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Anyone know how to do somatic hybridization?

Posted by MacDaddy z5b,z6a,z7 NY (My Page) on
Wed, Dec 3, 03 at 18:37

I was wondering if anyone has done somatic hybridization and how hard it is. What would be the cost of the equipment?


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RE: Anyone know how to do somatic hybridization?

Wow. I have'nt touched on this subject for over 20 years, but back when I was in college (late 70s, early 80s) it was an avenue of new possibilities. IF I remember right, the technique produced chimeras by using enzymes to remove the cell walls of plant cells in different species and then they were sort of smushed together in a centrifuge. The fused cells were then plated out and grown by tissue culture, and then hopefully some of the plants that developed were from the fusion of the two different species of cells. This could probably be done as a basement tissue-culture lab project, but I don't think it would be too cheap or easy. I could probably crack some books and find more info later.


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RE: Anyone know how to do somatic hybridization?

I haven't done it, but have done a lot of tissue culture and interact with some people who have. You definately need a species that some tissue culture protocols have been worked out with to be able to regenerate a whole plant from a single cell. As Agrinerd has said, cells are "mushed" together. What one needs to do is establish a cell suspension culture in liquid media. So in addition to standard tissue culture supplies, one needs appropriate flasks (125mL Erlanmeyers or about that size generally) and a shaker to keep the suspension moving so oxygen isn't too low.

Now the harder part comes. One needs to use enzymes to degrade the cell walls so they are gone and you have protoplasts remaining. Cell walls are something plants have and contain cellulose and other things that make the cells stiff and they need to be removed. Now you can mix the protoplasts from your two parents. The goal now is that not only the protoplasts fuse, but also the nuclei that are contained in them as well where the chromosomes are. Enzymes and I think even electical shock has been used to help fuse them. Even spontaneous fusing happens to some degree too, but at a low rate if I remember right.

At this point you have a mass of protoplasts that need to start growing again and forming cell walls. This can be tricky and they can take a long time to recover from all this stress, if they do at all. The cells are plated on solid media and hopefully they will continue to grow and shoots will regenerate and you can get plants out of culture into the "real world".

One isn't done yet.... The plants need to be characterized. Some may be fusion hybrids with a couple cells (or more than two) of just one of the parents. Some may not be fusion hybrids at all and would just be a parent. Some may have some combination of fused protoplasts of each of the parents and this is what you are seeking of course. Looking at morphology and chromosome number can help one determine which are somatic hybrids. There are many molecular techniques that can be used too to determine if both parents are represented in the offspring.

Hopefully this helps and doesn't sound too discouraging.

Sincerely,
David


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Heck, that sounds very discouraging.

Well the plants I was interested in working on was hardy bamboos. But I don't think that anyone has worked out the tissue culture mediums for these.

My idea was to cross very cold hardy runner called P. Nuda with P. Henon, P. edulis, and P. Vivax. The idea being to get a very hardy giant bamboo.

I also had the thought of crossing a water tolerant variety like our native switchcane with one of the giant hardy varieties to get one that could grow in soggy soil. Don't think it would probably work but would be fun to try.

However the amount of work involved does not sound promising.


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RE: Anyone know how to do somatic hybridization?

MacDaddy have you done a search on the plant TC listserve? I know several of the people there do work with bamboo. If you can't find answers doing a search, post your question, someone may be able to help you.

Here is a link that might be useful: TC list serve


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RE: Anyone know how to do somatic hybridization?

  • Posted by TQPL UK south (My Page) on
    Wed, Dec 10, 03 at 10:33

Hi MacDaddy,
Your kind of reaching for the sky working with Phyllostachys tc. As David has pointed out you need to find an 'easy' plant first. Unfortunately I don't think ptc listserve will be of much use even simple questions on Phyllostachys tc are not answered. (Most people that post info tend to work on other genera). Phyllostachys simple tc is difficult but not impossible. Work by Huang in Taiwan on P.aurea was the basis for the dubious patent held by WestWind Technologies TN.
---------------------------------------------------------------------- ----------
United States Patent 5,334,530
Woods, et. al. Aug. 2, 1994

---------------------------------------------------------------------- ----------
Method and media for the somatic embryogenesis and regeneration of bamboo
Abstract

The present invention provides a method and medium for the in vitro propagation of bamboo through organogenesis of vegetative explants from mature bamboo plants and somatic embryogenesis of the resulting organogenic callus. Optimal organogenesis is obtained by culturing vegetative explants from mature bamboo plants on first stage bamboo nutrient media comprising vitamins, MS salts, and supplemented with 1-naphthalemic acid, 6-benzylaminopurine and sucrose. Explants from the organogenic calli obtained from first stage media are cultured on second stage media (nutrient media comprising MS salts, vitamins, and supplemented with 2,4-dichlorophenoxyacetic acid, 6-benzylaminopurine and sugar) to induce somatic embryogenesis.

So with that techniqe patented you will have to find _another method_. Most bamboo tc is on tropical genera.

It is great to aim for the stars....but cracking simple Phyllostachy bamboo tc would probably pay enough for you to charter your own rocket!

You might heave better luck with finding Phllostachys in flower cycle and rely on conventional pollen dusting and embryo rescue if seeds abort. Seedlings might be a good starting point for in-vitro work anyway.

Regards
Alan


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RE: Anyone know how to do somatic hybridization?

hi,
if you have a method for making hybrids i was wondering can someone get a worldwide patent and the sort of money they would be looking at and could anyone please tell me which enzimes are used to break down cell walls.
regards
kevin


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RE: Anyone know how to do somatic hybridization?

Somatic hybrids don't always work out. I got the idea myself when I found out about polyployed daylilies. The main problem with cross species hybrids like mules is they are infertile because the chromosome numbers and kinds don't match. That wouldn't be a problem if one could only join the nucleuses of two species somatically. I had this idea about twelve years ago and thought it was original. I didn't get an opportunity to try. I decided last year to do more research and found out the idea had first been tried thirty or more years ago. It works better or worse depending on the species and tends to have a very high failure rate.


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RE: Anyone know how to do somatic hybridization?

MacDaddy,

"My idea was to cross very cold hardy runner called P. Nuda with P. Henon, P. edulis, and P. Vivax. The idea being to get a very hardy giant bamboo."

Sounds like a good idea.

"Somatic hybrids don't always work out. I got the idea myself when I found out about polyployed daylilies. The main problem with cross species hybrids like mules is they are infertile because the chromosome numbers and kinds don't match. That wouldn't be a problem if one could only join the nucleuses of two species somatically."

As a kid I ran into a similar problem with zinnias. Crossing diploids with tetraploids was difficult and yielded sterile triploids. I had some colchicine, but was unable to double a triploid to a fertile hexaploid. I think it should have been possible. But of course, you can't just keep solving sterility problems by doubling the chromosomes. You get an "unhealthy" large number of chromosomes in your genetic product. All living things seem to have a chromosome number that falls within a relatively limited range. There must be some kind of "survivability barrier" that prevents inordinately high chromosome numbers.

As I see it, somatic hybridization is just a kind of "low tech" genetic engineering. The "big boys" just insert a microprobe into a cell and squirt in the genetic material they want there.

I always wanted to cross zinnias and marigolds because zinnias had more colors and marigolds had better foliage. I tried a lot of cross pollinations, both ways, with no success.

Maybe we both need microscopes and some of those micropipettes. Although many people would be very nervous, probably rightfully so, if a bunch of amateur genetic engineers learned the tricks of the trade. But lay people are starting to learn to do tissue cultures.

-- Burton --


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Not to disillusion you . . .

. . . ,but you will also need either a airlift or impeller bioreactor to propagate plants from somatic embryos.

However, if you are still interested take a look at

Prutpongse, P. and P. Gavinlertvatana. 1992. In-vitro micropropagation of 54 species from 15 genera of bamboo. 'HortScience' 27:453-454

Rout, G.R. and P. Das. 1994. Somatic embryogenesis and in vitro flowering of 3 species of bamboo. 'Plant Cells Reports' 13:683-686

-Jay


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Perhaps it would be easier for me to fund research over at Stony

I did find this article based on the info you gave: http://www.geniaal.be/downloads/CRKeynotemicropropagation.pdf

Where would I go for this, the Stony Brook college library?


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RE: Anyone know how to do somatic hybridization?

Question,
This is probably a stupid question but here goes anyhow. Can you cross the 2 bamboos the old fashion way natural selection? Same family and species will cross right? Ruedee.


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RE: Anyone know how to do somatic hybridization?

Perhaps you could do it the old fashion way. Say one flowers on a 90 year cycle and the other on a 26 year cycle. So they flower syncronously every 1170 years. On average you would only have to wait 585 years before you could cross them.


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RE: Anyone know how to do somatic hybridization?

WOW!!
I see the need for speeding thinks up. They must spread in the wild through runners? Other wise there would be none? Like I earlier said this question may sound stupid and it did. Thanks for the info. Ruedee.


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RE: Anyone know how to do somatic hybridization?

Yes, they spread by underground runners called rhizomes.


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RE: Anyone know how to do somatic hybridization?

Hey there everyone,

I just stumbled upon this conversation while looking online for more info on bamboo micropropagation. I've been working on a project for a few months now that may interest you: I'm working on bamboo micropropagation from seed and vegetative tissue and in vitro flower monocultures. The project isn't finished yet so nothing is published yet but I can share preliminary results with people who are interested (easiest way would be to contact me on Facebook - see link below). A few weeks / months from now I will build a website to publish the results from the project.

So far I have done trials for different sterilization protocols and have found protocols for seed and vegetative tissue sterilization that achieved 100% success. Now that I have the sterilization down, I'm scaling the quantities up. Monday I inoculated inoculated 200 seeds of Chusquea culeou and next week I will inoculate probably around 100 tubes of vegetative tissue from Fargesia rufa, F. murieliae, Phyllostachys aureosulcata 'Alata' and maybe some Chusquea culeou.

It's always nice to chat with people interested in this kind of stuff so feel free to message me!

Cheers!

Here is a link that might be useful: https://www.facebook.com/Phyllostachys

This post was edited by PhyllostachysSam on Fri, Mar 14, 14 at 6:43


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