Hello everyone, i am using GFP as marker for transgenic plant confirmation but the problem is, whenever i check the GFP positive roots, there i also found too many fake/false roots (without GFP) and i need only the transgenic roots with GFP. So everytime i need to cut those negative roots that waste a lot of time. I always use antibiotics AS ( Acetosyringone ) and Timentin (TMT) (Ticarcillin disodium salt/potassium) in the MS medium when co-cultivating the explants but all in vein .
Is there any solution that i can only got positive roots with GFP?
Thanks
1. The roots did not detect for GFP can still contain nptII-gfp gene cassette (assuming you used nptII gene for selection), but for some reasons ony the gfp gene became silenced (not expressed). Therefore, the roots still can grow in antibiotic-containing MS medium, but without GFP detection.
2. You could use the GFP-positive roots to induce callus and regenerate plantlets from those calli. Then you should be able to obtain transgenic plants w/o chimeric. I know this will take sometime. However, again, some parts of these transgenic plants can become transgene silenced and no GFP produced in vitro.
Dear, Yuan-Yeu Yau i am using 35S promoter i don,t know either its specially for root formation, but i am using agrobacterium rhizogene for bacterial infection.
Hi Hamza,
as Yuan-Yeu mentioned, silencing is a possibility (the gfp gene should still be detectable by pcr).
But, are you also using antibiotic (NPTII, HPT?) selection in your transformation cassette? If so, there is also the possibility of incomplete T-DNA transfer. That happens fairly often, especially if the selectable marker is near the right border from where the transfer starts). Antibiotic resistant regenerants might end up without the gfp construct or with a partial, non-functional one. Selection works more efficient if the selectable marker is near the left border (and the gfp reporter near the right one) basically eliminating incomplete transfer events.
If you only rely on GFP for selection, that may prove difficult, depending on the plant species and tissue you work with. I tried to use GFP and eGFP in Vitis. Selection based on both flourescent proteins proved difficult (read "impossible") in Vitis due to weak GFP-flourescence and high endo-flourescence of regenerating calli and shoots.
Regards,
Klaus
Klaus Eimert Thank you so much sir for your kind remarks and guideline. Yuan-Yeu Yau sir your contribution for the problem is also appreciable.
I am compelled to chime in here. The 35S promoter was never great for me when I made composite plants, but I now know that the version I previously used lacked a translational enhancer (Tobacco Mosaic Virus Omega works quite well). Alternatively you might consider using the Arabidopsis thaliana Ubiquitin 10 promoter, or the Glycine max Ubiquitin 3 XL promoter (with the 5' UTR enhancing intron). Also, I would highly recommend that you replace GFP with tandem Tomato localized to the endoplasmic reticulum; tdTomato-ER will be considerably brighter, and targeting of this protein to the ER enhances brightness by taking advantage of the highly invaginated nature of this organelle.
I have to caution against the suggestion of trying to regenerate stable transgenic plants from hairy roots. If you are successful at doing this (and this is no guarantee, since it is apparently impossible in some plant taxa to force the hairy root out of the hairy root development pattern (our experience in soybean, 2 years of futile effort)), the resulting plants will be phenotypically aberrant due to the expression of the rol genes.
There are reports in the literature describing the successful use of herbicide/antibiotic mediated selection of transgenic roots. For this strategy to work you will need to modify your plasmid to carry a transcriptional unit that confers resistance to the transgenic roots against the selective agent of choice. The risk with this strategy is that each transgenic root is essentially an independent event (which, as the other respondents have previously noted could be a contributing factor to your observation of imperfect penetrance of the GFP expression phenotype) so just as the GFP expression was variable, so too will be the expression of the selection marker. There is also the issue of long-distance translocation of the selective agent to the plant shoot, which is in your case wildtype, with potentially negative consequences.
Composite plant work is hard work. In order to extract meaningful data one must make very careful, frequent, detailed observations, and it is absolutely necessary to have multiple biological replicates, as well as multiple experiments. My work that earned the cover of The Plant Journal in 2012 was the result of more than 9 independent experiments with more than 20 plants per treatment/experiment. My wife is a saint for having helped me nodule counting. I don't know what your biological question is that you are trying to answer, but you should prepare yourself for some very critical questions from reviewers upon publication submission if you haven't provided enough replicates and corroborative data.
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