title: Effect of Cryopreservation and Post-Cryopreservation Somatic Embryogenesis on the Epigenetic Fidelity of Cocoa (Theobroma cacao L.).
abstract: While cocoa plants regenerated from cryopreserved somatic embryos can demonstrate high levels of phenotypic variability, little is known about the sources of the observed variability. Previous studies have shown that the encapsulation-dehydration cryopreservation methodology imposes no significant extra mutational load since embryos carrying high levels of genetic variability are selected against during protracted culture. Also, the use of secondary rather than primary somatic embryos has been shown to further reduce the incidence of genetic somaclonal variation. Here, the effect of in vitro conservation, cryopreservation and post-cryopreservation generation of somatic embryos on the appearance of epigenetic somaclonal variation were comparatively assessed. To achieve this we compared the epigenetic profiles, generated using Methylation Sensitive Amplified Polymorphisms, of leaves collected from the ortet tree and from cocoa somatic embryos derived from three in vitro conditions: somatic embryos, somatic embryos cryopreserved in liquid nitrogen and somatic embryos generated from cryoproserved somatic embryos. Somatic embryos accumulated epigenetic changes but these were less extensive than in those regenerated after storage in LN. Furthermore, the passage of cryopreserved embryos through another embryogenic stage led to further increase in variation. Interestingly, this detected variability appears to be in some measure reversible. The outcome of this study indicates that the cryopreservation induced phenotypic variability could be, at least partially, due to DNA methylation changes.
  
  
  CONCLUSIONS
  Phenotypic variability observed in cryostored cocoa somatic-embryos is epigenetic in nature. This variability is partially reversible, not stochastic in nature but a directed response to the in-vitro culture and cryopreservation.
authors.affiliation: Faculty of Agriculture, University for Development Studies, P.O. Box TL 1882, Tamale, Ghana.
authors.forename: Raphael
authors.initials: R
authors.lastname: Adu-Gyamfi
authors.affiliation: School of Agriculture, Policy and Development, University of Reading, Whiteknights, Reading, Berks, RG6 6AS, United Kingdom.
authors.forename: Andy
authors.initials: A
authors.lastname: Wetten
authors.affiliation: Environmental Epigenetics and Genetics Group, Plant Research Centre, School of Agriculture, Food and Wine, Faculty of Sciences, University of Adelaide, Waite Campus, PMB1, Glen Osmond, SA, 5064, Australia.
authors.forename: Carlos
authors.initials: C
authors.lastname: Marcelino Rodríguez López
mesh_terms: D002099:Cacao / Q000166:cytology* / Q000235:genetics*; D015925:Cryopreservation; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D014644:Genetic Variation; D058439:Plant Somatic Embryogenesis Techniques; D012639:Seeds / Q000166:cytology / Q000235:genetics