Tissue culture hacks?

[seedjar]

So, I'm curious about tissue culture but I don't like handling chemicals - even garden store rooting hormone powders make me kind of uneasy. (Not to mention being on a budget.) Could I TC something easy, like Drosera roots, with just straight agar? How could I clean the samples before flasking, and how clean do they need to be? (I imagine the answer is somewhere in the neighborhood of really to super clean.) I remember klasac discussing this but I checked the thread and it didn't continue long. I know there are a number of you out there who are in the know.
Thanks everybody,
~Joe

 

[klasac]

Hi seedjar!
No, you cant make plants grow (from anything) just on pure agar. They need nutrition in order to grow and propagate. Imagine you living just off of water. You wouldnt last long Every TC medium contains basal anorganic salts, macroelements, then microelements and some has vitamins and hormones in it. You can successfully propagate some drosera on simple media without phytohormones, but never on plain agar. So if you dont feel comfortably handling chemicals, TC is not for you. For disinfecting jars and explants you will need to use some quite agressive chemicals, too.

 

[swords]

Joe, there's this excellent book here Plants From Test Tubes which explains "everything" you need to know on how to do things professionally or DIY.

I used to receive a huge catalog with lots of supplemental info from these guys:
http://www.phytotechlab.com/
They sell everything you could possibly desire for TC. Including small single-use kits with pre-measured chemical packets for CP (VFT) and Orchids as well as industrial size containers of chemicals Wistuba and BE probably use. IIRC they will also custom mix special media for you if you tell them what you want. Check out their site and see if they still do the catalogs, it was free and gives you a lot of info.

First things first, buy a large Pressure Canner/Cooker with a dial for sterilization. WalMart's PCs do not have dials (=useless ) I got a huge 21 quart PC at the farm store's canning dept for $90. This is your biggest investment for TC or doing anything that requires sterilization. Since you can't sterilize anything metal in a microwave and a microwave can easily overheat materials to the point of inactivity resign yourself to buying a proper PC right off the bat.

 

[Crissytal]

Why not just wear gloves when handling everything? That's what I do. I don't particularly like handling the stuff either, I'm yet to grow an 11th finger .

Like klasac said, there's more to it than just growing a plant in a jar. You need sugar, vitamins, hormones depending on what you are doing, micro and macro elements. I also use PPM (helps prevent airborne contaminates on the media) with all my cultures.

I would highly recommend getting a pressure cooker. I've never tried using the microwave for sterilizing media, but I have used it once for water. I will NEVER do that again. The microwave is just a huge pain all the way around.

swords, by dial do you mean a pressure gauge?

 

[EdaxFlamma]

You will also need to be very strict with overall cleanliness so as to not contaminate the vessels. In our lab we wash out all of our flasks with soap and water, RO water and then double distilled water. After that we autoclave our jars and media then pour the still liquid media into the flasks under a laminar flow hood. If we scratch our nose we have to rewash our hands haha. The biggest issue I see for a DIY type thing is keeping airborne particles from getting into your flasks and keeping your equipment (transferring tools, scalpels etc) clean. You can just do an alcohol dip but then the alcohol could potentially become contaminated for later dips, disregarding the sensitivity of some plants to it.

As for sterilizing the plants before taking cultures, that can get tricky just because again some plants can be sensitive. There are a lot of plant specific dips you can use but I know many people who use diluted bleach solutions for some of the more hardy plants (not CPs that is).

And if you do decide to play with TC in a home-type environment I will warn about dealing with contamination. Even though you probably won't, it is possible for mildly harmful bacteria/fungi to decide that your agar/plant is a nice new home. So if you do have a significant bit of contamination I would suggest something like a "double ziplock" deflasking. What you would do is keep the flask contained and place it in a ziplock bag (preferably 2). In with the flask I would pour in a nice little serving of bleach. Close the bag and then open the flask so that the open flask in contained in the bag but exposed to the bleach vapors. I would let it sit for a few hours just to make sure that it is adequately neutralized. If you want to be 100% sure (like me), after a while you could tilt the bag around to get some of the bleach inside your culture flask. We can simply autoclave our contamination but I would be wary about using a pressure cooker to sterilize a 100% contaminated flask, but then again I like to be 100% sure on everything haha.

But like the others I would definitely recommend grabbing a few books and doing a little extra research. All this being said it can be very fun and rewarding to see your cultures grow!

 

[droseraguy]

I'd go with the pressure cooker as well. Check some local farm auctions for them. I picked up 2 for $5 ! The one I use is giant and old but works well. It was the first heat-up and pressurization that was kinda nerve racking. I still have most of my fingers and have a cheap pressure cooker ta boot.

 

[seedjar]

Er, by agar I meant the premixed kit stuff you can get with some chemistry sets and educational toys and the like. I have some friends at the college who could probably help get me time with a vapor hood to do the flasking. I like to do things as simple as possible the first time, while I'm still learning. I have seen various homebuilt TC labs online and I feel comfortable with the ideas there, but I want to get it right the first time - early failures are bad for learning.
It's not so much that I'm uncomfortable with chemicals, Crissy, although I appreciate your warning. I just don't like fiddling with them - particularly hazardous organic chems that are often still dangerous even diluted. Once I get familiar with the process, I think I could maybe pull it off at home, but certainly not any time soon. For one, most of the tinkering I do at home happens in my kitchen. I'm having to scrimp and save just to do a little repainting at my house... building a lab won't be fitting into my budget any time soon.
Thanks for the tips guys - I'll definitely look into some books on the topic. Microbiology is big at my school so I'm sure there are a lot of good titles handy. I found some lab sheets for various high school and college bio courses online, and it sounds pretty straightforward to me.
~Joe

PS - Nice link swords - I didn't see the VFT kit though. It also crashed my browser, but I'll try checking it on the Windows machines at work tomorrow.

 

[swords]

Sorry about that! LOL

It's a been a couple years since I did anything so they may have different things nowadays. If you contact them they may still be able to make it for you or at least tell you what was in it so you can mix up your own. I'll scour my botany shelves and see if I have an old print catalog of theirs then you can ask them for the old VFT mix by product # (whatever it was).

Definitely buy a respirator and box of disposable gloves and tyvek arm covers. I wore a painters/concrete mixers double breather that really straps down good in several places over my head to avoid inhaling any sort of dust, fumes or exhaling any bacteria. The mask is only about $20 at home depot and gloves at pharmacy for $8 per 100.

The transfer box can be a simple clear sterlite container with a clear hood (to look down through) and 4" holes drilled into one side of it. There's a wire rack made for closets set on the floor of the box to keep tools up off the floor - that's all my hood was made of. I was going to do a whole box with a DIY HEPA filter but this worked out just dandy so why complicate? lol

 

[seedjar]

Thread resuscitation!
So I've got a access to a friend's flow hood now and also time on the one at the school if I work it right. He can walk me through preparing flasks but we need recipes for CPs. I've got easy access to seed from North American temperates, so that would be the most useful, but I'm definitely interested in Neps and other more exotic species as well. Are there any reliable recipes that use tissue other than seeds?
~Joe

 

[Crissytal]

Neps, Helis, Sarrs are near impossible to culture from tissue. Might as well label them as impossible for now. They have too much natural bacteria/fungus in their tissue. I would recommend starting from seed; it's slower, but is the best way.

 

[seedjar]

That's disappointing, but not too surprising. But I meant those as two separate inquiries - one to see if people had recipes for pitcher plants, another requesting non-seed methods for CPs in general.
~Joe