Effect of different cryoprotectant procedures on the recovery and maturation ability of cryopreserved Pinus pinea embryogenic lines of different ages

Abstract

Strategies for genetic improvement programs of Pinus pinea L, an important tree species of the Mediterranean ecosystem, are focused on increasing pine nut yield. Somatic embryogenesis and cryopreservation of elite genotypes are emerging as key components of advanced forest breeding programs. This study was carried out with embryogenic lines of different ages obtained from selected half-sib families of the species. The effect of three cryoprotectant procedures on the recovery and maturation ability was tested in embryogenic lines that showed different growth rate, two of them at different ages. In general, cryopreservation drastically reduced growth rates of frozen and rewarmed tissues; however, the use of 5% PEG–sucrose–DMSO dramatically increased growth rates of rewarmed embryogenic cultures. Overall, embryogenic lines of stone pine were suitable for cryopreservation. Seven out of eight lines were recovered, although the initial growth rates were variable. Five of six lines including the three oldest ones were recovered using 5% PEG–sucrose–DMSO. No relation was observed between age and growth rate of embryogenic lines and their response to cryopreservation. The line 2F47 showed the most stable response after long-term subculture and recovery after cryopreservation, at different ages. On the contrary, younger embryogenic lines either recovered after cryopreservation or did not, depending on the applied procedure. Maturation of some of the older lines was restored or enhanced after cryopreservation. Somatic embryos were obtained in three out of five tested embryogenic lines recovered from cryopreservation. However, only a few plantlets from cryopreserved lines were regenerated indicating the process must be optimized further before it is a practical adjunct to breeding.