Plant Engineering

[Ethan]

Greetings everyone!

I have not been on here is a while as I have been excessively busy with school and academic research. I am curious if anyone has any experience with engineering plants. My family has grown our own vegetables for as long as I can remember, and I would like to see if I can bring anything new to the garden. I realize that genetic engineering through Agrobacterium tumefaciens or gene transfection if a bit out of range for a DIYer, but there are some other techniques I am curious about. I know that there are several compounds that can be used to induce polyploidy. Some are hard to come by, but it seems that Oryzalin, a microtubule disruptor is sometime used as an herbicide. In fact, according to one study I came across, it may be more effective than colchicine, which appears to be the standard in the industry. Paper here: http://hortsci.ashspublications.org/content/43/7/2248.full

I know polyploid plants tend to have exaggerated fruits, but do you know of any other features I may turn up with if I attempt this (besides sterility with odd-number ploidies).

Another technique I am curious about is protoplast fusion. I was able to find some nifty instructions for home micropropagation by digging through some old posts: http://www.kitchenculturekit.com/StiffAffordablePTCforhobbyists.htm

That covers the culture part, but I am a bit lost on the whole fusion part. I did some protoplast experiments a while ago in a school lab, but I no longer have the protocol anywhere to be found. The basics seem pretty simple. Isolate the protoplast from cell walls by mechanical or enzymatic means, then you can use electricity or some chemical reagents to induce fusion. Would a crude lysate from some E. coli function as a decent substitute for cellulase? And what chemical means are there to fuse the protoplasts? Thanks for any help or direction you can provide!

-Ethan

[Avery louie]

You should try to get a hold of sung from genspace (check their website for contact info).  He probably knows something about this.

--A

-Ethan

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[Nathan McCorkle]

I remember in hybridoma techniques we fused mouse cells with
polyethylene glycol (PEG) of a few thousand molecular weight (MW), it
looks like it's the same for protoplasts:

a few paragraphs on protoplasts here:
http://www.rocw.raifoundation.org/biotechnology/MScBioinformatics/animalbiotechnology/lecture-notes/lecture-08.pdf

looks like a whole tutorial:
http://www.eplantscience.com/index_files/biotechnology/Plant%20biotechnology/In%20Vitro%20Culture%20Techniques/biotech_protoplast_fusion_hybridization.php

and finally a whole book with protocols references (published 1985):
http://pdf.usaid.gov/pdf_docs/PNABD686.pdf

got all these by googling:
fusion peg "plant biotechnology" protoplast filetype:pdf

also these:
http://www.btxonline.com/pages/FAQ.html#s

and another book, "Plant Cell Electroporation And Electrofusion Protocols":
http://depositfiles.com/files/37hsb7x03

On Thu, Jun 28, 2012 at 12:10 AM, Ethan <argen...@gmail.com> wrote:

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[Mega]

I'm also interested and wanna make some protoplasts some day... Maybe fuse an aplle tree with some tropical tree to make it winter-resistant and grow it in my garden :D

That'd also be the first step to agrobacterium transformation...



What I wonder:
I buy some MS medium. Plate the tissue (cells) on it.  Are the hormones for getting a whole plant (root formation, sprout,...)  in the medium or do I have to add them later??

[Conner Berthold]

I am also very interested in this topic. I have used tissue culture techniques before and I assume using similar protocols you would be able to manage it in a DIY setting. With regard to horomones the levels would have to be very low and watch out for side-affects (like how GA3 will cause rapid cell division but inhibit root growth.)

If interested in the TC side of it I would highley reccomend the book Plants From Test Tubes found here.

-Conner

[Dakota Hamill]

You might also want to check out the yahoo group hometissueculture, there are some pros on there and amateurs alike, and you can pick up some decent information, as well as see some neat photos.

http://tech.groups.yahoo.com/group/hometissueculture/

[Mega]

As for the plant engineering thing: 

You can fuse protoplasts to make chimeras of e.g. Pear+Apple or tomato & potato. 

But can you fuse plant protoplasts with fungus protoplasts? Has this been tried? 

[Ben Hunt]

I spent some time reading about modifying capsicum plants, and when you are figuring out whether you can do protoplast fusion, root culture, or any other micropropagation / transgenic methods, the first question is:

If: arabidopsis?

Yes.

Else:

Maybe. Probably not. Find a paper where someone demonstrated actually doing it.

If you really want to have a biotech party with some plants, get a wet lab setup and make some interfering RNA. No worrying about tissue culture, just inject it straight into the plant. Seems to work on a lot of plant types. I blogged about it a little bit but I am still working on my wet lab.

Good luck!

[Sebastian Cocioba]

If you guys need to know protocols for micropropagation of whatever species, just let me know and I will get on it. I do microprop for a living and media is cheap and I always have much left over. I can run hormone assays and protoplast fusions for anyone interested. I don't really trust many papers on microprop since few can be repeated. I just start with a large assay to find the proper concentrations of auxin/cytokinin for each microprop stage and only use papers to spot novel responses to odd concentrations and/or hormone varieties. Lemme know if yall need some data and/or tissue or whatever. I'm always eager to lend a hand. Happy hacking!

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[Matt Lawes]

Message for Ethan re Ecoli and cellulase (apologies if already addressed).  No E coli extract will have negligible cellulase. Growing a Trichoderma species on cellulosic material will probably work tho ... if you can't get cellulase per se.
>matt

Sent from my Verizon Wireless 4G LTE DROID

-----Original message-----

[Andreas Sturm]

Sebastian, 

that sounds cool :)

I'd be interested in weeping willows, because immagine one of those glowing. 

https://www.google.at/search?q=weeping+willow&hl=en&rlz=1C1GGGE_deAT388AT388&prmd=imvns&tbm=isch&tbo=u&source=univ&sa=X&ei=uQ-FULulJorWsga0tICoAg&ved=0CC4QsAQ&biw=1680&bih=959

[Rikke Rasmussen]

You could also solve the problem of daytime vs. nighttime gas exchange by using a CAM photosynthesizing plant - make a glow-in-the-dark Phalaeonopsis or Dendrobium orchid, remember to patent it, and voila (as they say). There are multiple articles on Agrobacterium-mediated transformation of orchids - and yes, do go with the Agro...protoplast fusion is nowhere near as trivial as it's being made to sound here - done on callus culture, and it is fairly easy to start one on solid medium w/ hormones from either seed or meristematic tissue. I did exactly this for my bachelor's thesis (Agro transformation of Dendrobium callus culture), so anyone looking for a walk-through is welcome to ask.

Apart from that, I still say the biggest problem is not transforming the plant, but figuring out  what to transform them with.

/Rikke

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[Cathal Garvey]

> remember to patent it

;_;

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[Rikke Rasmussen]

Joking, Cathal, joking.

It's a get-rich-quick scheme, and everyone knows you can't get rich without patents.

</sarcasm>

[Cathal Garvey]

Oh, you'd be within your rights in any case! "Making Cathal cry" isn't illegal..yet. :P

[Mega]

I'm gonna try to make protoplasts within the next week. I've got cellulase from my university, but there seems to be no pectinase left.

Can anyone guesstimate if this pectinase will work? http://www.ebay.at/itm/Pektinase-Enzym-Maischeverflussiger-50gr-/150737056744?pt=Spezial_Landwirtschaft&hash=item2318a0f3e8

It's for when you want to brew "beer" from apples and pears (we call that "fruit beer" Most in German).


And, especially: for how long the pectinase powder will work? Can I keep it 1 year without decaying? Because there's a lot of it in this package (that's fine because of shipment, but i it decays too quickly, it's wastement)

thanks ;) 

[Cathal Garvey]

Brewer's pectinase should work, but I imagine it's less pure than the
lab-grade stuff (if someone has access to both, a comparison would be
awesome!)

I've been thinking for a while now about bugging someone with more
protein-fu than I to help codify a method of separating cellulase from
those hippy digestive aid tablets you find in good hippy healthfood
stores; they often come with pectinase, cellulase, etc. etc., but also
lipase and a bunch of other potentially lethal *ases which render them
useless for plant protoplast engineering, most likely. A set of steps
you could follow to precipitate out unwanted proteins or otherwise
purify the cellulase/pectinase from these tablets/powders would be great
for DIY plant engineers!

Anyways, as far as viability is concerned trust the packaging. If you
need to test if your pectinase is working, you could always add some to
cloudy apple juice and see if it clarifies in a few hours or days! :)

Also FYI: in English Apple/Pear ferments are usually called "cider".

[Andreas Sturm]

Ah thanks.
Looked up cider, the german article in english say that cider is not wide-spread in Austria, we mainly have "Most" (which is closely related).
http://de.wikipedia.org/wiki/Most_%28Getr%C3%A4nk%29 for Most, there seems to be no adequate english homology, as there's not even a translation.

Doesn't relly matter, though, I don't drink that kind of stuff, neither beer, cider nor wine ;)


When applying just the cellulase and then vortex, will this set free at least some cells?  I'm gonna try that out anyway ;)


 

[Cathal Garvey]

It'll probably release some at least, or it'll weaken them for some
other process.. for full protoplasts though, you'll probably need pectinase.

Tip: protoplasts are apparently pretty fragile, so centrifugation could
potentially kill them. I've read about an old-school technique for
avoiding this, where you pipette a layer of sucrose-solution at the end
of the tube before layering the liquid to be centrifuged on top; this
creates a "density cushion" for your cells as they "land" on the surface
of this dense sucrose layer, and may lead to less cell rupture.

Could be bunkum nonsense, and could do nothing at all. Could even make
matters worse! But, there you go. :)

[Nathan McCorkle]

I've dissociated chicken heart before using trypsin, but they don't
have a cell wall so their main cell membrane may naturally be tougher.
I've also fused B cells before to make hybridomas and you've got to be
very careful with that, they're very susceptible to rupture. Same with
spheroplasts/protoplasts in e.coli... very fragile. I would imagine
you could just add whatever *-ase to some buffered media or even
growth media and wait, maybe stir gently with a pipet tip every 10
minutes for 30-60 minutes. Vortexing once a minute for 5 minutes would
also be a good 'other end of the gentleness spectrum' test to try.

got this by googling protoplast preparation, looks pretty good, says
to use shaking incubator for an hour or so
http://ivaan.com/protocols/128.html

here are relevant links too
http://en.wikipedia.org/wiki/Protoplast
http://en.wikipedia.org/wiki/Spheroplast

This looks OK too, $71 USD for 50mL
http://www.sigmaaldrich.com/catalog/product/sigma/V2010?lang=en&region=US

Viscozyme® L - A product of Novozyme Corp
General description:
Multi-enzyme complex containing a wide range of carbohydrases,
including arabanase, cellulase, β-glucanase, hemicellulase, and
xylanase

--
-Nathan

[Mega]

Got a new idea: 

What about retrotransposons? I read about them, and they seem to copy their coding sequence multiple times and integrate it into the chromosome. (like Copy and paste).

If the lux genes were within such a retorotransposon, that would give us multiple copies per cell, while just injecting the ~10 - 15 kbp !! 

I'm looking for annotated sequences, yet I haven't found any. But I'll keep going ;)