S. peruvianum & S. pennellii crosses

[andyb]

I've also been experimenting with cut-style pollinations, but for some runner x tepary bean crosses. To keep it simple to start with, I've mostly cut the style at an angle and brushed pollen on. Tonight, I put a tiny drop of water on the end of the cut style and tried to stick a lump of pollen onto the drop of water. It seemed like a good idea but it didn't work very well in practice. After reading the pamphlet you linked to, I might try mushing up a pollinated tepary stigma and using that to pollinate a cut style. Worth a try.

How are you applying the pollen in your crosses?

All of my attempts at style grafting have failed miserably, though I'm still trying. I'd guess that tomato styles would be even harder to graft than beans, since they're so delicate and easy to break off.

[keen101 (Biolumo / Andrew B.)]

Feb 7, 2018 1:55:10 GMT -5 andyb said:

I've also been experimenting with cut-style pollinations, but for some runner x tepary bean crosses. To keep it simple to start with, I've mostly cut the style at an angle and brushed pollen on. Tonight, I put a tiny drop of water on the end of the cut style and tried to stick a lump of pollen onto the drop of water. It seemed like a good idea but it didn't work very well in practice. After reading the pamphlet you linked to, I might try mushing up a pollinated tepary stigma and using that to pollinate a cut style. Worth a try.

How are you applying the pollen in your crosses?

All of my attempts at style grafting have failed miserably, though I'm still trying. I'd guess that tomato styles would be even harder to graft than beans, since they're so delicate and easy to break off.

So far just cutting them off straight and applying pollen to the end. Though i too may try the crushed up stigma idea too. Might work better if i crush up stigmas from both parents. I also think maybe i should only cut half way into the style and leave the rest like the paper suggests. Wonder how the heat treatment would work. Ya stigma grafting sounds like a really cool idea, but for tomatoes i don't think it would be particularly practical or easy. Still cool to know it is an option for other plants. Wonder if that would work on cucurbits?

[walt]

I have misgiveings about putting a drop of water on something and then pollen.  Pollen in general rapidly absorbs water and bursts.  It is a good way to clean a pollinating brush to make very sure old pollen won't still be on it.  Of course you have to let the brush dry completely before using it again.
I can't say for sure that all pollen reacts to water by bursting.  There is at least one aquatic plant that blooms under water and currents carry the pollen to other flowers of the same species.
Back about mid-1970s, some people were getting good results putting lily stigmatic exudate on cut styles then pollenanting.  Seems lily pollen exudate has the right ozmotic pressure to keep the pollen from bursting, and it has a balance of sugars and minerals to help feed the pollen until it has done its thing.
Never tried it myself.  But I remember Dr Cambel of KSU and Dr. James if CIMMYT talking about it after a siminar.  And this morning I forgot about a doctors appointment.  Strange what we remember and what we forget.

[andyb]

I've been cutting them at an angle, figuring that the larger surface area will hold more pollen grains. Just speculation, really.

Cutting half way into the style is a good idea. If nothing else, It would give some mechanical protection to the pollen on the cut. On my normal crosses, I've been closing up the flowers and clamping them with a cut-up piece of a twisty-tie to keep the environment moist and protected, but I've been worried that I would brush off whatever I put on the end of the cut style.

Seems like you could do all sorts of fun style grafting on cucurbits, since everything is so big.

walt Thanks for the input. I have enough other ideas to try that I'll skip doing more water droplet experiments for now.

[keen101 (Biolumo / Andrew B.)]

My experiments with cutting styles and tying to use domestic tomato and S. galapagense pollen seem to have all failed. However, two flowers [one flower] which have not specifically been marked as me having tried cross pollination have not dropped yet and seem to be larger than any up to this point. That is not to say i dd not try to cross these ones and forgot to mark them. Even though pure S. pennellii is allogamous self-incompatible (SI) it can also be facultative self-compatible (SC). I take that to mean that if no foreign pollen is accepted or received that it can revert and accept it's own pollen rather than die out and fail to produce offspring. I also remember reading that S. pennellii is something like 4% selfing even with SI. So it's hard to say what combination of SI has been restored in this particular potato-leaved F2 S. pennellii hybrid, but that is a possible explanation to these fruits developing (assuming that they make it to full maturity). So they could be selfed, hard to know.

I am working on an application to CSU, my local university, for a possible opportunity for a summer research position in an actual university lab and getting paid for it during the summer. I don't know if i will be accepted, but i hope i do. If i'm accepted there is a chance i could be working in the lab of Patricia Bedinger who is coincidentally working on studying interspecific reproductive barriers and wild tomatoes! How cool is that?! So that would be awesome, especially since it is very relevant to my tinkerings in this area already.

So because of my application i have been reading more about wild tomato compatibility mechanisms to refresh my current knowledge and i have been learning a lot of new stuff as well. I just happened by accident or coincidence on two papers co-authored by Bedinger which have been interesting but also enlightening and helpful.

vanderknaaplab.uga.edu/files/Bedinger_Sex_Plant_Review.pdf

academic.oup.com/jxb/article/64/1/265/632781

In particular though i found this little tidbit from the article "Developmental onset of reproductive barriers and associated proteome changes in stigma/styles of Solanum pennellii":

In the wild tomato species Solanum pennellii, both SI and self-compatible (SC) populations express UI when crossed with domesticated tomato, offering a useful model system to dissect the molecular mechanisms involved in reproductive barriers. In this study, the timing of reproductive barrier establishment during pistil development was determined in SI and SC accessions of S. pennellii using a semi-in vivo system to track pollen-tube growth in developing styles. Both SI and UI barriers were absent in styles 5 days prior to flower opening, but were established by 2 days before flower opening, with partial barriers detected during a transition period 3–4 days before flower opening.

so my new strategy will be to target earlier undeveloped flowers. It is possible that the two seemingly developing fruits were targeted by me quite early but if so i forgot to mark them. Regardless, targeting earlier undeveloped flowers in interspecies tomato crosses may be a way to help us overcome this issue sufficiently.

p.s. andyb they mention the use of the oil Trilinolein instead of water.

Trilinolein (50mg), a C18 lipid known to promote pollen germination (Wolters-Arts et al., 1998), was emulsified in 0.5ml of 14% sucrose and 0.01% boric acid (±3mM calcium nitrate) and used to compensate for the lack of exudate on stigmas of young pistils. Stigmas were painted with this emulsion using a camel-hair brush before applying appropriate pollen samples.

EDIT: so turns out one flower was sort of marked. So something i tried worked, i still kinda suspect i caught that one early.

[keen101 (Biolumo / Andrew B.)]

Feb 19, 2018 9:16:14 GMT -5 keen101 (Biolumo / Andrew B.) said:

Regardless, targeting earlier undeveloped flowers in interspecies tomato crosses may be a way to help us overcome this issue sufficiently.

p.s. andyb they mention the use of the oil Trilinolein instead of water.

Trilinolein (50mg), a C18 lipid known to promote pollen germination (Wolters-Arts et al., 1998), was emulsified in 0.5ml of 14% sucrose and 0.01% boric acid (±3mM calcium nitrate) and used to compensate for the lack of exudate on stigmas of young pistils. Stigmas were painted with this emulsion using a camel-hair brush before applying appropriate pollen samples.

I have supposition and idea that i am going to extrapolate from this. I could be way off or a complete nutter, but that's okay. I'm going to share my crazy idea anyway.

So ignoring any major chemistry errors i'm probably going to make: i'm going to say that linolein is basically the same as trilinolein. And i'm going to say that linolein is basically linoleic acid and glycerin. What proportions i don't know. And using the density of linoleic acid i calculated that the mix they are using in the above quote is about 50% Trilinolein and 50% (14% sugar and boric acid). According to wikipedia the following common oils have linoleic acid in the following concentrations:

Note: Unless cited, none of these percentages have been verified by scientific research.
Name % LA† ref.
Salicornia oil 75%
Safflower oil 74.62%
Evening Primrose oil 65-80% [19]
Poppyseed oil 70%
Grape seed oil 69.6%
Sunflower oil 65.7%
Prickly Pear seed oil 62.3%
Barbary Fig Seed Oil 65%
Hemp oil 54.3% [20]
Corn oil 59%
Wheat germ oil 55%
Cottonseed oil 54%
Soybean oil 51%
Walnut oil 51%
Sesame oil 45%

So, we could use common oil to create our own similar solution to compensate for the lack of exudate on stigmas of young pistils. If we use something like Walnut oil that has about 50% linoleic acid already and might be a fine substitute. If you think the 14% sugar is needed then you could use an oil with a higher concentration and add your own solution of sugar to it. Grape seed oil has a pretty high amount around 70%. In our bathroom we have some natural ear ache remedy that is grape seed extract and glycerin. I am not sure that grape seed extract is the same as grape seed oil however. But grape seed oil could be easily purchased from our nearest health grocery store. Or linoleic acid could be purchased online for expensive but affordable price as well.

Walnut oil is already my favorite oil to cook with.

So that's my crazy idea. To use some natural oil (maybe even with sugar) to help pollen stick to stigmas from young flowers (-5 days mature) for interspecies crosses since they lack the sticky substance to help them adhere and grow. andyb what do you think?

[andyb]

Heh. I saw your previous post this morning and navigated my way to that exact table on Wikipedia.

I ended up attempting seven runner x tepary crosses with severed styles. All seven flowers dropped off the plant without setting pods, while almost all of the crosses I attempted with my normal emasculation/pollination process set pods on that plant. When I looked at the cut end of the styles with a hand lens, they generally seemed dry, with very little pollen adhering. They also seemed to dry out pretty quickly. With the diagonal cut, the thin edge would shrivel within half an hour or so. When I tried smashing up some tepary stigmas/styles to try to get a paste or liquid, it just didn't work. The amount of plant material I was working with was really small and I think it was drying out pretty much instantly, before I could get it onto the cut style.

My organic chemistry is pretty weak, but it seem like messing around with a variety of different oils and emulsions and possibly glycerine is worth a try. At the very least, more pollen would stick to the cut end of the style. I found some boric acid at a hardware store, so I think I have everything I need.

I'm not going to have any bean flowers to work with for the next two months or so, as I try a grand de-fungus-gnatting of the house and then start the tomatoes and peppers for this year's garden. When I do, I think I'll try some more experiments. I'll be interested to hear what you figure out with your own experiments before then.

On a related note, I came across this talk by Dr. Charles Simpson, an emeritus professor at Texas A&M who has done a ton of work with peanuts and their wild relatives. In passing during the question and answer session, he mentions that he learned of a crossing technique that produced two seeds from five crosses of a particularly difficult wide cross that originally took 3500 crosses to produce one seed. I've been meaning to send him a note to ask him what the technique was: www.youtube.com/watch?v=-cDaMMjvrCY&t=2595s

[keen101 (Biolumo / Andrew B.)]

andyb, right at the very least plain oil would help more pollen stick, especially on early inmature stigmas/styles.
Cool. sounds good. i will continue my experiments. In the mean time you may find the following info useful and interesting.

Sounds like the boric acid is helpful in helping pollen grains germinate and grow. It also sounds like gibberellic acid may also do the same and perhaps boric acid is simulating this natural acid in this case. Boric acid should be easily obtainable, though i don't have any right now. Gibberellic acid is also easily obtainable (united nuclear is one source for it).

file.scirp.org/Html/17-2600922_35476.htm
www.scielo.br/pdf/rca/v48n3/1806-6690-rca-48-03-0496.pdf
Influence of Boric Acid and Sucrose on the Germination and Growth of Pollen

link.springer.com/article/10.1007/BF00132920
Break down of unilateral incompatibility through boric acid

link.springer.com/article/10.1007/BF00024772
Self-incompatibility has been partially overcome, in vitro by treating pollen and/or stigma with gibberellic acid

[andyb]

keen101 (Biolumo / Andrew B.) The original quote you posted mentioned an emulsion. I'm imagining using something like this technique:
livesimply.me/2016/08/12/smooth-homemade-lotion/
but with walnut oil, sugar, water, and ant poison (boric acid). I would store it in a locked container way, way away from anywhere a kid ever goes. I'm guessing it would go bad really fast, too.

It also refers to trilinolein as a substance that's known to promote pollen germination. Since boric acid seems to serve the same function, even if the walnut oil doesn't promote pollen germination its presence in the emulsion might provide the stickiness and moisturizing qualities that I think I need, while the boric acid might trigger pollen germination.

[philagardener]

Would it be worth checking pollen in these elixirs on a microscope slide to see if it actually is stimulating germination? Of course, there may be synergistic effects when it is applied onto the style (mixing with natural exudates) but it might be a quick way to see what is going on, literally.

[keen101 (Biolumo / Andrew B.)]

philagardener, not a bad idea. I have a microscope. But i wouldn't really know what to look for.

I guess something like this:

www.youtube.com/watch?v=xYD4Q77LYHo
www.youtube.com/watch?v=5udjZPeDeWM

www.youtube.com/watch?v=kBlg4sI_VyM

Doesn't look too hard. Guess i need to get some of those concave / well slides.

p.s. interesting facts, S. pennelli has the largest pollen grains of all the tomato species. S. habrochaites has the smallest. But both travel through far styles so grain size in tomatoes does not determine how far they can travel.

yeah, i'm thinking a good mixture would be something like Walnut or Grape Seed Oil, sugar, glycerin, and boric acid. But i don't know for sure. Still, i like the idea.

[andyb]

Most of the papers I've read (mostly found by keen101 (Biolumo / Andrew B.), thanks!) start with a section where the authors try out some pollen with their preferred elixir or a range of elixirs with various recipes in a model environment first, confirming the pollen germination with a microscope. Everyone seems to have a slightly different technique. After they confirm germination and good pollen tube growth, they move on to flowers on real plants.

Knowing myself, I'll probably take one or two shots in the dark and, when they don't work, let out a long sigh and run a proper experiment.

[philagardener]

A time lapse video can be a bit misleading with respect to how quickly things might happen, but the presence of elongated pollen tubes is pretty distinctive.

Take a look at the figures in this open access paper (they are of germinating tomato pollen):
www.frontiersin.org/articles/10.3389/fpls.2015.00391/full

[andyb]

I found a couple flowers on a P. coccineus plant and got excited. I spent two hours dusting off decades-old memories of lessons from high school chemistry, wrote out a bunch of conversion factors, and mixed solutions of fertilizer, sugar, and boric acid. A few final pours and I had 50 ml of in-vitro pollen germination solution ready to go. I got my microscope out. Got the slides out and...no cover slips. I searched the whole basement and they were nowhere to be found. Gaar.

Amazon is shipping me some, along with some well slides.

There are some more flower buds. Looks like the next batch will be open on Sunday.

[keen101 (Biolumo / Andrew B.)]

well, the good news is that i have at least two, probably 3 tomatoes developing from my original tinkerings. One of which is marked for sure. Not sure if they were successfull normal pollen dabs or cut styles or what. But probably with the what i am calling domestic tomato pollen (the one with possible S. cheesmaniae ancestry but is clearly mostly domestic). S. galapagense is also a possibility.

I tried a few with a random walnut oil mix last week. There may be some tomatoes from that, but not sure. Some of those clearly failed. Still, i got my Boric Acid in the mail the otherday, so i am ready for future experiments and oil protocols. Perhaps a technique even more useful with the S. sitiens and S. lycopersicoides plants. Pennellii and chilense are said to be good bridge species for them.