Cryo-conservation centres of excellence
Long-term storage of difficult (non-orthodox) seeds and plant tissues will be dependent on the application of low temperature science, particularly cryopreservation, which provides options for the storage of seeds, embryos, shoot-tips and totipotent cells.
Programmable freezer used to cool down the seeds to selected temperatures at a programmed rate before storing them in liquid nitrogen. (Photo: J. Nadarajan)
The proportion of the world’s flowering plants that produce non-orthodox seeds is still unclear. However, as the majority of species grow in the tropical moist forests, the number could be tens of thousands of species.
As with clonal species, cryopreservation is the only long-term option for banking. In addition to key forestry families producing non-orthodox seeds (much of the Fagaceae, Arecaceae, Dipterocarpaceae), numerous commodities also produce difficult-to-store seeds (coffee, citrus, tea, cocoa). Our studies aim to maximize survival of cryopreservation, using a range of materials (zygotic and somatic embryos, shoot-tips).
We have published two technical guidelines on cryopreservation as book chapters and c. 10 papers and trained about 30 students in cryobiology since 2006, primarily at the Univ of Kwa-Zulu Natal, RSA and at the Univ. of Bedfordshire, UK. Short-course delivery has been through the Fourth, Fifth and Sixth International Training Course on ‘In vitro conservation and cryopreservation of plant genetic resources’ in 2009, 2010 and 2011 (c. 12 students each) supported by the Indian Council of Agricultural Research's National Bureau for Plant Genetic Resources (India), Bioversity International (Italy) and the MSBP.
20 ferns from global biodiversity hotspots and small islands have been cryopreserved since 2006 using gametophytes as propagules at Conservation Biotechnology (Jodrell Laboratory). Work is in advanced stages to develop an efficient method for fern spore cryopreservation.
In addition to cryo facility enhancement in the Wellcome Trust Millennium Building, the core work for centre development was in South Africa, as summarised in the final report to a Darwin Initiative Project: Cryo-conservation Centre of Excellence for sub-Sahara Africa (CCESSA; 220 pp, two volumes). Highlights of CCESSA include:
- excised axes of 28 species cryopreserved
- development of a B.Sc. (Hons)-level cryobiology module and delivery to 16 students (UG, PG)
- support for four PhD and two Masters students
- four papers in international peer-review journals
- additional resource raised for the project was £88 k
Key findings include the identification of species-specific critical moisture contents to avoid ice formation in tissue on cooling, as determined by differential scanning calorimetry and using programmable freezing. Critically, such properties have been found to change with embryo developmental age (Fang et al., 2009; Hamilton et al., 2009).
In addition, for oil-rich seeds partially dried embryos were found not to survive anything but short-term storage at -20°C, possibly as a result of devitrification of the cytoplasmic glass and/or structural problems associated with high temperature-melting lipids. Ultralow temperature storage significantly reduces the risk of devitrification (Nadarajan et al., 2008).
Widescale application of cryopreservation methodologies has been shown to be possible with the amaryllids (Sershen et al., 2008). Long-term storage data under cryopreservation conditions are few (reviewed by Li and Pritchard, 2009), but are now emerging for citrus embryos. The research has contributed to the triennial targets of the International Seed Testing Association’s Seed Storage Committee: 2007-10 and 2010-2013.
In the future this work, which is part of the ‘frozen planet’ research theme, will expand with India and China in particular.
Project partners and collaborators
Erica Benson, Keith Harding (Kew Honorary Research Associates)
Andy Wetten (University of Reading)
Tiantian Zhang, David Rawson (Univ. Bedfordshire)
Sarah Ashmore, Kim Hamilton (Griffith University, QLD)
De-zhu Li, Hong-ying Chen, Wei-qi Li (Chinese Academy of Sciences, Kunming)
S K Malik, Rekha Chaudhury (National Board for Plant Genetic Resources, Indian Council of Agricultural Science)
Elena Popova (National Agrobiodiversity Center)
Pat Berjak, Norman Pammenter, N Sershen (Univ. Kwa-Zulu Natal, Durban)
Jong-yi Fang (National Pingtung Univ. of Science and Technology)
MSBP, UK Darwin Initiative (2005-08; £169 k; Project Leader – Hugh W. Pritchard) and collaborating institutes.
Key papers published since 2006 (top five papers only)
- Li D-Z, Pritchard HW (2009) The science and economics of ex situ plant conservation. Trends in Plant Science 14 (11), 614-621.
- Fang J-Y, Sacande M, Pritchard HW, Wetten A. (2009) Influence of freezable/non-freezable water and sucrose on the viability of Theobroma cacao somatic embryos following desiccation and freezing. Plant Cell Reports 28, 883-889.
- Hamilton KN, Ashmore SE, Pritchard HW (2009) Thermal analysis and cryopreservation of seeds of Australian wild Citrus species (Rutaceae): Citrus australasica, C. inodora and C. garrawayi. CryoLetters 30, 268-279.
- Sershen, Berjak P, Pammenter NW (2008) Desiccation sensitivity of excised embryonic axes of selected amaryllid species. Seed Science Research 18, 1-11.
- Nadarajan J, Mansor M, Krishnapillay B, Staines HJ, Benson EE, Harding K (2008). Applications of differential scanning calorimetry in developing cryopreservation strategies for Parkia speciosa, a tropical tree producing recalcitrant seeds. CryoLetters 29, 95-110.
Conferences and workshops:
- Invited lecture by Hugh Pritchard on ‘Frozen Planet: the biobanking of plants’ at the Society for Cryobiology 2010 Annual Meeting, Bristol UK.
- Invited lecture by Hugh Pritchard on ‘Biobanking of Plants’ at the 2010 Annual Biobanking Congress, London, UK
- Presentations at two Society for Low Temperature Biology Meetings, London, 2011
- GSPC, Target 8, Ex situ conservation of threatened species