Abstract
The date palm (Phoenix dactylifera. L) has a great ecological and socioeconomic importance in arid and semi-arid areas of the globe. This species displays a great diversity, with over 2,000 identified varieties, which is threatened by the large scale production of elite varieties. It is necessary to develop techniques allowing to conserve this biodiversity and to manage the production of in vitro propagated elite varieties. Cryopreservation (liquid nitrogen [LN], -196°C) is currently the only technique available ensuring the safe and cost-effective long-term conservation of this diversity. In this work, we compared the efficiency of two cryopreservation techniques, droplet-vitrification (DV) and D cryo-plate (DP), for the cryopreservation of proembryonic masses (PEMs) of two varieties of date palm, Sokary and Sultany. With DV, recovery of cryopreserved PEMs was nil without sucrose pretreatment (3 days, 0.5 M) and without treatment with PVS2 vitrification solution. After 15 to 120 min of PVS2 treatment, recovery was between 90.9-98.6% and 85.6-88.0% for Sokary and Sultany, respectively. Sucrose pretreatment led to 21.1% recovery of cryopreserved PEMs of variety Sokary without PVS2 treatment. Regrowth intensity of PEMs cryopreserved was generally lower in the Sultany variety compared to the Sokary variety. With DP, a positive effect of sucrose pretreatment on recovery of cryopreserved PEMs was observed. For Sokary, the highest recovery of PEMs (92.0 to 95.8%) after LN exposure was achieved for desiccation periods between 60 and 120 min. Recovery of cryopreserved PEMs of variety Sultany was between 67.0 and 74.6% after desiccation periods between 90-120 min. With DP, the regrowth intensity of cryopreserved PEMs was higher for variety Sokary compared to Sultany. The histological study performed showed that in absence of pretreatment, it was the loading treatment which induced the highest cell plasmolysis, with values of 26, 40 and 50% for meristematic, embryogenic PopI and PopII cells, respectively, compared to their initial state. Sucrose pretreatment induced a 50% plasmolysis only in PopII cells. The loading and vitrification treatments did not cause any additional plasmolysis. No plasmolysis was observed in meristematic or PopI cells. By contrast, the measurement of the surface of all cells revealed a significant decrease in the surface of meristematic cells after pretreatment, but no significant difference after the loading or vitrification treatments. In embryogenic PopI cells, an additional decrease in the cell surface was observed after the PVS2 treatment. The study of nuclei circularity showed a permanent deformation of nuclei of non-pretreated cells of the three cell types. In pretreated PEMs, only the nuclei of PopII cells displayed deformation. Finally, the study of the methylation degree in PEMs by immunolocalization revealed the absence of significant differences between the untreated controls and other experimental conditions. However, the differentiated tissues exhibited a higher percentage of methylated nuclei compared to embryogenic cells, while no stained nuclei were observed in meristematic cells. Our results allowed clarifying the effect of cryopreservation on the structural integrity and on the physiology of date palm PEMs. They also contribute to safeguarding of date palm biodiversity.