3. Immerse the seedling under sterile water, orienting the hypocotyl vertically to enable dissection (Fig. 1d, e).
4. Grip each cotyledon with a No 5.5 tweezer to put the seedling
under slight tension; then gently remove one cotyledon by
applying a downward force on this cotyledon (Fig. 1f–h).
Fig. 1 Procedure for dissection of seedlings. (a) A slit is made in a GM + Vitamin (GM + Vit) agar dish with a
sterile blade. (b) The seedling is then placed adjacent to the cut (dashed line). (c) The root of the seedling is
then pushed gently inside the cut. (d) The GM + Vit plate is filled with sterile water and (e) the hypocotyl
oriented vertically. (f–h) The seedling is dissected under water using two 5.5 forceps as described in the text.
Note dissected cotyledon (left) is shown floating next to the seedling to be imaged (right) in (g). (h) Once
dissected, the seedling can be imaged under the confocal using a water-dipping objective. (i) After imaging,
the water is removed and the seedlings transplanted to new plates which are then sealed with micropore tape
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Monica Pia Caggiano et al.