Tissue Culture Optimization and Construction of a Suitable Vector for Engineering Very Long-Chain Fatty Acids in Camelina sativa L.

Document Type : Original Article

Authors

1 Agricultural Biotechnology Department, Faculty of Agriculture, Azarbaijan Shahid Madani University, Tabriz, Iran

2 Department of Plant, Cell and Molecular Biology, Faculty of Natural Science, University of Tabriz, Tabriz, Iran

3 Department of Horticultural Sciences, University of Tabriz, Tabriz, Iran

10.22126/atic.2026.11724.1193

Abstract

Oil quality from oilseeds is determined by the fatty acid (FA) content. Fatty Acid Elongase 1 (FAE1) is an essential FA gene that has been genetically modified to change the constitution of fatty acids in oilseed plants. In this study, recombinant vector construction for genome editing of the CsFAE1B, CsFAE1A, and CsFAE1C genes using CRISPR/Cas9 was used to improve the quality of Camelina Sativa oil (which contains about 50% oil and 4% erucic acid). Additionally, camelina tissue culture optimization for recombinant vector transfer was performed concurrently. Finally, gRNA was designed to target three copies of the CsFAE1 genes, and after transfer to the pFGC-pcoCas9 vector, the recombinant vector was confirmed by PCR and enzymatic digestion. The tissue culture optimization results indicated that hormone ratios of 0.5 mg L-1 NAA and 3 mg L-1 BAP for cotyledon and hypocotyl in the Gamborg medium induced embryogenic calluses three weeks after cultivation. Additionally, hormone ratios of 0.5 NAA, 2 BAP, and 1 (mg L-1) Kin led to direct regeneration in cotyledon explants. In future studies, tissue culture optimization and recombinant vector construction for genome editing of FAEs genes could improve oil quality with the genetic transformation of camelina.

Graphical Abstract

Tissue Culture Optimization and Construction of a Suitable Vector for Engineering Very Long-Chain Fatty Acids in Camelina sativa L.

Keywords

Main Subjects

Full Text

  • Established an optimized tissue culture system for Camelina sativa from diverse explant sources.
  • Engineered a multiplex sgRNA to concurrently target the FAE1, FAE2, and FAE3 genes.
  • Developed a custom pFGC-pcoCas9-sgRNA vector to enable precise genome editing.
  • Achieved efficient genetic transformation, demonstrating a robust protocol for metabolic engineering in camelina.

References

Ahmad M., Waraich E.A., Hafeez M.B., Zulfiqar U., Ahmad Z., Iqbal M.A., Raza A., Slam M.S., Rehman A., Younis U. 2023. Changing climate scenario: perspectives of Camelina sativa as low-input biofuel and oilseed crop. Global Agricultural Production: Resilience to Climate Change. (pp. 197-236). https://doi.org/10.1007/978-3-031-14973-3_7 
Alberghini B., Zanetti F., Corso M., Boutet S., Lepiniec L., Vecchi A., Monti A. 2022. Camelina [Camelina sativa (L.) Crantz] seeds as a multi-purpose feedstock for bio-based applications. Industrial Crops and Products 182: 114944. https://doi.org/10.1016/j.indcrop.2022.114944
Balamurugan V., Abdi G., Karthiksaran C., Thillaigovindhan N., Arulbalachandran D. 2024. A review: improvement of plant tissue culture applications by using nanoparticles. Journal of Nanoparticle Research 26(8): 188. https://doi.org/10.1007/s11051-024-06103-2
Barlass M., Skene K.G. 1978. In vitro propagation of grapevine (Vitis vinifera L.) from fragmented shoot apices. Vitis 17(4): 335. https://doi.org/10.5073/vitis.1978.17.335-340 
Bashiri H., Kahrizi D., Salmanian A.H., Rahnama H., Azadi P. 2023. Control of erucic acid biosynthesis in camelina (Camelina sativa) by antisense technology. Cellular and Molecular Biology 69(7): 212-217. https://doi.org/10.14715/cmb/2023.69.7.34
Bashiri H., Kahrizi D., Salmanian A.H., Rahnama H., Azadi P. 2024. Engineering erucic acid biosynthesis in camelina (Camelina sativa) via FAE1 gene cloning and antisense technology. Cellular and Molecular Biology 70(7): 243-251. https://doi.org/10.14715/cmb/2024.70.7.35
Belide S., Petrie J.R., Shrestha P., Singh S.P. 2012. Modification of seed oil composition in Arabidopsis by artificial microRNA-mediated gene silencing. Frontiers in Plant Science 3: 168. https://doi.org/10.3389/fpls.2012.00168 
Birnboim H., Doly J. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research 7(6): 1513-1523. https://doi.org/10.1093/nar/7.6.1513
Cromwell C.R., Sung K., Park J., Krysler A.R., Jovel J., Kim S.K., Hubbard B.P. 2018. Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity. Nature Communications 9(1): 1448. https://doi.org/10.1038/s41467-018-03927-0
Ebrahimi V., Hashemi A. 2024. CRISPR-based gene editing in plants: focus on reagents and their delivery tools. Bioimpacts 15: 30019. https://doi.org/10.34172/bi.30019
Gamborg O.L., Miller R.A., Ojima K. 1968. Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research 50(1): 151-158. https://doi.org/10.1016/0014-4827(68)90403-5
Ghidoli M., Ponzoni E., Araniti F., Miglio D., Pilu R. 2023. Genetic improvement of Camelina sativa (L.) Crantz: opportunities and challenges. Plants 12(3): 570. https://doi.org/10.3390/plants12030570
Ghosh A., Igamberdiev A.U., Debnath S.C. 2021. Tissue culture-induced DNA methylation in crop plants: a review. Molecular Biology Reports 48(1): 823-841. https://doi.org/10.1007/s11033-020-06062-6
James D.W., Lim E., Keller J., Plooy I., Ralston E., Dooner H.K. 1995. Directed tagging of the Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene with the maize transposon activator. The Plant Cell 7(3): 309-319. https://doi.org/10.1105/tpc.7.3.309
Kim H., Lee W.J., Oh Y., Kang S.H., Hur J.K., Lee H., Song W., Lim K.S., Park Y.H., Song B.S. 2020. Enhancement of target specificity of CRISPR–Cas12a by using a chimeric DNA–RNA guide. Nucleic Acids Research 48(15): 8601-8616. https://doi.org/10.1093/nar/gkaa605
Li J.F., Norville J.E., Aach J., McCormack M., Zhang D., Bush J., Church G.M., Sheen J. 2013. Multiplex and homologous recombination–mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nature Biotechnology 31(8): 688-691. https://doi.org/10.1038/nbt.2654 
Li X., Jiang D.H., Yong K., Zhang D.B. 2007. Varied transcriptional efficiencies of multiple Arabidopsis U6 small nuclear RNA genes. Journal of Integrative Plant Biology 49(2): 222-229. https://doi.org/10.1111/j.1744-7909.2007.00393.x
Liu Q., He D., Xie L. 2019. Prediction of off-target specificity and cell-specific fitness of CRISPR-Cas system using attention boosted deep learning and network-based gene feature. PLoS Computational Biology 15(10): e1007480. https://doi.org/10.1371/journal.pcbi.1007480
Murashige T., Skoog F. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15(3): 473-497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Nishimasu H., Shi X., Ishiguro S., Gao L., Hirano S., Okazaki S., Noda T., Abudayyeh O.O., Gootenberg J.S., Mori H. 2018. Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science 361(6408): 1259-1262. https://doi.org/10.1126/science.aas9129
Ozseyhan M.E., Kang J., Mu X., Lu C. 2018. Mutagenesis of the FAE1 genes significantly changes fatty acid composition in seeds of Camelina sativa. Plant Physiology and Biochemistry 123: 1-7. https://doi.org/10.1016/j.plaphy.2017.11.021
Patra N., Barker G.C., Maiti M.K. 2025. Knockout of fatty acid elongase1 homeoalleles in amphidiploid Brassica juncea leads to undetectable erucic acid in seed oil. Plant Physiology and Biochemistry 222: 109679. https://doi.org/10.1016/j.plaphy.2025.109679 
Rezaeva B.R., Rutten T., Bollmann C., Ortleb S., Melzer M., Kumlehn J. 2024. Plant regeneration via adventitious shoot formation from immature zygotic embryo explants of camelina. Plants 13(4): 465. https://doi.org/10.3390/plants13040465
Sabbadini S., Capriotti L., Molesini B., Pandolfini T., Navacchi O., Limera C., Ricci A., Mezzetti B. 2019. Comparison of regeneration capacity and Agrobacterium-mediated cell transformation efficiency of different cultivars and rootstocks of Vitis spp. via organogenesis. Scientific Reports 9(1): 582. https://doi.org/10.1038/s41598-018-37335-7
Saraswat P., Chaturvedi A., Ranjan R. 2023. Zinc finger nuclease (ZFNs) and transcription activator-like effector nucleases (TALENs) based genome editing in enhancement of anticancer activity of plants plant-derived anticancer drugs in the OMICS era (pp. 281-293): Apple Academic Press.
Sinha S., Jha J.K., Maiti M.K., Basu A., Mukhopadhyay U.K., Sen S.K. 2007. Metabolic engineering of fatty acid biosynthesis in Indian mustard (Brassica juncea) improves nutritional quality of seed oil. Plant Biotechnology Reports 1: 185-197. https://doi.org/10.1007/s11816-007-0032-5
Wada N., Ueta R., Osakabe Y., Osakabe K. 2020. Precision genome editing in plants: state-of-the-art in CRISPR/Cas9-based genome engineering. BMC Plant Biology 20: 234. https://doi.org/10.1186/s12870-020-02385-5
Wang P., Xiong X., Zhang X., Wu G., Liu F. 2022. A review of erucic acid production in Brassicaceae oilseeds: progress and prospects for the genetic engineering of high and low-erucic acid rapeseeds (Brassica napus). Frontiers in Plant Science 13: 899076. https://doi.org/10.3389/fpls.2022.899076
Yemets A., Boychuk Y.N., Shysha E., Rakhmetov D., Blume Y.B. 2013. Establishment of in vitro culture, plant regeneration, and genetic transformation of Camelina sativa. Cytology and Genetics 47: 138-144. https://doi.org/10.3103/S0095452713030031
Agrotechniques in Industrial Crops

Articles in Press, Accepted Manuscript
Available Online from 19 November 2025

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