I have transfected Salvia milthiorrhiza plant using Agrobacterium tumefaciens cells carrying binary plasmid. At the level of Agrobacterium I confirmed presence of my binary vector by obtaining specific PCR product (250bp) and shadow of non-specific product 500bp in lenght. But after transfection, at the level of Salvia milthiorrhiza plant I have no specific product and strong non-specific product at 500bp. I am wondering what happened? Transfection did not heppened or maybe Salvia rearranged or excised from its genome transfected DNA fragment or my primers don't want to join to specific region for some reason?
1. Use the term 'transform', instead of 'transfect'.
2. After you inserted your gene-of-interest (GOI) into the binary vector, did you do a restriction digestion to make sure they were truly inserted? How did you do PCR to confirm your GOI was inserted in the vector? (one primer on the backbone and one primer in the GOI?)
3. It could be a failed transformation experiment as well.
I think that sequencing both the short 250 and long 500 products might help to understand what is happening, I do find it interesting that the larger band is 2x the smaller band and wonder if it is a dimer of the smaller amplimer
As Paul Rutland 's suggestion, you could have ligated 2 inserts in the vector (possible (1)), so PCR for the GOI won't tell you the structure of your ligation product. Restriction digestion will help. Link below just an example of ligating 2 inserts into a restriction site (sequencing confirmed). By the way, did you check at least 2-3 colonies? Probably you can find 'one insert' product (to check the theory of ligating 2 inserts into the vector)?
(1) https://www.researchgate.net/post/Is_it_possible_to_have_two_copies_of_my_gene_while_cloning_it_into_pcDNA31
You can also design the PCR primers outside the GOI, on the backbones, for amplification, if your GOI was correctly inserted. You should generate a ~500 bp amplicon (depending on the locations of the primers).
Yuan-Yeu Yau Paul Rutland
Thank you a lot for your suggestions.
The goal of my experiment is to overexpress gene which occurs naturally in my plant. So, to confirm transformation I should detect sequence foreign for my host plant. The sequence is promoter CaMV35S that controls my insert gene.
In my experiment I used brand new bought binary vector so I assume that its sequence is correct. I sequenced only my whole insert gene sequence.
If I did not manipulated CaMV35S sequence it should not be duplicated.
Malgorzata Majewska
1. Why did you detect (using PCR) the CaMV35S promoter region, not your gene-of-interest (GOI)? Is your GOI already exists in the plant? CaMV35S promoter (short for 35S promoter) derived from Cauliflower mosaic virus (CaMV), which is a plant pathogen. I don't know which plant species you are working on. But, it might already infected by the virus, and gave you false positive. (see attachment below)
2. Now I am a little clear about your experimental purpose. So, did you try to amplify the 'enhancer' region of the CaMV35S promoter? Current 35S promoter in many binary vectors have 'double' enhancers (man made) to enhance its performance. Some called it 'd35S'. If one enhancer is around 250 bp (PCR product), two copies will be 500 bp. But, I am not sure why they preferentially amplify the larger one (500 bp).
3. About using PCR to 'confirm' positive transformants: I really don't have faith in using PCR to screen/confirm putative transformants. Your plants might still have some resilient Agrobacterium which has not been eradicated by antibiotics. PCR can amplify the binary vector carried by those Agrobacterium. Does your T-DNA carries other screening protein genes? Such as 'GUS' or 'GFP' gene? You can use them for screening--GUS staining or GFP fluorescence.
Yuan-Yeu Yau
1. Yes, my gene of interest is native for my plant (Salvia milthiorrhiza).
2. I tried to amplify CaMV35S sequence, not engancer. Enhancer in my binary vector have 58bp and it comes from Arabidopsis thaliana.
3. That is a very valid point. But I made long selection, and after antibiotic was swiched off I did not have any infection.
My T-DNA did not carry GUS, EGFP, GFP. It contains: enhancer, CaMV35S promoter sequence, gene of interests, terminator form A. thaliana, NOS promoter and Neomycin/Kanamycin resistance gene.
Amy Johnson
It is a good idea for my next experiments, thank you.
Malgorzata Majewska
1. < 2. I tried to amplify CaMV35S sequence, not enhancer. Enhancer in my binary vector have 58 bp and it comes from Arabidopsis thaliana. >
I was talking about the 'native' enhancer in the CaMV35S promoter. There is one native enhancer in the original 35S promoter. Researchers had artificially added an identical native enhancer in the promoter region to boost its performance. The novel promoter was called double-enhancer 35S promoter (or d35S). What binary vector did you use? pCambia series?
2. By the way, you can design one primer from the 35S region and one primer from your GOI region for PCR, to avoid accidentally amplifying a 35S promoter of Cauliflower mosaic virus (CaMV), if the plants had already got infected by the virus.
Yuan-Yeu Yau
1. I didn`t know the fact. Thank you for explanation.
I use pRI201AN vector.
Malgorzata Majewska Ok, thanks. I checked pRI201AN ( https://www.novopro.cn/plasmid/V10821 ). It seems fine, a plant transformation vector (for Agrobacterium and dicot plant). The 35S promoter contains only one enhancer (according to the list from the website I listed above). I did see the Arabidopsis enhancer described at the website.
** I post the vector map for people who are interested to read.
Cereal crops have been the primary targets for improvement by genetic transformation because of their worldwide importance for human consumption. For a long time, many of these important cereals were difficult to genetically engineer, mainly as a result of their inherent limitations associated with the resistance to Agrobacterium infection and their recalcitrance to in vitro regeneration. The delivery of foreign genes to rice plants via Agrobacterium tumefaciens has now become a routine technique. However, there are still serious handicaps with Agrobacterium‐mediated transformation of other major cereals.
Amy Johnson
Plant cells are different from animal cells. Plant cell has a rigid cell wall. Agrobacterium is just an essential vehicle/tool to 'transfer' foreign genes (including CRISPR-Cas9 components) into plant cells. In plant biology fields, most researchers deliver CRISPR-Cas9 components into plant cells using Agrobacterium, especially for dicot plants. In other words, to do CRISPR technology on plants, Agrobacterium is still needed for most of plant researchers.Of course, In some cases, people can use other delivery means. For example, we can bombard CRISPR-Cas9 components (either in DNA form, or Protein-RNA complex form) into plant cells using a gene gun. We can also transfect plant protoplasts (plant cells w/o cell walls) with CRISPR-Cas9 components using chemicals. This is pretty similar to transfect DNA into animal cells.
Ayaz Latif Siyal Your post came from a paper abstract (see attachment). It published in 2006. The review paper probably was reviewing plant transformation techniques invented during 2000-2005. Today (2020), the advance of plant transformation technology after at least 16 years have greatly improved some important crop transformation protocols. For example, many rice and wheat can now be effectively transformed with pre-mature embryos.
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