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.
Table 1.
Description of cocoa samples used for MSAP analysis.
Table 2.
Sequence of oligonucleotides used for MSAP analysis.
Table 3.
Somatic embryogenesis/cryopreservation induced epigenetic polymorphisms.
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.
Table 4.
Somatic embryogenesis and cryopreservation induced epigenetic differences.