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Embryo cryopreservation of threatened ornamental species

Embryo cryopreservation of threatened cycads, recalcitrant palms and low temperature storage of orchids to complement conventional storage.

A cross section of a Zamia furfuracea seed (Photo: Jayanthi Nadarajan)

Many ornamental species are facing extinction due to over-exploitation, including cycads, palms, orchids and species particular to any country, eg New Zealand. The unique properties and beauties of these groups of species have led to their high demand in commerce. Horticultural interest has increased wild-sampling for landscaping and over-collection of these species. Illegal harvesting from natural populations has caused colony destruction and extinction of several wild populations and removal of seeds reduces opportunities for natural regeneration. The conservation effort on these species is further hindered by their under-explored seed biology.

The adoption of innovative ex situ conservation techniques, such as embryo cryopreservation, would provide an assurance for survival and would complement the in situ conservation effort on these species. This project focuses on developing embryo cryopreservation protocols for three main groups of species: cycads, palms and orchids.

Cycads

The cycads are remnants of the most ancient group of higher plants, with origins dating to the late Carboniferous period (300 MYA). Cycads comprise c. 300 species / subspecies in three families: Cycadaceae, Stangeriaceae and Zamiaceae.

Twenty three percent of cycad species are critically endangered or endangered and 15% are listed as vulnerable (IUCN Red List, 2010). All species are listed in Appendix I or II of CITES.

Cycad conservation and sustainable use through seed is hindered by the lack of research and baseline data on reproductive biology, pollen and seed storage behaviours and in vitro manipulations. Our results so far on five cycad species suggest that the seed are sensitive to desiccation, but with a few species surviving to 15% moisture content. Cryopreservation of cycad embryos has been initiated and our work is focusing on embryo size and cryoprotection.

Palms

There are approximately 2,300 taxa of palms of which 365 have been recently 'red listed' (IUCN Red List, 2011). Of these, 135 are either critically endangered or endangered.

As the availability of seed storage biology data is limited, we initiated a project of species screening a few years ago. To date we have worked on about 150 seed lots of a broad range of species. We have observed that intolerance to moisture content reduction is quite common in palm seeds, ie many species produce seeds that are non-orthodox and thus not suited to conventional seed banking.

Of the 125 species fully characterised, 81 were confirmed as having seeds sensitive to drying. On this basis, about two-thirds of palm species (c. 1600 species) may require ex situ conservation through cryopreservation. These taxa now form the target for our future cryo-conservation studies. 

Orchids

The Orchidaceae is the second largest plant family, with approximately 25,000 species, many of which exist in fragile habitats of high biodiversity. The recent IUCN Red Listing (2011) of 161 species found 104 to be either critically endangered or endangered. Wild collection and habitat degradation has put many of these and other un-reviewed species at extreme risk.

Orchid seeds are regarded as short-lived, with longevity dependent upon water content and storage temperature. Evidence suggests that this can be species dependent, for example, seed of several species of Coelogyne survive <12 months when dried and 'frozen' at -20°C. In contrast, other taxa either retain viability (e.g. Cattleya and Dactylorhiza), or demonstrate a variable storage response (e.g. Dendrobium).

Preliminary evidence would suggest that storage at ultralow (-70 and -196°C) temperatures may alleviate some of these problems exhibited at higher, but still sub-zero, temperature. To resolve the interdependence of storage temperature and water content, further species are being tested, and a better understanding of their biophysics is being sought, particularly in relation to the thermal properties of the seed lipids.

Other species

Cryopreservation research was also extended to New Zealand’s threatened species. Nearly 40 percent of New Zealand’s unique and diverse flora is either under threat of extinction or naturally uncommon in their natural habitat. Many New Zealand species are tropical analogues and show sensitivity to desiccation.

Species studied so far are Dysoxylum spectabile, the only endemic Meliaceae in the New Zealand flora, and Myosotidium hortensia (Boraginaceae). Myosotidium hortensia is an important ornamental from a monotypic genus, and is endemic to the Chatham Islands. These factors contribute to its assessment as being 'nationally vulnerable' (New Zealand Plant Conservation Network, 2010). The literature would suggest that the seed storage behaviour of D. spectabile is recalcitrant, while the storage physiology of Myosotidium hortensia is unknown, but preliminary studies on the latter species suggest that the dry seeds (~9% moisture content) start to lose viability at 5°C within 9 months storage.

The outputs of the project are being published in peer-reviewed journals. Two PhD students and many MSc and college-based sandwich course students and work experience students have been trained in the framework of our cryo work. Future work will concentrate on highly threatened cycads, embryo cryopreservation of recalcitrant palms (identified from our screen), and low temperature storage of orchid seeds to complement conventional -20°C storage.


Project partners and collaborators

Brazil

UNOESTE, Presidente Prudente

China

Shenzhen Fairy Lake Botanical Garden

South Africa

Lowveld National Botanical Garden
University of KwaZulu Natal

Thailand

Nongnooch Tropical Botanical Garden
Mahidol University

New Zealand

Massey University

Annex material


Key papers published since 2006:

  • Nadarajan, J., Wood, S., Marks, T.R., Seaton, P.T. & Pritchard. H.W. (2011) Nutritional requirements for in vitro seed germination of 12 terrestrial, lithophytic and epiphytic orchids. Journal of Tropical Forest Science 23: 204-212 (IF=0.5)
  • Seaton, P.T., Hu, H., Perner, H. & Pritchard, H.W. (2010) Ex situ conservation of orchids in a warming world. Botanical Review 72: 193-203 (IF=2.657).
  • Pritchard, H.W. & Nadarajan, J. (2008) Cryopreservation of orthodox (desiccation tolerant) seeds. In Cryopreservation Practical Guidelines. Reed BM (ed), Springer Verlag, pp. 485-501.
  • Seaton, P.T. & Pritchard H.W. (2008) Life in the freezer: orchid seed banking for the future. Orchids-The Bulletin of the American Orchid Society 77: 762-773.
  • Rakotondranony, G. L., Sacande, M., Wood, C. B. et al. (2006) Seed Storage responses in four species of the threatened genus Ravenea (Aracaceae). Seed Science and Technology 34: 513-517 (IF=0.631).

Conferences and workshops attended, including:

  • J. Nadarajan, S.R. Dandugula & H.W. Pritchard. Towards the conservation of cycads through cryopreservation. Symposium and Free Communications of Society for Low Temperature Biology, 6-7 October 2011, London.
  • C.R. McGill, M.J. Park, J. Nadarajan, W.M. Williams, B.R. MacKay & H.A. Outred. Seed Storage and Dormancy in Chatham Island forget-me not (Myosotidium hortensia (Decne) Baillon). Paper presented at 29th ISTA Seed Symposium, Cologne, Germany 16-22nd June 2010.
  • J. Nadarajan & H.W. Pritchard. Millennium Seed Bank- Working together to save plants worldwide. International Society for Biological and Environmental Repositories (ISBER) 2010 Annual Meeting, Rotterdam, 11-14th May 2010 (Invited talk).