Environmental and Community Biology - Fern Sporebank at the Royal Botanic Gardens Edinburgh

Environmental and Community Biology

Fern Sporebank at the Royal Botanic Gardens Edinburgh

Introduction

Fern dust was first investigated scientifically by Marcello Malpighi (1628 1694) who investigated microscopically the dark spots on the underside of fern leaves (Moran, 2004). The complete life cycle of the fern was not discovered until 1848 when Leszczyc-Suminski first described the archegonia as a female sexual organ and from this the life cycle of ferns as is it is today was deduced (Wagenitz, 1999). Ferns belong to the vascular plant group and possess true leaves but lack flowers and seeds. They possess free living 1n gametophytes which produce gametes by mitosis, often both sperm and egg on the same prothallus. A fertilised egg grows to become a 2n sporophyte, the stage universally recognised as being the fern. This produces spores in sporangia on the leaf underside (fig 1 + 2). There are two distinct types of spores, green and non-green. The green spores contain chlorophyll and are ready to germinate and grow as soon as they hit the ground. Unfortunately these spores are often short lived and lose viability within a few weeks although viability of up to a year has been recorded. Non-brown spores usually remain viable for a few months to several decades (Lloyd et al, 1970)

Fig. 1: The Lifecycle of Ferns (www.esu.edu)

Fig. 2: mature sporangium releasing spores (io.uwinnipeg.ca)

Ferns are not as valuable economically as seed bearing plants but some have historic medical values, Dryopteris filix-mas was once used as an anthelmintic to expel worms and is referred to in Nordic literature (Waller et al, 2001), some have been used as food such as Matteuccia struthiopteris, and some as an aid to nitrogen fixation but most are purely of horticultural interest as specimen and pot plants. Some ferns are now recognized as keystone species and support large varieties of invertebrate life (Ellwood et al, 2002)

Today 193 species of true fern (Pteridophyta) are listed on the IUCN Red List of Threatened Species (IUCN, 2007). As an aid to conversation it must be noted that spores can survive for significant amounts of time in the soil beneath a fern colony, only germinating when exposed to light, this is known as the soil spore bank and has implications in restoring lost species (Dyer, 1994). In order to ensure conservation of certain species, various organisations, usually botanical gardens now operate spore banks collecting and storing spores for use in the future similar to the seed banks held in the same institutions. Methods of storage vary greatly and affect the viability of the spores. Some spores have been known to germinate after 70yrs (Lloyd et al, 1970). The common variables among storage are moisture and temperature. Dry storage involves storing spores in glass vials, wet storage on agar plates and the temperature ranges from +20^oC to -80^oC. All these variables have effects on the viability of the spores (Quintanilla et al, 2002).

This study will look at the viability of spore samples subjected to differing storage methods taken from the Royal Botanic Gardens, Edinburgh. This is of importance as spores cultivated locally in order to replenish stocks has risks associated with the plants adapting to local conditions within three generations and losing their wild fitness. The Botanical Gardens are interested in the length of time these spores can be stored so as to limit the necessity to collect wild spores to a minimum.  

Spore Samples

Dry and cold stored at -20^oC. These spores have been less than ideally stored, often with extreme temperature fluctuations (unnoticed freezer breakages and transportation between stores).

Dryopteris affinis a southern and western European native: two samples, one from 1970 and one from 1990.

Athyrium filix-femina a temperate Northern Hemisphere native: sample collected 1986

Dryopteris filix-mas one of the most common ferns occurring in the Northern Hemisphere: sample collected in 1987

Cold Store at 4^oC These spores have been stored in spore envelopes and refrigerated

Woodsia ilvensis a small fern found on rocky outcrops above 350m. Currently there are less than 100 plants remaining in Britain distributed between 11 sites, 6 of which are in Scotland (UKBAP, 1998). There are samples from 3 sites: Feshie, Woodale and Moffat all collected in 2002.

Fresh Spores Stored Refrigerated

Todea barbara tropical rain forest fern native to Africa, Australia and New Zealand.

Method

1. The spores that have not been separated from their sporangia should first be collected using the method described by Dr. McHaffie. Removing the frond from the envelope, tipping out the large particles and collecting what remains.
2. The spores should now be placed in vials ready for sampling.
3. Again as described by Dr. McHaffie four or five layers of filter paper taped tightly over the end of the vial will serve as a screen to allow a steady flow of spores and prevent over sowing.
4. The spores should be sown onto nutrient agar plates by tapping the vial firmly ensuring the whole plate is evenly covered. This is aided by the filter paper screen.
5. The plates will then be left at room temperature and under natural light for 7 days.
6. Again as described by Dr. McHaffie, a count of the germination rate of the spores will be made by viewing the plate under a microscope and counting 100 spores. The amount that has germinated out of this 100 will give a percentage. In order to ensure accurate result the count should be made from the edge of the plate in straight line across the agar recording the germination state of every spore seen until 100 have been counted. This procedure should be repeated 3 or 4 times for each plate starting from different places on the plate edge. (I recommend that team members each prepare samples of all ferns in order to obtain the maximum amount of results.)
7. The percentage germination rate for each of the samples will be calculated and reported to the Botanic Gardens.

Notes on Method

I am unsure as to what medium to cultivate the spores on but a simple mix is described in The American Fern Journal: my laboratory has been successfully using a medium consisting of a solution of 15g of agar per litre of water in which is dissolved one commercial nutrient tablet. The resulting medium provides mineral nutrient yielding fern prothallia in numbers and size equal to those grown on conventional nutrient media requiring precise quantitative mineral additives.(Marengo, 1979)

It is also interesting to note that red light stimulates spore growth in the majority of fern species (Kiss, 1998) perhaps the germination rate would be increased if the plates were kept under a red light as opposed to natural light.

References

Dyer A., 1994, Natural soil spore banks - can they be used to

retrieve lost ferns? Biodiversity and Conservation 3, Dordrecht, Springer Netherlands

Ellwood M., Jones D., Foster W., 2002, Canopy Ferns in Lowland Dipterocarp Forest Support a Prolific Abundance of Ants, Termites, and Other Invertebrates

Biotropica Association for Tropical Biology

IUCN 2007. 2007 IUCN Red List of Threatened Species. <www.iucnredlist.org>. Downloaded on 08 February 2008.

Kiss H; Kiss J. 1998

Spore Germination in Populations of Schizaea pusilla from New Jersey and Nova Scotia International Journal of Plant Sciences, Vol. 159, No. 5.

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Lloyd R; Edward J. Klekowski, Jr. 1970, Spore Germination and Viability in Pteridophyta: Evolutionary Significance of Chlorophyllous Spores, Biotropica, Vol. 2, No. 2.

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Marengo N. 1979, A Simplified Nutrient Medium for Growing Fern Prothallia American Fern Journal Vol. 69, No. 4

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Moran R. 2002, A Natural History of Ferns, Portland: Timber Press

Quintanilla L., Amigo J., Panguae. , Pajaron S. 2002 Effect of Storage Method on Spore Viability in Five Globally Threatened Fern Species, Annals of Botany 90 V.4

Available at: https://aob.oxfordjournals.org/cgi/content/full/90/4/461

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UK Biodiversity Group, 1998, UK Biodiversity Group Tranche 2 Action Plans - Volume I: Vertebrates and vascular plants, Species Action Plan Oblong Woodsia (Woodsia ilvensis). Available online at https://www.ukbap.org.uk/GenPageText.aspx?id=52

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Wagenitz G. 1999, Botanical Terminology and homology in their Historical Context National Botanic Garden of Belgium

Waller P.,Bernes G,Thamsborg SM,Sukura A.,Richter SH,Ingebrigtsen K,Hglund J., 2000, Plants as De-Worming Agents of Livestock in the Nordic Countries: Historical Perspective, Popular Beliefs and Prospects for the Future, published online 2001

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