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

Author： Marum Liliana Estêvão Catarina Oliveira M. Margarida Amâncio Sara Rodrigues Lucília Miguel Célia

ISSN： 1742-0644

Source： Cryoletters, Vol.25, Iss.5, 2004-09, pp. : 363-374

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Abstract

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 Me₂SO in a mixture of PEG 4000 and sucrose (PSD solution), instead of Me₂SO 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% of the lines stored in liquid of nitrogen.