Long-term cryopreservation of embryogenic Pinus sylvestris cultures

Abstract

Long-term cryopreservation is important for producing clones from somatic embryogenesis (SE) since it maintains the regeneration ability of the SE lines during the field tests required for clone selection. In the present work, we report recovery, proliferation and embryo production capacity of embryogenic Scots pine (Pinus sylvestris L.) cultures following 2–12 years of cryopreservation. Altogether 108 SE lines from three donor trees were used as material. The SE cultures were originally cryopreserved using two different mixtures of polyethyleneglycol 6000, glucose and dimethylsulphoxide (PGD) as cryoprotectant. Good recovery rates (80–93%) were observed for the lines stored for up to 10 years whereas after 12 years recovery rate was lower (59%). The length of cryostorage did not affect the proliferation rate of the SE cultures. Percentage of the SE lines producing mature embryos varied from 56 to 100, without connection to duration of cryostorage. The average embryo production of the maturing lines varied, but not significantly, from 326 per gFW of tissue following 2 years of cryostorage, to 107–111 per gFW after 9–10 years, and to 55 per gFW after 12 years of storage. Genotypic variation and the age of the cultures at the moment of cryopreservation probably contribute to the observed differences in embryo production. Thus, long-term cryopreservation of Scots pine SE cultures seems possible using PGD as cryoprotectant, and good recovery combined with satisfactory embryo yield can be expected at least up to 10 years of storage.

Keywords:

• Maturation
• PGD mixtures
• Pinus sylvestris
• proliferation
• recovery

Acknowledgements

We would like to thank the personnel of the Punkaharju Research Unit and especially Taina Naukkarinen, Paula Matikainen, Airi Huttunen and Aila Viinanen for their technical assistance, and everybody involved in production and cryostorage of the SE lines used in the present study. We also thank the reviewers for their valuable recommendations. The Foundation for Forest Tree Breeding in Finland is acknowledged for the grant to M. Latutrie.