Cryopreservation of cocoa (Theobroma cacao l.) somatic embryos - is somaclonal variation an issue?

Increasing demand for improved yield, quality and stress and disease resistance in cocoa (Theobroma cacao L.) highlight the importance of securely preserving the diversity of the species for future breeding goals. Though vital, field collections are prone to losses through climatic catastrophes and the action of pests and diseases and the recalcitrant nature of cocoa seed with regard to storage make the establishment of a cryopreserved collection of key cocoa germplasm a sensible precaution. To this end approximately 600 accessions of cocoa are being cryopreserved at Reading University through the encapsulation-dehydration of floralderived somatic embryos (SEs) and a newly-developed PVS2-based approach. These vitrification-based procedures involve the rapid cooling of the prolific secondary SEs obtained from cultured cotyledonary explants of primary SEs. With a view to maintaining the genetic fidelity of cryopreserved plant germplasm, shoot tip culture has become the propagation system of choice. In the case of cocoa in vitro shoot regeneration has proved problematic though rapid clonal multiplication can be achieved via somatic embryos derived from the floral tissues. Primary SEs can be induced from petal bases and staminodes and multiplication rates can be further increased through the production of secondary SEs from cotyledonary explants of primary embryos. Due to concern about somaclonal variation arising through the protracted callus phase involved in the generation of these propagules, their genetic fidelity has been tested and primary SEs have been found to exhibit a significant number of mutations. In this study nuclear microsatellite-based screening has been applied to each of the cocoa linkage groups in SEs sampled from sequential stages of the cryopreservation procedure (i. e. following culture, sucrose pretreatment, dehydration over silica and thawing after storage in liquid nitrogen) and compared with profiles for the donor tree. For all 48 regenerants tested in duplicate none exhibited aberrant profiles with respect to the donor tree for any of the 12 microsatellites screened. Furthermore, the use of microscopy techniques involving only minimal sample preparation (i. e. confocal and environmental scanning EM) have provided convincing evidence that post-cryo’ regenerants are derived from epidermal rather than callus cells, minimising any additional mutation risk. We conclude that, within the limits of this test population, no gross chromosomal changes occurred during cryopreservation and given the recent demonstration of virus elimination as a result of somatic embryogenesis, secondary SEs constitute an acceptable target tissue for cocoa germplasm conservation.