Cryopreservation of embryogenic tissues from mature holm oak trees

The development of a vitrification method for cryopreservation of embryogenic lines from mature holm oak (Quercus ilex L.) trees is reported. Globular embryogenic clusters of three embryogenic lines grown on gelled medium, and embryogenic clumps of one line collected from liquid cultures, were used as samples. The effect of both high-sucrose preculture and dehydration by incubation in the PVS2 solution for 30-90 min, on both survival and maintenance of the differentiation ability was evaluated in somatic embryo explants with and without immersion into liquid nitrogen. Growth recovery of the treated samples and ability to differentiate cotyledonary embryos largely depended on genotype. Overall, high growth recovery frequencies on gelled medium and increase of fresh weight in liquid medium were obtained in all the tested lines, also after freezing. However, the differentiation ability of the embryogenic lines was severely hampered following immersion into LN. Two of the embryogenic lines from gelled medium were able to recover the differentiation ability, one not. In the lines with reduced or no differentiation ability, variation in the microsatellite markers was observed when comparing samples taken prior to and after cryopreservation. The best results were achieved in the genotype Q8 in which 80% of explants grown on gelled medium differentiated into cotyledonary embryos following cryopreservation when they were precultured on medium with 0.3M sucrose and then incubated for 30 min in the PVS2 solution. Explants of the same genotype from liquid medium were unable to recover the differentiation ability. A 4-weeks storage period both in liquid nitrogen and in an ultra-low temperature freezer at -80°C was also evaluated with four embryogenic lines from gelled medium using the best vitrification treatment. Growth recovery frequencies of all lines from the two storage systems were very high, but their differentiation ability was completely lost.