Importance of structural integrity of somatic embryos for long-term cryopreservation of cocoa (Theobroma cacao L.) germplasm

Abstract

Cryopreservation is a reliable means for the long-term conservation of plant genetic resources. It is of particular interest for cocoa (Theobroma cacao L.) whose seeds are recalcitrant to conventional storage methods and field collections susceptible to disease infestations. However, the encapsulation- dehydration procedure previously developed for cocoa somatic embryos has resulted in poor survival after retrieval from liquid nitrogen. To examine the causes of such failure, cocoa somatic embryos following each treatment step of the encapsulation-dehydration procedure were examined using a combination of confocal scanning laser microscopy and transmission electron microscopy. Results showed that the parenchyma’s cells of the hypocotyl and radicle were the major sites of injury possibly due to their large size and non-cytoplasmic nature, whereas the shoot meristem and provascular strand were well preserved throughout the treatments. In general, cell deformation and/or disruption was observed following the sucrose preculture, desiccation and freezing steps, with the extent of damage increasing at each treatment step. Post-thaw regrowth of injured embryos was possible through the proliferation of surviving cells, but these embryos often failed to convert into plantlets. The present study suggests that the maintenance of structural integrity of somatic embryos at each treatment step is essential for the successful cryopreservation of cocoa germplasm. Key words: Cocoa somatic embryos, confocal scanning laser microscopy, cryopreservation, structural integrity, transmission electron microscopy.