beth80721
Posts: 7
Joined: Mon Oct 15, 2007 6:24 pm

cryopreservation of plants-- need help with procedure asap!

Postby beth80721 » Mon Oct 15, 2007 6:30 pm

I am interested in doing a project with cryopreservation of plant seeds, but i am unsure about how to approach the procedure.

how do i properly store seeds in liquid nitrogen?? i have never used liquid nitrogen before, and do not know where to start.

thanks!
-liz

wildfirefox
Former Expert
Posts: 45
Joined: Tue Oct 02, 2007 3:27 pm

Postby wildfirefox » Tue Oct 16, 2007 4:13 pm

Hi Liz:

I don't know whether or not you already bailed on this subject? If your are:

1. Dip it in liquid nitrogen will kill it on the instant. Handling liquid nitrogen is extremely dangerous. You can accidentally lose your fingers, or any of your body parts without realizing it. If you don't, you can severely burn yourself.

2. To approach in cryogenic preservation, you have to bring the temperature surrounding the seeds to subzero, that means from -32 degrees C or lower, but at the same time, prevent the internal from overly frosted to the point the genetic materials die. In other words, freezer burn. Most seeds will not germinate around 28 degrees F. They stay in a suspended state until there's enough moisture, and warm enough temperature for germination, which is between 56 to an optimal 82 degrees. The shells of most seeds will protect the seeds throughout its long dormant period until the right conditions come about.

3. To experiment with this, why not perform a research on which plant seeds survive the longest in certain dormant temperatures. For plants to survive from 100 years to eons, a protective ice capsules (a small pocket of air surrounded by ice), similar to the arctic tundras and glacier areas, need to be formed around the seeds without killing the internal essential materials.

Hope this help.

Nam
Those who can see that do not exist are geniuses. Those who can see what exists are brilliant. Those cannot see what exists are ignorant.
- Lao Tsu

beth80721
Posts: 7
Joined: Mon Oct 15, 2007 6:24 pm

Postby beth80721 » Tue Oct 16, 2007 4:47 pm

Thank you so much for your help, I am indeed still planning on doing this project! I was thinking about varying the types of seeds, slow-cooling vs. direct immersion in the LN2.

You mentioned that I need to bring the temperature down to about -32 C, without killing the internal essential materials-- would you recommend a stabalizing agent, such as some kind of glucose mixture? or some kind of dehydrating procedure so that ice crystals do not form? (I have searched the internet a LOT for these, but can not find procedures on how to make either solution-- do you know a source?)

Thanks again for your help!

wildfirefox
Former Expert
Posts: 45
Joined: Tue Oct 02, 2007 3:27 pm

Postby wildfirefox » Wed Oct 17, 2007 8:27 am

Liz, you're becoming a scientist and an engineer altogether. Great deduction!

If you're planning to dip the seeds directly into LN2, you have to encase the seeds into a protective shell, and on top of that, a non-solidify liquid cellulose-glucose mixture, and in combination with even more advanced form of non-solidifying liquid called "liquid hydrofluorocarbon". This brings about the Shaffer's liquid equilibrium-suspension method, which NASA computes to be the long term suspended animation solution for deep space travel, preventing drastic aging issues as human travel through space.

To obtain this type of liquid mixture for experimentation can be a hair raising search. You can perform this task by using various materials available to you now.

Petroleum product (Vaseline)
Jello/Glucose gels
Ice/Water
Time capsules (a hard casing of plastic/metal/styrofoam/etc.)
Non-moist soil, mimicking the drought and freezing seasons of the tundras

There are many tricks without blow your budgets into the thousands of dollars.

Nam
Those who can see that do not exist are geniuses. Those who can see what exists are brilliant. Those cannot see what exists are ignorant.
- Lao Tsu

beth80721
Posts: 7
Joined: Mon Oct 15, 2007 6:24 pm

Postby beth80721 » Wed Oct 17, 2007 1:37 pm

I am still really having a hard time finding some kind of procedure to prepare the seeds for cryopreservation-- how and in what order might you suggest using the products you suggested? (the vaseline, glucose gels, water, time capsules, and soil)
Thanks again, your advice is incredibly helpful!!

wildfirefox
Former Expert
Posts: 45
Joined: Tue Oct 02, 2007 3:27 pm

Postby wildfirefox » Wed Oct 17, 2007 2:29 pm

Hopefully this might shed some light on how to begin. The processes is similar:

http://books.google.com/books?id=rLrqco ... LI5Fq1DSvE

http://www.fao.org/biotech/docs/lambardi.pdf

http://www.fao.org/biotech/docs/decarlo.pdf

Any gel like material can be preserved by first placing the seeds far in the middle as possible, away from any possible exposure or contact with LN2. Encapsulate the gel in a layer of protective layer, such as cellulose, plastic/metal/etc. to prevent the LN2 from destroying the gel material. Continually expose the frozen cryo-static materials in a deep freeze environment.

Same goes with soil. But with soil, you have to pack it to minimize any open gap between soil particles. That means you may need to use clay powder to add into the soil, and mix the soil with clay very thoroughly, then pack it down around the seeds.

Water requires you to create a bubble around the seeds. The air within the ice capsule acts as a buffer to minimize damages. But, creating a suspension within the ice capsule may be tricky, so you need to find a way to hold the seeds in place as the water freezes. Meshes or fine twines will do the trick.

Nam
Those who can see that do not exist are geniuses. Those who can see what exists are brilliant. Those cannot see what exists are ignorant.
- Lao Tsu

beth80721
Posts: 7
Joined: Mon Oct 15, 2007 6:24 pm

Postby beth80721 » Wed Oct 17, 2007 4:30 pm

I am so grateful for your help, and can't wait to get started on my project now!

beth80721
Posts: 7
Joined: Mon Oct 15, 2007 6:24 pm

Postby beth80721 » Thu Oct 18, 2007 5:04 pm

Okay, sorry now I have another question-- do you think that a non-LN2 storage system would work okay, as long as it reaches -20 degrees C? (such as http://www.amazon.com/Cryo-Safe-Maxi-Co ... 55&sr=1-41) or do you think that i should definately use something like this (http://cgi.ebay.com/Liquid-Nitrogen-Dew ... dZViewItem)? I am leaning towards the first one since my school is small and has very limited resources and i dont know any research scientists, so i am buying one myself haha.

thanks again!

Craig_Bridge
Former Expert
Posts: 1297
Joined: Mon Oct 16, 2006 11:47 am

Postby Craig_Bridge » Thu Oct 18, 2007 7:06 pm

What do you think you are buying? This is just a well insulated cooler. It does not supply the cold, just keeps something that is already cold from warming quickly. It does provide a handy test tube rack.

If you are only trying to get to around -20 C and hold if for a few hours, then a large picnic type plastic shell polyurethane insulated cooler filled with wadded up newspapers around a card board box holding six pieces of dry ice wrapped in freezer paper with your experiment in the middle would probably accomplish your goal with less expense as long as CO2 exposure is not a problem for your experiment. You might even find everything you need except for the dry ice at home.

CAUTION: Dry Ice must be handled with thick cotton gloves even when wrapped with a few layers of paper to prevent "burns". See http://en.wikipedia.org/wiki/Dry_ice for more information on its cooling properties.

Your going to have to know the total thermal mass of your experiment and the temperature difference and time duration and do some calculations to determine how much dry ice you need and how much insulation you need between your experiment and the dry ice to not over cool your experiment.
-Craig

Louise
Former Expert
Posts: 921
Joined: Mon Jan 16, 2006 2:17 pm

Postby Louise » Fri Oct 19, 2007 8:15 am

I work with liquid nitrogen daily. I'm not quite sure what it is you want to do...

If you intend to store something in liquid nitrogen for a long time (more than 1 hour), then you will need some sort of dewar designed for liquid nitrogen.

If you want to quickly freeze something, do some manipulations in liquid nitrogen for less than 1 hour, then you can use a cheap styrofoam cooler.

If you are transporting liquid nitrogen from one place to another, you will need a liquid nitrogen dewar designed for transport. Note, this isn't the same type of dewar as for long term storage. [I'm not sure where you are getting liquid nitrogen from...]

If you are not working at liquid nitrogen temperature, and are instead working at temperatures where you could use dry ice, Craig's solution is perfect.

If you can work with dry ice rather than liquid nitrogen, do so. Always work with the safer material. Liquid nitrogen can be very dangerous.

With both dry ice and liquid nitrogen be aware that in addition to the serious frostbite concerns, there is a suffocation hazard as these materials turn to gas and displace the air. Always work in a well ventilated room. Work with the minimum volume of material, and make sure someone is checking on you. Safety glasses are a must. Nitrogen spatters and jumps around a lot. Some materials also shatter from thermal shock upon freezing/thawing OR from the expansion of water upon freezing. I've seen people cut up from glassware that has shattered while they were freeze-thaw cycling.

Louise

MichaelD
Former Expert
Posts: 21
Joined: Tue Oct 02, 2007 10:52 am
Occupation: Research Scientist

Postby MichaelD » Fri Oct 19, 2007 8:30 am

Just to add to Louise's suggestions, a dry-ice/alcohol bath may also work well for your experiments and will be a little easier to handle than liquid nitrogen.

beth80721
Posts: 7
Joined: Mon Oct 15, 2007 6:24 pm

Postby beth80721 » Sun Oct 21, 2007 10:07 am

Thanks! does anyone know where i can get access to a LN2 dewar/ LN2/ all the accessories? i go to a very small school with pretty limited resources... i would be willing to pay for it myself (i saw a refurbished one for $500, but any cheaper would be really nice haha) if thats the only option..

Louise
Former Expert
Posts: 921
Joined: Mon Jan 16, 2006 2:17 pm

Postby Louise » Sun Oct 21, 2007 10:53 am

beth80721 wrote:Thanks! does anyone know where i can get access to a LN2 dewar/ LN2/ all the accessories? i go to a very small school with pretty limited resources... i would be willing to pay for it myself (i saw a refurbished one for $500, but any cheaper would be really nice haha) if thats the only option..


Where are you getting the liquid nitrogen from? They must have dewars and such that they maybe could loan you?

You didn't answer any of my questions though about what you really need to do. It seems silly to spend hundreds of dollars on a dewar if some dry ice and a $5 cooler would work. Similarly, if you just need a dewar for a few days, it might be easier to borrow one than if you need it for 3 weeks.


Louise

beth80721
Posts: 7
Joined: Mon Oct 15, 2007 6:24 pm

Postby beth80721 » Sun Oct 21, 2007 11:31 am

I am planning on doing a project about the cryopreservation of plant seeds, so I think that I would need to use subzero temperatures for more than an hour (probably about a week, i think). And I am looking into where to get LN2, but I am really just not sure!

Louise
Former Expert
Posts: 921
Joined: Mon Jan 16, 2006 2:17 pm

Postby Louise » Sun Oct 21, 2007 12:04 pm

beth80721 wrote:I am planning on doing a project about the cryopreservation of plant seeds, so I think that I would need to use subzero temperatures for more than an hour (probably about a week, i think). And I am looking into where to get LN2, but I am really just not sure!


Right, I knew this much, I was asking you to expand/provide more detail on what you've already said. My point is that you don't seem to have any concrete plan for this experiment. You are proposing to use a dangerous and expesive setup when a much cheaper alternative may work as well. What temperatures do you really need? There is a world of difference between "subzero" and -196 C (liquid nitrogen). wildfirefox's post to you seems to suggest that anything below 28 F (-2 C) will prevent germination.

Dry ice will allow you to get "subzero" temperatures (dry ice is at -78.5 C) and is easier to handle and buy than liquid nitrogen. Dry ice is often used with specific solvent to get certain temperatures, as MichaelD mentioned. For example, a mixture of dry ice and ethanol is -78 C, with water/glycerine it is -34 C. [Note, you cannot put these mixtures in styrofoam. You need a liner that won't react.]

So again, why are you so insistent on using liquid nitrogen, when you have no source to get it, none of the equipment needed to handle it, and (as far as I can tell) have a much cheaper and safer alternative in the form of dry ice? Why don't you come up with a more specific plan, and see if some of the suggestions you've gotten here can help you reach your goals. Then, post back here with some of these details.


Louise


Return to “Life, Earth, and Social Sciences”