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

Summary 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.


Figure S2
Plants from (a) RTx430 and (b) BTx642 transformed with pPHP81814. In both, plants null for Zm-Bbm and Wus2 (right) and plants retaining introduced Zm-Bbm and Wus2 (left) have shorter stature, twisted leaves and poorer seed set, compared to wild-type.

Figure S3
Schematic representation of the molecular components of constructs. (a) pPHP83911, used for generating pGL193 and pGL198, is a pENTR vector. (b) pPHP85425 is a destination vector for morphogene-assisted transformation (MAT). Molecular components between left and right borders are shown. (c) pGL193 is a pENTR vector for MAT-mediated editing with gRNAs. The remaining backbone part is from pPHP83911. (d) pGL198 is a pENTR vector for MAT-mediated editing with the maize ubiquitin promoter and maize codon-optimized SpCas9 for carrying gRNAs. The remaining backbone part is also from pPHP83911.

Figure S4
Two gRNAs targeting pds in pGL196; yellow rectangles indicate exons; light gray lines indicate intron regions. gRNA1.1 is located in the third exon of pds genes. gRNA1.3 is located in the fifth exon of pds genes.

Figure S5
Molecular cloning strategy for constructing sorghum CRISPR/Cas9 vectors. Cloning vector for gRNAs containing BsaI sites for sequential insertion of two or three gRNAs with tRNAs that forms polycistronic cassettes (gRNA-tRNA), driven by rice U3 promoter (Os-U3pro). The gRNA-tRNA and Cas9 expression cassettes were mobilized into pPHP85425 through Gateway recombination, generating pGL196 and pGL199 for Agrobacterium-mediated sorghum transformation.

Figure S6
Determination of transgene independent integration (TII) with adapter ligation-mediated PCR. Genomic DNA with a T-DNA insert (i) was digested with EcoRI and HindIII (ii). Adapters are ligated to digested sites creating adapter-flanked templates (iii). Only the longer arm of the adapter contains a sequence exactly matching the adapter primers. Adapter-to-adapter amplification does not occur due to lack of primer match sites on the shorter arm of adapters. If T-DNA is present in the template (black line, Fig. 4a.i), T-DNA primers (yellow arrow, left border; dark blue arrow, right border, Fig. 4 a.iii) will bind to their corresponding sites and initiate synthesis of a complementary strand (iv). PCR products will contain adapter primer-binding sites derived from the complement of the longer arm of the adapter.  MS salts plus vitamins, 4.43 g/L, thiamine-HCl 1mg/l, 2,4-D 1.5 mg/l, sucrose 68.5 g/l, glucose 36 g/l, acetosyringone 1 39.24 mg/l, pH 5.2.