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Doubled Haploids (Anther Culture) Introduction to concept/possible at Home?

TheTRPV1Agonist

TheTRPV1Agonist

TL;DR
  • Cross plants to make a hybrid
  • Grow the F1 plant from the seed
  • Use fancy techniques to grow a plant from pollen alone
  • More fancy footwork - now your seed is stable, do with it as you please. 
Result: a stable cross within just two generations (rather than the 6,7,8) and arguably a more targeted approach bringing your wildest crosses to fruition. 
 
WARNING: somewhat technical
 
Preamble.... 
Despite this is only my first year growing, I've been swept away by the beauty and diversity of the Capsicum genus and I've spent these past several months intensely researching some of the more technical details of the genetics of these plants. I'm so enthralled and can't wait to develop some crosses of my own. Though I don't have a background in botany or horticulture, I do in biomedical science, and so I've had a bit of a leg up in starting to get my head around things.
 
While I plan to cross by hand, admittedly there's a lot in the way of producing a new, stable cultivar. Following the initial dihybrid cross (selfing of the F1), there's a lot of variation in the F2. Say you're interested in 3 gene loci (Aa, Bb, Cc), then it follows that only 1/64 of your F2 will actually be homozygous recessive at the 3 loci (aa, bb, cc). In order to get your desired cross, you have to grow out a lot of seed, and to stabilise you need to repeat the process for a few generations, tolerating some subtle and not so subtle variation (obviously not for the fully recessive plant...but anyway) Don't get me wrong, there are probably a lot of happy accidents that come from this process, but surely there's a more targeted way of getting your desired cross (cue anther culture) 
 
 
Doubled Haploids/Anther culture... 
 
So I've been reading up on a technique used commercially, which involves the usual cross between the two parents, and growing out of the F1 seeds, but following this, the anthers are taken and cultured in such a way that what you get are immediately stable (homozygous) without any further crossing. To explain this genetically, take the AA;BB;CC x aa;bb;cc cross. Your F1 is just Aa;Bb;Cc and then there are 8 different F2 genotypes as a result of random assortment. So your pollen might have the following A;B;C / a;B;C / A; b; c / and so on. What the technique involves is then taking the pollen (or anthers) from your F1s and culturing them in such a what that you actually get a plant that germinates from this culture without having gone through sexual reproduction. The resultant plant is a hemizygote, or haploid, that has only 1 set of genes, instead of the usual 2 sets. With a chemical such as colchicine you can then initiate doubling of the genome into 2 sets (or more...) Ultimately, you get a plant that bares fruits with seeds that are all homozygous, depending on the genotype of the pollen (i.e. A;B;c becomes AA; BB; cc or a;B;C becomes aa;BB;CC, etc) 
 
Now for my question ... has anyone got experience with Anther culture or related techniques and has actually ever tried this at home? If so, what protocol did you follow? 
 
Though there are some specialised chemicals/materials involved, a cursory search has found that they are both not excessively costly or illegal to possess. 
 
If anyone is interested in the protocols I've come across I can include some references! 
 
 
 
 
 
 
 
b3rnd said:
Very interesting. What if the F1 doesn't show the exact characteristics you're looking for?
Click to expand...
 
This could very well happen, but hopefully with the traits I'm selecting for, which are pretty well described, I won't run into this. And the thing is the F1s will all actually look the same, it will be the plants from the anther culture (the F2s) that will differ. The beauty is, there will be a number of variants depending on how carefully I select the parents of the F1, but all will be stable. 
 
Here's an example  for one of the key traits I'm looking at: colour
 
As a brief overview there's 4 key ones for mature fruit colour, with different combinations giving rise to all the possible colours. In isolation:
  • yy produces yellow while Yy/YY produces red, red dominant over yellow
  • c1c1 reduces yellows and reds to about 1/10 - this is how you get orangey red and pale yellow; the dominant trait is no colour reduction
  • c2c2 is similar, but more significant reduction - oranges and lighter yellow 
  • clcl retains chlorophyll and so fruit is green (or brown/caramel/olive green when mixed with other colours) 
 
So say I really want a peach coloured pepper, but I'm happy to ignore other traits and let nature do it's thing... The genotype of a peach pepper is that it needs to be homozygous recessive for c1 and c2, and either homozygous/heterozygous for Y or Cl. I'll start with a white pepper (homozygous recessive for c1, c2 and y) and an Orange pepper (think Orange Hab - homozygous recessive for c2).
 
My cross is: 
  • yy; c1c1; c2c2; ClCl and YY; C1C1; c2c2; ClCl 
 
So my hybrids will be
  • Yy; C1c1; c2c2; ClCl (thats red + c2 pigment reduction, so orangey-red)
 
If my hybrid doesn't fit that description, then I know that something has gone wrong... But if it hasn't then I can grow the F1 seed and then the pollen will contain the following;
  • Y ; C1; c2; Cl (doubles to form orangey-red) 
  • Y ; c1; c2; Cl (double to form peach, bingo!) 
  • y ; C1 ; c2; Cl (doubles to form "lemon yellow" think Aji Pineapple) 
  • y ; c1; c2; Cl (doubles to form white) 
 
Now I haven't controlled for other traits so for each colour they could vary on pungency, size, shape, etc, etc. I suppose the more different the parents are, the greater the variability. So grow lots of them out, and just keep the one's you love, which theoretically won't change when you plant the seed again! 
 
I just was finishing a longer response when my computer crashed ( :violin:) , so I'm going to be briefer this time. Let me know if you have any questions I'd be happy to elaborate.
 
I am a tissue culture biologist, here are some things you might not have thought of that you will need:
Autoclave, Laminar flow hood, cartridge filtration kits: These are important for the same reason. The media you will use is all excellent breeding grounds for fungus and bacteria, and they will spread quickly and kill all of your tissue. There are guides online for how to use your oven as an autoclave, but sometimes you can pick them up on surplus from universities or hospitals for cheap. Laminar flow hoods are very expensive and usually custom. If you can't afford it I think there are guides online for building your own. Cartridge filtration will need to be ordered from a science company, but worth it for sterilizing some hormones and such directly into your already autoclaved medium before pouring plates.
 
In 2001 the Society for In Vitro Biology (of which I am a member) commissioned a review of biotechnology in Capsicum. There is a section on this subject. If you can get the paper free from a library go for it, but honestly it sounds like it might be worth buying for you. Let me know if you can't afford it.
https://link.springer.com/article/10.1007/s11627-001-0121-z
 
Another wonderful source is this chapter from Biotechnology in Agriculture and Forestry. I've never read it but still.
https://link.springer.com/chapter/10.1007%2F978-3-642-61499-6_19
 
The most recent work was in 2005, and honestly it seems like they might have thought of everything. This is a must read, as it is (imo) the current state of the art.
Supena, Ence Darmo Jaya, S. Suharsono, E. Jacobsen, and J. B. M. Custers. "Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper (Capsicum annuum L.)." Plant cell reports 25, no. 1 (2006): 1-10.
https://link.springer.com/article/10.1007/s00299-005-0028-y
https://drive.google.com/open?id=0B6l7HqYTqtv_NUU3R0RKRkVJUm8
 
 
 
 
 
Thanks Gorizza! Fortunately I've got access to all those articles via my university. I have read the last paper and also came across another (below) that is quite recent. Hopefully in the coming months I'll be able to evaluate each and see what's most feasible with my resources. Thanks for the extra reading, I'll let you know if I have any questions! 
 
Parra-Vega V., Seguí-Simarro J.M. (2016) Anther Culture in Pepper (Capsicum annuum L.). In: Germana M., Lambardi M. (eds) In Vitro Embryogenesis in Higher Plants. Methods in Molecular Biology, vol 1359. Humana Press, New York, NY
 
TheTRPV1Agonist said:
...but following this, the anthers are taken and cultured in such a way that what you get are immediately stable (homozygous) without any further crossing.
 
Click to expand...
 
I'm curious, what is the physical process/procedure for this? If you happen to know
 
 
 
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mrgrowguy said:
 
I'm curious, what is the physical process/procedure for this? If you happen to know
Click to expand...
 
I'm very new to this area so my understanding is limited. All I know is that a certain cocktail of chemicals can induce the pollen to form an embryo that will germinate. You can then induce aneuploidy, producing a diploid plant from your haploid with another chemical mediator (colchicine). As wiki puts it: 
 
"Since chromosome segregation is driven by microtubules, colchicine is also used for inducing polyploidy in plant cells during cellular division by inhibiting chromosome segregation during meiosis; half the resulting gametes, therefore, contain no chromosomes, while the other half contains double the usual number of chromosomes (i.e., diploid instead of haploid, as gametes usually are), and lead to embryos with double the usual number of chromosomes (i.e., tetraploid instead of diploid). While this would be fatal in most higher animal cells, in plant cells it is not only usually well tolerated, but also frequently results in larger, hardier, faster-growing, and in general more desirable plants than the normally diploid parents; for this reason, this type of genetic manipulation is frequently used in breeding plants commercially." 
 
https://en.wikipedia.org/wiki/Colchicine#Botanical_use
 
I hope that makes sense, happy to clarify if some of the terminology/concepts are not familiar. 
 
Nigel did some polyploid colchicine experimentation and posted the results in a thread here.
 
thehotpepper.com/topic/45601-secret-project/ It might not be what you are after but it might be worth sending him a PM.
 
TheTRPV1Agonist said:
 
I'm very new to this area so my understanding is limited. All I know is that a certain cocktail of chemicals can induce the pollen to form an embryo that will germinate. You can then induce aneuploidy, producing a diploid plant from your haploid with another chemical mediator (colchicine). As wiki puts it: 
 
"Since chromosome segregation is driven by microtubules, colchicine is also used for inducing polyploidy in plant cells during cellular division by inhibiting chromosome segregation during meiosis; half the resulting gametes, therefore, contain no chromosomes, while the other half contains double the usual number of chromosomes (i.e., diploid instead of haploid, as gametes usually are), and lead to embryos with double the usual number of chromosomes (i.e., tetraploid instead of diploid). While this would be fatal in most higher animal cells, in plant cells it is not only usually well tolerated, but also frequently results in larger, hardier, faster-growing, and in general more desirable plants than the normally diploid parents; for this reason, this type of genetic manipulation is frequently used in breeding plants commercially." 
 
https://en.wikipedia.org/wiki/Colchicine#Botanical_use
 
I hope that makes sense, happy to clarify if some of the terminology/concepts are not familiar. 
Click to expand...
 
That answers my question well enough, I at least understand the basic premises. Thank you very much for doing that for me!! I appreciate it!
 
 
 
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