As using A.Tumefacinies for inserting gene X and A.Rhizogenes for gene Y (in my case Y is for CRISPR cas9).
Abdelrahman Ahmed Khalifa The short answer is yes. You can prepare 2 Agrobacterium with its own binary vector (different gene on the vector) and then transform these 2 Agrobacterium to the plants. You can also transform 2 binary vectors into a Agrobacterium (Agrobacterium contains both vectors) for genetic transformation
Abdelrahman Ahmed Khalifa Now, I notice your sub-question < As using A.Tumefacinies for gene X and A.Rhizogenes for gene Y. >
The suggestion in my first answer is for both using A. Tumefacinies. Now, you want to do one in A.Tumefacinies and one in A.Rhizogenes. What kind of explants you are going to use? If succeed, some parts of the explants produce ' hairy roots' (induced from A. Rhizogenes ) and some parts of the explants will produce resistant callus (from A.Tumefacinies )?
Yuan-Yeu Yau
Thank you for your response, Taraxcum Koksaghyz is the plant that i want to do this project on it. As i want to use A. Rhizogenes for specific gene silencing by CRISPR technique considering that this gene is active in the roots. Then, Introduce specific gene by the means of A.Tumefacinies.
Abdelrahman Ahmed Khalifa I think sequential transformation is a more suitable way to do it. First, you transform explants with A. Rhizogenes for a specific gene silencing by CRISPR. The transformed hairy roots will come out, then you collected those induced hairy roots, and do a second transformation for the other gene with A.Tumefacinies. Hopefully, the roots can be easily transformed in your plant?
What kind of plant tissue you are using as explants?
Abdelrahman Ahmed Khalifa Is Taraxcum Koksaghyz roots easy for genetic transformation? Not every part of plants are suitable for genetic transformation.
Yuan-Yeu Yau Yes it has done before many times by the means of A. Rhizogenes.
Abdelrahman Ahmed Khalifa OK, get it. So, after you transform the plant roots with A. Rhizogenes, new 'hairy' roots will be induced (they are transformed with CRISPR machinery genes). And then, you are going to collect these hairy roots (with CRISPR machinery genes), and do a secondary transformation with A. tumerfacinies?
Abdelrahman Ahmed Khalifa But, do you know that the induced 'hairy' roots (not the original plant roots) are easy for secondary transformation? Find any report?
Yuan-Yeu Yau Actually that's a thing that i keep searching in, that if those fibrous roots can be transformed again in an easy transformation process but i have not reach to that yet.
Abdelrahman Ahmed Khalifa The right words should be 'hairy' roots, not 'fibrous' roots. My bad. I have changed them in my previous answers. In my opinion, it is hard to do a genetic transformation on these 'hairy' roots. Do you know any root-specific promoters for Taraxcum Koksaghyz ?
Yuan-Yeu Yau A specific promoter for the root is not in my hands right now but i think i can get it in the upcoming days.
Abdelrahman Ahmed Khalifa The reason I asked you about root-specific promoters is that you can also do it this way:
(1) transformation 1: use A.Tumefacinies --> binary vector: root-specific-promoter is hooked to Cas9 gene (for CRISPR) --> transform plants --> Cas9 only expresses in roots and CRISPR only the roots.
(2) transformation 2: Take transgenic plants (w/ single copy transgene) generated from transformation 1--> do a secondary transformation --> also use A. Tumefacinies; binary vector: 35S promoter hooked to your gene-of-interest
Different plant selection marker is needed on the vectors. For example, kan for one, and hygromycin for the other one.
Make sense to you?
Yuan-Yeu Yau Yeah I got you I think it is great idea of choosing the specific root promoter and I will take it in consideration very well. I am so thankful and grateful for you Dr. Yuan-Yeu Yau. You are really helped me in this.
Abdelrahman Ahmed Khalifa Later, I continued discussing this question with my colleague. Below is our discussions. Thought you might be interested in these info:
Me:
"A researcher wants to transform plant roots with A. Rhizogenes first with CRISPR components to silence a gene. 'Hairy' roots will be induced (they are transformed with CRISPR machinery genes). And then, He is going to collect these hairy roots (with CRISPR machinery genes), and do a secondary transformation with A. tumerfacinies to overexpress a gene-of-interest."
my colleague:
" The biggest downside of that experiment is that the Cas9 would still be present. For me, I would rather not have the editing enzyme stay in the plant, running amok, chewing on who knows what. How about putting the editing cassette in a inducible Cre lox configuration? You could generate the transgenic hairy roots, then induce the Cre to excise the editing region, leaving behind the overexpression cassette (assuming the edit has happened). Link a tdTomato marker to the Cas9 cassette so loss can be followed non-destructively. A small root sample that has lost the tdTomato can then be analyzed for the edit. This does beg the question, do you have good promoters to strongly (and simultaneously) drive the Cas9 and guide RNA expression (I guess some people do 2A fusions to get around needing multiple promoters)? "
Abdelrahman Ahmed Khalifa Then I proposed this construct for discussion. I am waiting my colleague's comment. Some info might be useful to you.
-lxp-P1/Cas9-P2/sgRNA-IP3/Cre-P4/SMG-loxp-
(1) P1, P2, IP3 and P4 are promoters. IP3 is an inducible promoter
(2) SMG: selectable marker gene
(3) Cre and loxP: derived from Cre/loxP site-specific recombination system
Steps:
1. transform plant
2. Plant selection (P4/SMG); P4 can be 35S
3. P1/Cas9-P2/sgRNA started to edit plants
4. (few days after), apply inducible agent on the plants, such as heat, for heat inducible promoter. Cre protein is produced to loop out the cassette.
5. Cassette -lxp-P1/Cas9-P2/sgRNA-IP3/Cre-P4/SMG-loxp- is looped out, left footprint -loxp-
**P1 can be a strong root-specific promoter, because he wanted the gene only in the plant roots silenced
**Not sure if the P1 (drive Cas9) will be equally effect when works in plant original roots and the Agrobacterium induced hair-roots, if the P1 is an root-specific promoter [the cassette is supposed to place in A. Rhizogenes ]
**Actually, a cassette [ -P1/Cas9-P2/sgRNA-P4/SMG- ] can also be segregate away in the edited plants (derived from the hairy roots) later without using Cre-lox strategy
Yuan-Yeu Yau Iam sorry but i am not familiar with the Cre-Lox system so i did some searches about it and got the main idea but i didn't understand well the point of " induce the Cre to excise the editing region, leaving behind the overexpression cassette (assuming the edit has happened). "
Abdelrahman Ahmed Khalifa < induce the Cre to excise the editing region, leaving behind the overexpression cassette >
No, no. The Cre protein will excise the whole cassette (everything between the two loxp sites) through recombination of these 2 loxp sites. There is nothing to do with your edited site/region.
So, in this case: -lxp-P1/Cas9-P2/sgRNA-IP3/Cre-P4/SMG-loxp- , after Cre catalyzing, 2 loxp sites go through site-specific recombination, the resulting product will be only -loxp-, left behind in the genome. The main goal to do that is to remove Cas9, sgRNA, SMG and Cre itself after editing is done. The constant expressions of Cas9, sgRNA, SMG and Cre can cause plant metabolic burden. The constant expressions of Cas9/sgRNA might cause unwanted knockout in the genome. The expression of SMG, make a lot of people afraid of transgenic plants.
But, as I mentioned in my previous answer, you don't need to involve the Cre-lox system in your construct. You can just do a cross to segregate the whole construct away. If your plant is a selfing plant, you can self them and chose the ones without the cassette from the selfed population. If your plant is an outcross plant species, do a backcross to remove the cassette. Backcross: Transgenic plant x wild type.
Any question?
Abdelrahman Ahmed Khalifa Looked at the attached picture (click on the picture to see the whole thing).
The first diagram. When the two loxp sites are placed in same orientation, after Cre-mediated recombination on these two loxp sites, only one loxp left (footprint) in the genome. The excised fragment (in-between the 2 loxp sites) are in circular forms and disappear in the cells later.
Yuan-Yeu Yau Yes I got you now, I will think what i will go through either Cre-lox system or crossing according to what will be better to the plant.
Again i thank you and your colleague for your cooperation with me in this. I will keep in touch with my updates if you want.
Yes co-transformation is quite possible. I did it in tobacco althought both strains were of Agro tumefaciens.
It does not be a infection on the same day.
I had a transgenic plant. From it I did use a leaf to be inserted with A.rhiz.
Got hairy roots. And from it new plantlets.
Later I need to examine which genes are transferred 8nto this new plantlet.
S. Wenkart So, you were saying that you obtained those 'hairy roots' (through A. Rhiz), and then regenerated them into plantlets? I am interesting to know what plant species was that, and what was the efficiency? Also, did you induce callus from the 'hairy roots' before you regenerated plantlets?
Dear Allah Bakhsh Yes, co-transformation is possible. But, in Abdelrahman Ahmed Khalifa case, it is better to take 'sequential transformation' approach. First, to obtain transgenic plants with roots having been CRISPR-edited (make sure the editing is there; in this case, silencing a root gene), then second transformation can be applied to the edited plants. He is going to use different Agrobacterium-- A.Tumefacinies for inserting gene X and A. Rhizogenes for the other gene. If he co-infect an explant with these Agrobacterium, he might get an explant with callus-resistant callus (from A.Tumefacinies) and antibiotic-resistant 'hairy roots' (from A. Rhizogenes).Besides, it seems to be harder for plantlets to grow if two antibiotics are used for selection simultaneously.
I got the hairy roots right? I left them grow under the normal used amount of light in the growth room at 25 C. On half of MS. The roots turned green. No callus and here and there they started to sprout shoots.
You are right: not every plant species would be so nice to go along with it.
Mine is Lisianthus. But a little desert species I would like to behave accordingly still refuses...
S. Wenkart Thanks for your answer to my question. Yes, not every plat species will work the same.
Abdelrahman Ahmed Khalifa and Yuan-Yeu Yau : I am the colleague of whom Yuan-Yeu speaks. Sorry I didn't get back to your proposed plasmid design (my wife and I just bought a condo, and I am in the middle of painting...a decent use of forced quarantine time, better than watching news reports).
Anyway, the loxP flanked design looks good to me, theoretically it should work.
Abdelrahman Ahmed Khalifa, now that I have read this line of Q&A, I'm wonder if we have made a far more complicated strategy than is really necessary. Do you need phenotypically normal plants at the end of the project? Hairy roots, composite plants (this is a wildtype shoot, with a population of transgenic roots) typically all have a phenotype that differs from the wildtype plant. It depends on your research question if using this particular tool will be the right choice for you. It might be better to simply go the "normal" transformation route for the gene editing work, using a disarmed Agrobacterium (this will ensure phenotypically "normal" plants), and take from these the null segregants (in which you have confirmed the gene editing result that you are trying to obtain) forward in the next round of transformation (I guess you want then to overexpress a mutant form of the gene that you have edited, but maybe it is something else?). Remember, hairy roots can be very weird.
The thing is that you never know in advance how the hairy roots will look like. E.g. got different coloured roots from Beta vulgaris, the red beet. Because you dont know how the Agro.rhiz. will split. Also some roots wete fast growers and others very lazy.
And because the rhiz. is splitting I took the chance. Because the first plasmid that I inserted, did not "work". And thus I hope both genes together will give another result than the first plasmid alone.
Maybe you think that is a strange aproach but sometimes you got to try.
So far there are plantlets and waiting for them to flower. They dont look small.
Ray Collier Thanks for your answer. Do you know if root promoters can express in the 'hairy roots' (induced by A. Rhiz.) as well as they express in regular roots? And reports?
Thats a difficult question for me...First, after 1982, the main interest in our lab had been, to start hairy roots in order to use them in a bioreactor for students to get their PhD's.
There was not the question, IF hairy roots can sprout into plantlets and if this is feasable, would they "remember" having been once a hairy root in order to produce a profound system of roots.
What I am working on just now, is exactly THAT question. If hairy roots have more of IAA in them, maybe they should develop into plantlets with more roots.
So far I did not observe this.
Yes it can be done by preparing 2 separate Agrobacterium and practicing sequential transformation.
This is problematic since there will be silencing of the Kan resistant gene from the two plasmids. Most importantly you need to use auxins and cytokinins to regenerate from explants transformed with A. tumefacinies, whereas the hairy roots are sensitive to auxins. In addition, if you use the two bacterial strains it is hardly possible to get plants with hairy roots since the two may arise from different cells. Regeneration of plants from hairy roots is very difficult and only a few articles reported that, in potato, tomato, Mexican lime, Medicago truncatula, Taraxacum kok-saghyz (TK, Rubber Dandelion) and etc.. here are some liks
Article CRISPR/Cas9 genome editing of rubber producing dandelion Tar...
https://pubmed.ncbi.nlm.nih.gov/15332121/
file:///D:/Academic/Zotero/Plant%20Regeneration%20From%20Hairy%20roots/Planta%20Volume%20223%20issue%206%202006%20[doi%2010.2307_23389376]%20Cynthia%20Crane,%20Elane%20Wright,%20Richard%20A.%20Dixon%20and%20Zeng-Yu%20Wang%20--%20Transgenic%20Medicago%20truncatula%20plants%20obtained%20from%20Agrobacterium%20tumefaciens-.pdf
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