I spent yesterday evening watching pollen grains start to grow tubes and then pop, spilling their guts out onto the slide. I'd adjust the formula, try again, and about 9 minutes later, "Pop!" out came the guts. It was the most fun I've had in a long time. Thinking on it this morning, I think I made a mistake with the amount of sucrose in the solution. Hopefully I have a flower or two to work with again tonight.
keen101 (Biolumo / Andrew B.), I imagine I'm going to have a fair number of posts on this subject, as I work my way through various species of beans and different techniques. It's pretty far from the main topic of this thread, but since we're both trying very similar overall techniques it's nice having the discussion in one place. Would you like me to create a new thread called "Advanced pollination techniques" or something, or are you fine with me continuing to post here?
Post by keen101 (Biolumo / Andrew B.) on Feb 27, 2018 1:29:22 GMT -5
andyb, thats fine. If you want to make a new thread you can or you can keep posting here for now. Whatever works best.
Interesting results with the pollen germinating and then exploding. would too much sugar cause that? almost sounds like killing bacteria with osmotic pressure and what i learned about in microbiology. Perhaps i'm not too far off?
I know with bacteria that if you have a too salty external environment it can shrivel a bacteria and sometimes they will die from a too salty external environment vs. internal. The opposite is a too un-salty environment where water basically goes into the bacteria filling it up like a balloon until it pops and dies. If something similar is happening with the pollen i would imagine that either you have too much water or too little sugar/salt. But maybe it's something else.
Try eliminating the water from your mix and consider replacing it with glycerin or just more oil and see if the exploding goes away.
I have misgiveings about putting a drop of water on something and then pollen. Pollen in general rapidly absorbs water and bursts.
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.
Post by keen101 (Biolumo / Andrew B.) on Feb 27, 2018 13:06:44 GMT -5
Here is a photo of some tomato pollen i looked at today. tried just using some oil and some sugar (did not measure it out). Did not observe any germination, but i'm also not sure how long it takes. Will look again later at the same slide. Perhaps some water is needed for germination? But since oil and water do not mix i'm not sure how mixing the two will work. To be honest though i'm not sure the sugar and oil mixed all that well if at all. I feel like one either needs to use all non-water mixes OR a buffered water solution to prevent osmotic pressure explosion but probably not both. But i only have a vague idea of what i am talking about so i could be completely crazy.
This photo is of the mostly domestic tomato that may or may not have S. cheesmaniae ancestry. I know habrochaites pollen is supposed to be smallest and pennellii is supposed to be largest, but i don't know if shape changes as well. Would be cool to collect pollen pictures of all the species as well.
keen101 (Biolumo / Andrew B.) that's a nice photo. I don't have a camera set up on my microscope, so I've just been taking pictures with my phone through the eyepiece. Works reasonably well.
First, here's a link to a short review article I found that describes the different compounds people generally use for in-vitro pollen germination media. It describes the function of each compound, which I think should be useful in figuring out which one to adjust when things don't work: In Vitro Pollen Germination - A Review
You'll definitely need some water to get the boric acid, sugar, and salts in solution. I imagine there are other solvents they would dissolve in, but the water is probably needed to trigger germination, as walt said. I'm starting my mini-project by just trying to get good pollen germination and growth in water solution. After that, I'm probably going to mess around with creating an emulsion using some sort of oil and that identical water solution. If that doesn't work, I'll start messing with the ratios again.
steve1 sent me a recipe from a paper about germinating P. angustissimus pollen (a bean wild relative.) I followed it reasonably closely, making substitutions as needed since I don't have a fully-stocked cabinet of chemicals, and got germination within a minute or two each time. Last night, I increased the sugar concentration and had much slower bursting. Still pretty poor pollen tube growth, though. The recipe from the paper is:
sucrose: 40% H3BO3: 400-500 mg / l CaNO3: 600 mg / l MgSO4: 400 mg / l KNO3: 200-400 mg/l
I made up stock solutions of 1% H3BO3 and 20% Shultz All-purpose Liquid Fertilizer. After making a solution with 20 g table sugar and < 30 ml water, I added 2.5 ml H3BO3 1%, 1 ml fertilizer 20% and topped up the total water to 30 ml. This solution doesn't have any Ca at all, which might be why they're still bursting. I picked up some Damp Rid (CaCl2) and I'm going to add some of it the next time around.
Since I didn't have cover slips, I used little squares of cling wrap. They worked surprisingly well. When I got some cover slips, it turns out that cover slips completely smash the pollen grains when used with a regular slide. I picked up some well slides that I want to try out, but the cling wrap trick is a good one. I saw a mention of Saran Wrap when I was skimming one of the papers you linked to, which is why I gave it a try in the first place.
So, here's a picture of some germinated pollen grains before they popped like balloons:
andyb, nice work so far. What pollen are you trying to germinate there? I'm looking for germ tubes, but can't see any. As I mentioned via pm, getting the recipe right is the issue. It does look like as you've already said you need more sugars or salts to stop the pollen imbibing too much and exploding. Cheers Steve
Oh, for those doing inter-specific crosses, be aware the incompatibility barriers can be temperature related too, so a higher or lower than normal environmental temperature can interfere with these barriers, and allow a cross. Also, cover your pollinated flowers. Just for general pea crosses pollination success is greatly increased. I tape petals over and wait until I can feel a pod form before removing the tape (or else the flower drops), but you could just as easily use small zip lock bags tightened around the petiole.
andyb , Have you tried cutting the styles low down on the runners? My guess is you want the distance the pollen tube has to travel not too many multiples of the length of the tepary style length.
And here is link to a paper with a tepary pollen growth recipe, and nice pics of the germed pollen.
steve1 these experiments are with runner bean pollen. I don't have any other plants growing right now, and I'm going to toss these plants in a week or so. For now, I'm mostly just practicing working with the chemicals and the microscope and working through how to optimize a pollen germination recipe.
Thanks for the link to the paper with the recipe for tepary pollen. I think I'll be attempting actual crosses with added goops and severed styles on runner x tepary crosses in about 2 months. I may also try some crosses with P. polystachios. My tests with applying tepary pollen directly to the end of cut runner bean styles all failed to set pods. For all of my bean crosses, I've been closing the petals back up and clamping the bud shut with a bent piece of twisty-tie.
The pollen in the picture I posted is actually germinated, though just barely. It's a lot easier to see through the microscope than in my low-quality photo. The lumps on the pollen grains are the beginnings of pollen tubes. Last night, I tried adding the CaCl2 and, despite an arithmetic error and adding about twice what I was aiming for, got pollen tubes that grew over the course of 90 minutes to a bit longer than one pollen grain diameter before bursting. Progress!
Switch gears a bit and pique your interest. Go to www.wildgardenseed.com/ and click on the peppers link. Little Tangerine and Stoplight Bells are new listings selected out of a cross I made in 2014 between Orange Bell and Little Bells. I grew the F1 seed in 2015 and had one very nice hybrid plant that produced very good red bell peppers. I sent Frank Morton about 100 seed which he then grew out in 2016 and 2017.
Orange Bell brings exceptionally good sweet flavor, crisp texture, and thick walls to the cross. It has negatives of being susceptible to several diseases and has very brittle stems that are easily broken.
Little Bells brings early maturity, better disease resistance, thin skin, and good flavor. It has negatives of relatively thin walls and is not quite as productive. From the offspring, blocky bell peppers in various shades of green, tangerine, medium orange, and various shades of red can be selected. I expect Frank to stabilize many different lines over the next few years.
Only complaint, he got my name wrong. I'm not Doug, I'm Darrel Jones
Post by Joseph Lofthouse on Apr 5, 2018 21:40:56 GMT -5
I had one plant of F2:[domestic X S. pennellii] which flowered this winter. I vibrated the flowers routinely. They didn't release pollen. They didn't set fruit. Be interesting to see how siblings grow this summer. Two other sibling plants that also grew in the house overwinter are runty, and haven't flowered yet.
Silt/clay, high-altitude, super-arid, sun-drenched, irrigated-desert garden. Cold radiant-cooled nights. ~100 frost free days. Grow most of my own locally adapted landrace seed. GDD10C ~1300. Author of Mother Earth News: Landrace Gardening Blog.
Post by keen101 (Biolumo / Andrew B.) on Apr 6, 2018 0:27:12 GMT -5
The one plant i have growing also does not appear to release pollen. But i finally got it to set a few fruits after weeks of messing with it trying to do manual crosses. Also will try to grow more siblings.
In addition i got seeds for another line of F1 pennellii from TGRC who has a different domestic tomato parent.
This might be slightly off topic, but has anyone focused on using S. peruvianum to introduce higher sugar content or unique flavor profiles to the domesticated tomato? It seems most of the wild species are used mainly for disease resistance, grafting, or technical science mumbo jumbo LOL I'm growing some S. peruvianum and they have the most amazing, mouth-watering-smelling leaves I've ever smelled! It almost smells like cinnamon with a background of some sort of plant secondary compound, the aroma was quite a surprise! I think Joseph mentioned the flavor of S. peruvianum was decent or better than any store bought tomato, does anyone else have experience/input on the flavor of S. peruvianum? Of course, with all the diversity, it's expected some will taste great while others won't.