Protoplast isolation succesful ;)

[Mega]

Hi,

As the pectinase had finally arrived, I mixed some cellulase (around 10-20 uL) with pektinase (just some with a spatula)  in an 1.5 mL eppi. Then I added 8% xylitol solution (isotonic, that the cells don't burst from osmotic stress. I had no manitol like the instruction video said so I had to use that).


Incubated for ca 90 minutes, and then microscoped some of it. Find the pictures attached ;)


I think I will let the enzymes work over night with the rest, and then centrifuge it, discard supernatant, add xylitol solution. And microscope again.


Does anyone know if there are some protoplasts in it? Highest zoom = 400

I belive to see there are some cells of which you see the chloroplasts. But the ''chloroplasts'' might be as well be small entire plant cells in a sugar coating? 

[Dakota]

Cool stuff.  I put them in an imgur gallery so people can view them a bit easier rather than unzipping the files or downloading 1 by 1 via google docs

http://imgur.com/a/rLtTy#0

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[Sung won Lim]

I think you induced too much stress in the cells. I don't see any protoplasts. Which plant are you working with, and do you know how large the protoplasts will be?

-sung

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[Andreas Sturm]

Oh, thank you ;)

Well, I'm actually not sure that all of those tiny fragments are protoplasts, but I saw a few which definitively were some.

Anyway, as I said, I'll have a look at them tomorrow again. With centrifugation to get better results. 

(But for transformation, do you really need protoplasts, or will those semi-protoplasts work in reduced efficiency?)

[Andreas Sturm]

I used Stevia plant's leafes. Becuase it's quite the only plant in our house that is still green, and has not big leaves such as orchids. 


No ones? there are a few circles... Hoped, they were protoplasts.

[Cathal Garvey]

Images 3 and 4 look like they /could/ be protoplasts?
I think it's usually best to digest cells to protoplasts right before
use, then transform and plate on "recovery" medium, though the precise
steps evade me. We only did it once in my undergrad years and I haven't
touched plants at a cellular level since.

Sung, you got any protocols to share from P.patens?
PS Andreas, I like the use of Xylitol instead of mannitol! It's a plant
sugar, so shouldn't be very toxic, and it's bacteriostatic against lots
of common bacteria, so might help avoid excess contamination if it works
well..

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[Sung won Lim]

They might be... But in my experience protoplasts tend to be quite unmistakable in their feature.

I had samples like yours when I first stared learning about extraction and they didn't quite work for transformation. In my case it was ratio of enzymes and excessive mechanical stress that threw things off, when I corrected them I got perfect protoplasts from patens and arabidopsis samples.

Good luck!

-sung

[Andreas Sturm]

Maybe it was too weak concentration (8% mannitol equals 8% xylitol?) and therefore the protoplasts bursted?

[Andreas Sturm]

Ah ok thanks :)

Well I think I've thrown in much enzyme. Much wil give you much :D
-> too much?

[Nathan McCorkle]

On Thu, Nov 22, 2012 at 1:09 PM, Sung won Lim <4phl...@gmail.com> wrote:

They might be... But in my experience protoplasts tend to be quite unmistakable in their feature.

I'm no expert, and I haven't ever made these with plant cells, but you should be looking for perfect spheres with green inside them. Google images of protoplast look pretty distinct compared to most of your images, I think you need to grind up your sample better, and gently centrifuge it after the digestion and look for floaters in the upper areas of the tube. 

[Xabier Vázquez Campos]

3 & 4 look more like a bubble rather than a protoplast, specially if you consider the refringency.and lack of greenish or any internal stuff

41 and 42 are clearly ghosts. 16 and 17 could be partially digested cells as they are not so "squared", but hard to tell without a good reference of the start material

There are a lot of debris and the free green small "pearls" in #16 I would say are chloroplasts