Scientists in Australia have developed a robust and reproducible genetic transformation system for Brassica carinata, an ancient East African oilseed that is rapidly emerging as one of the most promising non-food feedstocks for sustainable aviation fuel and renewable diesel. The new protocol, described in the journal Plant Molecular Biology, delivers stable transformation efficiencies of at least 5 percent across four genetically diverse genotypes, with the best-performing line reaching 12 percent, a substantial advance for a crop whose genetic improvement has long been hampered by genotype-specific, unreliable transformation methods.
Brassica carinata, commonly known as Ethiopian or Abyssinian mustard, is one of the oldest cultivated leafy vegetables and oilseed crops, originating in East Africa, particularly Ethiopia. In recent years it has attracted attention as a second-generation biofuel crop because its seed oil, rich in erucic and linolenic acids, can be converted directly into drop-in fuels that are compatible with existing engines and fuel infrastructure. Unlike conventional oilseed crops, carinata shows strong abiotic stress tolerance, supported by a deep taproot system, high water-use efficiency and effective nutrient uptake, which allows it to maintain stable biomass production in water-limited environments. When grown as a winter cover crop in double-cropping systems it can support soil health without compromising food yields, and its extensive root architecture contributes to soil carbon sequestration. The protein-rich meal remaining after oil extraction serves as a high-value livestock feed, while the oil has industrial applications ranging from biolubricants to bioplastics and biopesticides, giving the crop a broad bioeconomic footprint.
Despite this promise, the authors argue that B. carinata requires extensive genetic improvement of many agronomic traits before it can be widely deployed in agricultural systems, and that rapid improvement through gene editing or genetic modification demands transformation protocols that work reliably across a broad genetic base. Until now, transformation reports for the species have been scarce and confined to a narrow range of genotypes, mostly Canadian-bred lines such as C90-1089 and C90-1163. Earlier studies demonstrated that efficiency depends heavily on the explant type and the selection agent used: kanamycin selection yielded transformation rates of 22 to 50 percent, but switching to L-phosphinothricin collapsed efficiency to as little as 1 to 2 percent. A simplified floral dip approach achieved only about 1.5 percent efficiency, and a comprehensive regeneration screen of 51 accessions revealed wide variation in shoot production among genotypes. Protocols that work for one genotype often fail entirely for another, a problem well documented in other crops such as maize, leaving the species without a dependable platform for functional genomics or trait engineering.
To break through this bottleneck, a team led by Reshma Roy of the ARC Training Centre for Future Crop Development at the Australian National University, together with Srinivas Belide of CSIRO Agriculture and Food and collaborators at Nufarm, evaluated cotyledonary petioles from five diverse B. carinata genotypes: DHN1 and DHN2 obtained from Nufarm, ATC 94023 and ATC 94024 from the Australian Grains GeneBank, and line 52648 provided by the NSW Department of Primary Industries. Cotyledonary petioles excised from five-day-old sterile seedlings were chosen because they have historically performed best among explants in Brassica transformation. The researchers tested callus induction media supplemented with either 6-benzylaminopurine (BAP) or thidiazuron (TDZ) at 0.5 or 1.0 mg per litre, in combination with the antioxidants L-cysteine, ascorbic acid and glutathione, followed by sequential culture on shoot induction, shoot outgrowth and shoot elongation media before rooting and transfer to soil.
Regeneration efficiency varied substantially across the five genotypes and four cytokinin treatments. The genotype AGG2 proved the strongest regenerator, producing an average of 19.6 plus or minus 5.3 shoots per explant under 1.0 mg/L TDZ, with every explant producing shoots, while BAP treatments yielded around 12 shoots per petiole. DHN1 also regenerated vigorously, with 0.5 mg/L TDZ producing 12.5 shoots per explant. By contrast, DW3 and DHN2 produced fewer than five shoots per explant across all treatments; DW3 callused at nearly 100 percent but converted that callus into only 2.1 to 4.2 shoots, indicating that organogenesis initiation and shoot formation are separable steps. Tukey HSD post-hoc testing confirmed that TDZ treatments significantly outperformed BAP in AGG2, with pairwise comparisons showing p values as low as less than 0.001. The team notes that near-complete callus response and markedly higher shoot numbers than previous reports, which peaked at roughly six shoots per callus, likely reflect the inclusion of antioxidants, precise subculturing timing and optimized cytokinin choice.
A critical preliminary test used the binary vector p1305.1, derived from the pCAMBIA series, which carries a GUSPlus reporter gene containing a catalase intron driven by the CaMV 35S promoter. The intron prevents expression in Agrobacterium itself, restricting GUS activity to eukaryotic plant cells and ensuring that blue histochemical staining after X-Gluc infiltration reflects genuine plant-derived T-DNA expression rather than bacterial background. Transient GUS expression confirmed high transformability of the cotyledonary petioles in all four genotypes tested, with expression frequencies ranging from 96.4 to 100 percent, demonstrating that the Agrobacterium infection and T-DNA transfer steps were uniformly effective regardless of genotype.
For stable transformation the researchers used the binary vector pBSV-004 carrying the phosphinothricin acetyltransferase (PAT) gene, which confers resistance to glufosinate ammonium, deliberately choosing L-PPT selection because earlier work suggested it imposes a stronger, more discriminating pressure than kanamycin. Agrobacterium strain AGL1 cultures were grown to OD 600 of 0.45 in MS medium with 2 percent glucose, induced with 100 micromolar acetosyringone and 1 millimolar spermidine to activate vir gene functions, and used to infect freshly isolated petioles for five to six minutes before a two-day dark co-cultivation. Infected explants then passed through pre-selection, callus induction, shoot induction, shoot outgrowth and elongation stages on media containing 5 mg/L phosphinothricin, on which non-transformed wild-type controls bleached and shrivelled while transgenic tissue remained green.
The stable transformation results revealed a striking and biologically important finding: regeneration capacity did not predict transformation success. DHN1, a strong regenerator, achieved the highest efficiency at 12 percent, but DW3, which regenerated poorly, reached a respectable 9 percent, whereas AGG2, the best regenerator of all, transformed at only 5 percent. AGG1 initially transformed at just 1 percent, a failure traced to hyperhydration of the callus tissue. Drawing on prior experience in safflower and eucalyptus protocols, the team added iota-carrageenan and calcium chloride dihydrate to the callus induction medium for AGG1, which suppressed hyperhydration without harming regeneration and lifted that genotype’s efficiency to 5 percent. The authors conclude that genotype-specific factors governing T-DNA delivery, integration and survival under selection, rather than shoot production per se, are the principal determinants of transformation outcome, echoing earlier observations in canola where high regeneration is a permissive prerequisite but not a predictor of stable events.
Molecular validation underpinned the entire pipeline. All regenerated shoots were screened by PCR for the PAT gene, and transgene copy number in 28 independent T0 lines across the four genotypes was quantified by digital droplet PCR using the single-copy high-mobility group (HMG) gene as an endogenous reference. Copy numbers ranged from one to eight, with most plants carrying multiple insertions, a pattern typical of Agrobacterium-mediated transformation. Functional confirmation came from glasshouse spray tests in which T1 seedlings from three genotypes with differing copy numbers were treated twice with 0.05 percent glufosinate ammonium. Transgenic seedlings remained green and continued growing, while non-transformed wild-type controls developed chlorosis and died, demonstrating stable, heritable PAT-mediated herbicide resistance across generations. All PCR-positive shoots rooted successfully with no losses at the rooting stage, and plants were grown to maturity in the glasshouse.
The team believes this is the first study to systematically evaluate regeneration efficiency, transformation competence, molecular validation and trait expression across multiple B. carinata genotypes, and the resulting platform is expected to directly accelerate the engineering of climate-resilience traits such as drought, frost and salinity tolerance, along with herbicide resistance and improved oil content and composition. Because the protocol works with L-PPT selection, a marker compatible with many gene-editing and stacking strategies, and because it is validated across genotypes representing a broad genetic background, it removes a longstanding constraint on carinata breeding programs. As demand grows for low-carbon-intensity aviation fuel, tools that allow rapid trait insertion into this hardy, non-food oilseed could prove decisive in scaling its cultivation, and the researchers suggest the system now provides the framework needed for functional genomics and targeted trait improvement in a crop poised to move from the margins of agriculture to the centre of the renewable fuels transition.
Subject of Research: Development of an efficient and genotype-flexible Agrobacterium-mediated transformation and regeneration system for the second-generation biofuel crop Brassica carinata
Subject of Research: Biology
Article Title: Development of efficient Agrobacterium-mediated transformation of a second-generation biofuel crop, Brassica carinata
Article References: Roy, R., Kassety, S., Gororo, N., Bennett, R., Millar, A. A., & Belide, S. (2026). Development of efficient Agrobacterium-mediated transformation of a second-generation biofuel crop, Brassica carinata. Plant Molecular Biology, 116(3), Article 51. https://doi.org/10.1007/s11103-026-01717-7
Image Credits: AI Generated
DOI: 10.1007/s11103-026-01717-7
Keywords: Brassica carinata, Agrobacterium-mediated transformation, cotyledonary petioles, shoot regeneration, cytokinin, sustainable aviation fuel, glufosinate ammonium resistance, PAT gene, digital droplet PCR, transgene copy number, plant molecular farming, genotype-flexible transformation
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Alan Morgan. (September 11, 2026). Efficient Agrobacterium transformation developed for biofuel crop Brassica carinata. Scienmag. https://scienmag.com/efficient-agrobacterium-transformation-developed-for-biofuel-crop-brassica-carinata/
Alan Morgan. “Efficient Agrobacterium transformation developed for biofuel crop Brassica carinata.” Scienmag, 11 September 2026, https://scienmag.com/efficient-agrobacterium-transformation-developed-for-biofuel-crop-brassica-carinata/. Accessed 11 September 2026.
Alan Morgan. “Efficient Agrobacterium transformation developed for biofuel crop Brassica carinata.” Scienmag. September 11, 2026. https://scienmag.com/efficient-agrobacterium-transformation-developed-for-biofuel-crop-brassica-carinata/
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Tags: Agrobacterium-mediated plant transformationAgrobacterium-mediated transformation protocolsbioenergy crop breedingbiofuel crop developmentbiofuel crop improvement researchBrassica carinata genetic transformationdrought-tolerant oilseed cropsEast African mustard cropEast African oilseed cropsenvironmentally resilient biofuel cropsgenotype-specific transformation efficiencygenotype-specific transformation techniquesnon-food bioenergy cropsnon-food biofuel feedstocksplant genetic engineering advancementsplant molecular biology protocolsrenewable diesel feedstockrenewable diesel productionstable transformation efficiency in Brassicasustainable aviation fuel feedstock
