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.
Determining gene function is an essential goal for the key bioenergy crop, Sorghum bicolor (L.) Moench - particularly for genes associated with its notable abiotic stress tolerances. However, detailed molecular understanding of the genes associated with those traits is limited. This was made clear in our in-depth transcriptome studies in sorghum, which indicated nearly 50% of its transcriptome is not annotated. In this report, we describe a full spectrum of tools needed to transform sorghum in order to validate and annotate genes. Efforts began with modifying a transformation method that uses the morphogenic genes Baby Boom and Wuschel2 (Ovule Development Protein2) to accelerate transformation speed and expand amenable genotypes. In our experience, transforming RTx430 without morphogenic genes requires ~18 to 21 weeks, compared with ~10 to 12 weeks to generate T0 plants using methods with morphogenic genes. Utilizing morphogenic genes also allowed for the transformation of several sorghum genotypes not previously transformed or historically recalcitrant to transformation, i.e., rapid cycling SC187, stay-green BTx642, BTx623 and sweet sorghum Ramada. In order to validate candidate genes via engineering, while simultaneously introducing the morphogenic genes, a co-transformation strategy, termed altruistic transformation, was developed. To accomplish editing of the target gene, phytoene desaturase, novel constructs were created that also included morphogenic genes. To enable full characterization of transformed plants, we adapted techniques to determine copy number and independence of events at high-throughput levels. Through these efforts, we created a complete pathway from Agrobacterium infection to high-throughput molecular genotyping that can be used to ascertain gene function and expedite basic genetic research in this widely-grown bioenergy crop plant.
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