The survival and the genetic fidelity of coconut embryos after cryopreservation

Coconut (Cocos nucifera L) plays an important role in the socio-economic life of many millions of people in the tropical regions of the world. However, the genetic diversity of this species is being dramatically decreased by diseases, natural disasters and through land cleaning for the planting of the so called 'more valuable' crops. Coconut seed cannot be conserved by normal means as it is big and sensitive to drying (recalcitrant). Cryopreservation is an alternative conservation approach but as yet, this method is still poorly developed for coconut. Thus, there is a need to develop a new, more reliable cryopreservation protocol for this crop. A new cryopreservation protocol, based on a rapid tissue desiccation step was developed to produce a high plant recovery rate after cryopreservation. Embryos from four coconut cultivars (Malayan Yellow Dwarf - MYD, Nias Yellow Dwarf - NYD, Nias Green Dwarf and Sagerat Orange Dwarf) were rapidly desiccated for 8 hours (water content decreased from 80% fresh weight or 4.14 g g-1 dry weight to 19% fresh weight or 0.24 g g-1 dry weight). This dehydration pre-treatment prevented ice crystal. forming in the embryo cells during freezing and a differential scanning calorimetry analysis approach indicated that the glass transition temperature had been raised from ca. -123 to -48 0C. Upon recovery from liquid nitrogen, 24 hours later, the percentages of embryos that produced normal seedlings was high and varied from 21% in NYD to 40% in MYD. Most of the seedlings produced after cryopreservation could be field planted after a further 4 months growth in the glasshouse. In general, genetic fidelity testing undertaken on these plants showed that no morphological abnormalities could be detected. In addition, karyotype analysis also showed no ploidy level or gross chromosomal abnormalities to have taken place. While molecular analysis, using microsatellite and global DNA methylation techniques, revealed no significant differences in the genomic DNA isolated from seedlings that developed from cryopreserved or non-cryopreserved embryos. (Texte intégral)