The cryopreservation of Picea sitchensis germplasm

  • Samantha Gale

Student thesis: Doctoral Thesis


Picea sitchensis is an important tree species for UK forestry and is at the forefront of prototype clonal breeding programs. These can only be implemented using in vitro, culture therefore cryopreservation technology development is imperative such that elite germplasm can be conserved, without compromise to genetic integrity, whilst phenotypic validation of selected genotypes is undertaken.Three explant types were transferred from the Northern Research station, Roslin to the University of Abertay, Dundee where cryopreservation testing was initiated. Each explant comprised of different anatomical complexities varying from the simplest dedifferentiated embryogenic suspensor masses, to matured somatic embryos and whole tissue shoot-tip apices. Before cryopreservation was initiated in vitro cultures were stabilised and characterised at UAD. Shoot cultures showed different growth responses between genotypes and between culture locations, but biochemical profiling of oxidative stress markers, ethylene and DNA methylation did not confirm stress or epigenetic change as the cause of these differences and physiological recalcitrance.A cryopreservation protocol, using a programmable freezer (Planar), was successfully developed and is reported for the first time for P. sitchensis embryogenic suspensor masses (ESM). Post-LN survival rates of up to 100% were observed in several genotypes. Encapsulation-dehydration was successfully utilised to cryopreserve P. sitchensis mature somatic embryos reported for the first time. Recovered embryos were able to re-initiate into dedifferentiated non-embryogenic masses (NEM) (up to 100%) and embryogenic suspensor masses (ESM) (up to 20% post -LN) as a source of material to mass multiply cryopreserved clonal offspring. Major steps were progressed for the most recalcitrant conifer explant in the project, shoot-tip apices, with essential pre-treatment steps established. Critical cryogenic factors were determined through thermal analysis using Differential Scanning Calorimetry.The study concluded with the initiation of technology transfer of cryopreservation methods to the Northern Research Station to establish the UK’s first conifer cryobank. These systems were preliminary validated. Further implementation will proceed out with the timescale of this project but based on recommendations generated from this thesis.
Date of AwardNov 2005
Original languageEnglish
SponsorsEuropean Commission, UK Forestry Commission, Scottish International Educational Trust, Society for Low Temperature Biology & Plant Conservation Group

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