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A new GAL4-based feed-forward loop circuit enhances β-glucuronidase (GUS) reporter gene expression in leaves and stems of stably transformed sugarcane plants.
Datasheets relating to the article "Brachypodium SPEECHLESS2 promoter drives expression of a synthetic EPF to reduce stomatal density in sugarcane without pleiotropic effects" published in Plant Biotechnology Journal.
This project aims to study the microbial structure and potential functions of bacterial and fungal microbiomes in leaves, stems, roots, rhizospheres, and bulk soils of energy crops (oilcane) grown in greenhouses.
The expansion of sugarcane onto land currently occupied by improved (IMP) and semi-native (SN) pastures will reshape the U.S. bioenergy landscape. We combined biometric, ground-based and eddy covariance methods to investigate the impact of sugarcane expansion across subtropical Florida on the carbon (C) budget over a 3-year rotation. With 2.3- and 5.1-fold increase in productivity over IMP and SN pastures, sugarcane displayed a C use efficiency (CUE; i.e., fraction of gross C uptake allocated to plant growth) of 0.59, well above that of pastures (0.31–0.23). Sugarcane also had greater C allocation to aboveground productivity and hence, harvestable biomass relative to IMP and SN. Cane heterotrophic respiration over the 3-year rotation (903 ± 335 gC m −2 year −1 ) was 1% and 14% higher than IMP and SN pastures, respectively. These soil C losses responded largely to disturbance over the first year after conversion (1510 ± 227 gC m −2 year −1 ) but declined in subsequent years to an average 599 ± 90 gC m −2 year −1 —well below those of IMP (933 ± 140 gC m −2 year −1 ) and SN (759 ± 114 gC m −2 year −1 ) pastures—despite a significant 40%–61% increase in soil C inputs. Soil C inputs, however, shifted from root-dominated in pastures to litter-dominated in sugarcane, with only 5% C allocation to roots. Reduced decomposition rates in sugarcane were likely driven by changes in the recalcitrance and distribution rather than the size of the newly incorporated soil C pool. As a result, we observed a rapid shift in the net ecosystem C balance (NECB) of sugarcane from a large source immediately following conversion to approaching the net C losses of IMP pastures only 2 years after conversion. The environmental cost of converting pasture to sugarcane underscores the importance of implementing management practices to harness the soil C storage potential of sugarcane in advancing a sustainable bioeconomy in Southeastern United States.
ABSTRACT The expansion of sugarcane, a tropical high‐yielding feedstock, will likely reshape the Southeastern United States (SE US) bioenergy landscape. However, the sustainability of sugarcane, particularly as it displaces grazed pastures, is highly uncertain. Here, we investigated how pasture conversion to sugarcane in subtropical Florida impacts net ecosystem CO 2 exchange (NEE) and net ecosystem carbon (C) balance (NECB). Measurements were made over three full growth cycles (> 3 years) in sugarcane—plant cane, PC; first ratoon cane, FRC; second ratoon cane, SRC—and in improved (IM) and semi‐native (SN) pastures, which make up ca. 37% of agricultural land in the region. Immediately following conversion, PC was a stronger net source of CO 2 than pastures, indicating the importance of CO 2 losses related to land disturbance. Sugarcane, however, shifted to a strong net sink of CO 2 after first regrowth, and overall sugarcane was a stronger net CO 2 sink than pastures. Both stand age and low water availability during cane emergence and tillering substantially decreased its potential gross CO 2 uptake. Accounting for all C gains and removals (i.e., NECB), greater frequency of burn events and repeated harvest increased removals and overall made sugarcane a stronger C source relative to pastures despite substantial C inputs from the previous land use and a stronger CO 2 sink strength. Time since conversion substantially reduced C losses from sugarcane, and the NECB of SRC was similar to that of IM pasture but lower than that of SN pasture, indicating a rapid shift in the NECB of cane. We conclude that the C‐balance implications following conversion will depend on the proportion of IM versus SN pastures converted to sugarcane. Furthermore, our findings suggest that no‐burn harvest management strategies will be critical to the development of a sustainable bioenergy landscape in SE US.
Sugarcane ( Saccharum spp.) is an important biofuel feedstock and a leading source of global table sugar. Saccharum hybrid cultivars are highly polyploid (2n = 100–130), containing large numbers of functionally redundant hom(e)ologs in their genomes. Genome editing with sequence-specific nucleases holds tremendous promise for sugarcane breeding. However, identification of plants with the desired level of co-editing within a pool of primary transformants can be difficult. While DNA sequencing provides direct evidence of targeted mutagenesis, it is cost-prohibitive as a primary screening method in sugarcane and most other methods of identifying mutant lines have not been optimized for use in highly polyploid species. In this study, non-sequencing methods of mutant screening, including capillary electrophoresis (CE), Cas9 RNP assay, and high-resolution melt analysis (HRMA), were compared to assess their potential for CRISPR/Cas9-mediated mutant screening in sugarcane. These assays were used to analyze sugarcane lines containing mutations at one or more of six sgRNA target sites. All three methods distinguished edited lines from wild type, with co-mutation frequencies ranging from 2% to 100%. Cas9 RNP assays were able to identify mutant sugarcane lines with as low as 3.2% co-mutation frequency, and samples could be scored based on undigested band intensity. CE was highlighted as the most comprehensive assay, delivering precise information on both mutagenesis frequency and indel size to a 1 bp resolution across all six targets. This represents an economical and comprehensive alternative to sequencing-based genotyping methods which could be applied in other polyploid species.
Sugarcane (Saccharum spp.) is an important biofuel feedstock and a leading source of global table sugar. Saccharum hybrid cultivars are highly polyploid (2n = 100–130), containing large numbers of functionally redundant hom(e)ologs in their genomes. Genome editing with sequence-specific nucleases holds tremendous promise for sugarcane breeding. However, identification of plants with the desired level of co-editing within a pool of primary transformants can be difficult. While DNA sequencing provides direct evidence of targeted mutagenesis, it is cost-prohibitive as a primary screening method in sugarcane and most other methods of identifying mutant lines have not been optimized for use in highly polyploid species. In this study, non-sequencing methods of mutant screening, including capillary electrophoresis (CE), Cas9 RNP assay, and high-resolution melt analysis (HRMA), were compared to assess their potential for CRISPR/Cas9-mediated mutant screening in sugarcane. These assays were used to analyze sugarcane lines containing mutations at one or more of six sgRNA target sites. All three methods distinguished edited lines from wild type, with co-mutation frequencies ranging from 2% to 100%. Cas9 RNP assays were able to identify mutant sugarcane lines with as low as 3.2% co-mutation frequency, and samples could be scored based on undigested band intensity. CE was highlighted as the most comprehensive assay, delivering precise information on both mutagenesis frequency and indel size to a 1 bp resolution across all six targets. This represents an economical and comprehensive alternative to sequencing-based genotyping methods which could be applied in other polyploid species.
SUMMARY Sugarcane, which provides 80% of global table sugar and 40% of biofuel, presents unique breeding challenges due to its highly polyploid, heterozygous, and frequently aneuploid genome. Significant progress has been made in developing genetic resources, including the recently completed reference genome of the sugarcane cultivar R570 and pan‐genomic resources from sorghum, a closely related diploid species. Biotechnological approaches including RNA interference (RNAi), overexpression of transgenes, and gene editing technologies offer promising avenues for accelerating sugarcane improvement. These methods have successfully targeted genes involved in important traits such as sucrose accumulation, lignin biosynthesis, biomass oil accumulation, and stress response. One of the main transformation methods—biolistic gene transfer or Agrobacterium ‐mediated transformation—coupled with efficient tissue culture protocols, is typically used for implementing these biotechnology approaches. Emerging technologies show promise for overcoming current limitations. The use of morphogenic genes can help address genotype constraints and improve transformation efficiency. Tissue culture‐free technologies, such as spray‐induced gene silencing, virus‐induced gene silencing, or virus‐induced gene editing, offer potential for accelerating functional genomics studies. Additionally, novel approaches including base and prime editing, orthogonal synthetic transcription factors, and synthetic directed evolution present opportunities for enhancing sugarcane traits. These advances collectively aim to improve sugarcane's efficiency as a crop for both sugar and biofuel production. This review aims to discuss the progress made in sugarcane methodologies, with a focus on RNAi and gene editing approaches, how RNAi can be used to inform functional gene targets, and future improvements and applications.
Reliable predictions of climate change impacts on water use, irrigation requirements and yields of irrigated sugarcane in South Africa (a water-scarce country) are necessary to plan adaptation strategies. Although previous work has been done in this regard, methodologies and results vary considerably. The objectives were (1) to estimate likely impacts of climate change on sugarcane yields, water use and irrigation demand at three irrigated sugarcane production sites in South Africa (Malelane, Pongola and La Mercy) for current (1980-2010) and future (2070-2100) climate scenarios, using an approach based on the Agricultural Model Inter-comparison and Improvement Project (AgMIP) protocols; and (2) to assess the suitability of this methodology for investigating climate change impacts on sugarcane production. Future climate datasets were generated using the Delta downscaling method and three Global Circulation Models (GCMs) assuming atmospheric CO2 concentration [CO2] of 734 ppm(A2 emissions scenario). Yield and water use were simulated using the DSSAT-Canegro v4.5 model. Irrigated cane yields are expected to increase at all three sites (between 11 and 14%), primarily due to increased interception of radiation as a result of accelerated canopy development. Evapotranspiration and irrigation requirements increased by 11% due to increased canopy cover and evaporative demand. Sucrose yields are expected to decline because of increased consumption of photo-assimilate for structural growth and maintenance respiration. Crop responses in canopy development and yield formation differed markedly between the crop cycles investigated. Possible agronomic implications of these results include reduced weed control costs due to shortened periods of partial canopy, a need for improved efficiency of irrigation to counter increased demands, and adjustments to ripening and harvest practices to counter decreased cane quality and optimize productivity. Although the Delta climate data downscaling method is considered robust, accurate and easily-understood, it does not change the future number of rain-days per month. The impacts of this and other climate data simplifications ought to be explored in future work. Shortcomings of the DSSAT-Canegro model include the simulated responses of phenological development, photosynthesis and respiration processes to high temperatures, and the disconnect between simulated biomass accumulation and expansive growth. Proposed methodology refinements should improve the reliability of predicted climate change impacts on sugarcane yield.
The utilization of plants and other agricultural produce can partly offset petroleum dependency for energy requirements and can potentially provide sustainable solutions to global environmental problems. To this end, synthetic biology has shown great potential in developing transgenic bioenergy grasses such as sugarcane, sorghum, miscanthus, and energy cane that hyperaccumulate energy-rich lipid molecules in their vegetative tissues, such as leaves, stems, and roots. These perennial high-biomass transgenic C4 grasses are not targeted to grow on prime agricultural land and can be dedicatedly used to produce biofuels and other value-added bioproducts. Recently, sugarcane has been metabolically engineered to sequestrate carbon from juice towards biosynthesis of lipid molecules in the vegetative tissues. Transgenic sugarcane is referred to as “oilcane.” Transgenic sugarcane–oilcane has improved energy density due to an elevated lipid content in the vegetative tissues. Transgenic bioenergy crops cater to both cellulosic sugars and vegetative lipids, hence resulting in higher biofuel yield (biodiesel/renewable diesel and bioethanol) per unit area of cultivable land. In this book chapter, the genetic engineering of sugarcane and its bioprocessing are discussed to illustrate its development and use as an alternative feedstock for the production of biofuels (bio-jet fuel, biodiesel, and bioethanol) and value-added bioproducts.
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.
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 .
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.
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.
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.
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).
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.
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.