Morphogene‐assisted transformation of Sorghum bicolor allows more efficient genome editing

Abstract

Sorghum bicolor (L.) Moench, the fifth most important cereal worldwide, is a multi-use crop for feed, food, forage and fuel. To enhance the sorghum and other important crop plants, establishing gene function is essential for their improvement. For sorghum, identifying genes associated with its notable abiotic stress tolerances requires a detailed molecular understanding of the genes associated with those traits. The limits of this knowledge became evident from our earlier in-depth sorghum transcriptome study showing that over 40% of its transcriptome had not been annotated. Here, we describe a full spectrum of tools to engineer, edit, annotate and characterize sorghum's genes. Efforts to develop those tools began with a morphogene-assisted transformation (MAT) method that led to accelerated transformation times, nearly half the time required with classical callus-based, non-MAT approaches. These efforts also led to expanded numbers of amenable genotypes, including several not previously transformed or historically recalcitrant. Another transformation advance, termed altruistic, involved introducing a gene of interest in a separate Agrobacterium strain from the one with morphogenes, leading to plants with the gene of interest but without morphogenes. The MAT approach was also successfully used to edit a target exemplary gene, phytoene desaturase. To identify single-copy transformed plants, we adapted a high-throughput technique and also developed a novel method to determine transgene independent integration. These efforts led to an efficient method to determine gene function, expediting research in numerous genotypes of this widely grown, multi-use crop.

Optimized Transformation and Gene Editing of the B104 Public Maize Inbred by Improved Tissue Culture and Use of Morphogenic Regulators

“…Alternative approaches have been reported to exclude MR expression late in development. These approaches rely on transient expression of MRs without integration in combination with selection of the T-DNA of interest (Hoerster et al, 2020;Aregawi et al, 2021;Nelson-Vasilchik et al, 2022).…”

Comparing in planta accumulation with microbial routes to set targets for a cost-competitive bioeconomy

“…We assume the selected bioproducts can be accumulated in engineered biomass sorghum at a range of mass fractions (dwt%). Biomass sorghum was selected as a representative bioenergy crop because of its high biomass yield, its natural drought tolerance and water-use efficiency, and the relative ease with which sorghum can be engineered when compared to other bioenergy crops such as Miscanthus (37)(38)(39)(40). In our simulated biorefineries, the desired bioproduct accumulated in the engineered biomass sorghum is extracted up front (before downstream deconstruction and bioconversion).…”

Leaf transformation for efficient random integration and targeted genome modification in maize and sorghum

“…https://doi.org/10.1038/s41477-022-01338-0 was further extended to sorghum transformation 10,12 , sorghum genome editing 37 , maize public inbred transformation 14,15,38 and maize genome editing 15,35,36,39 . Recently, Wox5 has also been demonstrated to improve transformation in numerous wheat varieties, with potential application in other cereals such as Triticum monococcum, triticale, rye, barley and maize 19 .…”