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Author Topic: about how to breed seedless citrus varieties  (Read 424 times)

SoCal2warm

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about how to breed seedless citrus varieties
« on: June 26, 2017, 01:53:47 AM »
Here's a bit of information that will be very useful to anyone trying to breed new varieties of citrus and wants them to be seedless.

Crossing a tetraploid citrus with a regular diploid citrus will result in a triploid citrus. Triploids are generally sterile and seedless. This is a strategy that has been used in many cases to breed seedless citrus varieties (although there are other strategies).

It turns out that most regular citrus varieties—the ones that are polyembryonic producing clonally from seed—will convert to tetraploids at least once in a while. That is, if you grow 300 seeds, at least one of them will likely turn out to be tetraploid, even though it is otherwise a clone of its diploid parent. Tetraploids are often slightly bigger and slightly deeper in color than their corresponding diploid parent.

Some citrus varieties are more likely to convert to tetraploids than others. For example, out of 78 seeds grown from a Duncan grapefruit, 5 turned out to be tetraploids.
'Mapo' tangelo (7 out of 73) and 'Tardivo di Ciaculli' mandarin (2 out of 38) also had high rates of tetraploid seed. For Minneola Tangelo, 3 tetraploids were observed out of 166 seeds.
Something else notable, it appeared that plants producing fruit in colder conditions in marginal climatic areas had an increased likelihood of producing tetraploid seed.

Troyer citrange had very high rates of polyploidy, as high as 10-20 percent of the seedlings. (Carrizo was almost as high too)

Now, you most likely may not be able to know the chromosome count of any individual seedling, but if you grow enough of them, there should be some seedless plants two generations down the line. Assuming you try to prevent self-pollination. A much more convenient strategy is to rely on the blossoms of the tetraploid plants to pollinate other citrus varieties which are known not to be polyembryonic, usually mandarin but also pomelo.

If it's not polyembryonic, that means all the seeds that form inside the fruit are zygotic, formed by sexual recombination. If the variety also happens to be self-incompatible, it will require two different parents for fertilization, seed formation, and most likely fruit formation too. Unless the variety is parthenocarpic, meaning it will be capable of producing fruits without any fertilization, which many citrus varieties are. Yes, the terminology can get a little complicated and there are many aspects to breeding and growing different types of citrus varieties in orchards.

SoCal2warm

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Re: about how to breed seedless citrus varieties
« Reply #1 on: June 26, 2017, 02:04:17 AM »
"Artificial hybridisation to create triploids is a promising method for production of seedless cultivars in citrus and thus in mandarin improvement programmes. In fact the female and male gametes of triplod genotypes are sterile because of the distorted trim of chromosomes. Triploid hybrids can be produced by the cross between female tetraploid parents and diploid male parents. But by this kind of cross only few hybrids and many nucellar seedlings are produced due to the high polyembryony level of the tetraploid female parent. On the contrary if monoembryonic zygotic diploid genotypes as female parent and tetraploid genotypes as male parent are used, several immature seeds are produced. The immature embryos, rescued and grown in vitro, generate triploid hybrids producing seedless fruits."
http://www.fao.org/docrep/003/x6732e/x6732e12.htm

Solko

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Re: about how to breed seedless citrus varieties
« Reply #2 on: June 29, 2017, 04:38:23 AM »
Thanks for all the info.
Does anyone know if there is an easy 'homegrower' way to check if one of your plants is tetraploid? I do have an antique microscope and from what I remember from highschool I should be able to take a leaf, cut it and put in under glass and look for a cell that is about to divide. Could I then count chromosome pairs, or is that too difficult?
Any tips are welcome
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Ilya11

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Re: about how to breed seedless citrus varieties
« Reply #3 on: June 29, 2017, 01:21:17 PM »
Chromosome counting can not be done easily without special fixings, stains and rather powerful microscopes.
The easiest "kitchen" methods for ploidy determination is comparative measuring of leaf stomata replicas
with the help of nail polish (colorless) and a microscope (x40 - x100 will do). You apply the nail polish on a clean dry leaf, wait for it to dry and the peel it off. Afterwards you mount the peeled nail polish on a slide and cover with glass slip.
Best regards,
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SoCal2warm

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Re: about how to breed seedless citrus varieties
« Reply #4 on: June 29, 2017, 02:49:01 PM »
Basically you would need to grow about 300 seeds. Then when those seedlings first begin producing blossoms you would need to make sure they pollinate with a different tree (by clipping off their stamens before they form). Then, when they form fruits and produce seeds of their own, you would need to keep track of which seeds came from which tree. If any of this next generation of seeds turn out to produce trees with seedless fruit, then you know that their parent was a tetraploid (most likely). Then you can use the parent for future breeding attempts.
This process would require growing a lot of seedlings and is not the fastest.   

I'm also not entirely sure that a diploid-tetraploid seed will be viable without embryo rescue, but if the seeds of one plant consistently fail to germinate while others have no problem, then you have very likely identified a tetraploid.

"Triploid embryos are preferentially found in seeds between one-third and one-sixth smaller than normal seeds and these small seeds generally do not germinate under conventional greenhouse conditions. Embryo rescue from these small seeds is required to reach high germination rates"
(source: ‘Safor’ Mandarin: A New Citrus Mid-late Triploid Hybrid, José Cuenca)

"Triploid embryos were identified in normal and undeveloped seeds that did not germinate under greenhouse conditions.
[...]
Implementation of extensive citrus breeding programs based on 2x x 4x hybridizations requires (1) the
production of tetraploid genotypes to serve as male parents, (2) an effective methodology for the recovery of triploid
citrus plants from seeds that do not germinate under greenhouse conditions, [...] "
(source: Extensive citrus triploid hybrid production by 2x x 4x sexual hybridizations and parent-effect on the length
of the juvenile phase, P. Aleza)
« Last Edit: July 02, 2017, 05:23:24 PM by SoCal2warm »

Solko

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Re: about how to breed seedless citrus varieties
« Reply #5 on: June 29, 2017, 05:14:36 PM »
Ilya, that is very interesting, I never heard of that before. I just examined my microscope and since it is an antique one I think I can get up to 60x or maybe 100x.
That makes your method very interesting, except the plant I want to test is not citrus. Do you know wether your method works for other genera as well? I think I may have a tetraploid Pitanga, based on relative leaf size, vigor and growth rate, compared to the rest of the batch of seeds. I would like  to verify that using my microscope but I have no idea wether that is feasible or not.
I have the other seedlings to compare the plant with, so maybe your test will already give some results.
Have you ever done this kind of testing yourself? Or could you direct me to more reading on the web on the method you mention? All help is very appreciated.
Thanks!
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Solko

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Re: about how to breed seedless citrus varieties
« Reply #6 on: June 29, 2017, 05:27:24 PM »
Basically you would need to grow about 300 seeds.

Then when those seedlings first begin producing blossoms you would need to make sure they pollinate with a different tree (by clipping off their stamens before they form).

Then, when they form fruits and produce seeds of their own, you would need to keep track of which seeds came from which tree.

You are absolutely right, but since I already have problems tracking 6 flowers out of 24 on one little Ugni plant, pollinated with pollen from two different parents, you can imagine that your method, although completely sound, is ultimately not practical for me. When 300 trees are involved with several hundred flowers each and several different parents it just gets too complicated.

So a method in which you can just put material of a certain plant under the microscope and try to count chromosomes would be extremely attractive, it is fast, direct and simple. But probably too good to be true...

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Ilya11

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Re: about how to breed seedless citrus varieties
« Reply #7 on: June 30, 2017, 03:39:11 AM »
Ilya, that is very interesting, I never heard of that before. I just examined my microscope and since it is an antique one I think I can get up to 60x or maybe 100x.
That makes your method very interesting, except the plant I want to test is not citrus. Do you know wether your method works for other genera as well? I think I may have a tetraploid Pitanga, based on relative leaf size, vigor and growth rate, compared to the rest of the batch of seeds. I would like  to verify that using my microscope but I have no idea wether that is feasible or not.
I have the other seedlings to compare the plant with, so maybe your test will already give some results.
Have you ever done this kind of testing yourself? Or could you direct me to more reading on the web on the method you mention? All help is very appreciated.
Thanks!
It is working on any higher plant specie. The method is simple and is a part of practical botany courses in many universities.
stomata size measurement
It can be used even with very simple USB connected 200X microscope camera.
I posted some photo on Agrumes-Passion forum, but it is currently down.
Best regards,
                       Ilya

ricshaw

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Re: about how to breed seedless citrus varieties
« Reply #8 on: June 30, 2017, 11:25:56 PM »
Near where I live they net the seedless trees. The problem is cross-pollination between nearby seeded citrus and the seedless mandarins by bees causes both to grow seeds.

Solko

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Re: about how to breed seedless citrus varieties
« Reply #9 on: July 06, 2017, 12:16:43 PM »
Ilya, I have got the microscope operational again and will soon get the nail polish to make the leaf-prints.
It seems that a small magnification is actually more practical to be able to count leaf stomata, which is reassuring.

I couldn't find anything about ploidity in the document you linked to. Is it reasonable to assume that if I find a larger number of leaf stomata per area on one leaf than on the leaves of several other plants I want to compare it to, that that can be an indication of it being a tetraploid?

My microscope: an old Zeiss


Small magnification - I think I can actually see some leaf stomata


Large magnification, probably already to large to be able to count leaf stomata, but obviously still too small to be able to see chromosomes.

Thank you for any help.


Another layman question: do triploids always give seedless fruit after pollination, or do some species just abort the entire fruit since pollination doesn't work perfectly?
« Last Edit: July 06, 2017, 12:18:37 PM by Solko »
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SoCal2warm

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Re: about how to breed seedless citrus varieties
« Reply #10 on: July 06, 2017, 04:29:21 PM »
I know that triploid citrus and triploid cherry trees both produce fruit. In the case of cherries, the seed inside the fruit is sterile. Triploid citrus do produce seeds inside the fruit but not very many seeds, and the seeds are mostly shriveled up and very small, so they usually have very low viability rates, unless you can find the rare larger seed.

Solko

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Re: about how to breed seedless citrus varieties
« Reply #11 on: July 06, 2017, 05:34:14 PM »
SoCal2warm, It's interesting that not all species seem to do the same thing. Some triploid apples also produce seeds and actual viable seedlings, albeit in very small percentages.

And I seem to have have made the wrong assumption about the number of stomata per leaf area, I just found this document:

"In general, stomata and epidermal cell frequency per unit leaf area diminished while stomata guard cell length improved with an increase in ploidy" (Yuan et al. 2009, Ye et al. 2010).

http://www.scielo.br/scielo.php?script=sci_arttext&pid=S2197-00252013000400006

It seems the thing to look for in the leaf is not more, but less stomata per area and larger guard cells, when compared to normal diploid leaves...
Hmm, I'll try to make some prints this weekend an I'll test one of my suspected tetraploid leaves against a couple of normal ones. Even if they all turn out to be the same, I'll see if I can post some pictures for those who might find this kind of thing interesting and entertaining...



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Sylvain

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Re: about how to breed seedless citrus varieties
« Reply #12 on: July 06, 2017, 06:41:20 PM »
> It seems the thing to look for in the leaf is not more, but less stomata per area and larger guard cells, when compared to normal diploid leaves...
Yes, that's right.

Ilya11

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Re: about how to breed seedless citrus varieties
« Reply #13 on: July 08, 2017, 05:09:11 AM »
Solko,
The link was for replica technique. For effect of polyploidization on citrus stomata, see for example the following papers:
http://www.pakbs.org/pjbot/PDFs/40(4)/PJB40(4)1755.pdf
https://www.jstage.jst.go.jp/article/jsbbs1951/36/4/36_4_371/_pdf
Some triploids are giving occasionally seeds, but their number is very small.
Many diploid citrus varieties are able to form fruits without pollination, its prevention in this case is also giving seedless fruits.
Best regards,
                       Ilya

Sylvain

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Re: about how to breed seedless citrus varieties
« Reply #14 on: July 08, 2017, 06:54:19 AM »
There is a DIY way to obtain triploids. Just grow the small seeds that you usually discard.
They are hard to grow but a significant number are triploid.

There is a (nearly) simple way to obtain tetraploids using oryzaline.
Here I show how I did ( Sorry it is for register people only , it's in french and the picture are long to load...):
http://www.agrumes-passion.com/experimentations-agrumes-f70/topic4547.html

Solko

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Re: about how to breed seedless citrus varieties
« Reply #15 on: July 08, 2017, 10:28:45 AM »
Ilya et Sylvain, merci beaucoup!

Thank you very much for the documents and the links, I will study them carefully. I had already found some old publications online as well, but it is very interesting to see how far a DIY backyard grower is able to go.

I know colchicine is a pretty powerful hormone that can also interfere with our own cell division, and it needs to be handled with the utmost care. Is oryzaline less dangerous?

I speak French and will register for the agrumes-passion forum.

I am looking for tetraploids and multipliers in another genus from citrus, and these don't make small triploid seeds I think.

Thank you again for the great information!

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Sylvain

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Re: about how to breed seedless citrus varieties
« Reply #16 on: July 08, 2017, 12:30:11 PM »
> Is oryzaline less dangerous?
Yes much less dangerous but don't drink it. ;-)

Ilya11

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Re: about how to breed seedless citrus varieties
« Reply #17 on: July 08, 2017, 12:50:48 PM »
Both should be used with precaution, but actually colchicine is a registered human drug being used to treat gout and is available in pharmacy under prescription. It is a natural toxin and is acutely poisonous in higher doses.
http://www.webmd.com/drugs/2/drug-8640/colchicine-oral/details
Oryzaline is suspected to be human carcinogen and endocrine disruptor, but acutely it is less toxic when swallowed.
Best regards,
                       Ilya

Millet

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Re: about how to breed seedless citrus varieties
« Reply #18 on: July 08, 2017, 02:52:05 PM »
I've taken Colchicine for gout, the strength of the pill is 0.6 mg.   I do not know the strength needed for triplorid use, but one could dissolve enough pills.  As mentioned a lot of caution would be required when working with colchicine.
« Last Edit: July 08, 2017, 02:53:41 PM by Millet »

Solko

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Re: about how to breed seedless citrus varieties
« Reply #19 on: July 09, 2017, 12:59:58 PM »
I wasn't planning on working with colchicine, but it is good to know the risks and dangers. If Oryzaline is easier to obtain and less toxic, than I would like to try to create some tetraploids in the future. It is a subject that fascinates me. And I will be very careful with anything that can disrupt natural endocrine or hormone concentrations.

In many books it is mentioned that tetraploidity is so common among plants though, that even without these methods it should be quite likely for you to come across a natural occurring tetraploid sport or seedling in your lifetime career of gardening. I would like to be alert at that time and not miss those occasions when they occur. That is why I was looking for an easy way to verify if some plant may be tetraploid.
Of course in theory it should become clear as well eventually out of the crosses made with that plant. But that is a long method. Triploids could turn up in the crosses.

But thank you very much for pointing out this method. I have made my first leaf impressions and slides, and the results are pretty unexpected and lead me to want to make many more leaf impressions and leaf stomata counts.

I bought two kinds of colorless nail polish, of which one is clearly superior to the other for making leaf prints.
The magnification on my microscope is good enough, but it takes quite a lot of fiddling with the diaphragm in order to get a clear focused picture that is good enough to do the count.
The part of the leaf you look at makes quite a lot of difference, there are area's with very few leaf stomata and areas that are quite packed with them.

I decided to compare the areas that were maximally packed with them, and here are the first pictures:

I have used three plants: a mini pitanga, with very small leaves. A 'Big Red' pitanga, which has very large leaves and supposedly very large fruit. And then my own supposed tetraploid that has unusual vigor and very large leaves.
From the study I would have suspected that my own suspected tetraploid plant would have the smallest number of leaf stomata per area, and the Mini pitanga and Big Red would have more it less the same amount...

Here are the pictures:

Mini Pitanga: counted leaf stomata: 48

Mini Pitanga: counted leaf stomata: 53


Big Red Pitanga: counted leaf stomata: 35


Suspected Tetraploid: counted leaf stomata: 68

Suspected Tetraploid: counted leaf stomata: 60



From these results I should deduce that my suspected tetraploid is not at all a tetraploid, but just a very vigorous grower with large leaves, since it has an even higher count of leaf stomata than my control Pitanga- the mini Pitanga.

In the process though, it looks like the Big Red Pitanga could be a genuine tetraploid. I got seeds from this one from Cassio, and it would be interesting to make crosses with it in the future to see if any triploids turn up.

In the coming weeks I'll do some more counts of other different pitanga's I have and hope to get some more data and a sort of baseline number of leaf stomata.

Thanks again for pointing me in this direction, I'm having a lot of fun doing this!

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