Effect of cryopreservation and post-cryopreservation somatic embryogenesis on the epigenetic fidelity of cocoa (Theobroma cacaoL.)

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. Key message: 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.

Epigenetic and Genetic Integrity, Metabolic Stability, and Field Performance of Cryopreserved Plants

“…Further analysis showed that only 2.6-9.8% of the changes were induced by cryopreservation procedure and the rest were attributed to the in vitro culture processes. In vitro culture-induced DNA methylation was also found in the regenerants recovered from cryopreserved Carica papaya shoot tips [65] and Theobroma cacao somatic embryos [66]. DNA methylation levels were higher in in vitro stock somatic embryos of Theobroma cacao than those regenerated after cryopreservation [66].…”

“…In vitro culture-induced DNA methylation was also found in the regenerants recovered from cryopreserved Carica papaya shoot tips [65] and Theobroma cacao somatic embryos [66]. DNA methylation levels were higher in in vitro stock somatic embryos of Theobroma cacao than those regenerated after cryopreservation [66]. Therefore, the manipulations of in vitro culture procedures, particularly for regeneration via somatic embryogenic, are needed to reduce the probability of alternations in DNA methylation.…”

“…Such reversible epigenetic mechanism has also been noted in the regenerants recovered from cryopreserved shoot tips of Carica papaya [65], Mentha × piperita [70], and Actinidia chinensis var. deliciosa [71], and from the cryopreserved somatic embryos of Bactris gasipaes [72] and Theobroma cacao [66]. As speculated by Harding et al [54], changes in DNA methylation may be an adaptive response to oxidative stress induced during cryopreservation.…”

Cacao biotechnology: current status and future prospects

“…Because of the recalcitrant (do not survive drying) nature of its seeds, the germplasm of this allogamous tree crop must be conserved in field genebanks as a living collection (Motilal et al ., ) or by cryopreservation (Adu‐Gyamfi and Wetten, ; Adu‐Gyamfi et al ., ). The largest collections of cacao germplasm are those at the International Cocoa Genebank, Trinidad (ICG, T) (https://sta.uwi.edu/cru/index.asp) (2400 accessions) and at the Centro Agronomico Tropical de Investigación y Enseñanza (CATIE) in Costa Rica (https://www.catie.ac.cr/en/) (1146 accessions), with another collection (c. 400 accessions), the International Cocoa Quarantine Centre, housed at the University of Reading, UK (http://www.icgd.reading.ac.uk/icqc/).…”

“…Moreover, a study conducted by López and co‐workers in 2010 found that de novo genetic mutations and epigenetic modifications do not accumulate with ageing of in vitro induced cacao calli (López et al ., ); this conclusion is also relevant to a recent study of long‐term SE (Quinga et al ., ). Additionally, the SE system has been utilized in cacao germplasm conservation through cryopreservation (Adu‐Gyamfi and Wetten, ; Adu‐Gyamfi et al ., ) and in genetic transformation (Maximova et al ., ; da Silva et al ., ). Induction of cacao somatic embryos has been observed from a range of its tissues, that is zygotic embryos (Pence et al ., ), floral parts—petals and staminodes (Alemanno et al ., , ; Boutchouang et al ., ; Li et al ., ; Tan and Furtek, ), and nucellar tissues (Figueira and Janick, ).…”