Dear All,
We have acetocarmine and other staining methods, germination by hanging drop method and we can even stain the germinating pollen on stigma and visualise with aniline blue staining to test pollen viability. In some publications the results have been interpreted as pollen fertility. Can we use these same methods to confirm pollen fertility/sterility in interspecific/intergeneric crosses?
To put the question in a different way; is it correct to interpret pollen viability tests by staining and germination as pollen fertility in the case of interspecific, intergeneric hybrids?
As these pollen will have all sorts of problems at the time of fertilization and embryo formation, what would be the best way to study their fertility?
Thanks
The tetrasolium test was the best in my experiments.
Hauser, E.J.P. and Morrison, J.H. 1964. The cytochemical reduction of nitro blue tetrazolium as an index of pollen viability. American Journal of Botany, 51(7):748-752
and for comparison of methods: http://plantstress.com/methods/1%20Pollen%20Firmage-Dafni%202001.pdf
Hi Ranjith,
I would say they are different concepts regarding 'pollen viability' and 'pollen fertility (in interspecies cross)'. In short, pollen viability simply applies to if the pollens are still good to germinate, the ability to germinate. However, the pollen fertility probably involves complex 'genetic incomparability' between the pollen and the stigma (a pre-fertilization barrier) between the interspecies. So, a viable pollen does not mean it can be fertilized by the other interspecies. But, the other way around, the fertilization of interspecies definitely need a viable pollen.
I do not care for those old pollen viability tests, such as hanging drop or agar germination, as very little happens. My old professor (Bruce Knox) always insisted that these tests never germinate more than 10 - 15% of grains no matter how viable they were in a subsequent cross. I think he was right. As your lab is set up for fluorescence I'd rerun to standard fluorascein diacetate testing of sample pollen of the male donor(s). I have not used it for decades but it proved extremely effective in the following case where, in fact, we contrasted pollen viability of two parent species and the F1.
Bernhardt, P. & Calder, D.M. 1981. Hybridization of Ameyma pendulum and Amyema quandang (Loranthaceae). Bulletin of the Torrey Botanical Club 108: 456-466.
Recently our lab published a paper on a threatened, wild species that produces an enormous number of sterile grains. Using some of our techniques on pollen carried by pollinators (white light microscopy) and pollen deposited on stigmas (fluorescence) might offer more options.
Bermhardt, P., Meier, R. & Vance, N. 2013. Pollination ecology and floral function of Brown’s peony (Paeonia brownii) in the Blue Mountains of northeastern Oregon. Journal of Pollination Ecology. 1: 9 – 20.
Now here is another thing to consider in an interspecific cross that you know about already. What happens to the perfectly viable pollen tube of one species as it penetrates the pistil of the second? Does it grow normally within the style or ovary or are there continuous aberrations? Here is an example of aberrations in hand made interspecific pollinations (greenhouse) but enough pollen tubes reached the ovules to authenticate the repeated production of F1 hybrids under field conditions.
Edens-Meier, R., Westhus, E., Bernhardt, P. 2013. Floral biology of large-flowered Thelymitra species (Orchidaceae) and their hybrids in Western Australia. Telopea 15: 165-183.
Question has been answered well by Dr. Bernhardt. Pollen viability does tell you about whether pollen are functional but when it comes to actual crosses you have to factor in various other aspects. Different factors work at different levels starting from pollen tube germination, pollen tube growth, delivery of sperm cells in the right place, and eventual fusion of gametes. Easiest of the method which will answer most of these questions is Epifluorescence of pollinated gynoecium in time dependent manner. Please look at AoB PLANTS 5: plt015; doi:10.1093/aobpla/plt015. In this paper we actually followed the pollen tube growth through the gynoecium at different times after manual pollination. Delivery of gamete still does not ensure fusion of gametes and normal growth of embryo, but these are different questions than success of pollination. Hope this answer some of your questions.
A very fast and reliable analysis of pollen viability or germination is provided by impedance flow cytometry. The technology is label-free which means that there is no need for incubation with chemical dyes. The battery powered instrument can be used in the lab or in the field. Results are obtained within about a minute. See www.amphasys.com for more information.
See Article Impedance Flow Cytometry: A Novel Technique in Pollen Analysis
orArticle Impedance Flow Cytometry as a Tool to Analyze Microspore and...
I am also fully agreed with Dr. Yuan-Yeu Yau that pollen viability and pollen fertility are two different things.
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