Anyone Here into Micropropagation?

[migraineur]

Hi, guys.

Is anyone here into micropropagation?

I'm interested in starting but wanted to see if anyone could provide me with any hints or tips about where to start or get supplies etc. Below are some of the kits that I found whilst searching Google.

http://www.texashydr...?productid=2414

http://www.hometissueculture.org/

http://www.kitchencu...it.com/kits.htm

So, I've made a list of places for tissue culture supplies to make things easier for us. This list can also be used for mycology. It is like an updated and edited version of the mycology supply thread I made ages ago.

Plant Tissue Culture Supplies - http://www.austratec.com.au/

Lab Gear - http://www.wiltronics.com.au/

Lab Gear - www.labtek.com.au

Lab Gear - http://www.livingstone.com.au/

Lab/Myco Gear- This place sells agar, parafilm and other useful tissue culture supplies. The synthetic filter discs are very handy as well as the breathable foam plugs for making clean media etc - www.mycosupply.com

Cheap isopropyl alcohol and some other chemicals - http://www.altronics.com.au/

Mason Jars - This place sells proper mason jars. The synthetic filter discs from Mycosupply fit these jars perfectly which allows for contaminant free gas exchange. However, you could cut the discs up to make your own filter discs of various size for your own lids - http://www.redbacktrading.com.au/

Jars - http://www.plasdene.com.au/

Syringes - This is a Perth feed store that sells syringes in bulk which helps save money. It might sell some other handy equipment but I have not checked yet - http://www.bio-john.com.au/

Cheapest Pressure Cookers - Amazon.com

Edited December 13, 2012 by migraineur

[GoOnThen]

Cool links I will read through them later. This is something that I have some interest in and hope to attempt in the future.

Here are a couple of links

http://www.shaman-au...showtopic=26366

http://www.shaman-au...l=&fromsearch=1

http://www.shaman-au...=1

Monk has done some interesting stuff with cacti as well at the nook

http://www.shaman-au...l=&fromsearch=1

Una from the shroomery

http://www.shroomery...83/fpart/1/vc/1

There is some interesting Youtube vid's on TC as well

My memory isnt as bad as thought it was

Cheers

Got

[migraineur]

Thanks for the links. I was engrossed in the articles by Una. It had some links to books etc too.

[Darklight]

Hi, guys.

Is anyone here into micropropagation?

 

Happy to help, I've been running tissue culture experiments for over a decade, I'm right about now leaving to run a workshop with an Ag crew. Do a username search or search micropropagation on the forums

Best if you post yout questions here unless there's a need for confidentiality for some reason

Not sure if this should be in Sustainable Technologies tho, unless you're planning to do some conservation work on rare species or similar. Should we get this thread moved to Ethnobotany?

cheers

DL

[migraineur]

How did you get started and where do you get your supplies from?

I already have a pressure cooker and some other things that would come in handy for micropropagation such as a dissection type kit for taking tissue samples from mushrooms. I also have petri dishes and some other bits and pieces. A kit can be preferred for a beginner but sometimes you end up paying more as a "lazy man tax".

From what I've seen online some people use homemade glove boxes to keep things sterile but they can be inconvenient to work with and I am not sure how effective they will be for the hobby. I'd like to get my hands on a laminar flow hood but it seems to be a lot harder to get my hands on a small, well priced flow hood, especially in W.A.

HELP ME, DARKLIGHT!!! =P

Regarding the location of the thread, I was tossing up between Ethnobotany and Sustainable Technologies.

[Bigred]

im in the same boat i wish i could get my hands on a small amount of media so i can play around with it first

if any member can sell me a little i would be much appreciated

[Darklight]

How did you get started and where do you get your supplies from?

I already have a pressure cooker and some other things that would come in handy for micropropagation such as a dissection type kit for taking tissue samples from mushrooms. I also have petri dishes and some other bits and pieces. A kit can be preferred for a beginner but sometimes you end up paying more as a "lazy man tax".

From what I've seen online some people use homemade glove boxes to keep things sterile but they can be inconvenient to work with and I am not sure how effective they will be for the hobby. I'd like to get my hands on a laminar flow hood but it seems to be a lot harder to get my hands on a small, well priced flow hood, especially in W.A.

HELP ME, DARKLIGHT!!! =P

 

Yay! At last questions I can answer!

First- do a search for tissue culture or micropropagation posts on this forum, there have been a few. One is at:

http://www.shaman-au...showtopic=26366

I got started by doing a TAFE course in micropropagation about 25 years ago. There really is no substitute at all for the hands on learning experience. There are a million things that the textbooks don't tell you and some you *might* be able to get a grip on via Youtube re. sterile technique. If you know of anyone who does mushroom culture ask if you can drop round to get tips. Mushroom culture is an excellent start to learning sterile technique for higher species

Sorry, your dissection kit is most likely not going to have long enough forceps, which could cause contamination problems

Second- do *not* spend any money just yet, unless you're going to buy forceps and a scalpel blade holder or disposable blades and handles. There is absolutely no need to buy a flow hood until you are sure you're going to maintain an ongoing interest, and you don't know that- yet. Flow hoods are a bitch to move around and careless handling or storage can cause seals etc around filters to warp. They can be hard to fit through doors in some cases. Yes they're great, but for small scale work and learning, they're not necessary at all. Owning one will not improve sterile technique if you don't have that yet, and it will piss you off mightily until you actually have good tech, cos you'll be cogniscent as to how much money you've wasted for no results. Reward yourself with one when you are confident

Get a perspex tunnel instead of a flow hood, just to start. Don't even bother with a glove box yet til you are good at sterile tech.

http://www.shaman-au...showtopic=32095

They rock.

You can use small chinese food containers for TC if you sterilise them first, I don't do it with the lids on b/c I've found them to warp- I autoclave ( pressure cook ) lids and containers separately in bags and hot pour the media under sterile conditions, leave to dry and lid. Using a perspex tunnel this could be a bit cramped, use small bottles like 250ml and keep them warm ( and tightly closed! ) in a hot water bath while you're waiting for the other container's media to cool under the tunnel. Ideally you want a good amount of air space in your vessels, no more than 1/3 media to 2/3 air

I get my TC supplies from Austratec in Melbourne. But really, give your sterile tech a good workout before you spend $$. That goes for you too Big Red.

Try this instead, it uses readily available ingredients and will show you how your technique is progressing:

http://web.archive.o...for_home_~1.htm

If you give this a go and can establish sterile cultures, post pics here and I will send you some MS basal media, enough for 1L media. Start with something simple like Acacia seed using the above protocol and see how far you get

Be prepared to make a lot of mistakes and not mind. It happens to all of us

The protocol above is a very very general protocol and may not work for your species. If you want more specific protocols for your species, use google. Those protocols mightn't work either, but you'll need to establish that before you can proceed any further, and any failures can give a good idea of what to try next. Plants can be very very specific in their response to TC, the fact that there is a protocol published just shows that it's possible- it doesn't mean that media will necessarily apply to your particular population. But it gives you a starting point and that's important

Don't use all your starting plant material in your first experiment. Divide it into three, that way if something goes wrong or needs to be changed you have more material to start afresh

When you're a learner it's best to only have one piece of plant material per container. That way if one contaminates, it won't take the rest of your hard work with it. If you're starting with plants from a greenhouse ( instead of say, seed ) you can expect a 70% contamination rate, but that's cool, the other 30% will be the stock you get to replicate and you won't miss the initial loss after a while once the plants start to replicate

And if you're starting from plant material, pretreatment of the parent plant prior to cutting it up and bleaching it is a really critical point for success. Tell me if you're doing this and need advice on it, there are another few paragraphs outlining that which mightn't be relevant for your situation

Post pics of culture contaminants here as soon as they're visible- what they are and where they form on the media is an excellent guide to how they got there and how not to let it happen again

Take good notes, You simply can't include too much detail when you're starting out, you'll learn to weed out stuff that isn't important later. Even notes on your emotional state ( I have a few old pages headed Hangover ) or doubts you had on media components or things you thought you might have done wrong can be incredibly helpful, especially if you follow them up later with more notes ( ie, the container I mentioned earlier I thought I'd contaminated actually is still sterile! ) they're your notes, not a contender for the Man Booker Prize. Fill yer boots.

And take pictures, phone cameras are a beautiful thing if you can keep them stable at higher zoom

Don't run too many experiments at the beginning, no matter how keen you are. It can take 10 days-6 weeks for you to get any discernable result and you need to establish what works for you during this time. Running ten experiments in six weeks ( unless it's your full-time learning thing ) is *not* going to give you time to check the results and change them so you get success the next time, and you risk getting disillusioned in the meantime

Ah- bleach. It's not just bleach you know. You want the unscented stuff. And you want it reasonably fresh. Bleach from a 4yo bottle just isn't going to be as strong as it says on the pack. Most plant sterilising protocols use 1.5% bleach ( you can add a teeny drop of unscented detergent to it ). That's *final* concentration, not 1.5% of the bleach you get out of the bottle.So if your bleach is 4.2% when you open the bottle ( check the label )you'll want to use a bit more than 1/3 of it to 2/3 volume water

Coolwhite fluorescent tubes are the standard for TC lighting, but they need to be changed about every 3 months as a part of the spectrum that we're not usually attentive to does degrade after a bit and effects plant growth. Lots of people forget this, it effects some species more than others

Mmm. sterile water for rinsing bleach off your initial cultures, you can't have enough of it. If you're initiating new cultures make sure you sterilise it well in advance of your culture work so it's room temp when you start to sterilise. If you don't, and autoclave it with the rest of your media, you'll be waiting ages for the rinse water to cool, which is time where your work is potentially becoming harder- media sitting round etc. When you sterilise water for storage, seal the lid supertight by stretching ( clean, not from the garage ) electrical tape around the lid so no bugs can get it. And keep it in a temperature constant room if you can, temp changes cause things made from different materials ( like bottles and lids ) to expand and contract at different rates, which is how bugs get in

If you or Big Red give this a go and can establish sterile cultures, post pics here and I will send you some MS basal media, enough for 1L media ( 10ml media good for 30ml tubes x 100, or approx 40 petri dishes, it can go a long way when you're starting ).

My last words for this post. Do not spend too much money right now. Get your technique right first, you can do that on the cheap. Buying spendy gear will not increase your chances of success one whit. But taking time to get your technique right will do far more for your success than all the shiny toys ever made

OK, I've raved on enough, hopefully you'll find this useful

[migraineur]

I've done agar work and I still have some blue oyster mushroom petri dishes that I made from tissue samples. I have the sterile technique down pat because of the mycology work that I've done. I have bleach, isopropyl alcohol, an alcohol burner etc. I also had to make my own sterile water and sterilise my instruments in a pressure cooker. The kit I have also has some long forceps and other things such as a scalpel that uses disposable blades. The kit looks as if it was made for something else originally but was also good enough to use for mycology.

I have a glove box that I made but it could be bigger. It's a pain in the arse to work in whilst doing agar work. I thought using a tunnel that isn't sealed off better would make doing agar work worthless due to contaminants.

I bought my gear from http://www.wiltronics.com.au/ which has an awesome selection of science products. The agar I got from a proper myco shop so it was lab grade.

I need to learn learn about using the right hormones etc that apply to micropropagation and the individual plants. I'll see if I can find a course in W.A. Thanks for the link to the micropropagation supply shop. I'm sure that will come in handy.

[Bigred]

just a few questions

darklight what spectrum degrades as i work in a hydro shop and can get my hand's on a vast variety of lighting

what is Inositol tablets and is there a substitute ( from the recipe in your link )

can you buy cellus's of anyone same as you would buy sterile fungi culture's

thanks for all the advice dark light i already have a mycology setup so i have most the equipment

[Darklight]

what spectrum degrades as i work in a hydro shop and can get my hand's on a vast variety of lighting

I'm sorry, no idea- somewhere in the PAR spectrum

Plants in culture medium only photosynthsise at about 10% of their normal rate because in most cases a carbon source is provided, like sugar, for energy so they don't have to waste energy converting it from light. So normal coolwhite fluro tubes are fine for most things

what is Inositol tablets and is there a substitute ( from the recipe in your link )

I'm pretty sure you can get inositol as a powder from a health food supply place. Use 100mg, the same as in preprepared commercial TC media. I wouldn't substitute it until you're more familiar with using the standard media

can you buy callus's of anyone same as you would buy sterile fungi culture's

Yes you can, there is one place in Germany at least, but it would be prohibitive to bring them in via Customs. And I'm not sure if they'd be in a condition to regenerate or be a recongised cell line for production ( callus cultures can be tricky to work with for this, wouldn't recommend it until you have had a bit of experience, unless you'e happy just getting sterile replication of blobby material which is pretty cool in itself )

Might be better, cheaper, faster to make your own from your preferred species. Just use a basic medim and add 1-4mg/L 2-4D. You'll get callus that way quite often and you can work towards regeneration by switching it back next subculture to a medium without hormones, cut the sugar back to half and if that doesn't work dilute the media back to 50% the subculture after that

I have the sterile technique down pat because of the mycology work that I've done. I have bleach, isopropyl alcohol, an alcohol burner etc. I also had to make my own sterile water and sterilise my instruments in a pressure cooker.

I need to learn learn about using the right hormones etc that apply to micropropagation and the individual plants. I'll see if I can find a course in W.A. Thanks for the link to the micropropagation supply shop. I'm sure that will come in handy.

 

Bloke, you are so there, keep going! If your technique is OK you mightn't need the course ( but I'd reccomend it to anyone ).

Hormone selection is the thing which bugs all of us forever. It's not just the selection of one or two hormones, it can also be the ratio between two hormones that's important. Don't let this daunt you

Basic rule for Angiosperms ( before it gets messy but it often doesn't )

Auxins: increase internode length, encourage root formation and larger leaves ie: IBA, NAA, IAA. 2-4D is a synthetic auxin often used to establish callus.

Cytokinins: overcome apical dominance and encourage axillary branching for bulking up plant numbers ie: BAP, Kinetin

There are many other hormones you can play around with but even these five most common will give you sufficient variables to keep you experimenting for years. I haven't had to go too far off the reservation with these after all these years and they're my first try for any new species

I make up hormone stock solutions and freeze them for about 3 months, replacing them at the end of it. Some degrade faster than others ( IAA doesn't survive under lights for too long once it's in media ) - 3 months is a rule of thumb most labs stick by

To make up hormone stock solutions, I'd do it at 1mg/ml for easy dispensing. You can use other concentrations for your stock solutions ( ie 1uM/ml ) but the above is good to start.

Weigh your hormone into your storage tube at 1mg/ml. For a 10ml tube- usually enough to keep me going for 3 months, use 10mg. Dissolve it in 1 or 2ml of IM KOH ( for hormones listed here except 2-4D, I'd use 95% ETOH for that )until the solution is clear. Then add water to volume so the total volume is 10ml. Use it and freeze when not in use. Write the date on it so you know when to throw it out

Migraneur, just start and send pics here. Seriously.

[Bigred]

thanks mate im starting to get some supplies together i was planing on using the urine jars the ones i have survive the pc

so i think they would be suitable and will use a fluro to light it . What would be the best plant for me to begin with

thanks heaps

bigred

[Darklight]

im starting to get some supplies together i was planing on using the urine jars the ones i have survive the pc

so i think they would be suitable and will use a fluro to light it .

Standard use is two tubes in parallel. about 20-30cm above your cultures

Do the urine jars have transparent lids? If not, take them out of the autoclave and close the lids firmly and dry them on a slant. Put your plants in 1/2way down the media slope and rest them at an angle so light can get to your plant

What would be the best plant for me to begin with

You know, I'd start with Acacia seed. Not phlebophylla, but something like obtusifolia, acuminata, floribunda etc. Soak the seed in freshly boiled water overnight, then sterilise for 10min in 1.5% bleach + a drop of unscented detergent.

Rinse them three times with sterile water in your flow hood and if you can, let them dry on a paper towel in your cabinet which you saturated with 70% alcohol and let dry. Once the paper towel is un your cabinet and dry of the alcohol it should be sterile and will be a good place to dry off the excess water before you use the forceps to put them in your culture

Some acacias can even go from in-vitro cuttings from such sterile seedlings in media without hormones for a few generations, but I'd stick with the home-tek protocol from the UNE link above and include some growth factors like coconut water or banana in the media after the seed has germinated and has 2-3 sets of leaves

If you do get replication, you can go for callus from there using the leftovers once replication has given you sufficient numbers and tek confidence. Never tried callus culture with acacia spp

[GoOnThen]

Thanks to all of you for the great info that has been written so far in this thread and especially Darklight for all of the posts/ threads that you have posted previously.

I have also worked with agar successfully and have the sterile techniques sorted and have most of the equipment required as well. My plan had always been to learn as much as possible from working with mushroom agar cultures and then move on to tissue cultures but at the moment I don't have the time or space so I can only read with interest how others are progressing with there projects.

Migraineur

It will be good to meet you at the meet on the weekend and talk more about what your plans are. My brother did a basic TC course a few years ago in Perth so I will find out where it was and what was involved.

Cheers

Got

[migraineur]

So, I updated my first post to include a list of places where we can get supplies. I am not sure if it should have its own thread or something. Please feel free to suggest more links, information or edits that may be required or be helpful.

GoOnThen, I look forward to it.