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[FewTrueSeed]

I recently have embarked on the most difficult task of in vetro plant propagation. I have a small farm and we grow many vegetables as well as medicinal and culinary mushrooms. A great success at the farmers market. My tropical houseplants, cacti, and succulents have a short season outdoors for the summer before they must return to the indoors. (Zone4) My attempts with propagating several plants in vetro, including african basil, begonia, and African violet, has been difficult. My sterile space is amateur at best but works well for propagating fungi.

I was wondering what experience people might have with micropropigation, and if i could get some pointers on how to further improve my amateur method. Including Agar Recipie, sterilization technique, and workspace.

I am a new member and am very exited to share what i know as well as share some seeds.

-Olin

2 Likes happyconcacti and ONandONandON like this.

[Frog Pajamas]

Hi Tragicfalacygtr2,

Don't know about micropropagation, but welcome to STS! Feel free to hop on the Welcome and Intro area to introduce yourself, and we'll get you set up with full forum access. 

Peace,
Frog

[BubbleCat]

Hey there, I cant associate anything with "micropropagation" neither "in vetro". But I am familiar with in vitro propagation, tissue culture and culture. In the end no one can tell you the exact technique you will be working best with. But one could give you basic tips, like:

experiment.
Alcohol is, opposed to common belief, best for sterilising at ~70%, not 100%.
I use a type of bleach that will basically break down to salt and water.
Good agar medium starts with good agar, experiment how much agar you need to "bind" a certain amount of water, if it needs less agar for the same amount of water, its good.
Consider UV lamps (cheap and effective) in addition to regular sanitising your laminar flow hood / glovebox.
Use a pressure cooker for sterilisation, again its cheap and effective.
There are many accepted agar recipies out there, free and online, many named after its inventor, like here for example: en.wikipedia.org/wiki/Murashige_and_Skoog_medium others have just been developed by someone experimenting, sharing his results online, a lot in "forum recipies" is probably BS but they work some are easy some are difficult, some are best for rooting this, others good for germinating that.
Too find your recipe you either go with one of those if your application iscovered, if not look for the application closest to yours, then research the needs of the organism and adapt the recipe, experiment.

If we had more precise information on what youre trying to "micropropagate" and how exactly "micropropagating" differs from propagation in vitro maybe someone can be found who already has done exactly what you want to do and be more help.

Welcome !

1 Like happyconcacti likes this.

[gnosis]

Welcome to STS!!!!!!!!!!!!!!!!!!!!!!!!
[/ :)move]

[BubbleCat]

did the ones shown above (didnt reaslise them being there in the first place) show signs of fungal or bactwrial attack yet ? Are you working with calli ? ... uhm callus material

If the idea is just getting used to and doing some in vitro growing I'd advise on going with full cuttings or seeds. Or callus material, which is a step ahead.

[FewTrueSeed]

Im trying to get callus formation from the above explants. The medium is a very basic formula from the book PLANTS FROM TEST TUBES. I was able to form callus on tomato seed explants but eventually they were contaminated. The above explants are still sterile but have shown no signs of growth. Time to make a better medium i think. 

[BubbleCat]

Initial growth should be noticable disregarding of your medium choice, as long as its not terribly f****d up, like petrol + agar agar + ph'ed water would do. Obviously the growth would starve soon but the initial growth should show up in very simple media. The other question is, does the plant matter in there come with callus tissue or have the abillity to form roots, personally I dont thibk so. The best would be to make a callus explant, keep it sterile with bleach and let it sit on a shaker, or maybe a old beaten up washing machine then more callus material will be formed, it can be seperated into many sterile dishes and if the shaking stops the cells will orientate and form specialized cells and finally plants.

[modern]

Placing the callus in complete darkness for 2 weeks helps promote/force growth and is actually recommended with some plants.

I've had mixed results with microprop but I will likely attempt it again in the future. I've use a 'glove box' as the work space without contams so it is not an issue however space and movement is limited (sight also not the best)

[BubbleCat]

Sadly contams never are no issue, even the best of the professionals have a failure rate :/ but yeah, working concentrated and thinking your procedure through before attempting can result in very low contamination rates. Also if observed early its always worth a try to rescue the culture.

[FewTrueSeed]

I have very little to no contamination rate in propagating mycelium so it must be my sterilization technique. Although i do what i read to be recommended. 70 isopropal, bleach 10-15 minutes each with a sterile water bath.  Ill try again soon. I have had them in the dark for some time. Maybie my explants should be smaller.

[BubbleCat]

In the end you can make everything inanimate absolutely sterile with a good procedure, no question. It gets difficult when its about killing the live stuff on live things, that are supposed to stay alive. The more fragile your plant, tissue or seed material is the more tricky it gets, but experimentation is the way to go, keep records with each culture about how long something was submerged in what concentration of xyz, and always rinse well and several times with sterile water.
This will be really difficult if your contamination is in the plant, like with a virus, but even for that theres methods to get rid of it, altho I never tried, just read about the theory.

I made the experience that good care and maybe eventual cleaning of the stock plant prior to taking explants greatly reduces sterilising effort. Especially dust, soil splashing on the plant and overhead watering can make a plant rather dirty. Luckily, you can take advantage of the plant being strong and healthy by cleaning it shortly before taking your material and waiting for the plant to recover from cleanig. Then take explants and sterilise them conservatively.

Have you tried "coco - water" in your agar ? It could help induce callus growth cheaply.

[ONandONandON]

VERY NICE

AgarAgar is new to me, haven't even tried yet, but i wanted to say, maybe try anti-biotic agar, for a contamination problem.

[BubbleCat]

Go to the asia shop of your choice and ask for some. It can be od poor quality (taking more than expected to make a jelly) but that doesnt matter in our application, just mix cook pour and experiment for simple rooting and shipping of cuttings its enough to cook and pH some agar, sterilise that and the cutting of your choice and ship it safe from drought and infection in a testtube. Is a nice first try to get used to it.

[modern]

Give PPM a shot. It is $10 for 10 mL but you use it @ 1mL to 1L so it make quite a bit. You need sterile technique but this helps especially if you dont have a hood.

[BubbleCat]

whats that ? Nutrients ? Rooting 'mones ? For cuttings ?