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cenh3 null mutation for Cannabis?

mofeta

Member
Veteran
Recently a member here told me about some important developments in molecular genetics that I was unaware of. These developments were pretty well publicized (they were also discussed some here at ICMAG) because they significantly advance our understanding of genetics, and have important practical applications that could revolutionize the way we harness plants to do our bidding. I don't know how I missed this, it got under my radar somehow. Thanks to the member who brought this to my attention!

Here are the two papers that got national press a while back:



Haploid plants produced by centromere-mediated genome elimination

Synthetic Clonal Reproduction Through Seeds



The first paper, about haploid plants, tells how the authors developed a technique that would allow for the creation of plants that were perfectly homozygotic for all traits. This technique does not involve breeding over numerous generations, perfectly homozygous plants are created in a two-step process. This could be a gigantic breakthrough in plant breeding.

In the second paper, about the "clone seeds", the authors have successfully induced apomixis in plants that are not normally apomictic. This allows for the production of seeds that have not undergone the "card shuffling" of meiosis, and are identical genotypically to the parent. This too is a mind-blowing advance.

This thread is not to discuss the two papers above, but to discuss the key feature that both techniques rely on. Both techniques depend on the use of a mutant plant that the authors developed.



cenh3 null mutation

Plants with the cenh3 null mutation can fertilize another plant without passing on any of it's own genetic material! This mutant was developed while investigating the structure/function relationships of a single gene that codes for the centromere specific histone CENH3. It was not anticipated that this particular mutation would have the properties is does.

The creation of the cenh3 null mutation was first reported in this paper:



The Rapidly Evolving Centromere-Specific Histone Has Stringent Functional Requirements in Arabidopsis thaliana

(EDIT: This paper wasn't the first, the "Haploid plants produced by centromere-mediated genome elimination" was the first to mention the haploid inducer. I think when they discovered the haploid inducer in the process of doing the research for this "Rapidlly Evolving... " they published on the haploid inducer before they were done with the study. Sorry about the confusion on my part.)

This paper investigates how changes in the CENH3 gene affect its function. They wanted to find out what structural features of the resultant histone were the most important. The CENH3 gene in the test plants was replaced with CENH3 genes from other species (including humans) and artificially created chimerical CENH3. The results of these replacements would give clues to which portions of the gene conferred function.

In order to test these gene replacements, they had to have a plant that was a blank slate for this gene, so they knew it was the inserted gene that was being expressed. They were successful in creating this mutant, named cenh3 null. The original gene still exists in this mutant, but is truncated in a way that makes it completely non-functional.

In the course of using the cenh3 null mutant to probe CENH3 structure-related function, the researchers discovered (by accident) that it could fertilize another plant and produce offspring that contained none of the genetic material of the mutant!

Among other uses, this mutant can be used to produce haploid plants in one step, just by crossing the subject with the cenh3 null mutant. Because of this the researchers call the cenh3 null mutant a "haploid inducer". The haploid is then converted to diploid with colchicine or another ploidy multiplying substance to obtain a perfectly homozygous plant.

The significance of having haploid inducing plants is enormous.

For one thing, the diploid plants resulting from doubling the haploids are perfectly homozygous in a way that a conventionally bred plant could never be.

For another thing, once you have the haploid inducer for a species, it would be capable of producing haploids, and therefore homozygotic diploids of any individual plant in the species. You could take any plant with traits you liked, cross it with the haploid inducer, double the results and be left with a bunch of perfectly homozygous plants. The ones with traits you liked would be perfectly true-breeding in a way that a normally bred plant could never be.


The second paper about "clone seeds" uses the haploid inducer along with another important new mutant. I won't discuss this other mutant now, other than to say that it is a mutation that converts the process of meiosis to mitosis in the mutant. This produces seeds that bypass the allele shuffling of meiosis. If that doesn't blow your mind, nothing will!

In my next post I will explore how the researchers created this fabulous haploid inducer.
 

spurr

Active member
Veteran
Good post mofeta, thank you very much for your insights. Lets hope the person(s) working on this are successful. With people like you, and others, being kind enough to offer input I think he(they) might just have a chance at success ... only time will tell.

P.S. Damn you! Your thread drug (pun intended!) me out of non-public posting just so I could thank you. (... spurr now heads back to the nether regions of ICmag ;) ).

EDIT

@ All:


Here is a list of a few relevant papers for those interested:

  • Plant centromeres: structure and control
  • Haplodiy with histones
  • Haploid plants produced by centromere-mediated genome elimination
  • Meiosis-Specific Loading of the Centromere-Specific Histone CENH3 in Arabidopsis thaliana
  • Synthetic Clonal Reproduction Through Seeds


And here is the patent application by Dr. S.Chan and (Dr.?) R.Maruthachalam
Generation of haploid plants and improved plant breeding
Provisional application No. 61/248,996, filed on Oct. 6, 2009
www.google.com/patents/about/GENERATION_OF_HAPLOID_PLANTS_AND_IMPROVE.html?id=Lhp6AQAAEBAJ

:tiphat:
 
Last edited:

mofeta

Member
Veteran
Please make sure to make note of the edit in my first post if you missed it. It is an important correction.


Hi spurr-

Couldn't resist the bait, eh?
 

mofeta

Member
Veteran
How To Make a Diploid Inducing Arabidopsis

How To Make a Diploid Inducing Arabidopsis

How To Make a Diploid Inducing Arabidopsis


The short answer is "With a chemical mutagen." It's a little more complicated than that though, although it is relatively easy to induce the desired mutation, it is more difficult to render it useful.

The two main problems to overcome:

1) The researchers thought that the cen3h null mutation would be lethal to gametophytes (the cells that give rise to the gametes). It turns out they were right. Since gametophytes in diploid plants are haploid, the cenh3 null mutation would be impossible to pass on to progeny.

2) The chemical mutagen creates random mutations, and you would have to determine which plants have the desired cenh3 null mutation


The researchers came up with a clever solution to the first problem. They used a tetraploid Arabidopsis strain for the initial population. Tetraploid plants have diploid gametophytes. This means that a tetraploid that carries the cenh3 null gene could pass on the gene if the gametophyte was heterozygous for it. The presence of a normal CENH3 in the gametophyte allows for enough normal histone to keep the cell alive, and to produce a gamete. The teraploid is then reduced by crossing to normal diploid plants a couple of times to reduce the ploidy to diploid so it can be used in breeding.

The second problem was solved by using a process known as "TILLING". TILLING is an acronym for "targeting induced local lesions in genomes".

The first step in TILLING is to induce mutations. Ethyl methanesulfonate (EMS) is the preferred mutagen, for a few reasons. One reason is that EMS produces single point mutations as opposed to knockouts, the mutations are more subtle. Another reason is that the effect of EMS is very broad, and it is likely to produce mutations for the gene that is being targeted, its not picky. Lastly, the use of EMS is easy, it does not require complicated techniques like tissue culture.

The second step is the hard part. This is the screening of the mutated plants for the desired mutation. This is pretty complicated. I'll try to tackle the subject in my next post.
 

Chimera

Genetic Resource Management
Veteran
I never thought I'd see someone discussing TILLING on these boards; I remember when that paper came out.

Good stuff mofeta keep up the good work.... there might be hope for us yet!
Looking forward to the rest of your post.

-Chimera
 

mofeta

Member
Veteran
Thanks Chimera. It's nice to have a pro around to review our thoughts on things like this, to keep us out of the weeds so to speak.

TILLING is awesome, eh? There have been some significant refinements in the process since the original McCallum paper, I am going to post a link downthread to a great paper on an improved methodology.

I'll be counting on you to correct the errors I'm almost certain to make in my next post about the screening process, I think I understand it pretty well, but I'm not a pro so its it's likely I will screw part of it up.

Thanks,

mofeta
 
Saw this talked about by gojo and others over a year ago elsewhere... is there any discussion on reverse breeding anywhere?
 

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