I talked with one of my professor; he told that chimeric plants are mostly found in T0 generation only and the opportunity for finding a chimeric T1 generation plant is extremely rare or most of the times none. But in one of the paper they talk about a probability of getting a systemic contamination of Agrobacterium in to the T1 generation plants that may lead to chimerism which my professor says is not a very good chance. Please help me find out and also share references if you have any.
Also note that my plants are generated by in planta transformation.
I will be really grateful
Thank you
In our lab, We encounter some chimerism in Ag transformed To rice plants, but never came across any chimeric T1 plants.
Even without Agrobacterium contamination, T1 plants can still host chimeric cells. It depends on what construct you transfer into T0 plants. For example, if you transfer an auto-excision construct (containing Cre-lox system), -lox-IP1-Cre---P2-SMG-lox-, into T0, and while the Inducible Promoter (IP1) is not induced in some T0 plant cells, this 'un-excised' construct might transmit into T1. In T1, if IP1 works in some cells and not in other cells (depending on IP1 promoter strength), then some of the cells will have -lox- (excised form**) allele, and the others will still have the original -lox-IP1-Cre---P2-SMG-lox- allele. This results in 'chimeric' cells.
**When IP1 works, it expresses Cre protein. In turn, the Cre enzyme will recombine the two lox sites, and loop out (or excise) the DNA fragment between the two lox sites.
My guess, it depends on what kind of the referent genes and DNA-construction you use. Here can be a tricky one with transient expression, when in some cells the expression of the target gene is occurring still, and in others - is done. Also, it applies to constructions with site-specific recombinases like Cre, when the chimeric cells can persist even in T4 and further progenies.
1. We have seen some escaped (from antibiotic selection) Agrobacterium being carried over to progeny. The presence of Agrobacterium might still have the ability to transfer more T-DNA into some plant cells at the infected/contaminated area. And, if it does, then 'chimeric' nature will arise. However, I have not seen a report describing systematic study on this issue.
2. The paper attached did mention this possibility. Following statement is from the paper:
"Although antibiotics were used to eradicate Agrobacterium after infection, bacterial cells might still contaminate selected transgenic Arabidopsis and maize plants. To eliminate the possibility of contaminating Agrobacterium DNA falsely increasing the apparent T-DNA copy number, we hybridized several membranes with the Agrobacterium chromosomal picA gene. We did not detect a hybridization signal using this probe (data not shown)."
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Hi Anirudha
Some useful references on attachment 1
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC150518/
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2
https://onlinelibrary.wiley.com/doi/10.1046/j.1365-313X.1997.11061369.x/pdf
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https://bmb.natsci.msu.edu/_bmb/assets/File/Sharkey_lab/Selecting%20Homozygous%20Transformed%20Plants.pdf
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https://jag.igr.poznan.pl/2004-Volume-45/2/pdf/2004_Volume_45_2-127-144.pdf
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http://www.personal.psu.edu/mjg9/Guiltinan_Lab_Website/Manuscripts_files/Fisher%201995.pdf
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https://www.jstage.jst.go.jp/article/plantbiotechnology/33/5/33_15.0901a/_pdf
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https://www.ejbiotechnology.info/index.php/ejbiotechnology/article/view/v1n3-1/789
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https://www.plantphysiol.org/content/plantphysiol/120/3/653.full.pdf