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.

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.

Fig 1. Schematic representation of experimental design.

Somatic embryogenesis was initiated from staminodes (red squares) obtained from a single AMAZ 15 cocoa tree from the International Cocoa Quarantine Centre, Reading, UK. Triangles represent 24 samples collected from individual newly expanded leaves for DNA extraction from each group (Donor plant (Red) and somatic embryo derived plants (Green)). Orange boxes represent successive somatic embryogenic events (as described in [28]). Blue boxes represent a cryopreservation event (1 h in liquid nitrogen) of somatic embryos (as described [29]). Green boxes represent maintenance of somatic embryos in vitro on ED medium [28]. Horizontal bar represents accumulated time spend under in vitro conditions by the samples used in this study.

Fig 2. Effect of conservation method on somaclonal variation pattern in cocoa somatic embryos.

Principal coordinate analysis based on Euclidean Analysis of MSAP distances between 72 somatic embryos grouped by method of conservation and 24 leaf samples of the donor tree. MSAP profiles were obtained using methylation sensitive isoschizimers MspI (circles) and HpaII (rhomboids) and methylation insensitive enzyme EcoRI primer combinations and Hp1/Eco1 and Hp3/Eco3. Individual figures show PCoA analysis from MSAP profiles obtained using (A) both MspI and HpaII, (B) MspI only and (C) HpaII only. 1 h: SEs recovered from secondary SE after 1 hour storage in liquid nitrogen; Post 1 h: tertiary SEs generated from 1 h samples; Invitro: In vitro maintained secondary SEs, and Donor: AMAZ 15 cocoa ortet tree used to regenerate SEs. Hp or Msp preceding a treatment means MSAP profiles were generated using restriction enzyme HpaII and MspI respectively.

Fig 3. Somatic embryogenesis and cryopreservation induced between epigenetic variability.

(A) Bars represent epigenetic distance (PhiPT) between ortet tree samples and each of the in vitro treatments (Invitro: In vitro maintained secondary somatic embryos; 1 h LN: Secondary somatic embryos recovered from after 1 hour storage in liquid nitrogen and Post 1 h LN: tertiary somatic embryos generated from 1 h LN samples) calculated using 10.000 permutations and AMOVA analysis. (B) Bars represent the average epigenetic variability (mean sum of squares within population (SSWP)) between samples with the same origin (i.e. Donor, Invitro, 1 h LN and Post 1 h LN). PhiPT and SSWP values were calculated using GenAlex 6.1 software from MSAP profiles generated combining Hp3/Eco3 and Hp1/Eco1 selective primer combinations and restriction enzymes HapII (Blue) and MspI (red) as frequent cutters. All somatic embryos and ortet tree samples (24 per treatment) were initiated/collected from a single AMAZ 15 cocoa tree from the International Cocoa Quarantine Centre, Reading, UK. * Indicates significantly different (P>0.0002) PhiPT values between treatments.