Tisssue culture basic qestions ( long )

[Darklight]

' Mornin!

I've had a few enquiries about basic tissue culture these last few months, and am more than happy to answer them. However since a good reply can take quite a lot of time and I end up repeating myself over several emails, I decided its best to answer any basic questions on the public forums where more people can contribute and learn about the process.

So if anyone has any basic TC questions for me, feel free to ask them here or in Chill. And please, keep us up to date with the results. We don't need to know the species you're working on if it's a private project. But if you can post pics as well of any progress or problems it will help everyone interested understand more

Thanks to the kind person who allowed me to publicly post details of this correspondence ( you know who you are )

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I've done mycellium tissue culture and comparing this to plant tissue culture it doesnt seemmuch more different.

It is *very* different. You'll find out once you start mucking around with minutaea in formula :-)

Could you please check my plans and tell me if anything is wrong with them?

Tissue samples will be obtained from existing household plants.

Make sure they're not flowering

Then pretreat them. Put them somewhere dry-ish and apart from other plants, and water them at soil level ( not overhead ) for a week or two to minimise fungal attacks. About a week before you cut them, hit them with some systemic fungicide *at the appropriate rate for the species*. Systemic fungicide is the go as it permeates the whole plant, not just sitting on the leaf surface.

The night before you cut them to TC, hit 'em with the systemic fungicide again if its OK to do it once a week ( depends on the brand of fungicide )

Leave enough plant material on your parent plant so you can do another run if you have a problem, and enough extra to allow the parent plant to live!

MS medium will be purchased

Check- both for your plant and your records- whether you are buying MS Basal Media, or MS Basal Salts. For some stupid reason these are similarly named in every catalogue in the universe and are easy to confuse even for the most advanced TC people. They are NOT the same thing. MS Basal media has macro, micro elements and vitamins. MS Basal salts has only macro and micro elements.

If one fails to work you may want to try the other

and placed into jars and autoclaved (pressure cooked) similar to preperation of liquid culture for mycellium (dextrose-water).

Absolutely NOT. Sorry. Prepare as follows...for a 1L sample

* Put 700ml water in a beaker or something.

* Add the media ( MS is usually a preprepared powder, only sometimes liquid )

* Add any extras- vitamins and shit first, weird stuff like coconut milk, hormones last. If you're not using any of these, omit this step :-)

* Add sugar- if it isn't already in your preprepared media. CHECK THIS! If you add sugar it will be between 10-30g/L

* Top up to 1L with more water

* check pH. Usually pH is around 5.8, but if you're following a formula or established protocol it may have a different value. Adjust dropwise with dilute HCl to acidify or sodium hydroxide solution to basify. pH is important- below 5.5 and many gels won't set after autoclaving. Above 6 and the plant may find it difficult to uptake some elements in the manner you would like

* Add agar or gelling agent( if it isn't already in your preprepared media- ( CHECK! in some formula it is!) -unless you're doing liquid culture, in which case it prolly isn't used at all :-)

* Stir it when you dispense or it won't be even. If you have a stirrer plate, great! If not, do what I did before I got one and stir continuously while sucking media up into a 10ml syrings. This works just fine but is time consuming.

* Fill so the volume of media = 30% of total jar volume. This allows enough air to circulate

* Autoclave for however long your media volume dictates. 10ml volume won't require as much time as 100ml- there is a schedule so check it!

1L media should be able to do you for 125 x 30ml test tubes at 8ml tube. Store what you don't use of the unautoclaved media in the freezer for up to six months

(question, does MS medium in liquid form have to be diluted, or is it designed as a product to be used straight-out-of-the-bottle?)

MS is usually sold in powder form, as are many popular prepackaged media

Under sterile conditions (in a glove box) the plant samples will be sterilized in alcohol

If you like you can risne under running water for three hours ( or more )providing the flow is controlled so it doesn't cause tissue damage. A hose from a tap going into a jar with holes punched in the lid is fine where water supply is plentiful, you can take the runoff and water the garden with it.

Use 70% alcohol only. Metho is fine!

You don't have to put it in the sterile conditions until it comes outta the bleach. Just make sure your hands and tools are clean

and washed with bleach

How long? What percentage? 1.5% is standard, but other percentages apply. Use fresh bleach, the stuff goes off after a couple of years. If you can't find 1.5% bleach, dilute some heavier stuff. Make sure there are no perfumes or any other shit in it.

Chuck some unscented detergent in with it to help disperse the bleach around. A pinheads worth is literally all you need for about 500ml of 1.5% bleach

then distilled water.

Twice! Wash it twice with sterile distilled water!

and placed into the MS medium jars

Which you will have prepared in advance the night before so they are cool once your plant is ready to cut up ( a not uncommon scheduling problem lol, done it myself til I learned better )

These jars will be rocked (still working on building a rocking table) until calluses have formed. The calluses will be removed in sterile conditions via a coarse seive.

*sigh* you have this thing with callus culture, regen via somatic embryogenesis. It's a great idea, but as a beginner this is waaay too much to start with. Even now I only work with it when I absolutely have to, and as a research tool you have to spend f*n ages mucking about with hormone levels unless you get it first go. Can't claim huge amounts of success with it.

Really, I'd stick to straight axillary proliferation for your first effort. If you can get uncontaminated material past its second subculture at this stage in your career, you're flying, believe me :-) There is more than enough to confuse anyone going on just getting this far and keeping your work sterile.

If you get contamination, and you may well on your first run, you need to be able to identify its source and type. That way you can make improvements on your sterile technique and go again if you have to.

Or if you must do callus culture, stick to doing it on solid media for a bit. You need to learn to recognise the stages of callus culture, which is hard until you actually see the changes with your own eyes, and will be harder while your soup is swirling around.

Additionally, liquid culture makes it much more difficult to spot contamination- like heaps more. Add to that the different results you can get with different amounts of oxygenation- so you need to determine parameters for O2 input- and you're setting yourself up to fail

If I'm wrong, I will eat my own cooking. I'd eat my hat, but it's made of some kind of carbon fibre, and the visor is difficult to grate. I'd hate to see you go to so much extra trouble uneccessarily, the more ppl we have working on TC the better

OK, presuming you are going to continue with your callus culture/liquid thing, I'm almost at a loss. I work with callus either directly initiated at first culture onto solid media, or that has spontaneously arisen as a part of my research. Never seived it, and have only worked with liquid callus once- to no avail, so sorry, can't help here

The calluses (i assume they can be seperated into smaller sections with a sterile scalpel) are placed into the same MS medium that was used to grow the calluses in, except the addition of agar for solidification, inside tissue culture jars (approximatly 8.5cm tall and 2.5cm in diameter).

The callus pieces are allowed to settle for a while and then the appropriate hormones are then added to induce vegitative and root formation.

No, you'll need to resubculture them to a new media containing the hormones you are after. Adding hormones sterile after plant material has grown can be a bit unreliable at best, and at worst toxic to plants What sort of lights are you planning to use at the various stages? What light cycle?

Have I got anything wrong?

Not really, seems to be a bit missing, and a tad ambitious, but not actually wrong!

Good luck with it, take pics and post them to the forums or gallery and lemme know how you go!

If you get contam, take a pic *immediately* If you get it posted here before it takes over I might be able to point out where it came from and you can fix the problem next time. Most contam shows up in 3-7 days. And some contam can overrun media and explants within hours

[ 08. August 2004, 19:25: Message edited by: Darklight ]

[Darklight]

These calluses can then be split and placed into new agar/media jars to form more calluses, which can then be used to form more plantlets, yes?

Yes it does work, but you're obsessed and I don't like your chances, but you can try. I'd just hate to see you waste your time

I was considering using malt and coconut milk as i heard these contain good hormones for rooting and vegitation, but I have to look into it.

Malt is fine, but make sure you use lab grade coconut milk- not the stuff in tins which has had god knows what done to it. You can make your own coconut milk from green coconuts only but it needs preparation- heating and filtering. Do a google to find out prep methods. Sometimes lab grade coconut water can be hard to find for some reason

Remember you can try too much at once. Especially since you will be doing callus culture. Start one batch of culture in maybe two media- at the absolute most, with only one variable difference between them. Check to see how the results differ, and if they do. If they don't, pick the media you like the most and increase or change the variable.

Run more than five media and believe me you'll be consfused unless its your full time job and you keep excellent notes

I'll attempt using some relatively simple plant I'll get out of my garden, then work up to more interesting things.

That's the way

[Darklight]

 

quote:

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Remember you can try too much at once. Especially since you will be doing callus culture. Start one batch of culture in maybe two media- at the absolute most, with only one variable difference between them. Check to see how the results differ, and if they do. If they don't, pick the media you like the most and increase or change the variable.[/QB]

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Or you can do these spectacular broad spectrum experiments- you need both a lot of plant material and a lot of media, as well as sufficient experience with technique to ensure all your work remains sterile so you get good data on results.

Basically you make up a grid of variables, and a few tubes of each media for every place in the grid. I think Ron de Fossard's work on this is still the definitive template. If you have the time, the media, the plant material and the skills, many university libraries will carry his book.

[Guest electro]

thankyou very much for laying all this info out in such a nice readable and understandable form..

the effort is greatly appreciated... d's faq version 1.0

[Darklight]

electro:

thankyou very much for laying all this info out in such a nice readable and understandable form..

My pleasure

you are suggesting taking say 20 tubes, and filling them with media and 20 bits of the plant material, one per each jar?

You got it! Not just any plant parts- try transverse leaf segments a la psychotria cuttings, nodal sections and active growing points like the tips of branches.

Hardwoods require more sterilisation but for all species you don't have a protocol already published for it can be hit and miss. Try the above sterilisation regime described using 1.5% bleach, shaking between 3-10 min.

If you've overdone the bleach, within 24 hrs the chlorophyll will disappear from the green bits of the plant ( it may live anyhow ). If it is underdone you will get lots of contamination

125 test tubes is a helluva lot to fill with starting material, and if your formula is fairly basic, why not try as many different plant parts and sterilisation times and methods as is practical

[Stonehenge]

I would think a log would be essential for a project like that. Memory is not going to work very well and putting everything on the label is tedious. I'd say to make an identifying number and some basic info like what it is on the label and keep the bulk of your notes elsewhere. You would want to record what it is, how and how long it was sterilised and any other data like medium components, how medium was made etc. If you find some are working great and some not at all, you could find through your notes the common denominator(s) and refine your technique.

Stoney

[Darklight]

Stonehenge:

I would think a log would be essential for a project like that.

You're 100% right Stoney. Also a log is useful for returning to when you're looking back to see what worked when you're starting a related species.

I usually put a code on the test tube lid, two letters for species, and two letters or numbers to indicate media. Any more and you're pushing it for space on the lid! If you use chinagraph pencils you can rub the lettering off with your finger when the lid is ready to re-use, and if you get different colour pencils you can further differentiate between batches of media in the same sterilisation run, or over time.

EVERYTHING is important to note, especially when you start out. Parent plant condition and pretreatment, sterlisation lengths and protocols, lighting, temperature and transfer date, and anything else that may come to you while you're working ( such as potential muckups- useful to check how they fared ). As you progress you will evolve your own shorthand.

I link species and media type codes back to a spreadsheet, printed with the date and the media formulation, and link those back to the notes I took, and they can all sit happily together in a special folder.

Another hint is to take pictures as you go- esp if you have a digital camera. It's too easy to fail to notice that something has grown, or think you're getting one type of result when in fact over time your memory plays tricks on you. I set up a separate directory for each plant, and then subdirectories for each transfer date.

[caludia]

Hi DL, apologies if this Q has been answered somewhere else. I did UTFSE but i am not very bright. Do you recommend any intro books on TC, for someone unable to get trained at TAFE, uni or industrially? Something comprehensive.. someone actually worth buying when money is tight

thanks.

[Darklight]

MetalGuru:

Hi DL, apologies if this Q has been answered somewhere else. I did UTFSE but i am not very bright. Do you recommend any intro books on TC, for someone unable to get trained at TAFE, uni or industrially? Something comprehensive.. someone actually worth buying when money is tight    

Having said that, you can check out the website

http://www.kitchenculturekit.com/index.htm for a whole world of startup information- there's a shitload of good pages for all levels avail via the links too

If you want an acutal book you can't go past a local production by Taji, Dodd and Williams, published UNE Armidale Plant Tissue Culture Practice it's still useful to me on a daily basis as well as being a good beginners practical guide. Or you can get Plants From Test Tubes by Lydia Kyte which is good on theory but not as useful practically

Of course if you want cheap books, go to abebooks.co.uk or somesuch, if it isn't there whack it on your wish list and one prolly won't be far away

***edited to add book title*** kiss kiss goodnight

[ 25. February 2005, 22:09: Message edited by: Darklight ]

[caludia]

thank you for this info, DL. That website is fantastic! And thanks for the book recommendations.

On a personal note DL, what would you say is your greatest satisfaction or triumph so far, in TC? Is there a certain plant which you are extra proud of TCing, perhaps because of difficulty, or perhaps because of being a pioneer (or both)?