Megagametophytes of Douglas fir (Pseudotsuga menziesii) and hybrid larch (Larix x eurolepis) in culture: multiplication of neck cells and the formation of binucleate cells

To study the effect of culturing on megagametophytes of Douglas fir (Pseudo tsuga menziesii) and hybrid larch (Larix x eurolepis), cones were collected at the time of fertilization and the megagametophytes were removed, then p laced on medium. We used a modified Murashige and Skoog medium supplemented with 5% lactose and 10% polyethylene glycol 4000. A variety of cell types proliferated including prothallial, neck, and jacket cells. Some of these m ultiplying cells showed a binucleate condition. The prothallial cells of th e apex divided and expanded. The neck cells formed clusters composed of mor e cells than normally found in situ; though otherwise they showed ultrastru ctural similarity to neck cells in situ. These neck cells had large numbers of active Golgi complexes, numerous large and small vacuoles, coated vesic les, smooth vesicles, a well-developed endoplasmic reticulum, and thickened cell walls. These are the first reports of neck cell multiplication and in duction of a binucleate state for gymnosperm megagametophyte cells in vitro .