I've recently come across the method of agroinjection and vacuum infiltration of various plant tissues (seedlings, potato eyes, tomato fruit). These methods are new to me and very fascinating but I've been having a hard time finding answers regarding these methods.
1) Are these methods only for transient expression? Are the chances of creating a chimera, which then will produce transgenic seeds, extremely unlikely? Of course I would like to establish a stable transgenic line but transient expression might do for the experiment I have in mind.
2) Would multiple injections or vacuum infiltration sessions result in multiple integrations into the host genome? The outcome would probably still be a chimera, but I would think that multiple transfection rounds could greatly increase transgene expression, integration events and maximize the chances of producing seeds for a stable transgenic line. Is this a valid train of thought?
3) If my transgene encodes for a His-tagged protein and I am really only interested in purifying the His-tagged protein, transient expression alone can satisfy my needs. Is there an certain plant species or explant which is ideal for the production/purification of protein?
Thank you so much for any thoughts or suggestions while I continue to investigate this subject.
Yes, Agrobacterium injection or infiltration techniques can yield a stable transformed line. These techniques are commonly used for plant transformation and have been successful in generating stable transformed lines¹²³⁴.
For instance, an improved method of Agrobacterium syringe infiltration for transient transformation has been developed, which has been applied in the elucidation of gene function in poplar¹. This method has greatly improved the transient transformation efficiency of syringe Agrobacterium infiltration in poplar¹. The transiently transformed leaves could be used to generate stably transformed plants with high efficiency through callus induction and differentiation processes¹.
Another study reported that the Agrobacterium germ-line transformation method is a relatively new and immensely successful procedure for plant transformation³.
However, it's important to note that the success of these techniques can depend on various factors such as the species of the plant, the specific conditions of the experiment, and the genes being introduced. Therefore, while these techniques have the potential to yield a stable transformed line, the actual results can vary¹²³⁴.
(1) An improved and efficient method of Agrobacterium syringe infiltration .... https://bmcplantbiol.biomedcentral.com/articles/10.1186/s12870-021-02833-w.
(2) (PDF) Infiltration with Agrobacterium — the method for stable .... https://www.academia.edu/56492965/Infiltration_with_Agrobacterium_the_method_for_stable_transformation_avoiding_tissue_culture.
(3) Agrobacterium Germ-Line Transformation: Transformation of Arabidopsis .... https://link.springer.com/chapter/10.1007/978-94-011-5242-6_2.
(4) High-efficiency Agrobacterium-mediated transformation of .... https://www.maxapress.com/article/doi/10.48130/OPR-2022-0001.
James Carrillo If your task as specified in the #3 "If my transgene encodes for a His-tagged protein and I am really only interested in purifying the His-tagged protein, transient expression alone can satisfy my needs." - the answer is yes.
Exact plant species /plant part which is ideal for the production/purification of your protein can be found only by experiments. My colleagues used lettuce plants (commercial hybrids) grown in commercial hydroponics or tobacco.
For task #1 "Are the chances of creating a chimera, which then will produce transgenic seeds, extremely unlikely?" - sure, you create a chimera, and there is a chance to obtain transgenic seeds, but this is very far from the task of obtaining a stable transformed line. GM plants must have only a single insertion of T-DNA, the site of insertion into plant genome must be characterized and stability of the insertion and transgene expression must be tested in several generations of seed propagation. This part takes 95% of all work, so, transient expression looks more suitable for your purpose.
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