Breeding for improved beta-carotene
 

I plan to breed for improved beta-carotene in a paste tomato. I know to some degree the presence of the desired genes can be discerned by the color of the tomato, but I believe to be truly accurate, the tomato would have to be tested. Does anyone have an idea where this testting could be done, and how much it would cost (I'm far from that point, but would like to prepare). Also is there any 'home-made' or home chemistry testing that could be used? (Even if not as accurate, it would be useful for discriminating between segregating lines.)

Very easy to test for carotenes at home with commonly available materials.

Extract the pigments with a solvent.
Run a paper chromatography test.
If you keep the concentrations of your tests consistent, you would be able to dry the test strips, and compare them one against another to determine which has the highest concentration of carotenes.

You could even make it semi-quantitative by using β-carotene vitamin pills to make control solutions of known concentration.

For example: [URL]http://www.hsu.edu/pictures.aspx?id=1653[/URL] will give you ideas about what keywords to search for additional details. "Paper chromatography carotenoids" is a great search term.

Joseph is correct. Paper chromatography is a good way to separate plant pigments and can be done easily, it was one of the favorite lab exercises I used to teach. Making it quantitative will require some work and careful measurement. You should be able to get relative differences if not an absolute quantities. if you can get your hands on real chromatography paper that would be best. I would also recommend a dot instead of a line of extracted pigment, the smaller the better.

Depending on how fancy you want to get, old spectrometers can be found on Ebay for a couple hundred dollars...would be able to do quantitative analysis easily.

The attached doc is not mine but is a reasonable overview of the process.

Thanks! This is the kind of start I'm looking for.

Chris

You might start by finding the variety 97L97 which is a 40X carotene variety.

DarJones

Just googling the terms in this thread has made me smarter. Not that that's saying much, but still.

Would a "spectroscope" work for this purpose? Or does it have to be a spectrometer?

Also... it's truly amazing what you can buy on Amazon in the Industrial & Scientific section. :lol:

To get really accurate results would require a gas chromatograph. Fortunately, it is not necessary to get that detailed, a recent study showed that visual examination can lead to significant genetic gain when selecting for lycopene which just happens to be a red carotenoid.

Interesting, so you'd basically be looking for the orangest, most carrot-colored (or reddest, for lycopene) tomato you could get. In that case, could you use a digital camera plus color balance strip to make photos to compare to other attempts? Maybe use the one you referred to earlier and a carrot as reference points?

Thinking "cheap" and "no volatile chemicals" here.


Tl

I am breeding for high carotene corn and butternut squash. A visual test is sufficient. Of course I don't have red lycopene interfering with my color tests.

[IMG]http://i1074.photobucket.com/albums/w406/JosephsGarden/Plant_Breeding/Popcorn/popcorn-colored_zps9c1ade75.jpg[/IMG]

[IMG]http://i1074.photobucket.com/albums/w406/JosephsGarden/Plant_Breeding/butternut-yellow-to-orange.jpg[/IMG]

The other thing that might work well is a set of standard color strips like you might find at a paint store, or on the back of the box for a pool test kit.

[QUOTE=tlintx;362488]Interesting, so you'd basically be looking for the orangest, most carrot-colored (or reddest, for lycopene) tomato you could get. In that case, could you use a digital camera plus color balance strip to make photos to compare to other attempts? Maybe use the one you referred to earlier and a carrot as reference points?[/QUOTE]

I would have thought this too, however in doing online 'research' I've found that not all orange tomatoes are created equal. Anything in the 'yellow' to white (often noted as [B][I]r[/I][/B]) reduces all carotenoids.

There is an 'Apricot' gene (noted as [B][I]at[/I][/B]) that seems to show a very slight increase in beta-carotene.

There is a 'tangerine' gene (noted as [B][I]t[/I][/B]) that has reduced beta- and and increase in something called zeta-carotene (and other different carotenoids

And there is [B][I]Del[/I][/B] which has 'normal' beta but much increase delta-carotenoid (I can find information about beta (of course) and have found out that alpha carotenoid is considered 'half' as effective for conversion to vitamin A, but there doesn't appear to be any information on whether delta-carotenoid can be converted to vitamin A in the body.

The article about breeding these is here: [URL]http://www.genetics.org/content/62/4/769.full.pdf[/URL]

There is another article about breeding with [B][I]B[/I][/B] (high beta-carotene) and [I][B]Del[/B][/I] here:
[URL]http://www.genetics.org/content/56/2/227.full.pdf[/URL]

However there doesn't appear to be articles about breeding these newer very high carotene tomatoes with both [B][I]B[/I][/B] and [B][I]M[/I][/B][SIZE=1][B][I]OG[/I][/B] [/SIZE][the G should be subscripted] and how they interact with the other 'orange' genes. An article about the USDA introduction of breeding stock with these high carotene genes notes that [B][I]B[/I][/B] (which is dominant) and the dominant form of the linked modifier gene [B][I]M[/I][/B][SIZE=1][B][I]OG+[/I][/B] [/SIZE]reduces beta-carotene from 90% of the carotenoids to 50-60% and increases lycopene to less than 50% giving red-orange tomatoes. That article is here: [URL]http://hortsci.ashspublications.org/content/36/2/387.full.pdf[/URL]

The 'safest' way (it seems to me) to preserve high beta would be to breed the high betas only to red (or pink) varieties, and select them back to orange.

Or you could eat twice as much of the super 40x beta carotene tomato Fusion mentioned above. :lol:

Beats liver any day!

There is a possibility that I can get a Del tomato to sell plants for next year. I'll see what can be done.

Meantime, look up "Stommel carotene" if you want to read about some interesting breeding work.

As an fyi, ramping up beta carotene has a drastic impact on flavor.

[QUOTE=Fusion_power;362876]As an fyi, ramping up beta carotene has a drastic impact on flavor.[/QUOTE]

I haven't played with high carotene tomatoes, but I've certainly noticed improved flavor in muskmelons, squash, and carrots due to higher carotenes.

[QUOTE=joseph;362675]I am breeding for high carotene corn and butternut squash. A visual test is sufficient. Of course I don't have red lycopene interfering with my color tests.
[/QUOTE]

Plus you can get orange by more than one path. :?

But your corn is beautiful, as I remember, a local museum (Landis Valley) sold a form of popcorn that appeared 'pre-buttered'. I like your squash as well, however, as I'm the only one in my family who likes squash, I only inflict a small amount on them. :lol:

You might find this paper interesting. Think I have posted it before.

I am also looking to breed for improved beta- carotene. Maybe we could share info. Here is my latest, a F1 cross between a galapagos tomato and a pink heart. The plant is huge and the tomatoes are bigger then normal for a cross with a wild galapagos. Now I just have to select for orange colored fruit and increased size.
craig

[QUOTE=Minnesota Mato;363806]I am also looking to breed for improved beta- carotene. Maybe we could share info. Here is my latest, a F1 cross between a galapagos tomato and a pink heart. The plant is huge and the tomatoes are bigger then normal for a cross with a wild galapagos. Now I just have to select for orange colored fruit and increased size.[/QUOTE]

I'd be glad to share information (and maybe even breeding stock), but I won't get my F1 cross until next year, as I didn't have the tomatoes (of the right kind) or my plan worked out until this summer. So I using this summer as a 'practice' for tomato breeding [I'd estimate that only about 1/3 of my crosses are 'taking'. Is this fairly standard for a beginner at tomato crossing?.] I'm not familiar with the Galapagos tomato. What's it like?

What I learned so far (plus questins):
1) Not all orange colors are created equal
2) Elevated beta carotene results from the presence of the gene [B]B[/B] (The
tomato is orange-red) the highest levels result from the additional precense
of the modifier gene [B]M[SIZE=1]OG[/SIZE][/B] (and an orange color).
3) Question: will high levels of beta-carotene cause unfavorable flavors? Can
this be bred around?
4) [B]B[/B] is on the same chromosome as the gene for determinate growth and is
linked to it. Can this link be broken?

Next year I plan that my main tomato breeding will involve CaroRich, 97L97, and Opalka.

I prefer the taste of high carotene foods to low carotene versions of the same food.

Genes crossover between the paired chromosomes, so it's possible to break the linkage between two genes on the same chromosome. How frequently that happens depends on how close together the genes are located on the chromosome. If they are on opposite ends of the chromosome it's like they are not linked at all. If they are side by side then it would be much less likely to break the linkage. If you can find a gene-map containing both genes, and it has a centimorgan scale on it, then that is a measures the likelihood of the linkage being broken in a cross. 1 centimorgan = 1% chance. So if they are tightly linked you just have to grow more plants to break the linkage.

Here's an [URL="http://solgenomics.net/cview/map.pl?map_version_id=137"]example of a gene map[/URL]. In this particular cross, chromosomes 3 and 8 contain a QTL for carotene content. The B gene is located on Chromosome 6. So there are lots of opportunities for increased carotene content.

I would like to trade info and breeding stock. To be more precise the tomatoes come from the Galapagos islands but are called CHEESMANII. There are alot of cool things about cheesmanii but crossing with them is like starting from the beginning. Crossing to get the B gene is easy but keeping all the positives and getting a decent size is hard. You should google cheesmanii.
craig

[QUOTE=Minnesota Mato;363806]I am also looking to breed for improved beta- carotene. Maybe we could share info. Here is my latest, a F1 cross between a galapagos tomato and a pink heart.[/QUOTE]

Has your cross started to ripen yet? How does it seem so far?

I am already fermenting the seeds! I am going to try to plant a few and see what I can do before winter. Both of my crosses so far have been a nice deep orange. I need to start selecting for greater size now.

Odd result in the F2
 

I can't post pictures at this time but I'm having an 'odd' result in the F2. I crossed Opalka X 97L97. Opalka is a large pointed heirloom indeterminate paste type and 97L97 is a determinate paste colored orange because it carries the B gene.

The F1 seemed to be as expected, there was slight size differences in fruit size and shape between the plants, but all the plants were indeterminate, orange colored, and about the size of 97L97.

I expected significant variation in the F2. I got them in late after significant groundhog damage, so none have ripened yet. I expected size variation, and some shape variation but all 7 of my F2s appear to be small, nippled ,longer than wide fruit that are much smaller than either original parent. I guess I have a problem with cross pollination, but is there any other thoughts why I would be seeing this? I would think I would see more variation given the F1 parent was a hybrid. The fruits thus far seem almost identical like I was at an F5 or something.

Please post any ideas you may have.

Chris, I had similar unexpected results - where the F2 were all very very similar to my F1 and looked to be easy to stablilize that type, but then the F3 turned out to be a very mixed bag. :?!?:
Another one was at F4 and seemed to be quite stable, but then the F5 suddenly saw a load of segregation for shape. These were hearts that segregated into many subtypes including paste shapes, nipples or not, etc.

My impression is that the shape genetics of pointy fruit are very very complex. There may be some epistasis involved which prevents things being expressed as expected. Then with another shuffle, bingo out they come. :surprised: Pretty baffling.
I was reading about it this spring, there are ??? seven I think different genes for 'pointy'. So crosses of two pointy fruit won't necessarily segregate as expected if there were only one gene for that.

Thanks bower - that at least helps make some sense.

I believe I am going to revise my approach. I think I am going to do some (F1XF1) crosses and (F1XHibeta). The F1s are already (XHibeta).

[QUOTE=bower;662150]Chris, I had similar unexpected results - where the F2 were all very very similar to my F1 and looked to be easy to stablilize that type, but then the F3 turned out to be a very mixed bag. :?!?:
Another one was at F4 and seemed to be quite stable, but then the F5 suddenly saw a load of segregation for shape. These were hearts that segregated into many subtypes including paste shapes, nipples or not, etc.

My impression is that the shape genetics of pointy fruit are very very complex. There may be some epistasis involved which prevents things being expressed as expected. Then with another shuffle, bingo out they come. :surprised: Pretty baffling.
I was reading about it this spring, there are ??? seven I think different genes for 'pointy'. So crosses of two pointy fruit won't necessarily segregate as expected if there were only one gene for that.[/QUOTE]

Changing Approach
 

Well my F2's have mostly come in and their consistency is amazing (I earlier described them as 'nippled', but when fully developed they are an oval.

All the fruit are all ovals and about 1-[SIZE=1]1/2[/SIZE] inches long by about 1 inch. The are all orange with the skins blushed with a red hue. There is slight internal wall thickness variation, but is slight enough that I decided not to differentiate on that.

So all my F3 that have produced tomatoes so far (Plants 1 through 5), I decided not to keep separately, but am fermenting them together.

The Jaune Flamme X Shannon (plant 2) F2, though it was the shape and size I wanted, and appears very productive, was red, was very juicy, and the fruit walls were thin.

I might grow some out in 2018 to see if I get any differentiation, but I am planning several different approaches now.

One of the problems I'm fight is the following 97L97 has high Beta gene with the modifier gene (for higher beta carotene, but is determinate (but is a paste type)

Jaune Flamme has the high Beta gene and is indeterminate, but does not have the modifier gene (and is not a paste type).

The high Beta gene is on the same chromosome as the sp gene (Determinate) so is 'linked' to it (and is close enough that breakage between the two is fairly rare.

I'm looking to develop a paste that is indeterminate, and has both the high Beta and the Modiere


First, I'm planning (Jaune Flamee X Shannon) X (97L97 X Opalka) (and vice versa)

Second Backcross to High Beta (Jaune Flammee X Shannon) X 97L97
AND (97L97 X Opalka) X Jaune Flammee.

Any thoughts?

Thinking about the beta linkage... Last year I grew six F3 plants of a heart or paste shaped selection - from an F2 that was Beta/- (orange-red) and indeterminate. I expected to get 1/4 (roughly!) that was Beta/Beta and determinate because of the linkage (Zolotoe Serdtse parent line), but instead I had more Beta/- indeterminates again and no determinates at all in the six.

You made that great post about odds awhile ago, do you think you could up the ante just by growing more plants from your F2 or F3?:?!?: I thought the result was 'unlucky' for me but it's not out of the question with half dozen plants.

I also like your strategy of crossing unstable generations, and of crossing sp/Beta with indeterminate Beta, where both conditions are linked, what will happen? Would be nice to see the result of some larger numbers in that cross. :yes:

[QUOTE=bower;662747]Thinking about the beta linkage... Last year I grew six F3 plants of a heart or paste shaped selection - from an F2 that was Beta/- (orange-red) and indeterminate. I expected to get 1/4 (roughly!) that was Beta/Beta and determinate because of the linkage (Zolotoe Serdtse parent line), but instead I had more Beta/- indeterminates again and no determinates at all in the six.

You made that great post about odds awhile ago, do you think you could up the ante just by growing more plants from your F2 or F3?:?!?: I thought the result was 'unlucky' for me but it's not out of the question with half dozen plants.
[/QUOTE]

1) You've given me an interesting idea (more later) Thanks!
2) I started playing with some Punnett squares, assuming there is no breakage (Beta and self-pruning are very close on the chromosome). I run into problems with the existing F1's crossed together, I would get 3/4 orange-red and 1/4 red, but only 1/3 of the orange-reds have the indeterminate high beta with the modifier gene present but hidden. Think I'll still do it, but I'll have to grow out a good amount of progency of the 2nd generation to find what I'm looking for.
3) This is the 'good' idea you've given me. I'm going to cross my two beta varieties, and select for indeterminate + Modifier (3/16) Only then will I cross the resulting tomato with pastes.
4) The other crosses I talked about (F1 X high beta) I may yet pursue but I'll need to analyze the potentials with more Punnett squares)


[QUOTE=bower;662747]
I also like your strategy of crossing unstable generations, and of crossing sp/Beta with indeterminate Beta, where both conditions are linked, what will happen? Would be nice to see the result of some larger numbers in that cross. :yes:
[/QUOTE]

I'm going to probably do that as well (I also saved seed from the same F1 cross, but a different plant, so I can see if get a different result.

Linkage Blues (Back to the drawing board)
 

Wanted to post about what I've realized about the Hi-Beta breeding I've been attempting.

The tomato I'm trying to achieve is a high beta-carotene, indeterminate paste tomato.

At this time, I'm going to ignore the "paste" part and concentrate on achieving a high-beta indeterminate tomato.

However the gene for high-beta (B) is located on the same chromosome as the gene for indeterminate/determinate (which is noted as sp (self-pruning) so indeterminate is (sp+). The linkage is close enough that I've now realized that it is unlikely to break for the numbers of plants I can deal with (20+ with up to 45 or so if I grow no other tomatoes). So I have realized for breeding purposes I have to effectively treat the beta gene and it originating growth type as a unit.

There is a third gene I want for this tomato - designated as m[SIZE="1"]og[/SIZE]. This is a modifer gene that increases the beta carotene from 50-60% to near 90% only in the presence of the high beta gene. the one I want is the recessive form (I'm going to use just (m) as the designator for this gene in the further parts of this post.

So the tomato I want to ultimately acheive is sp+sp+BBmm

Again the genes are
sp+ - indeterminate
B - high-beta carotene (50-60% when alone)
m - in the presence of B increases beta-carotene to ~90%.

The main 4 tomatoes I've worked with thus far:

[U]97L97[/U] - A high beta-carotene breeding tomato genetics (spspBBmm) (it's determinate)
[U]Jaune Flammee[/U] - high-beta french tomato without the modifier gene: (sp+sp+BBMM)
[U]Opalka[/U] and [U]Shannon[/U] - for this purpose they have similar genetics: (sp+sp+bbMM)

All these tomatoes have the R (red) gene which I'm going to ignore for the moment, but I wanted to note that B does not 'work' in the presence of the other form of yellow/orange tomatoes that are caused by the recessive (r) gene.

The one advantage I have is the coloring of the tomatoes shows me the beta/modfier genetics:

[U]bb--[/U] (modifier gene doesn't matter the fruit will be red )[ATTACH]78879[/ATTACH]
[U]B-M-[/U] (high Beta without modifier gene with be orange-red (orange, then developing an red blush (although the blush can be slow to appear) [ATTACH]78880[/ATTACH]
[U]B-mm[/U] (High Beta with modifier gene will be orange and never develop a red blush)[ATTACH]78881[/ATTACH]

So a cross between 97L97 and Opalka yields an F1 Hybrid of sp+spBbMm [ATTACH]78880[/ATTACH]

and a cross with Jaune Flammee and Shannon yielded an F1 Hybrid of sp+sp+BbMM which is also [ATTACH]78880[/ATTACH]

I thought of crossing the two hybrids but the results were disappointing. Also in looking at the Punnet square that instead of the 8X8 square you'd think of with 3 separate genes it is much smaller because you're handling the sp gene and the beta gene as a locked unit:

[ATTACH]78882[/ATTACH]

So I considered taking either of my original F1s and BC to the other Beta

(97L97 X Opalka) X Jaune Flamme

[ATTACH]78883[/ATTACH]

The F2 generation look like this (depending on whether the seed you got is MM or Mm

[ATTACH]78885[/ATTACH]

I actually like the BC of the 97L97 back to the Jaune Flammee X Shannon slightly better:

[ATTACH]78884[/ATTACH]

And the resulting F2s:

[ATTACH]78886[/ATTACH]

So I'm going to do that and I'm also going to cross Jaune Flammee and 97L97:

The F1:

[ATTACH]78887[/ATTACH]

And the resulting F2:

[ATTACH]78888[/ATTACH]

And all this is long before I get to breeding toward a paste!!!!

Great stuff! Thanks for posting, I don't have much to add to your thoughts, but I think you plan forward sounds logical. I enjoy these crosstalk threads, they are interesting and make me think.

As an aside I'm also interested because I have a cross using Jaune Flammee as the pollen donor that I'll be growing out the F1 this year.

Keep us posted!