Spring Flowers and Pollen Germination

by Chris Thomas

Daffodil dwarf golden Berberis Mahonia Double cherry pollen Yellow tulip pollen Crack willow pollen

Introduction

One of the myriad threads of interest for a small group of researchers has been unravelling the secrets of pollen tube germination. At the turn of the last century, Jost 1905 (1) described the progress made in germinating pollen away from the plant stigma in simple sugar solutions or on various media. Since that time, much progress has been made on defining ever more precise conditions and requirements for pollen germination for specific species as summarised by recent review by Taylor & Hepler 1997 (2) and Blevins & Lukzszewski 1998 (3). In Thomas 2000 (4) &, Garrett 1999 (5) a study leading to the discovery of a simple and inexpensive method of achieving pollen tube germination on onion or leek epidermis was described. The method is suited for those wishing to have a simple indicator of pollen germination that is applicable to a wide variety of species, either for educational or for research purposes. The study has now been expanded to look at a larger range of plant with particular reference to differences within plants, between plants of the same species and between different species. Finally, the data will be viewed in the light of phylogenetic relationships.

Method

The epidermis of large onions purchased from the local Tesco supermarket was removed in strips and laid with inner side facing upwards on microscope slides. Pollen was dabbed onto the exposed inner surface of the onion epidermis from flowers picked within the previous hour. The slides were placed and kept in a humid box lined with wet tissue paper. The slides were viewed after 3h and overnight at 40x & 100x magnification using a light microscope. Germination was scored according to a scale of 1 to 6 as follows (4):

1 = no germination
2 = 1% to 2% germination
3 = 3% to 10% germination
4 = 11% to 25% germination
5 = 26% to 50% germination
6 = 51% to 90% germination
7 = 91% to 100% germination.

Pollen sampling was conducted in the following variations:
From several flowers from same plant.
Flowers from different plants of same species & variety.
Flowers from different varieties or closely related species.
A record was also kept of which onions were used over a given period.

Germinating pollen was photographed with transmitted light using a 10 x objective and 10 x eyepiece, an Olympus OM2 Camera and Tesco 400 ASA 35mm film. Images were transferred to CD as part of the Kodak Picture CD developing service or scanned into a PC and processed using both the Corel Photopaint software and Corel Photohouse.

The phylogenetic comparisons were drawn using models displayed by M. J. Donoghue (6) as part of the world wide web project 'The Tree of Life' for the upper part of the phylogenetic tree and information from the phylogenetic description of individual species listed in entries for the DNA database in EMBL(7). (The EMBL Nucleotide Sequence Database.)

Results & Discussion

The study encompassed 400 samples collected by the author and 15 received from John Garrett and covered at least 39 species and varieties. Most of the samples were collected over a three month period in Spring 2000. The Raw Data can be accessed here.

Simply plotting the number of samples for each pollen score showed a consistent pattern over time in that plants had pollen that either tended to germinate (score 7, equivalent to 90% or greater germination) or were very poor germinators (scores 1 to 3, equivalent to no more than 10% germination) as shown in Graph 3. The same overall trend held true when comparing germination on epidermis strips from the 6 different onions purchased and used consecutively over the three months (Graph 4).

Initially, several samples were assayed from a single plant, preferably from different flowers on the same plant. Of the thirty species studied in this manner and listed in table 1 and also Graph 2, only three showed variable pollination scores, Acer palmatum v purpureum, Malus spectabilis and Sinapsis arvensis. A further four species showed one major divergent datapoint of at least three taken, namely an Alnus plant, Crateagus laevigata, Salix caprea and a Viburnum species. Therefore in seventy five percent of species sampled, pollen from a single flower gave a good indication of the pollen germination for an individual plant. This could be of considerable importance where only a single flower is produced or where due to rarity, sampling has to be kept to a minimum.

The reproducibility of pollen scores also holds in most cases for different individuals of a variety or species as is illustrated in graph 1. The standard error of the mean was used only for a visual illustration of lesser or greater variation within a dataset (as, strictly speaking, the categorical data is not suitable for such calculations). However, the data does show again, that three quarters of the species/varieties looked at give consistent results

With some plants, variation between samples of same species at different days was measured. Slightly more variation was found but trends were holding. Exceptions were Furze, Viburnum, & flowering currant, however, this may be a reflection of next point

The best examples of variation between samples of closely related species/varieties and the most intensively studied were daffodils and cherries due to their abundance.

Daffodils showed clear and consistent variety differences. The most obvious correlation was lack of pollen germination with dwarfing. This may be of potential interest to daffodil breeders as pollen germination could be an easy method of rapidly detecting male sterile or poor pollen parent plants prior to crossing or for screening progeny. Lack of pollen germination was not only reflected in lack of abundant pollen tube growth from expanded grains, but also by the higher proportion of unexpanded grains. (See index of pollen germination images.)

Other cultivated monocotyledonous bulbs such as tulips and crocus also showed variety differences. (See index of pollen germination images.)

Looking at variation within a family, Salix provided a good example. Crack Willow showed good pollen germination whilst Pussy Willow and Golden Weeping Willow poor germination. (See index of pollen germination images.)

Within a genus, of e.g. the Family Rosaceae, the following results were observed. Wild cherries were abundant in hedges and on Milton Road, Cambridge there was an avenue of cultivated cherries. I found that whilst pink and white simple flowers showed pollen germination, double flowers (with two rows of petals) did not. This was perhaps more understandable as current models of flower gene expression show that in double flowers, the extra petals are likely to arise from the anther region. The high germination scores were also reflected in the other prunus species such as Sloe and Laurel.

In contrast, crab apples showed mixed germination, although this may be more due to suboptimal conditions as material highly variable even from the same tree.

This variation within a genus but consistency for a given species or variety was also reflected in the labiates when comparing the deadnettles and ground ivy (germination v non-germinating)

Whilst non-germinators included Magnolia & Eucalyptus, the Caryophyllidae (Compositae) were perhaps the most representative of a genus where pollen germination was not observed at all (see Chris Thomas 2000) (4).

I looked at the phylogenetic tree for plants encompassing the plants I had scored in this and previous work (Thomas 2000) to see if there were any obvious visual patterns emerging for pollen germination on onion epidermis (see Dendrogram). Within the Angiosperms, both the monocots and dicots had good pollen germinators, Magnoliales (one representative) did not. Within the monocots, both groupings sampled showed germination. Within the dicots sampled, the Myrtales, Ericads and Asterids did not show germination. Ranunculids, Eurosids I and II and Lamiids did show germination.

The simple and probably naive explanation that sprang to my mind was as follows. Pollen germination on onion epidermis or similar supports other than the recipient stigma is an ability present throughout the flowering plants. Where inability to germinate manifests itself, this could have several reasons. One is probably straight loss of function by mutation, breeding or natural selection. The second is likely to be an increased stringency in requirements for pollen germination to ensure more specificity. Naturally, known incompatibility systems spring to mind.

In conclusion, further detailed studies on the germination behaviour of pollen on onion epidermis have shown that germination is widespread with only a few of the genera sampled showing no germination to date. Results within a plant or between plants of the same variety are extremely consistent with the exception of a few species. However, pollen germination in one variety is not predictive of success in a closely related variety. This may actually make the method described here a useful simple tool for those with limited resources or budgets such as schools or small plant breeders to look at differences in pollen germination. A practical application could be the ability to distinguish individuals that have lost pollen fertility early on in crossing programs, either to retain or discard them.

Finally, the work also shows that you can have a great deal of informative enjoyment out of a project like this. I certainly did!

References

1. L. Jost [1905] - Zur Physiologie des Pollens. Berichte. Deutsche Botanische Gesellschaft, 23, 504-515.
2. L. P. Taylor & P. K. Hepler [1997] - Pollen Tube Germination and Growth, Annual Reviews in Plant Physiology and Plant Molecular Biology, 48, 461-491.
3. D. G. Blevins & K. M. Lukzszewski [1999] - Boron in Plant Structure and Function, Annual Reviews in Plant Physiology and Plant Molecular Biology, 49, 481-500.
4. C. J. R Thomas [2000] - Studies on Pollen Germination of 40 Plant Species on Sucrose-gelatine and on Onion Epidermis, Quekett Journal of Microscopy, 38, 463-472.
5. J. Garrett [1999] - Pollen Tubes - This'll make your Eyes Water! http://www.microscopy-uk.net/mag/artdec99/jgpollen.html
6. M. J. Donoghue. As part of the world wide web project 'The Tree of Life' (http://phylogeny.arizona.edu/tree/eukaryotes/green_plants/embryophytes/angiosperms/angiosperms.html
7. The EMBL Nucleotide Sequence Database http://www.ebi.ac.uk/embl/

 

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