Plant regeneration from ploidy-stable cryopreserved embryogenic lines of the hybrid Pinus elliottii x P. caribaea

Clonal breeding programs of the highly important interspecific hybrid Pinus elliottii var. elliottii and Pinus caribaea var. hondurensis require the establishment of robust cryopreservation protocols, with no genetic variability. Embryonal masses of this hybrid were successfully cryopreserved by a fast slow-freezing method. The protocol was optimized by testing the effects of dimethyl sulfoxide (DMSO) based cryoprotectant solutions, sugar cryoprotectants (e.g. sucrose or maltose, 0.4 M) and different times of pretreatments and pre-cooling storage. The addition of DMSO in a mixture of polyethylene glycol (PEG) 4000 and sucrose (PSD solution), instead of DMSO alone, was beneficial for recovery of cryopreserved cultures. This parameter, together with the time of pre-cooling storage (before plunging in liquid nitrogen), were the factors that most influenced the survival and regrowth rates of embryonal masses. A pretreatment combination of sucrose (0.4 M) and 5% PSD followed by a pre-cooling storage of 24 h allowed the cryopreservation and regrowth of embryogenic cell lines without major genetic variations (DNA-ploidy) or loss of embryogenic potential. Eight of the nine tested embryogenic cell lines survived to the cryopreservation procedures, however the genotype and treatment conditions clearly influenced the response. Somatic embryos maturation and conversion from recovered embryonal masses (EMs) took place according to our standard somatic embryogenesis protocol for Pinus, using modified Litvai (mLV) medium. This is the first report for this hybrid, and the proposed protocol is fast, robust, providing high rates of recovery and working with several genotypes. This protocol's suitability for industrial breeding programmes was also confirmed by allowing the regeneration of plants with no apparent variability from cryopreserved embryonal masses.