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Selfing and Crossing Techniques

For self-fertilizations, individual sexually immature gametophytes should be transferred to culture dishes (isolated) and watered (about 0.1-0.5 mL) with sterile distilled water.

Cross-fertilizations or hybridizations between specific strains are easy to accomplish if procedures are followed carefully and care is taken with regard to gametophyte age and developmental stage. Cultures to be used as male and female stocks should at 10-14 DFS (under standard culture conditions). At this age, the male stock should contain well defined spatulate males with numerous antheridia and the female stock should contain cordate (female) gametophytes with two to four archegonia and zero to few antheridia. Careful attention to gametophytic development age ensures high spermatozoid number and activity for the male parent and one to two receptive archegonia for the female with a minimal chance of selfing. In addition, all manipulations should be carried out in a reasonably rapid and efficient manner.

Set up females for crosses first. (Removal of cultures from a warm culture room to a cooler area can result in premature release of sperm and establishment of archegonial receptivity). Transfer females to crossing dish, be sure to place them on the agar surface with their archegonia facing up. Select only those females that are relatively flat and possess two to four archegonia.

Once all females are ready, prepare sufficient male stock cultures. Usually one large dish (60x20 mm) of a male stock is sufficient for 30 or more crosses. Add sterile distilled water to cover gametophytes on the dish. Monitor sperm release using a dissecting microscope. Maximum sperm activity should be evident within five minutes. With a sterile Pasteur pipet, collect the sperm suspension and avoid picking up gametophytes and other debris. Place two to four drops of sperm suspension on each female to be crossed. Use a microscope to check all attempted crosses. Be sure that archegonia are submerged and if necessary, anchor the female under the sperm suspension (use a sterile needle or probe). Remove any males that were carried in with the sperm suspension. You also should be able to observe large numbers of sperm swarming around receptive archegonia.

At three to five days following the crossing attempts, check the females for successful fertilizations as evidenced by obvious swollen regions (embryos) at the base of the archegonia. Discard all females that do not carry embryos. If the gametophytes, techniques and conditions used for crosses are ideal, 90-95% of crossing attempts can be successful.

Maintain the cultures until the sporophytes are large enough to be transferred to the greenhouse. Crosses must be confirmed by segregation from the F1 sporophyte or by use of a marker stock.