In 2016-2017 I was working with a strain of Agrobacterium tumefaciens, called EHA105 (agropine-type), that a guy from a lab in Portugal kindly sent me by mail, but the strain carried an incredibly resistant red yeast that made it impossible to get any results before the yeast proliferated,
The result is that I lost months and months of work, and abandoned that project with a very bad feeling.
I was wondering if any of you in this magnificent network would be so kind as to send me an aliquot of Agrobacterium EHA105 lyophilised or in culture medium, by regular mail. I really really appreciate this.
Thank you very much in advance.
J.D. Franco-Navarro Just wondering...why you have to use EHA105 strain, not other strains (such as GV3101....)? Is that better for citrus transformation?
Yuan-Yeu Yau
Transformation is done directly on stem pieces, and strain GV3101 produces tumours in aerial parts that differentiate and proliferate. Unlike GV3101, EHA105 has the particularity that in its plasmid (pEHA105), it carries the genes for the synthesis of agropine, and the tumours produced do not differentiate or proliferate.
Hood E.E., Gelvin S.B., Melchers S. & Hoekema A. (1993) New Agrobacterium helper plasmids for gene transfer to plants (EHA105). Trans Res, 2: 208-218.
J.D. Franco-Navarro
[1] The photos (both) you attached was from GV3101 or EHA105?
[2] Thanks for the paper. Isn't that these day's Agrobacterium-mediate plant genetic transformation system, the T-DNA in binary vectors are disarmed and the 'tumor'-producing gene has been removed? (I have not read the paper yet, not know what exactly they said yet)
"In genetic engineering, the tumor-promoting and opine-synthesis genes are removed from the T-DNA and replaced with a gene of interest and/or a selection marker, which is required to establish which plants have been successfully transformed" [1]
[1]https://en.wikipedia.org/wiki/Transfer_DNA#:~:text=In%20genetic%20engineering%2C%20the%20tumor,plants%20have%20been%20successfully%20transformed.
Yuan-Yeu Yau
[1] the previous photo was a transformation using EHA105
[2] Exactly, but the genetic background in which these cloning/transformation systems are embedded is important, and there are strains that cause more or less uncontrolled callus formation, or that are more or less suitable for certain species. In the case of CITRUS, the most optimal strain is EHA105.
By the way, 414 RG Score? Wow, is the highest I've ever seen. That means you did a great job, congratulations!
J.D. Franco-Navarro
1.
Isn't callus-forming mainly due to phytohormones we add inside the medium, less to do with Agrobacterium per se? (I need to check out more)
"Callus Induction by Plant Hormones. Auxin and cytokinin have been widely used to generate callus, but surprisingly little is known about how they induce callus at the molecular level. Several recent studies demonstrated that various regulators of lateral root development participate in callus formation on CIM." Article Plant Callus: Mechanisms of Induction and Repression
2. In-Planta transformation:
Have you tried *In Planta* transformation? It has been successfully used on Citrus species [See below article]. It seems to be much easier. No tissue-culturing is needed. You grow young seedling from seeds (Fig (a)). Cut the stem (b), add Agrobacterium to the wounds, co-cultivation for a length of time (c-d), clean, then add antibiotic and wait for shoots ("transgenic") to come out (f-h). (see attached Figure; from the article below). Article A simple and efficient in planta transformation method for p...
3. < .......That means you did a great job, congratulations! >
Thank you.
Reseach of Franco-Navarro is one of the best work on PLANT TRANSFORMATION, I fully recommend it
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