This is a busy season for me. I have not planted out any of my winter 2008 seedlings yet. I also have not yet started my pollinating in spite of the fact that many roses are having their first bloom cycle now. My time has been taken up collecting pollen for microscope examination and processing the resulting data. From time to time I will post the actual pollen pictures of roses of special interest at:
I decided to use a spreadsheet to find the average diameter and standard deviation (SD) for all of the pollen for a given rose. I realise that this will give an incorrect average and SD if the rose is a triploid and has actually yielded both diploid and tetraploid pollen, but a large SD will suggest that I do have a triploid. The spreadsheet can be observed at:
As of today, I have measured many more of my roses than I have recorded. I will be updating the spreadsheet as I have time.
The spreadsheet only identifies the rose by my internal number (for example - #14); so that you can easily see which rose that number corresponds to, I have entered the average diameter and SD into my “numbers used for seedlings and commercial roses in my notebooks” table:
Since most hybridizers will be interested mainly in the pollen diameters of commercial roses, I will add commercial roses to the above table as I have time.
Henry, you could save yourself a great deal of effort and improve your accuracy if you could hunt up someone at a university or industrial lab who has access to either a Malvern or Cilas particle size analyzer. These give not only the Mass Median Diameter (MMD) but a size distribution profile. They work best in exactly the size range of pollens.
These are very commonly used to measure powers such as paint pigments, pharmaceutical preps, even coal, so it should not be hard to find one. I’d even guess that a Malvern salesman would arrange to run a batch of pollens for you gratis.
Don, thank you for the suggestions, but one of my guiding principles, in my rose hybridizing experiments, has been to attempt to develop procedures / techniques that the amateur can use at home.
In this particular series of experiments, I am interested in seeing if a “used” relatively inexpensive microscope can provide information concerning whether a given rose produces a lot, some, or very little live pollen and whether the pollen produced is diploid and / or tetraploid.
In many cases, I have recorded the per cent of live pollen (as indicated by the acetocarmine stain procedure); but I have not yet posted these data. (From my literature search, it appears that other stains may be more accurate, but the preparation and storage of these stains do not lend themselves to home use.)
As an example of the use of the stain method, my observation of Fimbriata (hybrid rugosa, Morlet, 1891) pollen indicated “no” stainable pollen. I checked “Help Me Find” and found that no offspring have been reported. Please note: I plan on looking at a second batch of pollen to see if the first results can be reproduced.
I can certainly see where a do-it-yourself method would be useful to us amateurs. Any way the information is gathered, a database of pollen viability would be a real asset. I do admire your stick-to-it-ivness.
my observation of Fimbriata (hybrid rugosa, Morlet, 1891) pollen indicated “no” stainable pollen. I checked “Help Me Find” and found that no offspring have been reported.
That’s good to know. I have some Fimbriata pollen which I will use to test the hypothesis.
From my perspective, it’s worth trying because of the long odds and the interesting trait that Fimbriata has of fringed petals.
Percent stainable pollen is a great thing piece of information. Thank you for doiong that for us. In my Masters with potatoes I estimated pollen stainability in my wild species genotypes and hybrids. One thing that was important in that work was to get a uniform sample that was indicative of the clone. Using acetocarmine alone would result in the empty, aborted pollen grains and stainable grains not being evenly distributed over the slide. If my memory serves me well, more of the aborted grains moved to the edges of the slide. What I did to overcome this was to use glycerin. Just a small drop of it (the inexpensive kind from the pharmacy for dry skin) would solve the problem. After pollen and acetocarmine was on the slide I would put a drop of glycerin on the slide and then mix it well with a toothpick before putting the coverslip on. Pollen was much more evenly distributed and I would get a more uniform and accurate reading from the subsample I observed. If you are looking at all pollen across a slide you wouldn’t have to worry about it of course, but if you are observing a subset, it can be a factor. One thing that is really nice Henry is that with the glycerin your slide is preserved for a very long time. You can save it for months actually. You can prepare samples and look at them later or look at them just again in the future if you want to. If there is too much liquid the coverslip can slide if you use one of those slide boxes that have them go on end. Otherwise, I just stored my slides flat on cookie sheets. The cookie sheets I used had little lips or edges that were about a half inch high. I piled the cookie sheets on each other with each sheet going perpenticular to each other so the one on top rests on the lip of the one below so it doesn’t rest on top of the slides themselves.
Pollen germination assays are a nice compliment to the work you are doing too if you had the time to include them. It will give you a much better estimate of pollen viability. You will be seeing a biological response of pollen and they need to be alive for that to happen. Just some sugar water with a bit of boron (perhaps borax can do it) is all that is needed. This is especially useful for triploids as we see in Leen Leus’ thesis and the work of others. Triploids tend to have pollen of varaible sizes with low viability, although many do stain. Many are probably aneuploid and tend not to be viable and are not capable of germination. There’s an easier method I’ve been using now than the hanging drop method with special thick slides I wrote about in the RHA newsletter a few years back. Take a sealable box (like a plastic sandwich box with a lid) and line the bottom with moist paper towel. On that towel place your cover slips with a drop of the sugar water solution and pollen. Seal the top and wait for a couple to few hours. After that, take out the coverslips with the drop on them and invert it on your standard slide and observe. Pollen tubes of viable grains will have germinated and be 20 or more times long typically as the pollen grain is wide. The key to this and the handing drop method seems to be that the air must be very humid around the pollen. Due to surface tension, the pollen stays to the outside of the sugar water drop and is partially in contact with the air.
Hope these ideas help as you continue to refine your methods and answer in the best way reasonable the questions you are most interested in.
Thanks Dave. I have been stirring with an iron nail as some literature references suggested that.
I will not be doing pollen germination studies in the forseeable future as I have too many roses to measure the pollen of.
I do not grow Dornroschen, but I was given some of its pollen to test. There were very few unstained grains. The average diameter was 36.3 and the standard deviation was 3.6. This relatively large SD suggests that Dornroschen is a triploid. “HelpMeFind” lists 18 offspring, some as seed parent and some as pollen parent:
From its offspring I would of thought that it would be a tetraploid/
Dornroschen is extremely fertile both directions for whatever reason. It’s an easy seed parent.
It mildews here which has prevented me from utilizing it further. The only thing I put on it this season was Rugelda x R15. Dornroschen seems to give yellows. I got good hip set.
I had earlier mentioned that I was using “super-glue” to join together the slide and cover slip (or actually, in my case 2 slides (because I had been able to purchase slides so cheaply at a trucking salvage store)). Unfortunately, once a tube of “super-glue” is opened; it quickly dries out. This method was too expensive.
From my fishing reel maintence days, I thought of using a thin layer of silicone grease. I went to a “outlet type store” - Ollies, and found that they had “Connector and Spark Plug Boot Grease” 99 cents for a 7 gram tube. It is “100% silicon grease”. So far this method has proved satisfactory.
In my measurements, I am finding some diploid size pollen in what should be only tetraploid pollen and (in one case so far) larger sized when only tetraploid pollen is expected. This brings up the possibility of using a screen to “concentrate” the desired size pollen (see the following link and the link/papers referenced in that link: http://www.rosehybridizers.org/forum/message.php?topid=13995#13996 ).
I, and others, have spent a lot of effort trying to half the number of chromosomes in modern tetraploid roses by what is called “anther culture” ( "anther culture" - Google Search ). I, and as far as I know all others, have not been able to get the plants past the callus stage. Modern diploid hybrid teas are desired since they could be bred with diploid species roses to give fertile F1 offspring.
I also would like to concentrate the pollen that are larger than tetraploid. See the following link for an example:
Henry, you have probably already seen this paper but I mention it on the outside chance that you haven’t.
“Dispersal and size fractionation of embryogenic callus increases the frequency of embryo maturation and conversion in hybrid tea roses”, Kamo K, Jones B, Castillon J, Plant Cell Rep (2004) 22:787-792
They report a method for bumping up the regeneration rate from callus, abeit from two different tetraploid HTs.
Some fifteen years ago a french scientific team of INRA Frejus did produce a few dozens diploid roses from tetraploid modern florist HTs.
They got them from ovules fecundated with irradiated pollen, embryo grown in vitro. I saw the plants: many not vigourous, some freaky, a few strong and fertile if definitely thinner and smaller in all parts than tetraploid parent.
They were bred with diploid species and beside expected diploids progeny was quite varied in ploidy. Up to pentaploid from diploid parents.
A program discontinued. Meilland that sponsored may have the plants.