Assessment of alternative freezing methods for preservation at −80 °C of radiata pine embryogenic cultures: A six-year study

Somatic embryogenesis is an essential component of breeding programs for Pinus radiata aimed at implementing multi-varietal forestry. Coupled with this technique, the long-term cryopreservation of embryogenic cultures is necessary to maintain the viability of the cell lines, but this entails high maintenance costs. In this research we evaluated the application of a protocol for long-term storage at −80 °C in an ultra-low freezer to preserve several radiata pine embryogenic cell lines. Also, we studied the influence of several parameters to optimize the protocol, such as the effect of dimethyl sulfoxide cryoprotectant solution, the effectiveness of alternative freezing methods, the use of post thawing treatments and the addition of sodium butyrate at maturation stage. We found that the use of dimethyl sulfoxide cryoprotectant enhanced somatic embryo production; slow cooling was the only viable method for preserving embryogenic cell lines at −80 °C and the use of sodium butyrate was not highly effective to improve maturation and germination stages. Moreover, we have regenerated embryogenic cell lines up to their conversion into plants after six years of storage. In line with these findings, the protocol to storage in an ultra-low freezer represents an economical alternative to preserve somatic embryogenic cultures of Pinus radiata.