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Cryopreservation for the conservation of Grevillea species

Tan, Sze-Ping (1998) Cryopreservation for the conservation of Grevillea species. Honours thesis, Murdoch University.

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Western Australia has one of the most diverse and vulnerable flora in the world, with over 300 species on the rare and endangered list and over 1000 more prioritised for further action - about 40% of described species. Due to a lack of resources or protection in situ, most species must be conserved ex situ. Traditional ex situ methods are limited in their ability to store an adequately diverse pool of genetic material, while maintaining its genetic integrity. The advent of cryostorage as a means of genetic conservation potentially provides a highly secure means of storing genetic material in the long term. This study in particular investigated the suitability of cryopreservation as a method of long term conservation of somatic tissue of Western Australian species, with the aim of application to large groups of taxa. The broad aims of this study were to research the applicability of specific cryostorage methods to larger groups of taxa, in this case the genus Grevillea, a woody shrub genus.

The first section of this study investigated the applicability of the cryostorage protocol elucidated for G. scapigera on itself and four other species without modification: G. cirsiifolia, G. dryandroides, G. flexuosa and the hybrid G. bipinnitifida X biternata (BxB). Shoot apices were subjected to a modified vitrification protocol. All five species utilised survived the treatment with significant variation in success. Survival values ranged from 3.6% for G. BxB to 92.3% for G. cirsiifolia. Observations seemed to indicate it was not the freeze-thaw process itself that was responsible for low survival rates seen, and that it was more likely the contribution of the cyototoxicity of cryoprotectectant compounds used.

The age of the explants after subculture was investigated as a factor in improvement of the established protocol for broader applicability. Shoot apices were excised at 7, 14, 21 and 28 days after subculture for the abovementioned five species. All five species tested showed a significant increase in survival post-thaw when the shoot apices were excised at 21 days after subculture, as in the unmodified G. scapigera protocol. Histological studies through GMA sectioning showed no obvious differences in cell characteristics between the ages tested, and no gross disruption in cellular integrity was seen post-thaw. It was postulated that damage caused was at a biochemical level.

The preculture of cells in cryopreservation is a step performed to optimise tissue condition for survival of the freeze-thaw process. It was found that the survival of three species of Grevillea - G. cirsiifolia, G. dryandroides and G. BxB – could be improved by variation from the stipulated protocol in type or concentration of the compound used. G. dryandroides showed a significant increase in survival from 18% to 75 % when incubated on a preculture medium containing 1.2 M glycerol. G . cirsiifolia showed survival of 24 % when incubated with 0.4 M sorbitol. G. BxB however showed a survival at the stipulated 0.6 M sorbitol of 15.6 %. These results were believed to be due to differing requirements between the species tested.

The results of this study showed that a specific established protocol could be applied with minor modifications to a wider range of species. Although only intrageneric similarities in cryopreservation requirements were tested, there is no reason to doubt that protocols could be applicable to wider groups of species. More research is recommended, particularly in the fields of preculture and recovery post-thaw, to improve existing protocols for the purposes of conservation.

Item Type: Thesis (Honours)
Murdoch Affiliation: School of Biological and Environmental Sciences
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: Thank you.
Supervisor(s): McComb, Jen and Touchell, D.
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