Corrigendum: Mining the Utricularia gibba genome for insulator-like elements for genetic engineering.

[This corrects the article DOI: 10.3389/fpls.2023.1279231.].

Identification and characterization of a temperature sensitive chlorotic soybean mutant.

Screening a transposon-mutagenized soybean population led to the discovery of a recessively inherited chlorotic phenotype. This "vir1" phenotype results in smaller stature, weaker stems, and a smaller root system with smaller nodules. Genome sequencing identified 15 candidate genes with mutations likely to result in a loss of function. Amplicon sequencing of a segregating population was then used to narrow the list to a single candidate mutation, a single-base change in Glyma.07G102300 that disrupts splicing of the second intron. Single cell transcriptomic profiling indicates that this gene is expressed primarily in mesophyll cells and RNA sequencing data indicates it is upregulated in germinating seedlings by cold stress. Previous studies have shown that mutations to Os05g34040, the rice homolog of Glyma.07G102300, produced a chlorotic phenotype that was more pronounced in cool temperatures. Growing soybean vir1 mutants at lower temperatures also resulted in a more severe phenotype. In addition, transgenic expression of wild type Glyma.07G102300 in the knockout mutant of the Arabidopsis homolog At4930720 rescues the chlorotic phenotype, further supporting the hypothesis that the mutation in Glyma.07G102300 is causal of the vir1 phenotype.

A source of resistance against yellow mosaic disease in soybeans correlates with a novel mutation in a resistance gene.

Yellow mosaic disease (YMD) is one of the major devastating constraints to soybean production in Pakistan. In the present study, we report the identification of resistant soybean germplasm and a novel mutation linked with disease susceptibility. Diverse soybean germplasm were screened to identify YMD-resistant lines under natural field conditions during 2016-2020. The severity of YMD was recorded based on symptoms and was grouped according to the disease rating scale, which ranges from 0 to 5, and named as highly resistant (HR), moderately resistant (MR), resistant (R), susceptible (S), moderately susceptible (MS), and highly susceptible (HS), respectively. A HR plant named "NBG-SG Soybean" was identified, which showed stable resistance for 5 years (2016-2020) at the experimental field of the National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan, a location that is a hot spot area for virus infection. HS soybean germplasm were also identified as NBG-47 (PI628963), NBG-117 (PI548655), SPS-C1 (PI553045), SPS-C9 (PI639187), and cv. NARC-2021. The YMD adversely affected the yield and a significant difference was found in the potential yield of NBG-SG-soybean (3.46 ± 0.13a t/ha) with HS soybean germplasm NARC-2021 (0.44 ± 0.01c t/ha) and NBG-117 (1.12 ± 0.01d t/ha), respectively. The YMD incidence was also measured each year (2016-2020) and data showed a significant difference in the percent disease incidence in the year 2016 and 2018 and a decrease after 2019 when resistant lines were planted. The resistance in NBG-SG soybean was further confirmed by testing for an already known mutation (SNP at 149th position) for YMD in the Glyma.18G025100 gene of soybean. The susceptible soybean germplasm in the field was found positive for the said mutation. Moreover, an ortholog of the CYR-1 viral resistance gene from black gram was identified in soybean as Glyma.13G194500, which has a novel deletion (28bp/90bp) in the 5`UTR of susceptible germplasm. The characterized soybean lines from this study will assist in starting soybean breeding programs for YMD resistance. This is the first study regarding screening and molecular analysis of soybean germplasm for YMD resistance.

Mining the Utricularia gibba genome for insulator-like elements for genetic engineering.

Introduction: Gene expression is often controlled via cis-regulatory elements (CREs) that modulate the production of transcripts. For multi-gene genetic engineering and synthetic biology, precise control of transcription is crucial, both to insulate the transgenes from unwanted native regulation and to prevent readthrough or cross-regulation of transgenes within a multi-gene cassette. To prevent this activity, insulator-like elements, more properly referred to as transcriptional blockers, could be inserted to separate the transgenes so that they are independently regulated. However, only a few validated insulator-like elements are available for plants, and they tend to be larger than ideal. Methods: To identify additional potential insulator-like sequences, we conducted a genome-wide analysis of Utricularia gibba (humped bladderwort), one of the smallest known plant genomes, with genes that are naturally close together. The 10 best insulator-like candidates were evaluated in vivo for insulator-like activity. Results: We identified a total of 4,656 intergenic regions with expression profiles suggesting insulator-like activity. Comparisons of these regions across 45 other plant species (representing Monocots, Asterids, and Rosids) show low levels of syntenic conservation of these regions. Genome-wide analysis of unmethylated regions (UMRs) indicates ~87% of the targeted regions are unmethylated; however, interpretation of this is complicated because U. gibba has remarkably low levels of methylation across the genome, so that large UMRs frequently extend over multiple genes and intergenic spaces. We also could not identify any conserved motifs among our selected intergenic regions or shared with existing insulator-like elements for plants. Despite this lack of conservation, however, testing of 10 selected intergenic regions for insulator-like activity found two elements on par with a previously published element (EXOB) while being significantly smaller. Discussion: Given the small number of insulator-like elements currently available for plants, our results make a significant addition to available tools. The high hit rate (2 out of 10) also implies that more useful sequences are likely present in our selected intergenic regions; additional validation work will be required to identify which will be most useful for plant genetic engineering.

Engineering custom morpho- and chemotypes of Populus for sustainable production of biofuels, bioproducts, and biomaterials.

Humans have been modifying plant traits for thousands of years, first through selection (i.e., domestication) then modern breeding, and in the last 30 years, through biotechnology. These modifications have resulted in increased yield, more efficient agronomic practices, and enhanced quality traits. Precision knowledge of gene regulation and function through high-resolution single-cell omics technologies, coupled with the ability to engineer plant genomes at the DNA sequence, chromatin accessibility, and gene expression levels, can enable engineering of complex and complementary traits at the biosystem level. Populus spp., the primary genetic model system for woody perennials, are among the fastest growing trees in temperate zones and are important for both carbon sequestration and global carbon cycling. Ample genomic and transcriptomic resources for poplar are available including emerging single-cell omics datasets. To expand use of poplar outside of valorization of woody biomass, chassis with novel morphotypes in which stem branching and tree height are modified can be fabricated thereby leading to trees with altered leaf to wood ratios. These morphotypes can then be engineered into customized chemotypes that produce high value biofuels, bioproducts, and biomaterials not only in specific organs but also in a cell-type-specific manner. For example, the recent discovery of triterpene production in poplar leaf trichomes can be exploited using cell-type specific regulatory sequences to synthesize high value terpenes such as the jet fuel precursor bisabolene specifically in the trichomes. By spatially and temporally controlling expression, not only can pools of abundant precursors be exploited but engineered molecules can be sequestered in discrete cell structures in the leaf. The structural diversity of the hemicellulose xylan is a barrier to fully utilizing lignocellulose in biomaterial production and by leveraging cell-type-specific omics data, cell wall composition can be modified in a tailored and targeted specific manner to generate poplar wood with novel chemical features that are amenable for processing or advanced manufacturing. Precision engineering poplar as a multi-purpose sustainable feedstock highlights how genome engineering can be used to re-imagine a crop species.

Editing efficiencies with Cas9 orthologs, Cas12a endonucleases, and temperature in rice.

The advent of CRISPR-Cas technology has made it the genome editing tool of choice in all kingdoms of life, including plants, which can have large, highly duplicated genomes. As a result, finding adequate target sequences that meet the specificities of a given Cas nuclease on any gene of interest remains challenging in many cases. To assess target site flexibility, we tested five different Cas9/Cas12a endonucleases (SpCas9, SaCas9, St1Cas9, Mb3Cas12a, and AsCas12a) in embryogenic rice calli from Taipei 309 at 37°C (optimal temperature for most Cas9/Cas12a proteins) and 27°C (optimal temperature for tissue culture) and measured their editing rates under regular tissue culture conditions using Illumina sequencing. StCas9 and AsCas12 were not functional as tested, regardless of the temperature used. SpCas9 was the most efficient endonuclease at either temperature, regardless of whether monoallelic or biallelic edits were considered. Mb3Cas12a at 37°C was the next most efficient endonuclease. Monoallelic edits prevailed for both SaCas9 and Mb3Cas12a at 27°C, but biallelic edits prevailed at 37°C. Overall, the use of other Cas9 orthologs, the use of Cas12a endonucleases, and the optimal temperature can expand the range of targetable sequences.

An Agrobacterium strain auxotrophic for methionine is useful for switchgrass transformation.

Auxotrophic strains of Agrobacterium tumefaciens can contribute to the development of more efficient transformation systems, especially for crops historically considered recalcitrant. Homologous recombination was used to derive methionine auxotrophs of two common A. tumefaciens strains, LBA4404 and EHA105. The EHA105 strains were more efficient for switchgrass transformation, while both the EHA105 and LBA4404 strains worked equally well for the rice control. Event quality, as measured by transgene copy number, was not affected by auxotrophy, but was higher for the LBA4404 strains than the EHA105 strains. Ultimately, the use of auxotrophs reduced bacterial overgrowth during co-cultivation and decreased the need for antibiotics.

Transgenic Soybeans Expressing Phosphatidylinositol-3-Phosphate-Binding Proteins Show Enhanced Resistance Against the Oomycete Pathogen Phytophthora sojae.

Oomycete and fungal pathogens cause billions of dollars of damage to crops worldwide annually. Therefore, there remains a need for broad-spectrum resistance genes, especially ones that target pathogens but do not interfere with colonization by beneficial microbes. Motivated by evidence suggesting that phosphatidylinositol-3-phosphate (PI3P) may be involved in the delivery of some oomycete and fungal virulence effector proteins, we created stable transgenic soybean plants that express and secrete two different PI3P-binding proteins, GmPH1 and VAM7, in an effort to interfere with effector delivery and confer resistance. Soybean plants expressing the two PI3P-binding proteins exhibited reduced infection by the oomycete pathogen Phytophthora sojae compared to control lines. Measurements of nodulation by nitrogen-fixing mutualistic bacterium Bradyrhizobium japonicum, which does not produce PI3P, revealed that the two lines with the highest levels of GmPH1 transcripts exhibited reductions in nodulation and in benefits from nodulation. Transcriptome and plant hormone measurements were made of soybean lines with the highest transcript levels of GmPH1 and VAM7, as well as controls, following P. sojae- or mock-inoculation. The results revealed increased levels of infection-associated transcripts in the transgenic lines, compared to controls, even prior to P. sojae infection, suggesting that the plants were primed for increased defense. The lines with reduced nodulation exhibited elevated levels of jasmonate-isoleucine and of transcripts of a JAR1 ortholog encoding jasmonate-isoleucine synthetase. However, lines expressing VAM7 transgenes exhibited normal nodulation and no increases in jasmonate-isoleucine. Overall, together with previously published data from cacao and from P. sojae transformants, the data suggest that secretion of PI3P-binding proteins may confer disease resistance through a variety of mechanisms.

Fast neutron mutagenesis in soybean enriches for small indels and creates frameshift mutations.

The mutagenic effects of ionizing radiation have been used for decades to create novel variants in experimental populations. Fast neutron (FN) bombardment as a mutagen has been especially widespread in plants, with extensive reports describing the induction of large structural variants, i.e., deletions, insertions, inversions, and translocations. However, the full spectrum of FN-induced mutations is poorly understood. We contrast small insertions and deletions (indels) observed in 27 soybean lines subject to FN irradiation with the standing indels identified in 107 diverse soybean lines. We use the same populations to contrast the nature and context (bases flanking a nucleotide change) of single-nucleotide variants. The accumulation of new single-nucleotide changes in FN lines is marginally higher than expected based on spontaneous mutation. In FN-treated lines and in standing variation, C→T transitions and the corresponding reverse complement G→A transitions are the most abundant and occur most frequently in a CpG local context. These data indicate that most SNPs identified in FN lines are likely derived from spontaneous de novo processes in generations following mutagenesis rather than from the FN irradiation mutagen. However, small indels in FN lines differ from standing variants. Short insertions, from 1 to 6 bp, are less abundant than in standing variation. Short deletions are more abundant and prone to induce frameshift mutations that should disrupt the structure and function of encoded proteins. These findings indicate that FN irradiation generates numerous small indels, increasing the abundance of loss-of-function mutations that impact single genes.

Metabolomics Differences of Glycine max QTLs Resistant to Soybean Looper.

Quantitative trait loci (QTLs) E and M are major soybean alleles that confer resistance to leaf-chewing insects, and are particularly effective in combination. Flavonoids and/or isoflavonoids are classes of plant secondary metabolites that previous studies agree are the causative agents of resistance of these QTLs. However, all previous studies have compared soybean genotypes that are of dissimilar genetic backgrounds, leaving it questionable what metabolites are a result of the QTL rather than the genetic background. Here, we conducted a non-targeted mass spectrometry approach without liquid chromatography to identify differences in metabolite levels among QTLs E, M, and both (EM) that were introgressed into the background of the susceptible variety Benning. Our results found that E and M mainly confer low-level, global differences in distinct sets of metabolites. The isoflavonoid daidzein was the only metabolite that demonstrated major increases, specifically in insect-treated M and EM. Interestingly, M confers increased daidzein levels in response to insect, whereas E restores M's depleted daidzein levels in the absence of insect. Since daidzein levels do not parallel levels of resistance, our data suggest a novel mechanism that the QTLs confer resistance to insects by mediating changes in hundreds of metabolites, which would be difficult for the insect to evolve tolerance. Collective global metabolite differences conferred by E and M might explain the increased resistance of EM.

Removing politics from innovations that improve food security.

Genetically modified (GM) organisms and crops have been a feature of food production for over 30 years. Despite extensive science-based risk assessment, the public and many politicians remain concerned with the genetic manipulation of crops, particularly food crops. Many governments have addressed public concern through biosafety legislation and regulatory frameworks that identify and regulate risks to ensure human health and environmental safety. These domestic regulatory frameworks align to international scientific risk assessment methodologies on a case-by-case basis. Regulatory agencies in 70 countries around the world have conducted in excess of 4400 risk assessments, all reaching the same conclusion: GM crops and foods that have been assessed provide no greater risk to human health or the environment than non-GM crops and foods. Yet, while the science regarding the safety of GM crops and food appears conclusive and societal benefits have been globally demonstrated, the use of innovative products have only contributed minimal improvements to global food security. Regrettably, politically-motivated regulatory barriers are currently being implemented with the next genomic innovation, genome editing, the implications of which are also discussed in this article. A decade of reduced global food insecurity was witnessed from 2005 to 2015, but regrettably, the figure has subsequently risen. Why is this the case? Reasons have been attributed to climate variability, biotic and abiotic stresses, lack of access to innovative technologies and political interference in decision making processes. This commentary highlights how political interference in the regulatory approval process of GM crops is adversely affecting the adoption of innovative, yield enhancing crop varieties, thereby limiting food security opportunities in food insecure economies.

Regulatory approaches for genome edited agricultural plants in select countries and jurisdictions around the world.

Genome editing in agriculture and food is leading to new, improved crops and other products. Depending on the regulatory approach taken in each country or region, commercialization of these crops and products may or may not require approval from the respective regulatory authorities. This paper describes the regulatory landscape governing genome edited agriculture and food products in a selection of countries and regions.

Two efficient CRISPR/Cas9 systems for gene editing in soybean.

Genome editing using CRISPR/Cas9 has been highlighted as a powerful tool for crop improvement. Nevertheless, its efficiency can be improved, especially for crops with a complex genome, such as soybean. In this work, using the CRISPR/Cas9 technology we evaluated two CRISPR systems, a one-component vs. a two-component strategy. In a simplified system, the single transcriptional unit (STU), SpCas9 and sgRNA are driven by only one promoter, and in the conventional system, the two-component transcriptional unit (TCTU), SpCas9, is under the control of a pol II promoter and the sgRNAs are under the control of a pol III promoter. A multiplex system with three targets was designed targeting two different genes, GmIPK1 and GmIPK2, coding for enzymes from the phytic acid synthesis pathway. Both systems were tested using the hairy root soybean methodology. Results showed gene-specific edition. For the GmIPK1 gene, edition was observed in both configurations, with a deletion of 1 to 749 base pairs; however, the TCTU showed higher indel frequencies. For GmIPK2 major exclusions were observed in both systems, but the editing efficiency was low for STU. Both systems (STU or TCTU) have been shown to be capable of promoting effective gene editing in soybean. The TCTU configuration proved to be preferable, since it was more efficient. The STU system was less efficient, but the size of the CRISPR/Cas cassette was smaller.

Effects of type I Diacylglycerol O-acyltransferase (DGAT1) genes on soybean (Glycine max L.) seed composition.

Type I Diacylglycerol acyltransferase (DGAT1) catalyzes the final step of the biosynthesis process of triacylglycerol (TAG), the major storage lipids in plant seeds, through the esterification of diacylglycerol (DAG). To characterize the function of DGAT1 genes on the accumulation of oil and other seed composition traits in soybean, transgenic lines were generated via trans-acting siRNA technology, in which three DGAT1 genes (Glyma.13G106100, Glyma.09G065300, and Glyma.17G053300) were downregulated. The simultaneous downregulation of the three isoforms in transgenic lines was found to be associated with the reduction of seed oil concentrations by up to 18 mg/g (8.3%), which was correlated with increases in seed protein concentration up to 42 mg/g (11%). Additionally, the downregulations also influenced the fatty acid compositions in the seeds of transgenic lines through increasing the level of oleic acid, up to 121 mg/g (47.3%). The results of this study illustrate the importance of DGAT1 genes in determining the seed compositions in soybean through the development of new potential technology for manipulating seed quality in soybean to meet the demands for its various food and industrial applications.

Development of mPing-based activation tags for crop insertional mutagenesis.

Modern plant breeding increasingly relies on genomic information to guide crop improvement. Although some genes are characterized, additional tools are needed to effectively identify and characterize genes associated with crop traits. To address this need, the mPing element from rice was modified to serve as an activation tag to induce expression of nearby genes. Embedding promoter sequences in mPing resulted in a decrease in overall transposition rate; however, this effect was negated by using a hyperactive version of mPing called mmPing20. Transgenic soybean events carrying mPing-based activation tags and the appropriate transposase expression cassettes showed evidence of transposition. Expression analysis of a line that contained a heritable insertion of the mmPing20F activation tag indicated that the activation tag induced overexpression of the nearby soybean genes. This represents a significant advance in gene discovery technology as activation tags have the potential to induce more phenotypes than the original mPing element, improving the overall effectiveness of the mutagenesis system.

Identification of functional single nucleotide polymorphism of Populus trichocarpa PtrEPSP-TF and determination of its transcriptional effect.

In plants, the phenylpropanoid pathway is responsible for the synthesis of a diverse array of secondary metabolites that include lignin monomers, flavonoids, and coumarins, many of which are essential for plant structure, biomass recalcitrance, stress defense, and nutritional quality. Our previous studies have demonstrated that Populus trichocarpa PtrEPSP-TF, an isoform of 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, has transcriptional activity and regulates phenylpropanoid biosynthesis in Populus. In this study, we report the identification of single nucleotide polymorphism (SNP) of PtrEPSP-TF that defines its functionality. Populus natural variants carrying this SNP were shown to have reduced lignin content. Here, we demonstrated that the SNP-induced substitution of 142nd amino acid (PtrEPSP-TFD142E) dramatically impairs the DNA-binding and transcriptional activity of PtrEPSP-TF. When introduced to a monocot species rice (Oryza sativa) in which an EPSP synthase isoform with the DNA-binding helix-turn-helix (HTH) motif is absent, the PtrEPSP-TF, but not PtrEPSP-TFD142E, activated genes in the phenylpropanoid pathway. More importantly, heterologous expression of PtrEPSP-TF uncovered five new transcriptional regulators of phenylpropanoid biosynthesis in rice. Collectively, this study identifies the key amino acid required for PtrEPSP-TF functionality and provides a strategy to uncover new transcriptional regulators in phenylpropanoid biosynthesis.

Functional analysis and development of a CRISPR/Cas9 allelic series for a CPR5 ortholog necessary for proper growth of soybean trichomes.

Developments in genomic and genome editing technologies have facilitated the mapping, cloning, and validation of genetic variants underlying trait variation. This study combined bulked-segregant analysis, array comparative genomic hybridization, and CRISPR/Cas9 methodologies to identify a CPR5 ortholog essential for proper trichome growth in soybean (Glycine max). A fast neutron mutant line exhibited short trichomes with smaller trichome nuclei compared to its parent line. A fast neutron-induced deletion was identified within an interval on chromosome 6 that co-segregated with the trichome phenotype. The deletion encompassed six gene models including an ortholog of Arabidopsis thaliana CPR5. CRISPR/Cas9 was used to mutate the CPR5 ortholog, resulting in five plants harboring a total of four different putative knockout alleles and two in-frame alleles. Phenotypic analysis of the mutants validated the candidate gene, and included intermediate phenotypes that co-segregated with the in-frame alleles. These findings demonstrate that the CPR5 ortholog is essential for proper growth and development of soybean trichomes, similar to observations in A. thaliana. Furthermore, this work demonstrates the value of using CRISPR/Cas9 to generate an allelic series and intermediate phenotypes for functional analysis of candidate genes and/or the development of novel traits.

Genome-Wide Reinforcement of DNA Methylation Occurs during Somatic Embryogenesis in Soybean.

Somatic embryogenesis is an important tissue culture technique that sometimes leads to phenotypic variation via genetic and/or epigenetic changes. To understand the genomic and epigenomic impacts of somatic embryogenesis, we characterized soybean (Glycine max) epigenomes sampled from embryos at 10 different stages ranging from 6 weeks to 13 years of continuous culture. We identified genome-wide increases in DNA methylation from cultured samples, especially at CHH sites. The hypermethylation almost exclusively occurred in regions previously possessing non-CG methylation and was accompanied by increases in the expression of genes encoding the RNA-directed DNA methylation (RdDM) machinery. The epigenomic changes were similar between somatic and zygotic embryogenesis. Following the initial global wave of hypermethylation, rare decay events of maintenance methylation were observed, and the extent of the decay increased with time in culture. These losses in DNA methylation were accompanied by downregulation of genes encoding the RdDM machinery and transcriptome reprogramming reminiscent of transcriptomes during late-stage seed development. These results reveal a process for reinforcing already silenced regions to maintain genome integrity during somatic embryogenesis over the short term, which eventually decays at certain loci over longer time scales.

Genome-Scale Sequence Disruption Following Biolistic Transformation in Rice and Maize.

Biolistic transformation delivers nucleic acids into plant cells by bombarding the cells with microprojectiles, which are micron-scale, typically gold particles. Despite the wide use of this technique, little is known about its effect on the cell's genome. We biolistically transformed linear 48-kb phage lambda and two different circular plasmids into rice (Oryza sativa) and maize (Zea mays) and analyzed the results by whole genome sequencing and optical mapping. Although some transgenic events showed simple insertions, others showed extreme genome damage in the form of chromosome truncations, large deletions, partial trisomy, and evidence of chromothripsis and breakage-fusion bridge cycling. Several transgenic events contained megabase-scale arrays of introduced DNA mixed with genomic fragments assembled by nonhomologous or microhomology-mediated joining. Damaged regions of the genome, assayed by the presence of small fragments displaced elsewhere, were often repaired without a trace, presumably by homology-dependent repair (HDR). The results suggest a model whereby successful biolistic transformation relies on a combination of end joining to insert foreign DNA and HDR to repair collateral damage caused by the microprojectiles. The differing levels of genome damage observed among transgenic events may reflect the stage of the cell cycle and the availability of templates for HDR.

Embryogenic cell suspensions for high-capacity genetic transformation and regeneration of switchgrass (Panicum virgatum L.).

BACKGROUND: Switchgrass (Panicum virgatum L.), a North American prairie grassland species, is a potential lignocellulosic biofuel feedstock owing to its wide adaptability and biomass production. Production and genetic manipulation of switchgrass should be useful to improve its biomass composition and production for bioenergy applications. The goal of this project was to develop a high-throughput stable switchgrass transformation method using Agrobacterium tumefaciens with subsequent plant regeneration. RESULTS: Regenerable embryogenic cell suspension cultures were established from friable type II callus-derived inflorescences using two genotypes selected from the synthetic switchgrass variety 'Performer' tissue culture lines 32 and 605. The cell suspension cultures were composed of a heterogeneous fine mixture culture of single cells and aggregates. Agrobacterium tumefaciens strain GV3101 was optimum to transfer into cells the pANIC-10A vector with a hygromycin-selectable marker gene and a pporRFP orange fluorescent protein marker gene at an 85% transformation efficiency. Liquid cultures gave rise to embryogenic callus and then shoots, of which up to 94% formed roots. The resulting transgenic plants were phenotypically indistinguishable from the non-transgenic parent lines. CONCLUSION: The new cell suspension-based protocol enables high-throughput Agrobacterium-mediated transformation and regeneration of switchgrass in which plants are recovered within 6-7 months from culture establishment.