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Summary: A major advantage of using CRISPR/Cas9 for gene editing is multiplexing, that is, the simultaneous targeting of many genes. However, primary transformants typically contain hetero‐allelic mutations or are genetic mosaic, while genetically stable lines that are homozygous are desired for functional analysis. Currently, a dedicated and labor‐intensive effort is required to obtain such higher‐order mutants through several generations of genetic crosses and genotyping.We describe the design and validation of a rapid and efficient strategy to produce lines of genetically identical plants carrying various combinations of homozygous edits, suitable for replicated analysis of phenotypical differences. This approach was achieved by combining highly multiplex gene editing in Zea mays (maize) with in vivo haploid induction and efficient in vitro generation of doubled haploid plants using embryo rescue doubling.By combining three CRISPR/Cas9 constructs that target in total 36 genes potentially involved in leaf growth, we generated an array of homozygous lines with various combinations of edits within three generations. Several genotypes show a reproducible 10% increase in leaf size, including a septuple mutant combination.We anticipate that our strategy will facilitate the study of gene families via multiplex CRISPR mutagenesis and the identification of allele combinations to improve quantitative crop traits. [ABSTRACT FROM AUTHOR]

In Zea mays, two ENOD40 homologous were identified that show only 30% of sequence homology to each other. We identified line e40-mum1 carrying a Mu transposon inserted in ZmENOD40-1, the maize gene that has the highest homology to leguminous ENOD40. The insertion causes a dramatic reduction of the ZmENOD40-1 transcript level. Irrespective of this, homozygous e40-mum1 plants are still able to interact with mycorrhizal fungi. Furthermore, no phenotypic aberrations correlated to the presence of e40-mum1 have been identified and therefore it is suggested that Z. mays ENOD40 genes are functionally redundant despite their strikingly low homology. [Copyright &y& Elsevier]

The effect of osmotic stress (−0.35 MPa) on the cell water balance and apical growth was studied non‐invasively for maize (Zea mays L., cv. LG 11) and pearl millet (Pennisetum americanum L., cv. MH 179) by 1H NMR microscopy in combination with water uptake measurements. Single parameter images of the water content and the transverse relaxation time (T2) were used to discriminate between the different tissues and to follow the water status of the apical region during osmotic stress. The T2 values of non‐stressed stem tissue turned out to be correlated to the cell dimensions as determined by optical microscopy. Growth was found to be strongly inhibited by mild stress in both species, whereas the water uptake was far less affected. During the experiment hardly any changes in water content or T2 in the stem region of maize were observed. In contrast, the apical tissue of pearl millet showed a decrease in T2 within 48 h of stress. This decrease in T2 is interpreted as an increase in the membrane permeability for water. [ABSTRACT FROM PUBLISHER]

Ensuring food security for an ever-growing global population while adapting to climate change is the main challenge for agriculture in the 21st century. Although new technologies are being applied to tackle this problem, we are approaching a plateau in crop improvement using conventional breeding. Recent advances in CRISPR/Cas9-mediated gene engineering have paved the way to accelerate plant breeding to meet this increasing demand. However, many traits are governed by multiple small-effect genes operating in complex interactive networks. Here, we present the gene discovery pipeline BREEDIT, which combines multiplex genome editing of whole gene families with crossing schemes to improve complex traits such as yield and drought tolerance. We induced gene knockouts in 48 growth-related genes into maize (Zea mays) using CRISPR/Cas9 and generated a collection of over 1,000 gene-edited plants. The edited populations displayed (on average) 5%–10% increases in leaf length and up to 20% increases in leaf width compared with the controls. For each gene family, edits in subsets of genes could be associated with enhanced traits, allowing us to reduce the gene space to be considered for trait improvement. BREEDIT could be rapidly applied to generate a diverse collection of mutants to identify promising gene modifications for later use in breeding programs. BREEDIT is a gene discovery pipeline that combines multiplex CRISPR/Cas9 genome editing of whole gene families with crossing schemes to improve complex quantitative traits. [ABSTRACT FROM AUTHOR]