GENERAL GYNOGENESIS PROCEDURES
Gynogenesis experiments are usually conducted using unfertilized ovules or ovaries, although entire
immature flower buds have been cultured in a few species. It is easier to dissect ovaries than ovules
without damaging the female gametophyte. However, in polyovulate ovaries, it may be advantageous
to excise the ovules so that they can be in direct contact with the culture medium.
Inflorescences must be collected before pollen shed, unless the species is highly self-incompatible
or a male-sterile line is used. In developing a gynogenesis protocol for a species, it may be
necessary to collect explants from several days before anthesis to just before anthesis. As discussed
earlier, the stage of microspore development is sometimes recorded as an indicator of developmental
stage of the female gametophyte. Procedures used for determining the stage of microspore development
are described earlier in this chapter.
Disinfestation varies depending on species, growing conditions of explant source, and choice
of explant. Woody plant material often requires longer disinfestation times and/or stronger sterilizing
agents than herbaceous materials. Tissue from greenhouse-grown plants is usually easier to disinfest
than that of field-grown plants. If ovules are to be cultured, a harsh surface sterilization procedure
should be applied to ovaries. It should not be necessary to disinfest the ovules, since they are
presumed to have been removed from a sterile environment inside the ovary. Commonly used
sterilizing agents and disinfestation times are presented in Chapter 3 of this book.
Techniques used for the excision of ovules depend on the arrangement of ovules within the
ovary. Care must be taken not to let ovules dry out during excision. A solid medium is typically
used for gynogenesis experiments; choice of culture vessel depends on size of explant. Disposable
petri dishes work well for culturing ovules of small-seeded polyovulate species, whereas test tubes
may be preferable for large ovaries.