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Tissue culture "directed study"!

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Eats genetically engineered tomatoes
[/begin useless background material] There's an uncommonly used way to sleep in at my school: Directed study! Basically, you come up with a subject that you're interested in, and if you find a teacher willing to give and grade assignments, you can make it a class. Put it during first hour, and do the work later, and BAM! You got some extra REM coming your way!

...Originally, and I'm being totally serious here, I tried to get approval to do a directed study in injecting chicken fetuses with food coloring to see if the chicks change color.

They GRANTED IT.

But my parents told me I should do something actually scientific, so I got permission to change "...injecting chickens with food coloring" to "Plant tissue culture".

Again, they granted it.

[/end useless background material]

So here I am. I've gotten my kitchen culture kit, I've read obscene amounts of information on the subject, and I'll be attempting my first "experiment" on Tuesday. I'll be doing everything in a lab at school, using a microwave to sterilize my unicorn vessels (and of course PPM).

My first experiment, which will also serve to get the hang of sterilizing stuff, will be comparing VFTs in different concentrations of MS medium (1/3rd, 2/3rds, and full concentration), to demonstrate if carnivorous plants prefer lower nutrient levels.

Comments, questions, or seasoned-veteran tips are welcome!

Thanks, happy growing! :boogie:
 
Dude, you should have sticked with the thing with the chickens...

A purple chicken sounds pretty scientific, scientifically awesome!
 
Dude, you should have sticked with the thing with the chickens...

A purple chicken sounds pretty scientific, scientifically awesome!

I have a feeling my parents just didn't want me raising chickens in the basement..
.__.
 
Inject the vft shoots with food coloring:)
 
I too would have gone with the chickens or perhaps even glow in the dark goldfish. That said, I will save you some time by suggesting that you don't use full-strength salts on any CP. There are already plenty of studies online that illustrate how poorly they perform under those conditions. A better experiment would involve quite the opposite: how dilute a media would still support germination and growth?
 
I too would have gone with the chickens or perhaps even glow in the dark goldfish. That said, I will save you some time by suggesting that you don't use full-strength salts on any CP. There are already plenty of studies online that illustrate how poorly they perform under those conditions. A better experiment would involve quite the opposite: how dilute a media would still support germination and growth?

I will probably do that too! As it is, I have to come up with enough experiments to satisfy my teacher over the next seven months, so I'll likely do it separately. Dilute concentrations also sound easier to do than stronger ones because I imagine contamination would be a little less of an issue.

Also, here's hoping this project goes better than my "FOWRL" project... >_>

Happy growing!
 
Dilute concentrations also sound easier to do than stronger ones because I imagine contamination would be a little less of an issue.
Happy growing!

The risk of contamination would be the same at any concentration of media. Sterile technique is the best and only insurance . . .
 
I meant that since I'm using PPM, less of a food source might mean the few surviving bacteria/fungi would have less of a chance to replicate. No idea how valid that is, of course.
 
I meant that since I'm using PPM, less of a food source might mean the few surviving bacteria/fungi would have less of a chance to replicate. No idea how valid that is, of course.

The same amount of sucrose is generally used, regardless of the lowered concentration of the media salts. I made a batch of 20% MS the other day and still used 30 grams of sucrose for the liter . . .
 
  • #10
The same amount of sucrose is generally used, regardless of the lowered concentration of the media salts. I made a batch of 20% MS the other day and still used 30 grams of sucrose for the liter . . .

...Really? I actually had no idea! The booklet that came with my kit didn't say that, and I never found that out anywhere else!

WOW. You just saved me a whole lot of trouble. :lol:

Edit: I'm confused. for carnivorous plants, it says to make a 2 liter batch with 1 liter of MS and the normal 2 tablespoons of sugar...

---------- Post added at 03:46 PM ---------- Previous post was at 03:35 PM ----------

Okay, I'm confused. checking a couple of other sources, they say not to dilute the sugar. But the kitchen culture kit packet says to dilute the sugar. What gives?
 
  • #11
For carnivorous plants, growers typically use, at the most, half-strength; and they far more commonly use 1:3, 2:5 -- even 1:10 strength media for some germination. For Murashige & Skoog (MS) Basal Medium w/ Vitamins, a full-strength batch would be 4.43 grams / liter -- far too strong for use on CPs. For the 1:5 batch, I used 0.89 grams and 30 grams of sucrose (I vary the sugar from time to time, down to 25). A good scale is a must in the long run . . .
 
  • #12
I'll have access to a precision scale, but the sugar thing bugs me... no pun intended. If carnivorous plants grow on the same amount of sugar, but a lower level of nutrients, do they just absorb less sugar? if so, why have it at the same concentration? Or do the plant structures actually use a lower than normal amount of nutrients, and somehow compensates by using extra energy?

it just doesn't make sense to me, since my sources say different things.

Thanks for the help!
 
  • #13
I'll have access to a precision scale, but the sugar thing bugs me... no pun intended. If carnivorous plants grow on the same amount of sugar, but a lower level of nutrients, do they just absorb less sugar? if so, why have it at the same concentration? Or do the plant structures actually use a lower than normal amount of nutrients, and somehow compensates by using extra energy?

it just doesn't make sense to me, since my sources say different things.

Thanks for the help!

The plants are sensitive to the concentrated media salts -- not so to the sucrose. Also, overly-dilute media is occasionally difficult to adjust in terms of pH.

Keep in mind that the plant species you desire to TC are endemic to acidic, nitrogen-poor environments; and that the original media formulations by Muroshige and Skoog were used for the micro-propagation of tobacco -- a far different plant in terms of cultivation than, say, a Venus flytrap.

Over the years, I can not recall ever making a liter of media with a sucrose level less than 25 grams, regardless of the concentration of the media salts . . .
 
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  • #14
This sounds really cool! Good on your school for giving you guys a chance to explore the things that interest you most and just think, maybe you'll discover something mind blowing that will change the world of cps as we know it! I wish I could be there to watch it all :boogie:
Jenn
 
  • #15
How long does it take to see any hint of contamination? I thought I might prepare a few jars of medium without plants, as a control to see if I can even sterilize stuff correctly. If they stay good, I can just use them later.

Thanks!
 
  • #16
You should have gone with the first one, because hey, chicken fetuses.
 
  • #17
You should have gone with the first one, because hey, chicken fetuses.

It's really not as cool as I made it out to be... you just inject the eggs with food coloring around day 12. And it's done extensively, so it's not like I wouldn't know what happens.

Then again, I wanted to try injecting a couple with glow in the dark food coloring. Now THAT would have been awesome.
 
  • #18
AARGH!!!! :headwall:

If there are three lessons I learned about tissue culture, today, they are:

1: It takes a lot, lot longer than you think it will.

2: The person who said "add a drop of soap to the bleach" is a sadistic psychopath.

3: Seeds are tiny.

I was able to prepare the media without any problems, mixed and sterilized fine. But when I actually went to do the seed transferring work, I realized *facepalm moment* that I had no type of strainer or anything for the seeds! I had already wiped down my area and all my tools with alcohol, so I thought I would just have the seeds loose in the bleach, alcohol, and peroxide, and I would just transfer them with a dropper.

Transferring the VFT seeds from the alcohol to the bleach went OK. Start ten minute timer. Swirling the container reveals... THE SOAP FOAMS UP. It becomes an impenetrable mass and I have NO idea where the seeds are. Eventually, after much frustration to try and just get a few, I give up on them. I wrap the containers I set aside with with plastic wrap, to be used later if they turn out to be sterile.

I also had a few jars set aside for D. filiformis seeds. This time, I made a solution of bleach without soap, and instead of having them loose, I just plonk the whole paper envelope in the alcohol, then bleach, then peroxide. I open up the packet, and... How am I supposed to transfer these seeds? They're TINY! I struggle to pick up some seeds with forceps and put them in the media... and eventually I do. Sort of. I must have had each container open for a full minute before I manage to shove the seeds down way further into the medium then they should be.

I think my seeds are just as likely to turn into dragons as they are plants...
 
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