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DOE OSTI2025

Gene editing to enhance biotic stress tolerance in sugarcane

Sugarcane (Saccharum spp.) contributes approximately 80% of global sugar production and 40% of biofuel while serving as a promising feedstock for bioproducts (Brant et al., 2025). However, productivity faces mounting challenges from biotic threats including fungal, viral and bacterial diseases (Rott, 2018). Traditional breeding approaches are severely constrained by sugarcane’s complex polyploid genome containing 10–12 copies of "hom(oe)ologous genes within an approximately 10 Gb genome (Healey et al., 2024), extending breeding cycles to 12–15 years. Genome editing has revolutionized crop improvement by enabling targeted modifications without necessarily introducing foreign DNA, potentially circumventing regulatory hurdles while accelerating variety development (Li et al., 2022a). For sugarcane, these technologies offer unprecedented opportunities to enhance biotic stress resilience while improving yield and quality. This article examines current progress and prospects for developing biotic stress-tolerant sugarcane through gene editing, emphasizing technical advances, promising gene targets, and strategic approaches for trait stacking.

CRISPR-Cas systems↗

DOE OSTI2026

Deacetylation and Mechanical Refining Pathway for the Bioconversion of Sugarcane Bagasse

Advancing lignocellulose biorefining is imperative for the deployment of cellulosic (2G) biofuels. This work investigates the tailoring of the alkaline deacetylation and mechanical refining (DMR) pathway for the bioconversion of sugarcane bagasse. Experiments are conducted at laboratory and pilot scales, varying the pretreatment conditions (70–92 °C; 48–100 g NaOH /kg) and the mechanical refining technologies (PFI and disk refining). The pretreatments selectively solubilize acetyl groups (> 86%) and lignin (10–63%) while mostly preserving structural carbohydrates in the solid phase. Enzymatic hydrolysis generates hydrolysates of clean sugars (glucose and xylose), with sugar yields increasing up to 81% for glucose and 89% for xylose in response to delignification and mechanical refining. Biochemical methane potential assays reveal specific methane productions of up to 568 NmL CH₄ gVS⁻¹ for alkaline liquor monodigestion and 344 NmL CH₄ gVS⁻¹ for co-digestion with sugarcane vinasse from the conventional (1G) sugarcane ethanol, indicating a strong potential for bioenergy recovery from this process stream. Synergies are identified in integrating 1G ethanol, 2G DMR processing of bagasse, and anaerobic co-digestion of 1G vinasse and 2G DMR alkaline liquor. This technology enables sugarcane biorefineries to enhance the co-production of ethanol, methane, and concentrated streams of CO 2 .

09 BIOMASS FUELS↗

DOE OSTI2021

Soybean fallow and nitrification inhibitors: Strategies to reduce N 2 O emission intensities and N losses in Australian sugarcane cropping systems

The Australian sugar industry is facing mounting pressure to reduce the nitrogen (N) losses and nitrous oxide (N 2 O) emissions associated with N fertiliser use. Research has shown that N 2 O emissions from sugarcane (Saccharum officinarum L.) cropping systems can be reduced with the use of fertilisers coated with nitrification inhibitors, or by sowing legume crops in the fallows between sugarcane crop cycles. However, the efficacy of these two N management strategies across different climatic zones is still unclear, as results from field studies have been contradictory. The objectives of this study were therefore to use the DayCent model to assess the long-term effects of the two strategies (separately or in combination) on N 2 O emissions, N losses and yields. The model was parameterised using data from eight field experiments (39 treatments) conducted across four of the five main districts of the Australian sugarcane industry. A series of long-term scenarios embracing a range of N fertiliser rates (0- 160 kg N ha -1 applied as urea), climatic regions (Tropics and Subtropics), soil textures (fine- and coarse-textured soils) and N-loss risk scenarios (crop fertilised well before or close to the on-set of the wet season) were tested for both N management strategies. Simulations identified that the combined use of soybean (Glycine max L.) fallows and N fertiliser coated with the 3,4-dimethylpyrazole phosphate (DMPP) nitrification inhibitor was the most effective strategy to maintain or even increase current yields while significantly reducing N 2 O and cumulative N (mainly due to nitrate) losses. When used alone, the soybean fallow strategy on average reduced N losses over the entire crop cycle by 29 % compared with current industry best practice. However, this strategy showed a limited capacity to reduce N 2 O emissions over the entire cane crop cycle (median: -3% compared with current industry best practice), or even increased them in scenarios at high-risk of N losses in the Tropics. The use of urea coated with DMPP alone allowed current fertiliser N rates to be reduced by 30 % without any substantial yield penalty. In environments prone to high N losses, DMPP urea abated overall N losses and N 2 O emissions by 41 % and 30 %, respectively. This was the first study to use such an extensive dataset to parameterise a biogeochemical model for yields, N 2 O emissions and N losses in sugarcane cropping systems. Finally, the findings will be instrumental in guiding future research and policies designed to improve the profitability and environmental sustainability of this industry.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2024

Identification and functional analysis of strigolactone pathway genes regulating tillering traits in sugarcane

Abstract Saccharum officinarum and Saccharum spontaneum are two fundamental species of modern sugarcane cultivars, exhibiting divergent tillering patterns crucial for sugarcane architecture and yield. Strigolactones (SLs), a class of plant hormones, are considered to play a central role in shaping plant form and regulating tillering. Our study highlights the distinct tillering patterns observed between S. officinarum and S. spontaneum and implicates significant differences in SL levels in root exudates between the two species. Treatment with rac-GR24 (an artificial SL analog) suppressed tillering in S. spontaneum. Based on transcriptome analysis, we focused on two genes, TRANSCRIPTION ELONGATION FACTOR 1 (TEF1) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1), which show higher expression in S. spontaneum or S. officinarum, respectively. While the overexpression of SoCCA1 did not lead to significant phenotypic differences, overexpression of SsTEF1 in rice stimulated tillering and inhibited plant height, demonstrating its role in tillering regulation. However, the overexpression of suggests that SoCCA1 may not be the key regulator of sugarcane tillering. Yeast one-hybrid assays identified four transcription factors (TFs) regulating SsTEF1 and four and five TFs regulating SsCCA1 and SoCCA1. This study provides a theoretical foundation for deciphering the molecular mechanisms underlying the different tillering behaviors between S. officinarum and S. spontaneum, providing valuable insights for the molecular-based design of sugarcane breeding strategies.

Qi, Yiying↗

DOE OSTI2021

Data from Managing Flowering Time in Miscanthus and Sugarcane to Facilitate Intra- and Intergeneric Crosses

Miscanthus is a close relative of saccharum and a potentially valuable genetic resource for improving sugarcane. Differences in flowering time within and between miscanthus and saccharum hinders intra- and interspecific hybridizations. A series of greenhouse experiments were conducted over three years to determine how to synchronize flowering time of saccharum and miscanthus genotypes. We found that day length was an important factor influencing when miscanthus and saccharum flowered. Sugarcane could be induced to flower in a central Illinois greenhouse using supplemental lighting to reduce the rate at which days shortened during the autumn and winter to 1 min d-1, which allowed us to synchronize the flowering of some sugarcane genotypes with Miscanthus genotypes primarily from low latitudes. In a complementary growth chamber experiment, we evaluated 33 miscanthus genotypes, including 28 M. sinensis , 2 M. floridulus , and 3 M. ×giganteus collected from 20.9° S to 44.9° N for response to three day lengths (10 h, 12.5 h, and 15 h). High latitude-adapted M. sinensis flowered mainly under 15 h days, but unexpectedly, short days resulted in short, stocky plants that did not flower; in some cases, flag leaves developed under short days but heading did not occur. In contrast, for M. sinensis and M. floridulus from low latitudes, shorter day lengths typically resulted in earlier flowering, and for some low latitude genotypes, 15 h days resulted in no flowering. However, the highest ratio of reproductive shoots to total number of culms was typically observed for 12.5 h or 15 h days. Latitude of origin was significantly associated with culm length, and the shorter the days, the stronger the relationship. Nearly all entries achieved maximal culm length under the 15 h treatment, but the nearer to the equator an accession originated, the less of a difference in culm length between the short-day treatments and the 15 h day treatment. Under short days, short culms for high-latitude accessions was achieved by different physiological mechanisms for M. sinensis genetic groups from the mainland in comparison to those from Japan; for mainland accessions, the mechanism was reduced internode length, whereas for Japanese accessions the phyllochron under short days was greater than under long days. Thus, for M. sinensis , short days typically hastened floral induction, consistent with the expectations for a facultative short-day plant. However, for high latitude accessions of M. sinensis , days less than 12.5 h also signaled that plants should prepare for winter by producing many short culms with limited elongation and development; moreover, this response was also epistatic to flowering. Thus, to flower M. sinensis that originates from high latitudes synchronously with sugarcane, the former needs day lengths >12.5 h (perhaps as high as 15 h), whereas that the latter needs day lengths <12.5 h.

Feedstock Production↗

DOE OSTI2024

The extent of multiallelic, co‐editing of LIGULELESS1 in highly polyploid sugarcane tunes leaf inclination angle and enables selection of the ideotype for biomass yield

Summary Sugarcane ( Saccharum spp. hybrid) is a prime feedstock for commercial production of biofuel and table sugar. Optimizing canopy architecture for improved light capture has great potential for elevating biomass yield. LIGULELESS1 ( LG1 ) is involved in leaf ligule and auricle development in grasses. Here, we report CRISPR/Cas9‐mediated co‐mutagenesis of up to 40 copies/alleles of the putative LG1 in highly polyploid sugarcane (2 n = 100–120, x = 10–12). Next generation sequencing revealed co‐editing frequencies of 7.4%–100% of the LG1 reads in 16 of the 78 transgenic lines. LG1 mutations resulted in a tuneable leaf angle phenotype that became more upright as co‐editing frequency increased. Three lines with loss of function frequencies of ~12%, ~53% and ~95% of lg1 were selected following a randomized greenhouse trial and grown in replicated, multi‐row field plots. The co‐edited LG1 mutations were stably maintained in vegetative progenies and the extent of co‐editing remained constant in field tested lines L26 and L35. Next generation sequencing confirmed the absence of potential off targets. The leaf inclination angle corresponded to light transmission into the canopy and tiller number. Line L35 displaying loss of function in ~12% of the lg1 NGS reads exhibited an 18% increase in dry biomass yield supported by a 56% decrease in leaf inclination angle, a 31% increase in tiller number, and a 25% increase in internode number. The scalable co‐editing of LG1 in highly polyploid sugarcane allows fine‐tuning of leaf inclination angle, enabling the selection of the ideotype for biomass yield.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Variation in sugarcane biomass composition and enzymatic saccharification of leaves, internodes and roots

The composition of biomass determines its suitability for different applications within a biorefinery system. The proportion of the major biomass fractions (sugar, cellulose, hemicellulose and lignin) may vary in different sugarcane genotypes and growth environments and different parts of the plant. This study investigated the composition of mature and immature internodes, roots and mature leaves of sugarcane. Internodes were found to have a significantly larger alcohol-soluble component than leaves and roots. The primary difference between the immature and mature internodes was the ratio of soluble sugars. In mature tissues, sucrose content was significantly higher, whereas in immature internodal tissues there was lower sucrose and heightened concentrations of reducing sugars. Carbon (C) partitioning in leaf tissues was characterised by low levels of soluble components and high “other” and cell wall fractions. Root tissue had low ratios of soluble fractions relative to their cell wall contents, indicating a lack of storage of soluble carbon. There was no significant difference in the ratio of the major cell wall fractions between the major organ types. Characterisation of individual non-cellulosic monomers indicated leaf and root tissues had significantly higher arabinose and galactose fractions. Significantly larger proportions of syringyl lignin compounds and the hydroxycinnamic compound, p-coumaric acid were observed in mature internodal tissues compared to the other tissue types. Tissue-specific differences in composition were shown to greatly affect the recalcitrance of the cell wall to enzymatic saccharification. Overall, this study displayed clear evidence of the differential partitioning of C throughout the sugarcane plant in specific organs. These organ-specific differences have major implications in their utility as a bioproduct feedstock. For example, the inclusion of trash (leaves) with the culms (internodes) may alter processing efficiency.

09 BIOMASS FUELS↗

DOE OSTI2025

Data for "Evaluating the industrial potential of emerging biomass pretreatment technologies in bioethanol production and lipid recovery from transgenic sugarcane"

The selection of pretreatment methods is critical to achieving high product yields during bioconversion of lignocellulosic biomass. Hydrothermal, soaking-in-aqueous ammonia, and ionic liquid pretreatment methods are viable candidates for minimizing sugar decomposition, permitting the effective hydrolysis of structural carbohydrates, and producing a fermentable substrate suitable for achieving industrial ethanol titers and yields. In this study, the effect of these three pretreatment methods on non-modified sugarcane cultivar CP88-1762 and two transgenic lipid-accumulating sugarcane lines, oilcane 1565 and oilcane 1566, were investigated and compared in terms of lipid recovery, sugar yield, and ethanol yields within the lignocellulosic biomass conversion pipeline. Fed-batch enzymatic hydrolysis at high solid loading yielded hydrolysates capable of supporting industrial bioethanol titers across all conditions. The highest sugar yields were obtained on ammonia-pretreated biomass hydrolysate (253.73 g L−1), followed by hydrothermally pretreated hydrolysate (213.10 g L−1) and ionic liquid-pretreated hydrolysate (154.20 g L−1). Commercially viable ethanol titers of 100.62 g L−1, 64.47 g L−1, and 52.95 g L−1 were achieved from ammonia, hydrothermal, and ionic liquid pretreated hydrolysate with the corresponding ethanol productivities of 2.08 g L−1 h−1, 0.53 g L−1 h−1, and 0.36 g L−1 h−1. The lower acetic acid concentration in ammonia-pretreated hydrolysate may have enhanced its fermentability relative to the hydrothermal pretreatment condition, as indicated by the differences in ethanol titer and productivity. Lower sugar yields and ethanol productivities under the ionic liquid conditions likely resulted from the inhibitory effect of cholinium lysinate. Oilcane 1565 and oilcane 1566 bagasse accumulated over 16- and 3 times higher lipids than the non-modified sugarcane CP88-1762. The total fatty acid content in the oilcane samples was reduced in ammonia and ionic liquid-pretreated bagasse relative to the hydrothermal pretreatment condition. While all pretreatment techniques tested are industrially viable, the observed differences in titer, productivity, and lipid content indicate that careful selection and validation of upstream processing methods can contribute to improved economic and environmental outcomes.

biomass analytics↗

DOE OSTI2022

Transcriptome and metabolome integration in sugarcane through culm development

Abstract Sugarcane (Saccharum sp.) is a tropical and subtropical C4 plant with a high photosynthetic and carbon assimilation efficiency that stores sucrose. Culm biomass is also composed of bagasse fibre, a by‐product of the sugarcane industry. This high‐yielding grass, high in sucrose and lignocellulosic biomass, is considered an optimal feedstock as an alternative to fossil fuels and to produce a broad range of high‐value biomaterials. The ideal sugarcane production system would optimise the relative production of sugar and these new products. Multi‐omics correlation analysis was used to generate a global view of the essential metabolic pathways identifying critical genes involved in carbon partitioning during different stages of development. This research employed an unprecedented metabolic and transcriptomic dataset of 360 samples from a selection of 1440 culms of 24 genotypes at five different development stages. Chemical composition and metabolome analysis showed an increase through the culm development of lignin, sucrose, carbon, and amino acids such as aspartic acid, serine, alanine, methionine, threonine 3‐cyano‐L‐alanine, and citric acid. Transcriptome analysis revealed functionalities such as transcription, nucleotide transport and metabolism, and the biosynthesis of amino acids that are highly activated during the immature stage and highly down‐regulated during the most mature age.

Perlo, Virginie↗

DOE OSTI2025

Synthetic feed-forward loop circuit boosts transgene expression in sugarcane

Bio-based products derived from natural plant-derived materials offer a promising alternative to petroleum-based products, which are essential for achieving global sustainability (Gupta et al. 2022). Sugarcane provides 40% of the world’s biofuel (Brant et al. 2025). Recently, metabolic engineering of sugarcane for hyperaccumulation of biomass oil is emerging as a strategy to elevate the crop’s energy content (Cao et al. 2023; Maitra et al. 2024). Synthetic transcription factors offer a powerful tool for modulating entire metabolic pathways by enabling fine-tuned activation or repression of specific genes (Liu and Stewart 2016; Hooghvorst and Altpeter 2023). However, the application of these technologies faces challenges, including the availability of well-characterized genetic building blocks for precise manipulation of gene expression, and the inherently challenging properties of gene expression in highly polyploid crops like sugarcane (Liu and Stewart 2016).

60 APPLIED LIFE SCIENCES↗

DOE OSTI2025

Evaluating the industrial potential of emerging biomass pretreatment technologies in bioethanol production and lipid recovery from transgenic sugarcane

The selection of pretreatment methods is critical to achieving high product yields during bioconversion of lignocellulosic biomass. Hydrothermal, soaking-in-aqueous ammonia, and ionic liquid pretreatment methods are viable candidates for minimizing sugar decomposition, permitting the effective hydrolysis of structural carbohydrates, and producing a fermentable substrate suitable for achieving industrial ethanol titers and yields. In this study, the effect of these three pretreatment methods on non-modified sugarcane cultivar CP88-1762 and two transgenic lipid-accumulating sugarcane lines, oilcane 1565 and oilcane 1566, were investigated and compared in terms of lipid recovery, sugar yield, and ethanol yields within the lignocellulosic biomass conversion pipeline. Fed-batch enzymatic hydrolysis at high solid loading yielded hydrolysates capable of supporting industrial bioethanol titers across all conditions. The highest sugar yields were obtained on ammonia-pretreated biomass hydrolysate (253.73 g L −1 ), followed by hydrothermally pretreated hydrolysate (213.10 g L −1 ) and ionic liquid-pretreated hydrolysate (154.20 g L −1 ). Commercially viable ethanol titers of 100.62 g L −1 , 64.47 g L −1 , and 52.95 g L −1 were achieved from ammonia, hydrothermal, and ionic liquid pretreated hydrolysate with the corresponding ethanol productivities of 2.08 g L −1 h −1 , 0.53 g L −1 h −1 , and 0.36 g L −1 h −1 . The lower acetic acid concentration in ammonia-pretreated hydrolysate may have enhanced its fermentability relative to the hydrothermal pretreatment condition, as indicated by the differences in ethanol titer and productivity. Lower sugar yields and ethanol productivities under the ionic liquid conditions likely resulted from the inhibitory effect of cholinium lysinate. Oilcane 1565 and oilcane 1566 bagasse accumulated over 16- and 3 times higher lipids than the non-modified sugarcane CP88-1762. The total fatty acid content in the oilcane samples was reduced in ammonia and ionic liquid-pretreated bagasse relative to the hydrothermal pretreatment condition. While all pretreatment techniques tested are industrially viable, the observed differences in titer, productivity, and lipid content indicate that careful selection and validation of upstream processing methods can contribute to improved economic and environmental outcomes.

09 BIOMASS FUELS↗

DOE OSTI2022

Thermochemical Conversion of Sugarcane Bagasse: A Comprehensive Analysis of Ignition and Burnout Temperatures

The Brazilian sugarcane industry generates a significant amount of waste each year, which should be properly analyzed and studied to allow an adequate recovery and application supported by the best understanding of its properties. The present work reports the ignition and burnout temperatures of sugarcane bagasse (SCB) obtained after performing a thermal analysis using four different heating rates. The intersection method (IM) and deviation method (DM) were employed to approach the ignition and burnout temperatures of the sugarcane bagasse. The ignition temperatures of the SCB measured from IM are between 250 and 263 °C, and their burnout temperatures are between 357 and 377 °C. The ignition temperature was in the range of 205 °C to 236 °C for the DM. IM is recommended for determining the ignition and burnout temperatures. In TGA, the heating rates in the range of 10 °C·min−1 and 15 °C·min−1 are suggested due to their accuracy and the contribution to timesaving in the analysis.

Morais, Leandro C. de (ORCID:0000000314685453)↗

DOE OSTI2024

Characterization of Levan Fructan Produced by a Gluconobacter japonicus Strain Isolated from a Sugarcane Processing Facility

During raw sugarcane processing, a significant portion of lost sucrose is attributable to microbial degradation. Sucrose consumption by many bacteria is also linked to the production of exopolysaccharides (EPS) such as dextrans and fructans. These resulting EPS cause operational challenges during raw sugar manufacturing. Here, we report the characterization of EPS from a fructan-forming Gluconobacter japonicus bacterium that we previously isolated from a Louisiana sugarcane factory. The genome sequencing revealed the presence of two encoded levansucrase genes, lsrA and lsrB. One levansucrase, LsrB, was detected in the secreted protein fraction of G. japonicus LASM12 by QTOF LC-MS. The spotting assays indicated that G. japonicus produces EPS using sucrose and raffinose as substrates. The G. japonicus EPS correlated with levan fructan commercial standards by 1H-NMR, and with the characteristic carbohydrate fingerprint region for FTIR spectra, confirming that the G. japonicus EPS is levan fructan. The glycosyl composition and glycosyl linkage analysis revealed a linear β-2,6-fructofuranosyl polysaccharide with occasional (5.7%) β-2,1-fructofuranosyl branches. The gel permeation chromatography of the levan fructan EPS showed two main peaks at 4.5 kDa and 8 kDa and a very minor peak at 500 kDa. G. japonicus was identified as a producer of levan fructan. These findings will be useful for future studies aimed at evaluating the impact of levan fructans on sugar crop processing, which have been historically underestimated in industry.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

The genomic footprints of wild Saccharum species trace domestication, diversification, and modern breeding of sugarcane

Sugarcane is a major crop of unclear origins due to its complex polyploid interspecific genome. We analyzed genome ancestries using whole-genome sequence data from 390 representative accessions based on repeated k-mers and chloroplast phylogeny. The results provided evidence that Saccharum officinarum was domesticated in the New Guinea region from the S. robustum wild species and revealed that its genome is a mosaic involving different S. robustum subgroups. We discovered a wild Saccharum contributor to most modern cultivars, likely originating from East Melanesia. We highlighted two early centers of sugarcane diversification associated with human transport, one in continental Asia through hybridization with different S. spontaneum subgroups and one in the Melanesian and Polynesian islands via hybridization with the discovered ancestor and Miscanthus. Finally, we revealed the genome ancestry of modern cultivars, highlighting untapped wild Saccharum diversity as a source of alleles for breeding programs.

Garsmeur, Olivier [CIRAD, Montpellier (France). Ag↗

DOE OSTI2025

Adapting C 4 photosynthesis to atmospheric change and increasing productivity by elevating Rubisco content in sorghum and sugarcane

Meta-analyses and theory show that with rising atmospheric [CO 2 ], Rubisco has become the greatest limitation to light-saturated leaf CO 2 assimilation rates (A sat ) in C 4 crops. So would transgenically increasing Rubisco increase A sat and result in increased productivity in the field? Here, we successfully overexpressed the Rubisco small subunit (RbcS) with Rubisco accumulation factor 1 (Raf1) in both sorghum and sugarcane, resulting in significant increases in Rubisco content of 13 to 25% and up to 90% respectively. A sat increased 12 to 15% and Rubisco enzyme activity ~40% in three independent transgenic events of both species. Sorghum plants also showed increased speeds of photosynthetic induction and decreased bundle sheath leakiness. These improvements translated into average increases of 15.5% in biomass in field-grown sorghum and a 37 to 81% increase in greenhouse-grown sugarcane. This suggests a potential opportunity to achieve substantial increases in productivity of this key economically important clade of C 4 crops, future proofing their value under global atmospheric change.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Identification and analysis of stem‐specific promoters from sugarcane and energy cane for oil accumulation in their stems

Abstract Considerable recent progress has been achieved in bioengineering oil accumulation in the vegetative tissues of plants, opening an opportunity for large scale production of biodiesel, jet fuel, lubricants, and high‐value lipid bioproducts. For the highly productive C4 crops, such as sugarcane, energy cane, Miscanthus, and fiber sorghums, the bulk of the biomass is the stem. However, little success has been made in accumulating oil in the stem. Since engineering a trait with a constitutive promoter often results in pleiotropic effects that counter trait improvement, identification of stem parenchyma‐specific promoters is a prerequisite for efficient use of the ample photoassimilates stored in mature stem parenchyma cells. In this study, we first identified two TST genes encoding homologues of tonoplast sugar transporters that were strongly and almost exclusively expressed in the stems of canes via a combination of RNA‐seq atlas analysis, in silico analysis of a sugarcane genome, phylogenetic analysis, and quantitative PCR analysis. They were further confirmed in the pith parenchyma cells of the mature stem by RNA in situ hybridization. When fused with the β‐Glucuronidase (GUS) reporter gene, the promoters of two alleles, TST2b‐1A and TST2b‐1C , from one TST gene demonstrated that they could drive the GUS expression exclusively in the stem in Arabidopsis.

09 BIOMASS FUELS↗

DOE OSTI2021

Multiallelic, Targeted Mutagenesis of Magnesium Chelatase With CRISPR/Cas9 Provides a Rapidly Scorable Phenotype in Highly Polyploid Sugarcane

Genome editing with sequence-specific nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), is revolutionizing crop improvement. Developing efficient genome-editing protocols for highly polyploid crops, including sugarcane ( x = 10–13), remains challenging due to the high level of genetic redundancy in these plants. Here, we report the efficient multiallelic editing of magnesium chelatase subunit I ( MgCh ) in sugarcane. Magnesium chelatase is a key enzyme for chlorophyll biosynthesis. CRISPR/Cas9-mediated targeted co-mutagenesis of 49 copies/alleles of magnesium chelatase was confirmed via Sanger sequencing of cloned PCR amplicons. This resulted in severely reduced chlorophyll contents, which was scorable at the time of plant regeneration in the tissue culture. Heat treatment following the delivery of genome editing reagents elevated the editing frequency 2-fold and drastically promoted co-editing of multiple alleles, which proved necessary to create a phenotype that was visibly distinguishable from the wild type. Despite their yellow leaf color, the edited plants were established well in the soil and did not show noticeable growth retardation. This approach will facilitate the establishment of genome editing protocols for recalcitrant crops and support further optimization, including the evaluation of alternative RNA-guided nucleases to overcome the limitations of the protospacer adjacent motif (PAM) site or to develop novel delivery strategies for genome editing reagents.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

CRISPR/Cas9-Mediated Multi-Allelic Gene Targeting in Sugarcane Confers Herbicide Tolerance

Sugarcane is the source of 80% of the sugar and 26% of the bioethanol produced globally. However, its complex, highly polyploid genome (2 n = 100 – 120) impedes crop improvement. Here, we report efficient and reproducible gene targeting (GT) in sugarcane, enabling precise co-editing of multiple alleles via template-mediated and homology-directed repair (HDR) of DNA double strand breaks induced by the programmable nuclease CRISPR/Cas9. The evaluation of 146 independently transformed plants from five independent experiments revealed a targeted nucleotide replacement that resulted in both targeted amino acid substitutions W574L and S653I in the acetolactate synthase (ALS) in 11 lines in addition to single, targeted amino acid substitutions W574L or S653I in 25 or 18 lines, respectively. Co-editing of up to three ALS copies/alleles that confer herbicide tolerance was confirmed by Sanger sequencing of cloned long polymerase chain reaction (PCR) amplicons. This work will enable crop improvement by conversion of inferior alleles to superior alleles through targeted nucleotide substitutions.

(Saccharum hybrid complex)↗