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ssdd
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sad.gif posted on 13-5-2007 at 16:56
Plant Culture Problems


I have been trying the past weeks to culture some plants on a gel based medium. I know that agar is the standard medium to use here but I have none available to me, so I have been trying to use a gelatin based medium. I am mixing 1 cup of water, some Glycerin, sugar, some urea, small amounts of magnesium sulfate and what ever hormones are necessary at the time. It forms the gel in the container and the plants seem to take well to it. I have it in a sterile positive pressure hood that I built and the plants and containers were sterilized before they were used with a 10% bleach solution.

The issue that I seem to be encountering is that after about 2 days of the plant part being on the medium a massive amount of water begins to form, the gel under the water stays intact and the plant continues to respond. There doesn't seem to be any infection of the gels. When to much water builds up (every day or so) i dump off the layer of water, sterilize the plant and place it back onto the gel. This has happened with every sample I have done so far and the plant seem to be still growing. Is this from the plant resperating out water? Or is something else happening here?

Thanks for any input anyone may have
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nitro-genes
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[*] posted on 14-5-2007 at 06:15


At higher temperatures and high humidity there will always be some condensation water when kept in a closed dish, but not in these amounts you are talking about. Best would be to switch to agarose (not as expensive as agar) and not hard to purchase.

I dont think you have an infection, gelatin gelled mediums are just not stable enough and phase seperation is very common. Especially when you have large amounts of ionics and other hydrogen bonding molecules like glycerin and sugar the protein will "salt out" pretty fast and loose its water upon which the water liberated will form a second layer on top. With agar and agarose this is much less of a problem even at higher temperatures, that is why they are solely used instead of gelatin as a medium.

Two days would be pretty fast to be caused by an infection, but gelatin beeing a protein IS susceptible to certain peptidases produced by numerous bacteria and fungi, so this would make long term growth on gelatin difficult anyway.

[Edited on by nitro-genes]
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ssdd
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[*] posted on 14-5-2007 at 14:23


Quote:

gelatin gelled mediums are just not stable enough and phase seperation is very common. Especially when you have large amounts of ionics and other hydrogen bonding molecules like glycerin and sugar the protein will "salt out" pretty fast and loose its water upon which the water liberated will form a second layer on top.



Hmmm you seem to be right. My guess would be that once this layer has formed there is very little that keeps this breaking down from speeding up. (if I am wrong please correct me) By "salt out" are you referring to the loss of water in the proteins?

Perhaps it is also the temperature I am keeping the mix at, I'll try refrigerating the water to see if it solidifies. It may also be neat to introduce a bit of moldy bread to see if I get the same effect with an intentional infection.

Does anyone know the proper ratios of nutrients and sugars that should be added to about 1 cup of medium? I have been guessing as I go, I know it is specific to the plant I am working with but is there a ball park estimate that most plants should be able to use. I have some other materials available to me if anyone feels I am missing something.

** After some reading on gelatin, let me post what I have found so far:

It has a melting point close to that of the human body temperature. (This medium would suck during the summer!)
It also has 9 Amino Acids in it that are essential to humans.
I'm out of time I'll post more later! :cool:

[Edited on 14-5-2007 by ssdd]
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[*] posted on 14-5-2007 at 17:33


Sorry for the double post but here is more Gelatin data for those interested...

Melting Point: < 35*C
The following Amino Acids are present: glycine 21 %, proline 12 %, hydroxyproline 12 %, glutamic acid 10 %, alanine 9 %, arginine 8%, aspartic acid 6 %, lysine 4 %, serine 4 %, leucine 3 %, valine 2 %, phenylalanine 2 %, threonine 2 %, isoleucine 1 %,hydroxylysine 1 %, methionine and histidine <1% with tyrosine < 0.5 %
The data above is from here: http://www.gelatin.co.za/gltn1.html

From just this small bit of data it seems as if gelatin based gels would be great for short term growth of different fungi and bacteria.
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[*] posted on 15-5-2007 at 07:00


For plants is usually used an agar gelled Murashige and Skoog salts based medium. (MS)
The composition of MS is listed below, some of the trace elements could easily be left out I think by using sterilized tap water as tap water will already contain a lot of these trace elements. only when you want to experiment with plant tissue cultures addition of sucrose and vitamins is necessary and usually root inducing hormones are added as well. For growing seeds you can best leave out the sucrose and vitamins, since it will make the medium less specific for plants and infections will become more likely...

MS medium: http://www.usbio.net/Product.aspx?ProdSku=M9503

I doubt though you could grow a large plant on a cup full of gelled medium as the roots would be pretty deprived of oxygen this way. For small plates this is less of an issue since the O2 can diffuse more easily over these small distances.

For fast growing bacteria and fungi cultures gelled media are not as good as liquid media. Gelled media are very good however for stroring isolated bacteria cultures very long without the need for a -80 degree fridge. For this take about 5 ml's of gelled media and inocculate the bacteria you want to preserve in the middle of the gel with a sharp needle (so without damaging the gel too much). Than grow for a couple of days at the optimum temperature of the bacteria until a nice colony has formed and store it at 4-7 deg. C. Stores for months...

[Edited on by nitro-genes]
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[*] posted on 15-5-2007 at 17:04


Just a quick update:

Since I have so much gelatin laying around I decided to try some experiments before I switch over to Agar.

1. I intentionally infected a gelatin mix to see if I get the water effect.
2. I made a batch with nothing growing on it, I sealed it and left it sitting to see if I get this water effect that way.
3. Lastly I tried making a more stable gelatin by adding a small quantity of flour to the boiling water, so far it seems to be a more solid gel than the mixes with out it.

Ill post further updates as they come.

Anyone have a cheap source of agar b y the way?

Thanks nitro-genes for the info you've provided. ;)
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[*] posted on 15-5-2007 at 17:47


Quote:
Originally posted by ssdd
Anyone have a cheap source of agar b y the way?


Try one of those vegetarian-health-freak-shops...;)
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[*] posted on 18-5-2007 at 02:14
Update


So I did a close inspection of my cultures and medium today.

The one that was infected with a damp bit of bread is thoroughly infected with both fungus and bacteria, but I am not seeing any degeneration of the gelatin based gel in this jar.

The jar that was sealed to be air tight also has had no breaking down of the gel.

The plant one that I originally had posted about is still producing massive amounts of water, but it seems as if just today the white fungus infection from the bread culture managed to spread its way over to this jar. (Which Im not sure how this happened because they have lids that are kept lightly on, and they are all in my positive pressure hood) :o

I am going to research where I can and see why the plants seem to be the only thing that causes this problem.
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[*] posted on 18-5-2007 at 02:50


A probability is that the root iself secretes some hydrolases or proteases that break down the gelatin. This is not unlikely, it is known plant roots exudate a whole range of anti-microbial and regulating compounds acting to protect them against potential pathogens. Hydrolases and proteases could possibly aid in doing so as well be involved in nitrogen take-up from protein rich substrates...
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[*] posted on 18-5-2007 at 08:48


Quote:

A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain. -Wikipedia


Yea seems you must be right. And it so happens that I had posted all that info earlier about all the amino acids gelatin contains.

Do you think that a gelatin mix would work better for perhaps bacteria culturing. As I have seen it works very well for fungal culturing, but i havnt a clue as to how to get a pure culture of fungus or bacteria going... Time to read on it I guess.

Thanks again for that input.
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[*] posted on 11-6-2007 at 07:18


Also, plants transpire in order to get the goodies from the gel to the leaves- they take up water from the medium and let it evaporate from the leaves.
Medium-grown plants never get very big, as they suffer from too much water on their roots, to little transpiration, etc.

For contamination I wouldn't really worry about bacteria, they are not very often autotroph- worry about green things growing in there- they appear often only after a week or two, but are a right pain in the but- once you see them you're too late, and you can throw away your culture.

Re-sterilising plants can't be good for them either, just pour off the water and get the lid back on, if this is done relatively quick it shouldn't cause too much problems...

What plants are you cultivating, and why do you want them sterile?




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[*] posted on 13-6-2007 at 08:01


I had been attempting to cultivate Pansies (or Viola tricolor hortensis)

I was using them as a test plant to get my gels properly mixed and to see if they would even work. Later on I plan to grow more complicated things like toad lillies and violets. Just trying this out of curiosity of plant chemistry, I find it facinating.

I dont need the plants to get too big, and it seems at some point they could be slowly introduced to the outside. (Where perhaps some hormones could be used to get a larger plant e.g. Gibberellic acid. )

Next time I'll have to try just pouring off the water to see what happens, I was hoping to keep them sterile to simply avoid contamination of my culture, some where I had read that this was the right thing to do...

-ssdd
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[*] posted on 14-6-2007 at 00:18


You can order plant agar and the like quite easily btw, from a company called 'duchefa' - they do everything when it comes to plant tissue culture, and will probably also sell to individuals. At the scale you're working at you can get along with really small amounts, 500g of plant agar will probably last you for ages.
I'm quite sure you'll have less problems with agar then with gelatin, its a lot more stable.
To whoever the person was that suggested that agarose is cheaper then agar- its not! Agarose is the pure compound that's used for DNA gels etc, while normal agar is less pure, but good enough to grow plants on.

I advise to do some research to the plants you want to cultivate, in articles you're probably able to find some information about the medium people generally use.

MS medium is the bog-standard thing, but not suitable for all plant species, as it is quite a salty medium- for Arabidopsis (that little shitty plant 90% of plant research is based on) grows well in 50 times diluted B5 medium for example.

To get plants from tissue culture to the real world is not very easy, as the plants are relatively weak after being pampered by living on a gel, at best you'll keep the air humidity very high for the first weeks by putting a cover over them, and gently let them get used to their new environment.

Let us know how you get on...

[Edited on 14-6-2007 by Taaie-Neuskoek]




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[*] posted on 14-6-2007 at 02:13


Thank You!

Duchefa seems to be a neat company with exactly what I am looking for. But I wonder about their prices since they are not posted... Also would I need to buy the MS medium with the agar, or do they come premixed.

I did some reading and the MS medium is the type of medium that would be used for most of the things I am doing.

Yea, it seems trying to get the plant back into the real world is where you are likely to kill it. From what I gather the stomata are always open when they are in a culture and must slowly be introduced to normal humidity or you dry them out.

Thanks again.

-ssdd
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[*] posted on 14-6-2007 at 07:04


you need to click on 'locations' and then 'usa' (presuming you're american)- select the product you want and there's the price.

I would advice to request the catalogue though, even if it only was because of the nice photographs in it (its free).




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[*] posted on 30-9-2007 at 17:35


Hello all, I have recently gotten the chance to do some tissue culture work here at school with the botany prof.

He said before we get started to find some background information. So I was wondering if anyone knew of a good reference to tissue culturing they could refer me to, or if they have it post it in references?

I recently ordered what I thought would be an interesting plant to try this with: Nepenthe Spathulata Do you think this will be a decent plant to work with?

Lastly I was asked to compile a list of chemicals that may be needed. I have Gibberellic Acid, Indole Butyric Acid, and some nutrient mixes, other than agar what might I need?

Thanks
-ssdd




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[*] posted on 30-9-2007 at 20:54


The plant culture gel recipe at the top of this thread seems to be missing phosphate and potassium.

Try this Google search for good prices on agar-agar
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