That's a great question. Yesterday I took some viability data on some of my stored pollen which was stored at different temperatures. I did an in vitro germination test. All this is is putting pollen in a drop of sucrose and boric acid and in a few hours record under a microscope the percent that germinated and produced pollen tubes. The technique uses a hanging drop from the cover slip over a depression in a thick slide.

There are multiple factors involved in if a species stores well and for how long. From my experience I would suggest a general protocol of freezing pollen and doing so without dessicant. If pollen is too dry and then frozen it has reduced viability as well. In addition, it is generally recommended to take pollen in very dry air and slowly bring the humidity up which is a hastle as well. So, just airdry it in containers that can be sealed (film canisters...), seal the containers, and then put it in a typical -20C freezer one has at home. Allow the vial to warm up before opening it to prevent condensation on the warming pollen when you are ready to use it.

Binucleate pollen stores better than trinucleate pollen. So, things in the rose family which has binucleate pollen will store frozen like this relatively well. Corn and other trinucleate pollen species does not store well. Pollen starts out with one nucleus and then divides once to make two and then one of those two divides again to make three in the end. One of the three is called the pollen tube nucleus and the other two are generative nuclei. One fertilizes the egg and the other fertilizes the central cell to form the endosperm. Some species are at the binucleate stage when the pollen is mature and then that last division occurs within the pollen tube after pollen germination. These are those species that store better and then trinucleate pollen species have all these divisions already done in the mature pollen grain and typically do not store well for more than a few days.

Frozen pollen of many species can stay viable for years.

Sincerely,
David

Hi David,

I enjoyed your posting and your "general rule." I have a follow up question. Do you know of some online reading that discusses the notion (storing pollen) further?

Cordially,

Conroe Joe

Excellent post David!

Now may I ask how the exact sucrose/Boric acid solution is built?? Is there some website that explains all that?

Ever contemplated creating multinucleate pollen by way of treating a budsite with oryzalin or trifluralin?

Thanks in advance!

To make the sucrose / Boric acid solution just add 7.5 grams of sucrose and 20 milligrams Boric acid to 500 mL of distilled water. Microbes will start to grow in a few days even in the fridge, so I like to just make up a fresh batch whenever I run a test.

That's a great question about multinucleate pollen. Well, I don't have any info about how treating pollen with antimitotic agents can alter pollen storability, but there is some info out there on the process. Kato, a scientist in Japan if I remember right, published some studies on treating corn tassels with trifluralin and preventing the last post meiotic mitosis. So, the original nucleus in the microspores (to become the pollen) divides once to make two copies and then one of them divides again for three total. The final division is interupted and then one has a haploid nucleus (n) and a 2n nucleus in the pollen. The n one seems to serve as the pollen tube nucleus helping lead the way of the pollen tube through the style to the ovule. The generative nucleus now which normally divides to make two copies is now just one cell with twice as many chromosomes (2n). One generative nucleus typically fertilizes the central cell to make the endosperm (nutritive substance for the embryo) and the other the egg which them grows into the embryo. What Kato found was that with only one generative nucleus he would sometimes get it to fertilize the central cell and the egg would develop unfertilized and be haploid or if the egg was fertilized and one has a triploid corn embryo it often would die since there would be no endosperm. He also tried follow up pollinations with normal pollen and sometimes another pollen grain would deliver a generative nucleus to fertilize the central cell and a triploid offspring could be recovered. There are more variations too he describes in his publications. Very intriquing concepts- this is the first place I learned about heterofertilization (two pollen grains participating in fertilization).

Sincerely,
David

P.S. I don't know of any good summary of pollen storage techniques summarizing multiple species besides just the general idea of binucleate pollen storing better than trinucleate and if they can endure freezing that's better for long term storage. There are a lot of papers published comaring various treatments of pollen handling storage and the resulting viability of the stored pollen looking at just one species at a time. Maybe if someone hasn't done it a good review article summarizing the primary literature would be very valuable indeed.