Abstract
Plant transformation has become an important technology for the large-scale production of a number of goods, ranging from vaccines and pharmaceutical products to biofuel. A plant that is frequently used for this purpose is Nicotiana tabacum due to several advantages: it can be easily regenerated, its genome is completely sequenced and genetic transformation is straightforward. We describe here the most common method for tobacco genetic transformation, which involves Agrobacterium tumefaciens. Many plasmid vectors are compatible with this bacterium, allowing the transformation of a wide range of plant species, as well as the insertion of more than two genes of interest in the plant genome. Furthermore, the capability to transfer a single copy DNA fragment to its host reduces the probability to switch off the gene or genes of interest. Here, we present a simple and reliable methodology to transform tobacco leaf explants using Agrobacterium tumefaciens to insert two genes of interest.
Keywords: Agrobacterium tumefaciens, Genetic transformation, In vitro culture
Background
Plants have been exploited as expression bioreactors for vaccine antigens, antibodies for immunotherapy, nutritional supplements, enzymes, and even peptides of interest with industrial applications (Sharma and Sharma, 2009). They require relatively low financial investment to conduct initial studies, constituting a large advantage for biotechnology entrepreneurs (Yao et al., 2015). After the first plant transformation reports (Fraley et al., 1983; Herrera-Estrella et al., 1983), protocols have not changed a lot using Agrobacterium as a method for this purpose; nevertheless, other methods have been developed to enhance transformation efficiency, such as biolistics. Currently, tobacco is among the most important plant platforms for protein expression; its cells, as other eukaryotic cells, can perform post-translational modifications to the proteins of interest and tobacco plants can be easily regenerated by organogenesis or embryogenesis; additionally, its genome is completely sequenced. Current protocols allow for the insertion of multiple transgenes, requiring only one selectable marker (Wang, 2015). Our protocol is based on Curtis et al. (1995) and involves co-cultivation Agrobacterium-Nicotiana tabacum, like another protocol with algae (Pratheesh et al., 2014) or fungi. We were able to transform Nicotiana tabacum with a binary plasmid that contained two expression cassettes for the simultaneous expression of two viral antigens and confirmed that both genes were expressed by RT-PCR analysis in transformed plants.
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This work was supported by SEP-CONACYT grant number 83863. Also, I would like to thank Ph.D. Asdrúbal Burgos for critical review of this manuscript. Authors declare that they have no conflicts of interest.
References
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