Most likely, some reagent in your PCR mix got contaminated by your plasmid, I wouldn try new batches of PCR mix, nucleotide triphosphates and DNA polymerase, and use pipette tips with filter plugs to avoid further contamination.

I agree with Pierre Béguin . As confirmation and during your clean up procedures you could run water as a blank which will also give you an amplified band if you do have contamination. Another area of contamination is the pipettes which will need stripping down and cleaning. As a start while waiting for new reagents borrow pcr reagents and plasticware and pipettes from another researcher and set up your test pcrs in another area of your lab while you clean up your area,change plasticware and reagents. You can get rid of the problem by designing a new primer set where one primer is outside of your current amplimer then the contamination will not amplify but that does not address the problem of how you got the contamination in the first place.

It means you have contamination. Toss ALL of your PCR reagents: water, primers, TE for diluting primers, enzyme, etc. Clean your work area really well too.

Start again with entirely new reagents.

Also, don't use the same micropipettes to load gels that you use to set up PCR reactions. That's the easiest way to get contamination and it's really hard to get rid of.

Hafiz Muhammad Rizwan

• That happens quite often. Except pipetman/reagents contamination mentioned, other factors can cause that too.
• Binary vector pCAMBIA1301/1302 are dedicated for Agrobacterium-mediated genetic transformation. After transformation, some resilient Agrobacteria (escaped from agrobacterium-killing agents) should still present. These Agrobacteria harbor the vector and can cause PCR false positives.
• I usually let the putative transgenic lines grow into plantlets and screen with GUS staining or GFP. Even for GUS staining, I test them several times at different locations of their leaves to make sure they are truly transformed. And use Southern blotting for final confirmation and find out transgenic lines with a single copy of the transgene.

Pierre Béguin Paul Rutland Katie A Burnette Yuan-Yeu Yau Thanks a lot for your answers,

I tried again by using the same reagents and material but this time I run the PCR at 26 cycles instead of 34 cycles (attached image file is the 26 cycle results), then I haven't got the bands in negative control samples, but the transformed plants show bands, so PCR conditions also matter for detection?

Yuan-Yeu Yau I will also double check by GUS and GFP staining,

I did the transformation of an empty vector (pCAMBIA1301/1302), so for GFP; will its location-specific or not? Can you please guide about GFP staining for empty vector (without target gene); as its my first time to do

Thanks

If this is pcr contamination then yes ..a small amount of contamination will be invisible for many cycles and only show at high cycle number. A water blank will distinguish between simple post pcr contamination and the very good explanation by Yuan-Yeu Yau

Hafiz Muhammad Rizwan < for GFP; will its location-specific or not? > ...

• I checked out the vector pCAMBIA1302 (see attachment). gfp gene is driven by 35S promoter, so it is not tissue-specific expression (or "location-specific"). But, you might encounter some transgenic lines with gfp gene silencing (which has been reported) or chimeric plants (ex. a single transgenic plant derived from 2 cells, one cell is transformed, and the other one is not). In these cases, some parts of the transgenic plants will either lack of gfp gene to express, or with gfp gene but no expression (gene silencing).
• GFP (Green Fluorescent Protein) is not for *staining*. GFP is a florescent protein. You need a florescence microscope to see it (see next post).

Hafiz Muhammad Rizwan Attached is one example of gfp gene expression in callus. After transformation, you can move your explants (ex. callus...) under a GFP microscopy for observation. You can see the 'transformed' callus with bright green and those not transformed callus. You can remove the transformed callus to another plate for further growing and regenerate it into a whole plant.

photo from: Article Factors Influencing Genetic Transformation and Plant Regener...

You should double check anout the primer designing and also PCR confition.

Paul Rutland thanks,

I will try by water blank then update,

Most Tanziman Ara Thanks , i will try it

Yuan-Yeu Yau Thanks a lot for your guidance,

I am using pCAMBIA1301/1302 vectors for overexpression study for cuticle wax genes, so can use the same vectors for transient expression? or need to design another vector for transient expression; if yes, then which vectors to recommend?

For cuticle wax gene overexpression study, which model plant is recommended; Tobacco/Arabidopsis?

Hafiz Muhammad Rizwan

1. Yes, you can use the same vector pCAMBIA1301 or pCAMBIA1302 as backbone. You can replace screening markers with your gene-of-interest. In your case, remove the gus gene from pCAMBIA1301 or remove the gfp gene from the pCAMBIA1302, and stick your cuticle wax gene into it. Your gene will be driven by a 35S promoter. Then, it is ready for transient expression. Of course, after that, you won't have a screening marker (GUS/GFP) for screening.

2. There are two common ways to do *gene transient expression* in plants: (1) PEG-mediated plant protoplast transformation, and (2) Agroinfiltration [where you inject Agrobacterium (harboring your binary vector) directly into plant leaves (see attachment)]. If you plan to do Agroinfiltration, you should use binary vectors (such as pCAMBIA vectors); they have the T-DNA borders (LB & RB) flanking your gene-of-interest for T-DNA transferring. For PEG-mediated protoplast transformation, you can use either *binary vectors* or *non-binary vectors*. Agroinfiltration is easier than PEG/protoplasts method, because PEG-mediate method needs to isolate protoplasts for experiment material.

3. The photo shows you how to do Agroinfiltration: 'Agrobacterium harboring your binary vector' in a syringe, push it into a tobacco leaves, mark the damped areas (for later use), observation in 2-3 days

Hafiz Muhammad Rizwan < ....For transient expression, which vectors to recommend? For cuticle wax gene overexpression study, which model plant is recommended, Tobacco or Arabidopsis? >

1. I have checked both pCAMBIA1301 and pCAMBIA1302 binary vectors. Attachment shows pCAMBIA1301 map. Both use kanamycin for bacterial selection (cloning stage), both use hygromycin for transgenic plant selection, both use 35S promoter for driving their screening gene (gus or gfp). And, both size is around 11k bp. So, there is not much different. You can use either one. Or, you can choose one which has better restriction sites for your cloning (to remove the screening gene and add your gene in).

2. Either model plant (tobacco vs. Arabidopsis) has its advantages or disadvantages. Arabidopsis takes less space and has short life cycle. But, for Agroinfiltration, tobacco plant is the #1 choice, because of its large leaves.

3. I wonder how would you use *transient expression* method to observe the phenotype change due to overexpression of your cuticle wax gene? The protoplasts are single cells and no cell wall. By using Agroinfiltraion, the injected areas will become dying in a few days. Unless you can see a result in 1-2 days if your gene is expressed by then....Or, are you looking for organelle-targeting of your gene (then, you have to create a fusion protein)??

Greeting

I think your problem is that you have some bands when you do not expect to see them.

One of my friends faced such a problem in a project. I asked her to change all of the materials and even the bench. But the band was seen again. I suggested her to wash all of her equipment like the racks(the tool that she used to put her vials during doing PCR test) and the samplers. Her problem solved. As alcohol can not remove DNA I asked her to immense her racked in Whitex overnight. And to open Eppendorf samplers, immense different parts in Whitex for several hours and assemble them. And calibrate them before using them again. Her problem solved.

Best wishes

Mahdieh Mehrab Mohseni Yuan-Yeu Yau Paul Rutland Most Tanziman Ara Katie A Burnette Katie A Burnette

Thank you very much for your suggestions on my question. I tried blank water to make sure, so the water blank shows bright bands which ensured that the reagents are contaminated. Then I tried again by using the new reagents with blank water, then it has no bands. But when I tried new reagents with negative control; the 35S promoter shows smear bands. so I tried to use 35S promoter forward and GFP/GUS reverse primers, so my problem was solved, the negative controls not giving bands now by using the one primer from the promoter and one from the screening gene.

But now having a new problem about GUS staining results (for double confirmation):

As some transformed plant samples showing the GUS bands ( used new reagents) and when I did the GUS staining by 1-3mm size leaf samples, kept in GUS solution for 24 h at 37 C, then washed several times with 70% Ethanol, till the solution was transparent and leave samples having no visible chlorophyll;

But after that there has no GUS reporter gene assay ( no any visible blue color, after GUS staining they not showing any GUS assay), so what should do?

Yuan-Yeu Yau

Sir; for overexpression, I have constructed both vectors pCAMBIA1301 and pCAMBIA1302 by cloning my Gene of interest into the MCS (lacZa) region of pCAMBIA1301 and pCAMBIA1302 by using BamHI and Xbal enzymes, so that's ok? so; can use those constructs for transient expression or not?

or it's mandatory to replace the screening marker (GUS/GFP) with my GOI?

as you mentioned above.

Yuan-Yeu Yau

Sir, I am working on two fruit varieties, in which one is susceptible to pathogen and another one is resistant, and wax genes are up and highly expressed in resistant variety, so now I have selected

those genes which have a direct or in-direct role on cuticle wax biosynthesis or Very long-chain acids biosynthesis (which are important for wax) and want to check overexpression in susceptible one, and also a comparison of overexpression in both varieties,

Along with overexpression work, I am considering to work on the function of those genes about the stress or pathogens behaviour/resistance, so for transient expression, I am considering to use the passionfruit at the ripening stage as mentioned reference article (The R2R3 MYB transcription factor MdMYB30 modulates plant resistance against pathogens by regulating cuticular wax biosynthesis) used the apple for transient expression and studied the expression of MdMYB30 was increased or repressed using viral vector-based transient transformation to confirm the function of MdMYB30 in enhancing resistance against pathogens in apple. First, they detected the transcription of wax-related genes at the injection point, and three wax-related genes were positively correlated with MdMYB30 expression (Fig. 7b).

So what you recommend about that consideration?

Hafiz Muhammad Rizwan < But after that there has no GUS reporter gene assay ( no any visible blue color, after GUS staining they not showing any GUS assay), so what should do? >

Did you use a positive control for GUS staining? Do you have known GUS-positive plants or seeds? any species. You can use them as positive control to see if your staining solution is good.

Hafiz Muhammad Rizwan

Greetings

I think you have washed your DNA with chlorophylls. Maybe the size of your leaves' samples is too small. Maybe it is better to perform PCR on unwashed leaves a couple of times. Do you extract your DNA by kit or manual protocols?

If you are using a kit, you should wash your beads tow time with nearly 50% injection water which is preheated to 70 oC. When we use the buffer, it helps to preserve our DNA from degredation, but it prevents PCR performance.

You should do PCR as soon as possible, I mean without freeze tawing the DNA. Because your DNA is very sensitive. I hope you can set up your test very soon.

Regards

Yuan-Yeu Yau

Sir; While using the MCS of pCAMBIA 1301/1302 vectors, whether, it is necessary to clone the insert of interest along with the promoter and terminator or not?

as I already inserted my GOI in MCS and constructed the vector

Yuan-Yeu Yau

Sir, I did not use any positive control while GUS staining, so I will try to get positive from other labs,

if; in case I don't found any known positive clone for GUS staining, so is there any other way to solve this problem

Mahdieh Mehrab Mohseni Yes; extracted DNA by CTAB method ( the concentration was 100-200ng with 1.2-1.5 A260/.A280 , and I used unwashed leave samples for DNA extraction and for PCR. so the quality is ok or not for only PCR detection?

by freezing and thawing DNA also degrated?

Greetings

Yes. Of course, it is enough. PCR test is a test for amplifying the DNA that exists in our samples. If the negative and positive controls work correctly, your protocol is not important. In fact, there are thousands of different manual protocols or kits. No matter which of them is using.

I hope you reach the desired consequence. Based on my experience you should reach it. Please let me know the consequence of your test.

Regards

Hafiz Muhammad Rizwan < Is it necessary to clone the gene of interest along with the promoter and terminator or not? >

Yes, your gene has to be in-between a promoter and a terminator. You need a "plant" promoter, meaning the promoter can work in a plant. The d35S promoter in pCAMBIA1302 works very well in plants.

Hafiz Muhammad Rizwan < In case I cannot find any known positive plant for GUS staining, so is there any other way to solve this problem. >

Yes, preferably, use GUS positive plant tissues or seeds as a control. You can ask around from other labs to request a such sample. If you cannot find one, I would use a little bit Agrobacterial clones [fresh, harboring pCAMBIA1301 (with a gus gene) or your cloned binary vector] as a 'control' and check it out..

Mahdieh Mehrab Mohseni Yuan-Yeu Yau I have optimized the PCR and now the negative and positive controls are working properly, those GUS samples showing the specific bands when I did the GUS staining ( i have used the known GUS positive plants, to make sure the GUS staining solution works properly), but I not got the GUS staining results in my samples ( which have specific bands), as i used the 35S forward and GUS/GFP reverse primer,

1- so what should i do to solve that GUS staining problem, even kept in the GUS staining solution at 37 C for more than 24 h, and washed many times with 70 % ethanol, negative control, and my sample showing the same colors after staining ( transparent),

2-I have BLAST 35S, GUS, and GFP primers (23-25 bp) on NCBI by specifying the organism as Passiflora edulis, so the BLAST results show > 80-95 % identity, and I have tried BLAST by using different region and whole regions of 35S, GUS and GFP on NCBI, so it shows identity, so what's that mean?

Yuan-Yeu Yau sir; while working on gene overexpression so a gene is transformed without a promoter, the gene will work for overexpression?

Hello

I am answering the question " while working on gene overexpression so a gene is transformed without a promoter, the gene will work for overexpression? "

Based on my knowledge, not only it won,t overexpress, but it will not express at all. Even when you have a good promoter, it is possible to not express. Because of the effect of silencers.

Hafiz Muhammad Rizwan < GUS staining ( I have used the known GUS positive plants, to make sure the GUS staining solution works properly), but I not got the GUS staining results in my samples >

Can you post a picture of this result? I want to see the "GUS positive plants" result. Thanks.

Hafiz Muhammad Rizwan

No. Unless, by any chance, this gene is randomly inserted behind a plant endogenous gene promoter. But, the chance is slime (with only a handful of bona fide transformants obtained).

Hafiz Muhammad Rizwan

Probably you did not get transformed plants at all, if the 'positive-control' plant tissue was stained blue and yours didn't......? gus is driven by d35S. It should work, unless they encountered gene silencing. But, only a small potion of plants will encounter gene silencing, not every one. How many putative transgenic lines do you have?

Hafiz Muhammad Rizwan

I asked a professor from USDA about the *lacZ-MCS* region on pCAMBIA1301/2. He confirmed: "The lacZ is a prokaryote promoter. Chances of it working in plants is slim to zero." The lacZa gene in the MCS is used for blue/white (colony) cloning. You can clone a *promoter::GOI* fragment into the MCS site if you want to keep all the other pCAMBIA1302 gene functional. Here, I attach a document as an example. You can see how s/he cloned a *promoter::GOI* into MCS site of pCAMBIA1301. The author cloned his/her X gene into 35S-Terminator in a plasmid (pRT101) first to form 35S-X-Terminator, then s/he cut 35S-X-Terminator fragment out and inserted it into the MCS site on pCAMBIA1301 (see attachment).

Mahdieh Mehrab Mohseni ok sir thank you very much

Yuan-Yeu Yau < Can you post a picture of this result? I want to see the "GUS positive plants" result. >

Sir, I have attached the gel results and the GUS staining pictures of only those plants which have visible GUS bands., as tried many times there GUS PCR and then after did GUS staining, so attached pictures are GUS staining picture of +ve known GUS samples, transformed samples in Gus solution (after 24h), _ve control plants, and transformed plants after 48 h in Gus solution and washing with ethanol,

Yuan-Yeu Yau

i have 30 plants in total

Hafiz Muhammad Rizwan

1. Looks like your GUS solution works pretty well.

2. In your picture, are plants in 24-h GUS solution treatment the same as plants in 48-h GUS solution treatment? Are they from the same plants?

3. Were those putative transgenic plants (in your picture) grew better in hygromycin-selection medium compared to those non-transformants? Do you have pictures?

Hafiz Muhammad Rizwan

Last time, I suggested that you could use Agrobacterium (with pCAMBIA1301) as GUS staining positive control, if you cannot find a known GUS+ plants as a control. But, later I found out the gus gene in pCAMBIA1301/2 has been engineered to contain a Catalase gene *intron* (see attachment). So, the gus gene should not express in bacteria (including Agrobacterium). It can only express in plants after the intron is removed. I am glad you found GUS+ plants as a control.

Yuan-Yeu Yau

Yes, sir, those are growing better, and have dark green as compared with non-transformed.

i attached the pictures

No, I meant while they were in hygromycin selection medium. Did they grow better in the selection medium (due to hygromycin-resistance), compared to those non-transformants? Did you use hygromycin (not kanamycin) for selection right?

I had one more question for you (above): " In your picture, are plants in 24-h GUS solution treatment the same as plants in 48-h GUS solution treatment? Are they from the same plants? "

Hafiz Muhammad Rizwan

Yuan-Yeu Yau

some died and some survived,

" In your picture, are plants in 24-h GUS solution treatment the same as plants in 48-h GUS solution treatment? Are they from the same plants? "

Yes sir, they from same plants,

I found a paper with Passion Fruit transgenic plants GUS staining. The variety used in the paper probably is different from yours. But, that should not cause big difference. Their GUS staining results were presented in the attachment.

• From the paper, I think they used transgenic plants from *tissue-culturing* stage for GUS staining, not the soiled ones. It should be easier for staining, because the wax of leaves should not form while they are in the tissue-culturing boxes, which is easier for GUS solution to penetrate. But, the transgenic plants in soil should also show some degree of blue color too, especially at the cut edges.

• Article A simple and fast Agrobacterium-mediated transformation syst...

Hafiz Muhammad Rizwan

Take a few roots for GUS staining from putative transformants (+ at least 2 negative-control plants). Treat them in GUS solution overnight. let's see what is going to happen. They don't have chlorophyll and easy to spot while it is blue.

Yuan-Yeu Yau thank you, sir, i will try to do GUS staining by using the roots,

before not used the roots as afraid to lose the transgenic lines, as limited transgenic lines,

another thing, if I will use the leaf as explants of those transgenic lines and try to develop callus through tissue cultures, so will they have GUS in callus?

Hafiz Muhammad Rizwan

1. Now, you have plenty of roots. You can use them.

2. Yes, the transformed callus will stain blue (with gus gene) [fig. c] or show fluorescence (with gfp) [fig. g], see attachment.

Yuan-Yeu Yau what will be recommendations about qPCR for transformed plants?

Hafiz Muhammad Rizwan

You should test your putative transgenic lines 1-2 more runs for GUS staining, using different leaves (young leaves). Are those putative transformed plants (in your picture) transformed with only empty vector pCAMBIA1301 (no GOI wax gene in it), right? You can try qPCR, because you don't have much choice now.

Yuan-Yeu Yau Are those putative transformed plants (in your picture) transformed with only empty vector pCAMBIA1301 (no GOI wax gene in it), right?

Yes sir, transformed with only empty vector pCAMBIA1301 (no GOI wax gene in it)

Hafiz Muhammad Rizwan What is the purpose of transforming only empty vector? Did you use it for practicing and learning?

Yuan-Yeu Yau

sir, at that time pCAMBIA constructs with my GOI were not ready but i was having time, so i tried to optimize the condition for transformation by using the empty vector through in planta method, so if that method is successful with high transformation efficiency so will use that method along with tissue culture/ ectopic expression for the overexpression study. so that method is easy as compared with the tissue culture

OK, understand. So, you did not use conventional method. That's probably the reason you did not get positive transformants (according to current evidence). So, have people used and published so-called 'in planta' method for passion fruit transgormation? Hafiz Muhammad Rizwan

Yuan-Yeu Yau

Sir, I haven't read the publication about passion fruit in-planta method, but there are publications in other plants (such as citrus, etc.).

Hafiz Muhammad Rizwan The in-planta transformation method is amazing (see attachment), without tissue-culturing. But, probably not every plant species can do the same. After I read the paper, I think the critical point is at the selection procedure. ex. Hygromycin or kanamycin selection. If the selection procedure is not proper, then many seedlings come from it will be *escapees* (escaped from selection). You also got many nice seedlings from the wounds as in Fig. h for your passion fruits?

Yuan-Yeu Yau

Sir, the publication reference you attached about citrus is also from our university student,

I have attached the figure of my seedling also,

Sir, if in case the antibiotic is not specific then what will be ?

Is there any relationship of an antibiotic with genes or the vectro>?; while using for bacterial or plants selections,

Hafiz Muhammad Rizwan

Can you elaborate what do you mean by 'not specific'? Not specific to what?

< Is there any relationship of an antibiotic with genes or the vector>?; while using for bacterial or plants selections >

What antibiotic did you use for your E. coli (harboring pCAMBIA with your wax gene) selection during cloning step? What antibiotics did you use to select Agrobacterium (after pCAMBIA plasmid were transformed into its competent cells)? What antibiotic did you use for transgenic plants selection during in-planta transformation? (I want to know your answer first.)

Yuan-Yeu Yau

sir, for example; a vector T-DNA region contains Hyg antibiotic, so which specific concentration need to use such as 50mg, or 100mg, etc and it depends on what?

Hafiz Muhammad Rizwan

Every plant species (even different varieties among a species) response to a specific antibiotic or herbicide differently. Most researchers have to test it out with a series of antibiotic/herbicide concentration. The citrus in-planta paper has suggested a specific concentration could be used for different antibiotics/herbicide. But, that is for citrus, not passion fruit. If the concentration is too high, it can kill all shoots, including the transgenic ones. If the concentration is too low, many shoots form will be non-transgenic, the so-called *escapees*. Of course, you can test the specific concentration they suggested first.

You have not answered my second question above.

Yuan-Yeu Yau sir, for PMD-T19 cloning, used Ampicillin as an antibiotic, and for agrobacterium used Kanamycin, and for transgenic plants selection during in-planta transformation used Hygromycin

Hafiz Muhammad Rizwan Yes, that is basically correct. Agrobacterium needs more than one antibiotic for selection, in addition to kan.

So, back to your question; " The answer is yes and is important. The 'hygromycin-resistant gene' will be used to produce protein which can detoxify hygromycin. So, transgenic plants with that gene can survive (under certain range of concentration) hygromycin selection.

Hafiz Muhammad Rizwan

Grow more plants, and probably you can sacrifice a few plants, and test the GUS staining at the 'f' stage in the attachment. To see if any callus is stained blue.

Yuan-Yeu Yau Sir, I solve the problem and GUS staining is ok,

I used the New GUS solution (from another lab) and kept it for 3-4 days and then after do the washing so now have good results,

Last time i did washing after 24 hours and also after 48 hours, so the results were not good, so maybe the passion fruit leaves were thick and need more time in GUS solutionm, ,

(attached the file)

Hafiz Muhammad Rizwan

That looks very good. Congratulations! How much concentration of hygromycin did you use for selection? Is the transformation efficiency high?

By the way, to increase the staining efficiency, you can put your leaf discs inside a plate with GUS staining solution (without a lid), and put the plate into a vacuum device (see attachment), and vacuum for a few minutes. It pushes the GUS solution into the plant tissue. After that, you can put it away for 1-2 days.

Yuan-Yeu Yau That looks very good. Congratulations!

Thank you very much, sir,

How much concentration of hygromycin did you use for selection?

100 mg/L Hygromycin

Is the transformation efficiency high?

Still, the experiment is in progress, after completion of the experiment can say about it.

By the way, to increase the staining efficiency, you can put your leaf discs inside a plate with GUS staining solution (without a lid), and put the plate into a vacuum device (see attachment), and vacuum for a few minutes. It pushes the GUS solution into the plant tissue. After that, you can put it away for 1-2 days I will try it

Thanks a lot for your guidance sir

Yuan-Yeu Yau Sir,

In Figure A, by using the 30 PCR cycles and the primers were as (35S promoter F primer + GUS R primer = 1607 bp, also same PCR conditions for GFP 30 PCR cycles, 35S promoter F primer + GFP R primer =951 bp ), In Figure B, only the PCR cycles were changed to 26, others conditions were same as like Figure A,

Fig A at 30 cycles no -ve control bands, as well as the GUS light bands and GFP 1st sample no bands, 2nd light band, 3, 4,5, bright bands, 6 no band, But in Fig B 26 cycles, GUS 1st sample no bands but 2, 3, 4, 5, bright bands, and 6th no band, GFP all samples, show bands as well as the -ve controls also,

Then I re-designed the primers and did PCRs, the results are in fig C and D,

The problem is that sometimes target samples show no band along with -ve controls ,but sometimes -ve controls show bands and the target no bands, even I have tried by using all-new reagents and even changed the PCR conditions., so still the problem is not solved through PCR,

But the GUS staining has good results, so can you please help me to check the primers whether the primers are not specific,

I have attached the primers details and the sequence which I have used.

Thanks a lot

Yuan-Yeu Yau Sir, can you please share a standard protocol for plant tissue preparation to visualize GFP?

As I also have seedlings with pCAMBIA1302 -GFP and want to do GFPvisualization,

Thanks sir

Hafiz Muhammad Rizwan Sorry to reply your earlier question < In Figure A, by using the 30 PCR cycles and the primers were as .....>

We discussed before, your PCR might be contaminated. Since your GUS staining worked out great. You should stick to this assay for checking out your bona fide transgenic plants. Most paper reviewers don't care too much about your PCR results (due to false positives). They are more interested in your Southern results (how many copies of transgene in each individual transgenic plants), GUS and GFP results, phenotypes, whether your transgenic plants are fertile or sterile, and if the new phenotype can pass to next generations, and whether these transgenic lines becomes gene-silencing (GOI) in later generations. Southern results also provide the proof that the foreign DNA fragment has successfully inserted into the plants' genomes.

Hafiz Muhammad Rizwan < Sir, can you please share a standard protocol for plant tissue preparation to visualize GFP? >

Because you just want to see if the gfp gene expresses, you can just view your tissue under a fluorescence microscopy. There is no standard protocol (depending on which microscope you have, you need to learn how to use it to obtain a nice picture). Unless you want to view GFP at specific subcellular organelles, gfp tagging experiments. Then, it needs a specific microscopy ("Confocal Microscopy") and a standard protocol to prepare tissues before you go viewing it. But I don't think you are doing that. Here is a paper for your reference (see below link). You can use it as an reference, although it mentions 'quantification GFP'. I think yours is easier-just want to see if the GFP present. Paper attached.

Article Quantification of GFP Signals by Fluorescent Microscopy and ...

Yuan-Yeu Yau < Most paper reviewers don't care too much about your PCR results (due to false positives). They are more interested in your Southern results >

Sir, you mean that I should also need to do a Southern-blot analysis of transformed plants. ?

Yuan-Yeu Yau

Thank you very much, sir,

I have tried using the Fluorescence microscope DMI8, so took an image showing the blue spots in the GFP transgenic plant's leaf samples,

Attached "GFP image" file showing the blue spots are from the GFP transgenic plant's leaf samples with fluorescent, while the "GFP leaf sample" image file is the same GFP transgenic plant's leaf samples without fluorescent,

Hafiz Muhammad Rizwan < Sir, you mean that I should also need to do a Southern-blot analysis of transformed plants? >

1. Yes, you should do Southern-blotting. That is an important piece of proof that your foreign gene has inserted into a genome. And tell people how many copy of the transgene is in every individual transgenic plant. Once we submitted an transgenesis paper with GUS and kanamycin (nptII gene one the transgenic cassette) selection proofs, but no Southern data. The reviewers returned our paper for Southern data.

2. You told me once that this primary experiment is just to see if the new transformation strategy works on Passion fruits. In this experiment you did not transform your gene-of-interest into your plant yet. You only transformed an empty vector into your plants. You can wait to perform Southern analysis on the following experiment which has your GOI in it. I know that the new transformation technique has not used on Passion Fruit. So, if you want to publish a small paper about it, you should include Southern analysis.

Hafiz Muhammad Rizwan < Attached "GFP image" file showing the blue spots are from the GFP transgenic plant's leaf samples >

• The color looks wield to me. And, why was there only one small spot on an entire leaf? The whole leaf should glow with fluorescence. The spot is probably some kind of contaminated 'object'.
• The green chlorophyll can affect you to see GFP fluorescence. Can you try using a root (no chlorophyll) sample to see for now. Put a transgenic root and a negative control root (side-by-side) and look under the microscope.
• Do you have 'positive' GFP samples? If not, you should ask around to see who has. Even seeds (with gfp gene inside) would work. Take a look at the gfp-positive tissues under YOUR microscope, to see what those positive tissues supposed to look like under your microscope. Then, you will have an idea.

Hafiz Muhammad Rizwan

You can see your own posts: the upper one (from the microscope company) is right color. Your color is like blue, not green.

Hafiz Muhammad Rizwan Attached is a slide from my old cotton transformation project. The good GFP color should look like that. The color in the picture was not not doctored or photoshopped. They are somatic embryoids regenerated from cotton calli. Of course, the GFP is much easier to spot because embryoids are not green like mature leaves. You can also cut a small section of your putative transgenic plants, and induce calli from them. Then, look for GFP on those calli, which is colorless and easy to see GFP.

Yuan-Yeu Yau

• The color looks wield to me. And, why was there only one small spot on an entire leaf? The whole leaf should glow with fluorescence. The spot is probably some kind of contaminated 'object'.

Sir, i will try again

• The green chlorophyll can affect you to see GFP fluorescence. Can you try using a root (no chlorophyll) sample to see for now. Put a transgenic root and a negative control root (side-by-side) and look under the microscopy.

I am worried as the plant roots will affect and maybe plant will die, so thats why used leave sample

• Do you have 'positive' GFP samples? If not, you should ask around to see who has. Even seeds (with gfp gene inside) would work. Take a look at the gfp-positive tissues under YOUR microscopy, and get an idea what those positive tissues supposed to look like under your microscopy. Then, you will have an idea.

I am trying to find the +ve GFP samples, but these days due to the Chinese new year holidays so students went back home,

Yuan-Yeu Yau You can see your own posts: the upper one (from the microscope company) is right color. Your color is like blue, not green

Sir, thanks i will check about it

Yuan-Yeu Yau

Thanks a lot, Sir for the suggestion,

Southern-blotting for all the transformed plants for example, if I did transformation in 30, or more 100 or less, etc, so should do Southern-blotting

for all? or only for selective plants?

2. You told me .......................So, if you want to publish a small paper about it, you should include Southern analysis.

Yes, sir, I want to make that publication about a protocol on passion fruit, because GOI transformation still needs time.

Yuan-Yeu Yau

Sir, if cut a small section of putative transgenic plants for calli induction will need sterilization?, so while sterilization of those small sections (explants) with 1-2% NaClO/75% Ethanol will have effects on GFP or not?

Hafiz Muhammad Rizwan < Southern-blotting for all the transformed plants? For example, if I did transformation in 30, or more 100 or less, etc, so should I do Southern-blotting for all? or only for selective plants? >

No, not all of them. Too much work. You do GUS or GFP screening first on the T0 putative transgenic lines, and select those GUS- or GFP-positive transgenic lines to do Southern blotting later. Eventually, you want to use the transgenic lines with 'single-copy' transgene for T1 generation test. Transgenic lines with multiple copies of transgenes might encounter 'gene-silencing'.

Hafiz Muhammad Rizwan < Sir, if cut a small section of putative transgenic plants for calli induction will need sterilization?, so while sterilization of those small sections (explants) with 1-2% NaClO/75% Ethanol will have effects on GFP or not? >

Yes, you need to sterilize your explants. To induce calli, first you choose and cut some small explants (depending on which explant is the easiest to induce callus ex. leaf or stem or root...for passion fruits [check literature]), then sterilize them, then place them in callus-inducing medium. After certain days, the callus might form from cut edges. Then look them under your microscope. If you don't sterile your explants, and put them on medium, it will become contamination with bacteria and other dirty stuff and kill the explants.

Hafiz Muhammad Rizwan Do you know passion fruits can produce at least small amount of callus clumps? Otherwise, you won't have enough materials to view. This paper (tissue culturing of passion fruits) said they obtained transgenic plantlets through directly' shoot organogenesis'. I did not see obvious callus forming in their paper. Figure is from the paper (below link). Please check it out. Article In vitro regeneration of Ugandan passion fruit cultivars fro...

Yuan-Yeu Yau

Sir, yes; can produce callus, attached is my tissue culture experiment picture about passion fruit callus and shoot regeneration,

I have been trying to optimize in vitro regeneration protocol for passion fruit (by using different explants and PGRs); which can be useful for transformation also in future.

Hafiz Muhammad Rizwan Ok, good. How long does it take for the calli to grow to that size shown in the picture?

Yuan-Yeu Yau Ok, good.

Thanks, sir

How long does it take for the calli to grow to that size shown in the picture?

More than 2.5 months

Hafiz Muhammad Rizwan Ok. And how long you can start to see the callus forming?

Yuan-Yeu Yau how long you can start to see the callus forming?

within a month

Hafiz Muhammad Rizwan Ok, let's take a look at 2 and 3 weeks after you start callus induction. Also, continue trying other tissues. The microscope user's instruction is at:

https://www.bostonind.com/leica-dmi8-widefield-live-cell-time-lapse-motorized-fluorescence-microscope

Yuan-Yeu Yau ok sir, thanks a lot

Yuan-Yeu Yau

Sir, from the previous discussion about the Transient expression by using pCAMBIA1301 and pCAMBIA1302 with wax/fatty acids target genes driven by 35S promoter.

1- which Tobacco variety to recommend a) Nicotiana tabacum, b) Nicotiana benthamiana? or both are ok? 2- While Agroinfiltration: how much volume should inject at a time and for one leave how many points?

3-Agrobacterium harboring my binary vector' in a syringe, push it into tobacco leaves, mark the damped areas, so it also needs to adjust the OD and which OD recommended for high efficiency?

Hafiz Muhammad Rizwan

1. < which Tobacco variety to recommend a) Nicotiana tabacum, b) Nicotiana benthamiana? or both are ok? >

Both are ok. I use Nicotiana tabacum. Worked fine with me.

2. < While Agroinfiltration: how much volume should inject at a time and for one leave how many points? >

Depending on the leaf size. I think I used 0.5 mL (it has been a while ago; you can review some literatures). I used 2 spots at 10 cm leaves. You can also use 1 spot per leaf if you wish if you have many leaves to play. I won't inject too many spots on a leave. The leaves might not survive.

Hafiz Muhammad Rizwan

3. < 3-Agrobacterium harboring my binary vector' in a syringe, push it into tobacco leaves, mark the damped areas, so it also needs to adjust the OD and which OD recommended for high efficiency? >

Yes, you need to adjust the OD. And what solution you should use. Since it has been a long while ago for me. I need to dig out those info somewhere in my notebook. It might take a while to get back to you. You can simply find the protocols online. Many people are doing it.

**What are doing transient experiment for? Interesting to know.

Yuan-Yeu Yau Thank you sir,

For that point has not decided yet, but according to the reference articles such as in Apple; they used Apple fruit along with pathogen for transient expression so my study is almost the same, so maybe I will try on Tobacco leaves, along with pathogenic fungus for there expression and observe its influences, and also passion fruit,

so do you have any recommendations for me about transient expression, ?

Your suggestions are highly appreciated

Hafiz Muhammad Rizwan < ....pathogen for transient expression so my study is almost the same, so maybe I will try on Tobacco leave >

I am a little bit confused. What did you mean by "pathogen for transient expression"? Do you mean *challenge* the fruit plants with a specific pathogen, than see some specific gene expression? I think they are two things. One is for "challenge a plant with a pathogen" and another one is "Agrobacterium infiltration". You need to define them well, so we can understand your research goal.....So, what tis the research goal before I can answer your question above?

Yuan-Yeu Yau

Sir, The objectives are as follows:

A) Overexpression of wax/fatty acids related genes, their expression, role in cuticle wax/fatty acids biosynthesis and comparison among two varieties,

B) Role of those genes in biotic/abiotic stresses,

< ....pathogen for transient expression so my study is almost the same, so maybe I will try on Tobacco leave >

Transient expression using the passion fruit/ Tobacco by challenging with a pathogenic fungus, and check the behaviour of genes against pathogens whether those genes will contribute towards fruit cuticle wax biosynthesis or not.

Such like the following reference

• DOI: 10.1186/s12870-019-1918-4 (

The R2R3 MYB transcription factor MdMYB30 modulates plant resistance against pathogens by regulating cuticular wax biosynthesis)

Hafiz Muhammad Rizwan

You have several things are going on, that was why I was a bit confused from your questions. You have:

(1) overexpression of genes (*stable transformation*)

(2) Binary vector Agroinfiltration (*transient expression*)

(3) *challenge* your plant 'passion fruit' and 'tobacco to see some genes' expression profile (nothing to do with genetic transformation)

Is the fungus you plan to use for challenging tobacco is a tobacco fungus (cause harm to tobacco)?

Yuan-Yeu Yau

Sir: The fungus also a source of tobacco leaf spots for example Alternaria alternate or Fusarium which causing wilt of Tobacco,

such fungus which influences both (Passion fruit and Tobacco ) will select and us.

Hafiz Muhammad Rizwan Thanks for the info.

Yuan-Yeu Yau Welcome sir,

Thank you too for nice sharings,

Sir, Can you please answer my following question, Thanks

https://www.researchgate.net/post/Can_use_Unigenes_from_RNA_sequences_for_overexpression_studies_transformation_without_requiring_a_specific_plant_genome