Leslie, I wanted to thank you for writing this article. The main take-away that I got from it that I didn't know before was the temperature at which pollen becomes viable and the temperature at which the pistils shut down. I didn't realize the pollen and stamen juices were quite so touchy, I thought my pollinations weren't taking simply because they were too thirsty. (It might still have been that, too, but atleast now I know to pay more attention to the temperature as well.)

Hi,
The temps aren't cut and dried. I've rec'd one message saying I was dead wrong in what I wrote about when they work and when they don't. I'm going on what I've been taught and my personal experience. Take it with a grain of salt.

Still, it's something good to go on. There might be something to it, or maybe it's simply correlational and not causal, but if it helps my crosses take better at all, it was good advice.

I think the problem with temperatures, to me anyway, is whether they are being measured in the sun at daylily flower height or, as in official weather forecasts/reports etc., in the shade at a specified height above the ground. The "official" temperature for any given hour in the shade is going to be quite different from what a daylily flower in the sun is experiencing.

Also perhaps there are variations according to cultivar and maybe those working in the south have, over time, developed cultivars that function at higher temperatures than daylilies in the north?

Oscie Whatley, in an article on hybridizing, re-published on the AHS web site here:
http://www.daylilies.org/Whatl...

"Temperature: Probably all varieties have a shut down temperature for the pod parent. I am not sure of the low side but the high side seems to be around 90 degrees F for tetraploids and a little higher for diploids with some variation for each variety. I believe pollen has a higher range but haven't tested it."

I checked when Hemerocallis lilioasphodelus flowers here for the last couple of years and typically gets loaded with bee pods, and the temperature only reached 80F or slightly above as an afternoon high temperature on a few days during those flowering periods.

Toru Arisumi of the USDA, Beltsville, Maryland did some studies on the cultivar 'Purity'. He pollinated at 75F and 85F and maintained the plants at those temperatures until seed harvest. He said "Seed set was good at 75F and poor at 85F".

There is an article on hybridizing in the American Hemerocallis Society's 2002 Daylily Handbook called "Breeding of Diploid Daylilies". Quote: "........crosses should be made as soon as the anthers containing the pollen have opened and the pollen is plainly visible. Crosses may be made earlier. By squeezing the anthers end to end, the anthers may be made to open and the pollen used. A dry morning with a temperature of 72F is ideal. Early pollination is advised because pollen dries rather quickly on a hot day and by noon it may have blown away, shaken out, or been knocked off by bees or other insects." The author goes on to say that pollination is better before the temperature gets to 90F, and says "it is estimated that three hours below 90 (optimum 72 to 80F) are required for the pollen tube to begin growing down the style into the ovary".

So there seems to be more or less a consensus on the high temperature, but not so much for the low. It's noon here on 1st August, the sun is shining and the temperature is currently 74F. If the stigmatic fluid didn't start until 80F then no pollen should be activating here yet today where the "normal" high for the time of year is 78.8F. We'll find out since I was out there happily slopping on pollen an hour or so ago

Edited to add this link to the AHS PDF "Daylily Fertility Tips" which gives opinions of Darrel Apps, Pat Stamile, Karol Emmerich, Jack Carpenter, Curt Hanson, Dan Trimmer, Melanie Mason, Maurice Dow and others:
http://www.daylilies.org/2007D...

Great info Sue. Thanks!

Lalambchop1 said:Great info Sue. Thanks!

You're very welcome. It was good of you to take the time to write this and pass on your experiences. I was also curious about the drying up of stigmatic fluid statement because I'd never heard of this before. So I went out mid-afternoon here yesterday and brought in some styles to look at under the microscope. At least at that time there was still plenty of presumably stigmatic fluid on the stigmas (and we never got to our predicted high, it clouded over and started to rain putting an end to my stigma/style collecting - the temp maxed out at 25.5 C (just under 78F). I tried to take some pictures but they're not as sharp as I would like, here's one:



Interestingly Mike Huben, a "northern" hybridizer, wrote a few years back in an article ".......Some varieties also remain receptive well into the senescence of the flower, and can be pollinated even after the bloom has closed."
http://world.std.com/~mhuben/p...

I wish I'd read that before because I'd wanted to put some pollen on a daylily that had lots of flowers the day before but none the day I had the pollen I wanted to use. I thought about trying it anyway and should have!

Wow! What an amazing picture.

I've done some reading about the stigmatic fluid. I know Dan told me that if it dries and seals over it won't accept pollen and I've found that to be true. One some pistils where I've seen what looked like a dry end I've swiped pollen over the tip and it literally would not stick. One of the articles from AHS archives says that in very high heat the fluid will sometimes dry out or not be made. My experiences of hybridizing are in areas where summer temps. regularly reach the mid-90's and sometimes rise above 100. I think this may explain why I've seen "dry" tips and others haven't.

Does anyone know what causes *curling* of the stigma? Is this related to hot weather as the flower is forming (maybe the day or two before?) or even the day of? Maybe it's lack of water (which could be related to the heat)? Or is it just a random deformity? Has anyone successfully set pollen on a curly or zig-zag stigma?

I think maybe you mean the style rather than the stigma (the style is the long tube with the stigma at one end and the ovary at the other, all comprising the pistil)?:

http://www.daylilies.org/ahs_d...

The style normally curves so I assume you mean more than normal, and spiraling of the style where it wasn't earlier in the day is a sign of an aging flower according to some research in Russia. Can you post a picture of what you're seeing? I think I've seen them start out that way and put it down to a developmental deformity but don't really know why.

Incidentally, that same research found that native daylilies had stigmatic fluid between one and a half and two hours after the anthers opened.

Yes! Sorry, wrong terminology, I did mean the style.

When you say an aging flower, do you mean the bloom, or the actual fan itself? The curly style was a one-time thing (so far) on my Jungle Beauty plant I just purchased in the last few weeks. It was the first issue with a style that I've had on her (she's probably bloomed 8-10 times since I've had her). I wish I had taken a picture, but I was in a hurry.

That's interesting about the stigmatic fluid time frame and the anthers. Good to know!

The bloom. Apparently even before the tepals have started to shrivel at the end of the flower's life the pistil becomes deformed or spirals, effectively pulling back the stigma to the level of the anthers. I've seen the occasional style that doesn't follow a normal curve right from the beginning, but I can't say that I've paid a lot of attention as I don't so much hybridize as experiment I did do a bit of dabbing this year but I've no idea where I'm going to put the seedlings if I get any Hitherto most of my experiments have been with daylily seed dormancy and I don't keep those seedlings.

I would post your question in the regular daylily forum too.

Lalambchop1 said:Wow! What an amazing picture.

I've done some reading about the stigmatic fluid. I know Dan told me that if it dries and seals over it won't accept pollen and I've found that to be true. One some pistils where I've seen what looked like a dry end I've swiped pollen over the tip and it literally would not stick. One of the articles from AHS archives says that in very high heat the fluid will sometimes dry out or not be made. My experiences of hybridizing are in areas where summer temps. regularly reach the mid-90's and sometimes rise above 100. I think this may explain why I've seen "dry" tips and others haven't.

Thank you - I was hoping to get some better pictures today but it's been raining so that's out the window. Just wondering, which AHS archives did you see the article in, the AHS web site or the AHS Robin?

In my experience viable pollen does not need moisture to 'stick' to surfaces. I have had anthers accidentally touch and pollen stick to dry styles, filaments, petals and sepals. I tried a test just now and had viable pollen 'stick' to dry petals, sepals, daylily leaves, filaments and styles.

I tried a different test as well. I opened daylily buds that were several days away from opening by themselves (a test that Sue suggested). The buds were approximately half-size and tightly closed. I used pollen expected to be viable and had no difficulty with it 'sticking' to the stigma. There would not have been any stigmatic fluid or any other liquid present. The pollen adhered to the dry stigmatic surface.

Whenever one does a garden test of the pollination process one has three possibilities. Any problem present may be caused by the pollen, by any part of the pollination process due to the pistil/female tissues or due to both.

Stout pollinated his flowers and then collected the pistils. He examined those pistils microscopically to see how far the pollen tubes had moved in the channel. To conclude that stigmatic fluid dried and blocked the growth of pollen tubes one would have to repeat his experiments and examine pistils microscopically and see a plug at the opening and no penetration of pollen tubes into the channel. Without doing that or something equivalent we are left with what are guesses or assumptions about what is happening.

If pollen is not sticking to a stigma, whether the stigma is obviously moist or seemingly dry, possibly the problem is not with the stigma but with the pollen?


The photo is of an immature (by several days) pistil (from a bud cut open today) and pollinated with viable pollen, showing the pollen sticking normally to the dry stigma.

Sue, here's a link to the article in which I read about high temps. It was originally published in the Journal. http://www.gracegardens.com/gg...

Maurice, that is interesting about Stout's studies. I'd like to replicate them.

Lalambchop1 said:that is interesting about Stout's studies. I'd like to replicate them.

It is possible to read Stout's scientific paper if one joins JSTOR (free) at http://www.jstor.org/stable/24....
You will be able to read how he examined the pistils and replicate his studies. Studies in other species have found that the effects of temperature on pollination are genotype-dependent so repeating his studies on several different modern diploid and tetraploid cultivars would be useful.

I have copied the relevant section of his Materials and Methods from his daylily paper directly below:
"In obtaining material for study, as a rule as many as 50 flowers of a single clone that had opened under
glassine bags were pollinated at one time, usually at or near 9:00 A.M. It is at this time that the anthers of
most flowers of the diurnal daylilies are fully dehiscing and the pistils are receptive to compatible
fertilizations. At intervals thereafter, at least three flowers were removed and their pistils preserved for
study. For premature pollination, delayed pollination and pollination of the flowers of night-blooming sorts,
applications of pollen were made at other hours.
In the study of the pollen tubes in the pistils the paraffin method was utilized, but mostly a method of direct
dissection and staining was employed as already described in considerable detail (Chandler, 1931; Stout,
1931b). The pistils are preserved in a solution of 100 cc. of 70%, alcohol and 6 to 8 cc. of commercial
formalin (37%). In staining, the pistils are dissected and a few drops of aceto-carmine (saturated solution in
45% acetic acid) are made to flow the entire distance of the style. After a few seconds a drop of magenta
(1% aqueous solution) or a drop of aceto-carmine to which has been added a trace of ferric acetate is
placed on the style. The excess stain is removed with absolute alcohol. Glycerine and a cover glass
complete a preparation which may be kept for study for a period of several months.
With the use of a microscope the extent of the growth of the pollen tubes in the various pistils is determined
and tabulated. If tubes extend into the lower part of the style the ovary is also sectioned and stained. By
this method it is rather easy to determine the position of the ends of the pollen tubes, their distribution in
the pistil, and their appearance. The pistils of the various kinds of daylilies differ somewhat in length. For
example, the styles of the H. thunbergii range from 7 to 8 cm. in length, and those of the H. fulva clone
‘Europa’ range from 9.2 to 9.6 cm. in length. In plotting the extent of pollen tube growth in these plants the
style of a pistil is divided into ten equal units and thus the data for numerous pistils differing somewhat in
length may be presented in the same type of graph."

Stout references a previous publication in 1931. That paper is also available on JSTOR and I have copied the appropriate section of those Materials and Methods directly below:
"The paraffin method of study was utilized, but for most of the studies a
method of direct dissection and staining was developed that is simple, rapid,
and especially reliable for tracing the continuous course of the pollen tubes.
As soon as the pistils were removed they were placed in a killing solution of
100 cc. of 70% alcohol and either 6 or 8 cc. of commercial formalin (37%).
With such preservation the pistils remained in excellent condition for study
for at least a year, but in most cases the material was studied as soon after
preservation as was convenient.
The dissections of pistils were made as follows: a pistil was placed on a
glass slide and slit with a razor blade lengthwise into halves which were then
arranged with the cut surfaces uppermost. Sometimes a half was also sectioned.
Another method used was to slit the pistil its entire length with a
fine sewing needle mounted in a wooden handle. The sections were made
either at right angles or parallel to the central placenta as desired.
Numerous stains were tested in various combinations and with various
methods of application before a satisfactory differential stain for the pollen
tubes in the pistils was obtained. The method found to be most fully satisfactory
for Brassica is as follows. Aceto-carmine solution made by saturating
warm 45% acetic acid with carmine is dropped on the dissected pistils
and allowed to remain for a few seconds, after which a few drops of a
saturated aqueous solution of aniline blue are added. After a few seconds,
depending on the density of stain desired, the excess stain is drawn off either
with a pipette or with blotting paper. A few drops of aqueous solution of
magenta red applied before the aniline blue sometimes improves the differentiation
of the pollen tubes. Any desired degree in the density of the stain
is then obtained by washing with absolute alcohol applied from a pipette.
A drop of glycerine and a cover glass complete the preparation.
With the use of a microscope the extent of the growth of the pollen tubes
in the various pistils was determined. If any tubes were found in the extreme
lower part of the style the ovary was also sectioned and stained. By
this method it was rather easy to determine the position of the ends of the
pollen tubes in the pistil and their appearance."

The paper by Chandler, "A method for staining pollen tubes within the pistil" is not available for free. It may be possible to read it through the library of a university near you. It can be found at http://www.tandfonline.com/doi...

If you are interested in examining the effects of temperature on garden hybridizing then if you wish to tease apart the importance of temperature on pollen viability versus the importance of temperature on pistil viability you would need to do two sets of experiments. In one set you would need to keep the temperature the pistil experienced the same while varying the temperature that the pollen experienced and then in a second set of experiments keep the temperature the pollen experienced the same while varying the temperature that the pistil experienced.

Thank you for posting all this useful information! I am having much better luck this year pollinating in the morning (as opposed to when I get home from work).

I am considering getting a microscope for the purpose of checking pollen viability and hybridizing curiosities such as the ones being discussed in this thread. Any recommendations on brand/type/where to buy?
@sooby Forgive me if this is a dumb question, but did you take that photo with a microscope?

@Jessie6162 yes, it was taken through one eyepiece of a stereomicroscope set at 20X, I may have had some zoom on the camera, not sure. I just play around with the zoom until I can get a decent picture. I use this microscope way more often than a monocular microscope, it's so easy to just plonk something down on the stage rather than make a slide, plus the specimen can be examined from several angles. My model only does 20x and 40X, it was relatively inexpensive. I'm using it mainly to identify insects and plant problems though, so I'm no expert on what you'd need for your purpose. Perhaps @admmad can help there. One thing I would suggest is to get one with sub-stage lighting, I wouldn't want to be without that. Can't help with where to buy, I could if you were in Canada I rather fancy one of those digital microscopes which would seem easier than trying to combine a microscope with a digital camera, but have no idea which would be good enough. I did try someone else's once but it didn't give a sharp enough image for my liking. One thing I've found with my set-up is that the digital camera has to have a lens small enough to fit the microscope eyepiece.

Jessie6162 said:I am considering getting a microscope for the purpose of checking pollen viability and hybridizing curiosities such as the ones being discussed in this thread. Any recommendations on brand/type/where to buy?

I wish I could provide some help but I have not been able to use either of the two old microscopes I have with a camera to take photos.
I also am thinking of buying a more modern microscope that can have a camera attached.
I have been looking on ebay.
The price range for microscopes is very large; perhaps the most important decision is how much one is willing to spend.

I was thinking, Maurice, that you might have some ideas on the magnification Jessie might need for what she wants to do? I know I germinated some daylily pollen on onion skins years ago and took some pictures but I don't remember which microscope I used. One can boost the microscope magnification with the camera to some extent but there's still a big difference between my dissecting microscope at 20X/40X and my monocular at up to 1,000X. I rarely use the maximum on the mono because it needs oil for that. For some things the lower power stereo just isn't good enough and it would be nice to have a higher power stereomicroscope, but for most of my purposes the one I have is perfectly adequate. Do you want to be able to take pictures, Jessie?

In a file I have a statement that diploid pollens have an average diameter of about 100 microns and tetraploid pollens have an average diameter of about 140 microns. My scanner picks up individual pollen grains on leaves and petals; its specs indicate that it can do high resolution scans of 4800 dpi but I know that is an over exaggeration. I would think that a maximum magnification of 500 or 1000 would easily be high enough. I suspect that attaching a camera is one feature that would be important.