'Transient' protoplast transformation or 'stable' protoplast transformation?

Hi Yau,  its for transient transformation.

I checked this paper and looked how they prepared root material for protoplast isolation (see attachment p.385). You can follow their method to prepare the root material for later protoplast isolation. Then either use their enzyme solution or solution regularly used for Arabidopsis leaf protoplast isolation (http://www.nkato.biology.lsu.edu/methods/protoplastisolation.html ) to digest those root tissues. You should be able to get protoplasts from roots. You probably will not see green-colored chloroplasts, which present in leaf protoplasts (see Figure 1), from root protoplasts (non-photosynthetic green tissue).

Once you obtain root protoplasts, the protocol needed for root protoplast transfection should be similar to the protocol used for leaf protocol transformation.

Hi Yuan,

Thanks for the information

I already have standardised protocol to isolate root protoplasts.  The problem is transformation. I tried to use the same protocol that has been used of leaf protoplasts but it is not working. :(

Are you using a GFP, GUS...screenable marker or something else?

Yes, I am using GFP reporter. I used 35S:GFP as a control to check the transformation efficiency. After transformation, I observed hardly 2-3 transformed protoplasts!

So, you did successfully transformed protoplasts. Not many, but 2-3. The transformation is not high. I remember that I did not get high transformation efficiency for Arabidopsis leaf protoplasts, too. I used eGFP. Is your protoplast quality high (ie. many intact protoplasts)?

hi :)

If you have a high quality of protoplast, I assume your problem comes from the transformation step where you use PEG solution to open up your protos. I also had this problem before, hence I only make the PEG solution fresh with PEG 4000 (mine is from Fluka and works better than the other brands we have in our lab) and just before adding it to my protos (I do not even use the ones I made one day ago). Also, i believe the timing is very important. U should pick a time when the PEG can open the protos and before it breaks them all (i keep it for about 1.5-2 minutes for leaf protos but u should try about root protos yourself).

I hope you can get good results next time :)

Cheers

@ Shirin,

Thanks for your inputs.

1. What is your transformation frequency? Do you see many GFP-transformed cells or just a few cells under fluorescent microscope?

2. I did not do many batches of Arabidopsis protoplast-transformation. But, in general, I feel my transformation frequency is low. I used pre-stored (-20C) sterile PEG4000, not fresh. Maybe, this is one of the causes for low efficiency?!

3. Your suggestion seems that the PEG can break the protoplasts in a short time, and suggested use 1.5-2 mins for them mix together. However, in tobacco protoplast transformation, co-exist of protoplasts and PEG for 25 min seemed to have no problem (protocol suggests to mix them, and stand for 25 min before next step).

@yuan,

Hello :)

1. Yes you should see a high transformation frequency ( I usually repeat the experiment till i get a higher frequency- 1 out of 10-20 is a good number), this way you can trust your GFP signals better

2. When I started doing this experiment, the only two tips my senior gave me along with the protocol was: always make the enzyme solution fresh, always make the PEG solution fresh. Well I have tried storing the PEG solution at -20 for the next day but usually I get the best result when i make it fresh. You can also try this and see if it makes any difference in the outcome.

3. Yeah, for the arabidopsis proto, the PEG solution starts breaking the protos if you keep them for longer times. Hence I add the PEG to the proto+plasmid mixture and after 2 minutes, i start adding the W5 buffer every 2 minutes. maybe it's a different story about tobacco. The arabidopsis protocol has 2-3 times washing with W5 solution to remove the PEG as much as possible before leaving the protos for expressing the protein.

I hope this could help :)

@ Shirin and Yuan.

Thank you so much for the input.

 For leaf protoplast isolation and  transformation  this protocol works very well http://molbio.mgh.harvard.edu/sheenweb/reprints/ProtoplastNP07updatedOct13.pdf 

I will try to standardise protocol for root protoplast transformation. Here the problem is, since root PPs are transparent,  once I add the PEG I cant see them!

next week I will try again with some modifications and I will update!

Thanks

Mohan

@ Shirin,

1 out of 10-20 is a pretty good frequency. Thanks for sharing your experience. I think mine is about 1 out of 50-100.

@ Mohan,

Yes, we will wait for your update. Thanks.

By the way, why are you using root protoplasts, instead of leaf protoplasts, for transfection? Except the root protoplasts lack of plastids (chloroplasts)?

I found one answer about my question above from the attached paper.

The authors mentioned the reason why do people use 'ROOT' or suspension culture cells from Arabidopsis for protoplast production (see below, yellow highlight in the article). Is this one of your reasons?

"A low autofluorescence of cell suspension and root-derived protoplasts is of particular importance, when light emitting enzymes, such as the green fluorescence protein (GFP) or luciferases, are being used as reporter proteins in nondestructive in vivo gene expression assays."

@ Shirin,

Have you ever done protoplast transformation with electroporation, instead of PEG? Using electroporation for protoplast transformation is possible (see attached paper). However, some people suggested that the survival rate for electroporated protoplasts is low. I have not done it, so I am just wondering.

This may be interesting to protoplast people:

Attached is a 2016 paper about protoplast isolation. The interesting thing about this paper is that they can conduct a low-cost protoplast isolation. They used non-lab grade cell wall enzymes. See below, this is what they done:

1. .... cost reduction of 1000-fold compared to previous methods of protoplast isolation in switchgrass, with a cost of $0.003 (USD) per reaction for mesophyll protoplasts and $0.018 for axenic cell culture-derived protoplasts. 

2. ...... food-grade cell wall degrading enzymes Rohament CL, Rohament PL, and Rohapect UF may provide a low-cost alternative to lab-grade enzymes for protoplast isolation (Buntru et al. 2014, 2015). To test this hypothesis, isolation of protoplasts from switchgrass leaf tissue was tested using Rohament CL, Rohapect 10L, and Rohapect 

Root protoplasts is a quite good alternative to leaf protoplasts in some species, but not for Arabidopsius: the population have high heterogeneity and only few cell types are able to respond. Moreover, to compare with Arabidopsis leaf, root cells have very different cell wall thickness in different root zones.

Of course, one can use activated root tissue (after induction of cell division in pericycle, but it is not any more pure root protoplasts.

https://www.jove.com/video/1673/fluorescence-activated-cell-sorting-of-plant-protoplasts