating fungal disease of small-grain cereals that results in severe yield and quality losses. FHB resistance is controlled by resistance components including incidence, field severity, visual rating index, Fusarium damaged kernels (FDKs), and the accumulation of the mycotoxin deoxynivalenol (DON). Resistance conferred by each of these components is partial and must be combined to achieve resistance sufficient to protect wheat from yield losses. In this study, two biparental mapping populations were analyzed in Canadian FHB nurseries and quantitative trait loci (QTL) mapped for the traits listed above. Nine genomic loci, on 2AS, 2BS, 3BS, 4AS, 4AL, 4BS, 5AS, 5AL, and 5BL, were enriched for the majority of the QTL controlling FHB resistance. The previously validated FHB resistance QTL on 3BS and 5AS affected resistance to severity, FDK, and DON in these populations. The remaining seven genomic loci colocalize with flowering time and/or plant height QTL. The QTL on 4B was a major contributor to all field resistance traits and plant height in the field. QTL on 4AL showed contrasting effects for FHB resistance between Eastern and Western Canada, indicating a local adapted resistance to FHB. In addition, we also found that the 2AS QTL contributed a major effect for DON, and the 2BS for FDK, while the 5AL conferred mainly effect for both FDK/DON. Results presented here provide insight into the genetic architecture underlying these resistant components and insight into how FHB resistance in wheat is controlled by a complex network of interactions between genes controlling flowering time, plant height, local adaption, and FHB resistance components.

Summary Recent advances in genome engineering technologies based on designed endonucleases (DE) allow specific and predictable alterations in plant genomes to generate value‐added traits in crops of choice. The EXZACT Precision technology, based on zinc finger nucleases (ZFN), has been successfully used in the past for introduction of precise mutations and transgenes to generate novel and desired phenotypes in several crop species. Current methods for delivering ZFNs into plant cells are based on traditional genetic transformation methods that result in stable integration of the nuclease in the genome. Here, we describe for the first time, an alternative ZFN delivery method where plant cells are transfected with ZFN protein that eliminates the need for stable nuclease genomic integration and allows generation of edited, but not transgenic cells or tissues. For this study, we designed ZFNs targeting the wheat IPK1 locus, purified active ZFN protein from bacterial cultures, complexed with cell‐penetrating peptides (CPP) and directly transfected the complex into either wheat microspores or embryos. NGS analysis of ZFN‐treated material showed targeted edits at the IPK1 locus in independent experiments. This is the first description of plant microspore genome editing by a ZFN when delivered as a protein complexed with CPP.

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