Kitchen tissue culture tek

[Darklight]

I get quite a few requests for beginner's TC information, and rather than repeat myself I thought I'd post some basic stuff here.

This recipie will let *anyone* get started cheaply and without buying bucketloads of equipment and chems before they know what they're in for To test your skills re contamination and technique when you start, it absolutely can't be beat.

You WILL need an autoclave or pressure cooker, and pH tape or a pH meter. Sterilise your rinse water, optional paper towells and anything else at the same time as you sterilise your media or you'll be waiting hours when you're halfway through the procedure ( do it occasionally meself ). And the bleach thing could be more accurate- MOST protocols specify 1.5% bleach- the scentless basic supermarket bleach is preferred. So check your bleach container to see what the proportion of NaOCl is and adjust accordingly. Oh and ditto on the scentless thing for the detergent. Black and Gold unscented is fine

Once you have that under control, start looking at purchasing proprietary powdered media and some hormones

This is taken from " Plant Tissue Culture Practice" by Acram Taji, William Dodd and Richard Williams, University of New England Press, 1992. Once you've mastered the kitchen tek I also recommend you get this book- it contains a useful culture checklist to see what's wrong or right with yer offspring. And a whole world of useful stuff, I think it was about $30 when I got it but it may be available secondhand

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SIMPLE AND PRACTICAL METHOD FOR PLANT TISSUE CULTURE

Method 1

The following method is used by people who have no access to a research laboratory. All the ingredients can be purchased using a supermarket, chemist and a health food shop. The recipe is as follows:

1. Tap water: 2 cups

2. Table sugar: 1/4 cup

3. Stock: 1/2 tablespoon all purpose 10:10:10 (N.P.K.) water soluble fertilizer in 1 L H20: 1 cup of stock

4. Inositol tablets (500 mg): 1/2 tablet

5. Vitamin tablet with thiamine: 1/2 tablet

6. Agar flakes: 4 tablespoons

Mix the ingredients in a saucepan and gently boil until the agar has dissolved, stirring continuously. Dispense into empty babyfood jars or any other suitable jars, using a ladle, so that the medium is about 2 cm deep. Cover and process in a pressure cooker. Cook for 15 minutes after the pressure is reached. Wrap forceps and razor blades in aluminium foil and sterilize in the pressure cooker at the same time.

Sterile water is needed for rinsing plant material and sterile paper towelling to be used as a clean surface to work on. The manipulation can be accomplished within the fold of a towel while the folded half forms a protective hood which is effective against air borne contamination. When the operation is completed, the towel can be discarded and a new sterile surface selected from the sterile supply.

All plant materials can be sterilized in diluted domestic bleach, for example White King (1/4 cup of White King + 3/4 cup of water + 1 drop of detergent - detergent acts as surfactant). Put plant pieces in a jar containing the bleach for 10 - 20 minutes. Agitate frequently. Discard the chlorine solution and rinse plant pieces with sterile water. Small pieces, 2-3 cm long with a few leaves can be cut and transferred to agar medium. If the leaves are too large either remove them or cut them to 1/3- '/2 the size. Store jars at room temperature away from direct sunlight.

Seeds can be surface sterilized as above and germinated under aseptic conditions.

All instruments can be dipped in methylated spirit and flamed on a candle.

ASEPTIC SEED GERMINATION

Simple and Practical Method II

Select sound seeds and surface sterilize them as follows:

1. Place seeds in a glass jar containing dilute methylated spirit ('/2 cup of methylated spirit +'/2 cup of water). Agitate for 30 seconds.

2. Discard the alcohol and cover seeds with dilute white king ('/4 cup of domestic bleach + 3/4 cup of water + 1 drop of detergent), for 15 minutes.

3. Drain the bleach. Rinse seeds in sterile water once and soak in sterile water overnight.* Cover jars with their lids or gladwrap and store in a clean place.

4. Next day, transfer 5 seeds to each baby food jar containing medium (follow instructions from "Rough and Ready Method for Plant Tissue Culture I").

5. Store some of the jars in a dark cupboard and some in light (Temp. 25C +/-2C). Compare the rate of germination and subsequent growth of the seedlings after a week and again 2 weeks later.

* If using Acacia or Sturt's Desert Pea seeds, apply boiling water before soaking seeds overnight. This will loosen up the seed coat and aid germination.

PRODUCTION OF CALLUS/SHOOT/ROOT

Simple and Practical Method III

1 cm segments of hypocotyl or the shoot tip from seedlings germinated as in the above section ("Aseptic seed germination") can be grown to produce callus/shoot/root using the following medium:

To the basic medium as in Simple and Practical Method for Plant Tissue Culture - Method I, add '/2 cup of coconut milk and '/2 teaspoon of malt. Replacing the coconut milk with 1'/2 cup of green tomato puree or '/2 cup of freshly squeezed orange juice may produce different responses. Ensure that the pH of medium is always between 5 and 6 using narrow range pH tape or pH meter and adjust as necessary.

[ 14. June 2004, 17:48: Message edited by: Darklight ]

[mescalito]

Excellent thanks DL

[Salviador]

Yeah thats cool, i never knew it was so simple Well in theory anyways. I will give that a go.

[Darklight]

The theory is very simple, and easy to follow up on via observation. The reality of course is veeery different Start this lot off if you're interested and get back to me with problems- some are lucky enough not to have any and they get the sterilisation tek no worries( Daniel )

Use a stir plate when dispensing if you can, or stir constantly between dispensing so media is evenly mixed when it goes into tubes for autoclaving. A 10ml/20ml syringe with no needly makes an excellent substitute pipette and can be reused providing it is washed carefully immediately afterwards.

Oh, and one piece of plant only per tube when initiating cultures from whole plants- that way if you get a clean one, it doesn't get contaminated by its neighbour

None of this guarantees success, but axillary buds from a non-flowering plant in active growth carefully pretreated stands a higher chance of success of successful decontam and establishment- once you have that you get to muck about with the zillion different possibilities offered by media mixes

[Jojobafruit]

On top of things as always. Great info i think anyone could use this info for their advantage. Thanks you so much for posting this information for us. GROW ON PEOPLE !! Peace. jojoba

[bloodbob]

Once the thing has grown in your TC check out

http://www.shaman-australis.com/Website/Cu...reInfoIndex.htm

[Darklight]

Good point bloodbob- deflasking is as subject to experimentation as any other stage in the process. What works for one species/ facility might not work for another. Good to remember

[woof woof woof]

Hello Darklighty,

I am interested to know more about growth media.

for example if I was to do something like http://www.mushmush.nl/?page=homegalleryti...n_zien_vanaf=12

cactus callous culture

Are there any special things you recommend adding to the growth media? Like what type of hormones or vitamins?

Your professional opinion would be greatly appreciated.

[ 17. October 2004, 13:56: Message edited by: brian ]

[mesq]

wow hi brian long time no see

[woof woof woof]

hello mesqualero! Yes, I am still around,... unfortunately have not as much time as I used to. I am going back to school now and working.... ( paharmacy assistant is what I am going to school for ) Muhahhahaha

It is a very nice education btw. I learn alll about basic / standard laboratory procedures. Chemistry. I also learn how to cook medicines. Muhuahahahah And of course also allot about the medicines them selves. how they are or can be administered and what happens to them in the body.

Being involved with ethnobotanicals has given my a good head start on most of my classmates. ehhh also because I am the oldest in my class But it is fun and it suits me well. it is however very though combining work and school. I work 35 hours a week,.... and go to school from 7:30 am to 2:40 pm ( with hour & a half break divided over 2 periods) then I usually start work from 4 to 10pm. On Fri and Sat i work 8 hrs.

so all that is keeping me tied up these days.

I still find the time to lurk here at SAB from time to time. Hard to leave this place of insperation!

cheerios all!

brian

The job of a pharmacy assistant is to be able to prepare medicines

[Darklight]

brian:

Hello Darklighty

Sweetie! Good to see you back! Sorry for the delay in replying, tho, slack of me

Are there any special things you recommend adding to the growth media? Like what type of hormones or vitamins?

Everyone seems hung up on the callus culture thing. While its the fastest way to regenerate serious numbers of plants, it can also take the longest to work out a successful media for. There are literally millions of possible combinations! And only a handful of these will regenerate your callus back to whole plants.

Having said this ( yet again ) I would feel like a total fool if someone aced it the first or second go ( which is possible ) if I hadn't tried to be of assistance. If you insist on trying ( someone else was interested too a while back ) you should stick to solid ( agar based ) media initially as liquid media is harder to spot contaminants on, and these can kill your babies.

Once you have sterile cutlures initiated, such as a handful of sterile seedlings ( often easiest ), take one or two of these and initiate callus culture with a tiny amount of 2-4D- 1or 2mg/L is usually more than enough. When you have lots of undifferentiated cells clumps ( blobby stuff that isn't any kind of recognisable plant part ), transfer these to fresh media containing things like small amounts of kinetin or IAA. Or maybe even try regenerating into whole plants using no hormones at all- it can also work

To make up your plant hormones you will need to store the parent chemicals properly, find out what they are soluable in ( usually ethanol or 1N NaOH ) and toss 5-6ml of that onto say 100mg of your hormone and dissolve completely. Wear the usual protective gear, gloves and shoes etc all the while.

Top up to 100ml with water and dispense into smaller containers ( 1ml centrifuge tubes are great but 5-10 ml is OK too ) and bag for freezing. When you need an amount of the hormone, just unfreeze the minumum you need. Most stock hormones don't keep much longer than 3-6 months in the freezer.

If you have kept spare sterile cultures lying around after your first attempt, pat yourself on the back, because at some stage if an experiment goes wrong ( often ) you can go back and have new starting material to work with

After four years I still don't have a reliable protocol for regenerating Mammallaria craigii from callus, and I must have run through 20-30 different media types. Now I settle for doing heaps of callus culture and deflasking the odd random regeneration. I have maybe ten viable plants.

Go for it and good luck cob. All the best with your studies too

[ 24. October 2004, 07:37: Message edited by: Darklight ]

[woof woof woof]

ha ha ha ,... Hi DL,... after reading your reply I have realized I have some more studying to do on the subject. Also especially with gathering the required products and the preperation. I underestimated it.

Thanks again for your elaborate reply.

Doing frankenstein thingies to plants can be so much fun.

B.

[woof woof woof]

http://www.omnisterra.com/botany/cp/slides/tc/tc.htm

helpfull site on this subject

[Darklight]

brian:

helpfull site on this subject

Hey sweetie it is. good one!

These type of sites put things into perspective nicely.

TC at home is far from impossible and a lot of people ( me included ) have had success working there. All it is is a fiddly process which requires mutliple steps and attention to detail- like so many other things.

Piss easy, well yes, once you get the hang of it, and if you're lucky with your species. But not if you're slack, given to cutting corners, or prone to giving up easily. It's worth investing the time, and around $100 will get you started- less if you can get a perspex hood and already own a pressure cooker