Recovery of cryopreserved embryogenic cultures of maritime pine--effect of cryoprotectant and suspension density.

Embryogenic cultures were obtained from seeds of open-pollinated maritime pine trees representing part of a breeding population. The aim of the present study was to develop and optimize a protocol for cryopreservation of Pinus pinaster somatic embryogenic tissue. The density of the embryogenic suspension and the type of pre-treatment significantly affected the recovery of cryopreserved embryogenic cultures, as evaluated by their survival and regrowth rate. An initial density of the suspension culture of 250 mg/ml improved the regrowth rate of the embryogenic lines. Pre-treatment with maltose 0.4 M significantly increased the regrowth rate when compared to the other tested carbohydrates. Also, the addition of DMSO in a mixture of PEG 4000 and sucrose (PSD solution), instead of DMSO alone, at the same final concentration, was clearly beneficial for recovery of cryopreserved tissues. This improved method for the cryopreservation of P pinaster embryogenic of cultures allowed the successful recovery of 97 percent of the lines stored in liquid of nitrogen.