Direct genetic transformation of plants: Techniques, applications, and biotechnological perspectives
DOI:
https://doi.org/10.33158/ASB.r266.v12.2026Keywords:
Modern agriculture, resistant cultivar, gene expression, genome editing, recalcitrant species, pest resistance, stress tolerance mechanisms, higher yieldAbstract
Direct plant genetic transformation is an important tool in plant biotechnology, allowing the introduction of genetic material into plant cells without the use of intermediate organisms, such as Agrobacterium tumefaciens. Among the main techniques used are biolistics, electroporation, and polyethylene glycol (PEG)-mediated transformation, widely applied in plant breeding and genetic engineering studies. These methodologies enable the development of plants with desirable agronomic characteristics, including resistance to pests and diseases, tolerance to abiotic stresses, and nutritional improvements. In recent years, the integration of genetic transformation techniques with modern gene editing tools, especially the CRISPR/Cas9 system, has significantly expanded the applications of plant biotechnology, allowing precise modifications to the plant genome. Furthermore, new approaches involving nanotechnology and ribonucleoprotein delivery are contributing to increased efficiency in genetic transformation and reduced random integration of exogenous DNA. Despite technological advancements, challenges related to cell regeneration, genetic stability, biosafety, and regulation still limit the widespread use of these technologies. In this context, this review addresses the fundamentals, techniques, biotechnological applications, advantages, limitations, and future perspectives of direct genetic transformation of plants, highlighting its importance for the sustainable development of agriculture and plant biotechnology.
Downloads
References
Altpeter, F., Springer, N. M., Bartley, L. E., Blechl, A., Brutnell, T. P., Citovsky, V., Conrad, L. J., Gelvin, S. B., Jackson, D. P., Kausch, A. P., Lemaux, P. G., Medford, J. I., Orozco-Cárdenas, M. L., Tricoli, D. M., Van Eck, J., Voytas, D. F., Walbot, V., Wang, K., & Stewart, C. N., Jr. (2016). Advancing crop transformation in the era of genome editing. The Plant Cell, 28(7), 1510–1520. https://doi.org/10.1105/tpc.16.00196
Baltes, N. J., & Voytas, D. F. (2015). Enabling plant synthetic biology through genome engineering. Trends in Biotechnology, 33(2), 120–131. https://doi.org/10.1016/j.tibtech.2014.11.008
Bélanger, J. G., Copley, T. R., Hoyos-Villegas, V., Charron, J.-B., & O’Donoughue, L. S. (2024). A comprehensive review of in planta stable transformation strategies. Plant Methods, 20, Article 79. https://doi.org/10.1186/s13007-024-01200-8
Birch, R. G. (1997). Plant transformation: Problems and strategies for practical application. Annual Review of Plant Physiology and Plant Molecular Biology, 48, 297–326. https://doi.org/10.1146/annurev.arplant.48.1.297
Brookes, G., & Barfoot, P. (2020). GM crop technology use 1996–2018: Farm income and production impacts. GM Crops & Food, 11(4), 242–261. https://doi.org/10.1080/21645698.2020.1779574
Browning, K. S., & Bailey-Serres, J. (2015). Mechanism of cytoplasmic mRNA translation. The Arabidopsis Book, 13, e0176. https://doi.org/10.1199/tab.0176
Cardi, T., Murovec, J., Bakhsh, A., & et al. (2023). CRISPR/Cas-mediated plant genome editing: Outstanding challenges a decade after implementation. Trends in Plant Science, 28(10), 1144–1165. https://doi.org/10.1016/j.tplants.2023.05.012
Chen, K., Wang, Y., Zhang, R., Zhang, H., & Gao, C. (2019). CRISPR/Cas genome editing and precision plant breeding in agriculture. Annual Review of Plant Biology, 70, 667–697. https://doi.org/10.1146/annurev-arplant-050718-100049
Christou, P. (1995). Strategies for variety-independent genetic transformation of important cereals, legumes and woody species utilizing particle bombardment. Euphytica, 85(1–3), 13–27. https://doi.org/10.1007/BF00023926
Daniell, H., Kumar, S., & Dufourmantel, N. (2005). Breakthrough in chloroplast genetic engineering of agronomically important crops. Trends in Biotechnology, 23(5), 238–245. https://doi.org/10.1016/j.tibtech.2005.03.008
Doudna, J. A., & Charpentier, E. (2014). The new frontier of genome engineering with CRISPR-Cas9. Science, 346(6213), 1258096. https://doi.org/10.1126/science.1258096
Food and Agriculture Organization of the United Nations. (2022). The state of food and agriculture 2022: Leveraging automation in agriculture for transforming agrifood systems. FAO. https://doi.org/10.4060/cc2459en
Fischer, R., & Buyel, J. F. (2020). Molecular farming – The slope of enlightenment. Biotechnology Advances, 40, 107519. https://doi.org/10.1016/j.biotechadv.2020.107519
Gao, C. (2021). Genome engineering for crop improvement and future agriculture. Cell, 184(6), 1621–1635. https://doi.org/10.1016/j.cell.2021.01.005
Gelvin, S. B. (2003). Agrobacterium-mediated plant transformation: The biology behind the gene-jockeying tool. Microbiology and Molecular Biology Reviews, 67(1), 16–37. https://doi.org/10.1128/MMBR.67.1.16-37.2003
Jones, H. D. (2015). Regulatory uncertainty over genome editing. Nature Plants, 1, 14011. https://doi.org/10.1038/nplants.2014.11
Lee, K., & Wang, K. (2023). Strategies for genotype-flexible plant transformation. Current Opinion in Biotechnology, 79, article number 102848. https://doi.org/10.1016/j.copbio.2022.102848
Lodish, H., Berk, A., Kaiser, C. A., Krieger, M., Bretscher, A. Ploegh, H., Martin, K. C., Yaffe, M., & Amon, A. (2021). Molecular cell biology (9th ed.). New York, USA: W. H. Freeman/Macmillan Learning.
Lv, Z., Jiang, R., Chen, J., & Chen, W. (2020). Nanoparticle-mediated gene transformation strategies for plant genetic engineering. The Plant Journal, 104(4), 880–891. https://doi.org/10.1111/tpj.14973
Maliga, P. (2004). Plastid transformation in higher plants. Annual Review of Plant Biology, 55, 289–313. https://doi.org/10.1146/annurev.arplant.55.031903.141633
Manghwar, H., Li, B., Ding, X., Hussain, A., Lindsey, K., Zhang, X., & Jin, S. (2020). CRISPR/Cas systems in genome editing: Methodologies and tools for sgRNA design, off-target evaluation, and strategies to mitigate off-target effects. Advanced Science, 7(6), 1902312. https://doi.org/10.1002/advs.201902312
Mathur, J., & Koncz, C. (1998). Establishment and maintenance of cell suspension cultures. In J. M. Martinez-Zapater & J. Salinas (Eds.), Arabidopsis protocols (Methods in Molecular Biology, Vol. 82, pp. 27–30). Humana Press. https://doi.org/10.1385/0-89603-391-0:27
Naqvi, S., Zhu, C., Farre, G., Ramessar, K., Bassie, L., Breitenbach, J., Perez-Conesa, D., Ros, G., Sandmann, G., Capell, T., & Christou, P. (2009). Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways. Proceedings of the National Academy of Sciences of the United States of America, 106(19), 7762–7767. https://doi.org/10.1073/pnas.0901412106
Nekrasov, V., Wang, C., Win, J., Lanz, C., Weigel, D., & Kamoun, S. (2017). Rapid generation of a transgene-free powdery mildew resistant tomato by genome deletion. Scientific Reports, 7, 482. https://doi.org/10.1038/s41598-017-00578-x
Reyna-Llorens, I., Ferro-Costa, M., & Burgess, S. J. (2023). Plant protoplasts in the age of synthetic biology. Journal of Experimental Botany, 74(13), 3821–3832. https://doi.org/10.1093/jxb/erad172
Schiemann, J., Dietz-Pfeilstetter, A., Hartung, F., Kohl, C., Romeis, J., & Sprink, T. (2019). Risk assessment and regulation of plants modified by modern biotechniques: Current status and future challenges. Annual Review of Plant Biology, 70, 699–726. https://doi.org/10.1146/annurev-arplant-050718-100025
Souza Junior, H., & Gonela, A. (2025). DNA methylation: Regulation of gene expression and applications in plant breeding. Agronomy Science and Biotechnology, 11, 1–16. https://doi.org/10.33158/ASB.r244.v11.2025
Squire, H. J., Tomatz, S., Voke, E., González-Grandío, E., & Landry, M. (2023). The emerging role of nanotechnology in plant genetic engineering. Nature Reviews Bioengineering, 1, 314–328. https://doi.org/10.1038/s44222-023-00037-5
Tester, M., & Langridge, P. (2010). Breeding technologies to increase crop production in a changing world. Science, 327(5967), 818–822. https://doi.org/10.1126/science.1183700
Tsai, S. Q., & Joung, J. K. (2016). Defining and improving the genome-wide specificities of CRISPR-Cas9 nucleases. Nature Reviews Genetics, 17(5), 300–312. https://doi.org/10.1038/nrg.2016.28
Vasil, I. K. (1988). Progress in the regeneration and genetic manipulation of cereal crops. Bio/Technology, 6(4), 397–402. https://doi.org/10.1038/nbt0488-397
Villiger, L., Joung, J., Koblan, L., Weissman, J., Abudayyeh, O. O., & Gootenberg, J. S. (2024). CRISPR technologies for genome, epigenome and transcriptome editing. Nature Reviews Molecular Cell Biology, 25(6), 464–487. https://doi.org/10.1038/s41580-023-00697-6
Voinnet, O. (2009). Origin, biogenesis, and activity of plant microRNAs. Cell, 136(4), 669–687. https://doi.org/10.1016/j.cell.2009.01.046
WHO - World Health Organization. (2022). Food safety aspects of genetically modified foods. Geneva 27, Switzerland: World Health Organization. https://www.who.int
Woo, J. W., Kim, J., Kwon, S. I., Corvalán, C., Cho, S. W., Kim, H., Kim, S. G., Kim, S. T., Choe, S., & Kim, J. S. (2015). DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nature Biotechnology, 33(11), 1162–1164. https://doi.org/10.1038/nbt.3389
Yang, S. H., Kim, S. W., Lee, S., & Koo, Y. (2024). Optimized protocols for protoplast isolation, transfection, and regeneration in the Solanum genus for the CRISPR/Cas-mediated transgene-free genome editing. Applied Biological Chemistry, 67, article number 21. https://doi.org/10.1186/s13765-024-00870-1
Yong, J., Wu, M., Carroll, B. J., Xu, Z. P., & Zhang, R. (2024). Enhancing plant biotechnology by nanoparticle delivery of nucleic acids. Trends in Genetics, 40(4), 352–363. https://doi.org/10.1016/j.tig.2024.01.005
Zaidi, S. S.-E.-A., Mahas, A., Vanderschuren, H., & Mahfouz, M. M. (2020). Engineering crops of the future: CRISPR approaches to develop climate-resilient and disease-resistant plants. Genome Biology, 21, 289. https://doi.org/10.1186/s13059-020-02204-y
Zhang, H., Zhang, J., Wei, P., Zhang, B., Gou, F., Feng, Z., Mao, Y., Yang, L., Zhang, H., Xu, N., & Zhu, J.-K. (2014). The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnology Journal, 12(6), 797–807. https://doi.org/10.1111/pbi.12200
Zhu, J.-K. (2016). Abiotic stress signaling and responses in plants. Cell, 167(2), 313–324. https://doi.org/10.1016/j.cell.2016.08.029
Downloads
Published
Issue
Section
License
Copyright (c) 2026 Helio Souza Junior, Maria Fátima Pires Silva Machado

This work is licensed under a Creative Commons Attribution 4.0 International License.