Cryopreservation of Pinus radiata embryogenic tissue: effects of cryoprotective pretreatments on maturation ability

The goal of the study was to quantify the effect of different cryoprotective pretreatments on the maturation ability of cryopreserved Pinus radiata embryogenic tissue. Fourteen cell lines were evaluated under five pretreatments: (1) Dimethyl sulfoxide 5% (v/v); (2) Dimethyl sulfoxide 10% (v/v); (3) Dimethyl sulfoxide 5% (v/v) and 0.09M of l-proline; (4) Dimethyl sulfoxide 10% (v/v) and 0.09M of l-proline; (5) 0.09M of l-proline and compared to embryogenic tissue that was not cryopreserved (control). The cell lines were thawed at 37 degrees C to evaluate the recovery percentage and determine the maturation ability. The recovery rate of all cryopreserved material was significantly lower than the control and material treated with 0.09M of l-proline, in absent dimethyl sulfoxide, showed the lowest levels of recovery. Regarding the number of embryos, an interaction between pretreatments and the cell lines under evaluation was identified. Moreover, morphological differences in the plants being treated with the most concentrated Dimethyl sulfoxide pretreatment was observed. At the microscopic level, no damage or morphological changes in proembryogenic masses were observed during the post-cryopreservation evaluation. Therefore, the best pretreatment was 5% Dimethyl sulfoxide supplemented with 0.09M l-proline, allowing the most growth recovery of thawed samples and more cotyledonary embryos (maturation ability). The evaluated responses in presence of cryoprotective elements were genotype dependent.