Progress Towards Cryopreservation of Axillary Meristems of Pinus Radiata and Cheesemania Sp

Abstract

The ability to field-test specific lines of Pinus radiata before selection and clonal production of planting stock offers the opportunity to significantly increase the rate of genetic improvement. Identification of superior field performances may not be effective until the trees are up to 15 years old. However, the growth performance of clonal material produced by cuttings from parent stock more than 4 years old is reduced. Tissue culture techniques for Pinus radiata using mature zygotic embryos are well developed and routinely used for the preparation of planting material. These established cultures can also be maintained by repeated subculturing. But as a holding technique such a practice is expensive, liable to losses through contamination and introduces the possibility of unwanted genetic change. The ability to preserve such tissue cultures using cryopreservation techniques would eliminate all the above difficulties and simultaneously allow an unlimited period for field testing and selection. This thesis reports the successful development of a protocol for the cryopreservation of cotyledon tissue from zygotic embryos of Pinus radiata. Zygotic embryos were dissected from mature seed and preconditioned for seven days with 0.3 M sorbitol. Following preconditioning, embryos were precultured for 20 minutes with 0.255 M sorbitol plus 15% DMSO. Embryos were then cooled at approximately 1 °C min-1 to -30 °C and immersed in liquid nitrogen. Cotyledons from zygotic embryos which had been cryopreserved for up to seven weeks showed meristematic tissue formation seven days after thawing. Meristematic tissue formed on up to 84% of cryopreserved genotypes in comparison to 92% of the non-frozen controls. Shoots produced from cryopreserved cotyledons subsequently formed roots and grew normally (15-25 cm after 3 months). Pinus radiata shoot tissues grown in vitro, which were preconditioned and precultured with a range of sorbitol and DMSO concentrations, formed undifferentiated callus following cryopreservation in liquid nitrogen. A critically endangered New Zealand Cheesemania species was successfully cryopreserved using a vitrification solution designated PVS2. On thawing, axillary meristems produced shoots without an intervening callus phase. Plants from the tissue were successfully established in the glasshouse.