Laser-Assisted Microdissection and subsequent DNA methylation analysis of CPR4 sequences in synergid cells from mature embryo sacs. (A) Dissection of the synergid cell from a mature embryo sac; an 8 μm section through an ovule bearing a mature embryo sac prior to laser microdissection with the MMI CellCut Plus instrument. The following abbreviations are used: S, synergids; E, egg cell; C, central cell. (B) The ultraviolet laser beam has been applied in order to isolate the synergid cells (arrow). The laser cut has a diameter of 1–2 μm. (C) The synergid cell has been removed with an MMI isolation cap. (D) DNA methylation analysis. Cytosine methylation of the CRP4 coding sequence was studied in synergid cell (S) and leaf genomic DNA (L), in both endogenous and transgenic contexts, using enzymes sensitive to CG/CHG methylation (HpaII) and CHH methylation (DdeI). The CRP4 coding region has two sites for HpaII and three sites for DdeI. Disappearance or reduced levels of a fragment after digestion with a given enzyme indicate loss of methylation at that site. Undigested input DNA is included at the far right.