To evaluate variation within maternal trees, SSR-PCRs were performed on DNA samples from 20 phenologically dispersed leaves from ortet trees (LCTEEN 37/I, LCTEEN 162/S-1010, SC3, and SIAL93) using primers mTcCIR15, mTcCIR3, mTcCIR17, mTcCIR10, mTcCIR6, mTcCIR7, mTcCIR1, mTcCIR8, and mTcCIR61 (Supporting Information Table S1) according to the protocol of Lanaud et al. (1999). In all cases, one primer was 5′-end-labelled using 6-carboxyfluorescein (FAM), 6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein (HEX) or NED (Applied Biosystems, Inc, Carlsbad, California, USA) to allow product detection during capillary gel electrophoresis on an ABI 373XL (Applied Biosystems, Inc, Carlsbad, California, USA). Variation among regenerated plants was assessed using the same primers and conditions applied to template DNA extracted from a random selection of in vitro regenerants (LCTEEN 37/I (13 samples), LCTEEN162/1010 (180 samples), SC3 (20 samples), and SIAL93 (20 samples)). Products were visualized on the resultant electropherograms and compared to identify cases of de novo slippage mutation. Profiles were only considered to provide evidence of novel genetic change among the regenerants when both replicate DNA extractions exhibited the same deviation from the maternal SSR profile. Regenerant ‘off types’ were then re-examined along with 51 randomly selected samples from the ‘wild-type’ regenerants in which no deviations were noted from the parental profile using the following additional set of SSR primers: mTcCIR19, mTcCIR18, mTcCIR2, mTcCIR26, mTcCIR25, and mTcCIR24.