Simple setup for growing sporophytes without a greenhouse – observation of sporophyte development and spore production
A plastic soda bottle makes a Mini-terrarium for the culture of C-Fern sporophytes
Although most C-Fern investigations concentrate on gametophyte development and sexual reproduction through the early sporophyte stage, (14-21 days following sowing; DFS), extended observations of sporophyte development, including the production of the next generation of haploid spores, are possible.
Under appropriate culture conditions, sporophytes with mature spores can be grown in about 90 DFS. For the large-scale culture of sporophytes, a warm and humid greenhouse is needed. However, the culture of individual sporophytes can be accomplished in a Mini-terrarium or other suitable vessel.
The following instructions are provided for growing individual sporophytes in 8 ounce to 2-L clear plastic beverage bottles, e.g. a soda bottle.
1. One to 3 weeks after water has been added to the gametophyte culture to facilitate fertilization, young sporophytes should be transferred individually to separate 60×15-mm Petri dishes containing Basic C-Fern Nutrient Medium. The transfer of sporophyte can be made with a clean toothpick or probe so that the root end of the sporophyte is imbedded in the agar and the first leaf is above the agar. Use care to avoid contamination.
2. Place these isolated sporophyte cultures in the Culture Dome or Growth Pod under the same lighting and temperature conditions that were used for gametophyte cultures. Sporophytes should remain under these conditions or 2-4 weeks. Usually, it is not necessary to add water, but if the culture begins to dry out, use a pipet to add sterile distilled water as needed.
3. When isolated sporophytes have produced several roots and leaves, they can be transferred to a bottle terrarium. If the sporophyte is small enough, both it and the soil mix (see step 4) can be pushed through the mouth of the bottle and planted and adjusted with a long narrow stick. Otherwise, take a circular cut 2 inches below the neck. Cut only 3/4 of the distance around the bottle so that there is a ‘hinge’ of plastic left. The top can now be lifted to provide a larger opening for planting.
4. Place 2 cups of pre-moistened (damp, but not wet) ProMix® Potting Soil in the bottom of the bottle. Add 4-6 medium-sized beads of Osmocote® 4-14-14 Fertilizer and mix them into the top 2-inch of potting mix. Take a shallow concave cavity (2″ deep — 1″ diameter) in the center of the mix.
5. Carefully remove a sporophyte from its petri dish by lifting the agar out of the dish, roots, and all. A flat knife or thin spatula works well for this. Remove as much agar as possible without damaging the roots and place the sporophyte (roots down!) in the cavity made in step 4. Gently cover the roots with potting mix and lightly press down on the surface around the sporophyte to give it a firm footing in the potting mix.
6. Add enough distilled water to thoroughly moisten the potting mix and help displace any air spaces between the roots and soil mix. The mix should be moist but not soaked, although excess water will not typically harm C-Fern. Subsequent watering can usually be made as needed, usually on a weekly basis. Use distilled or bottled drinking water, if available.
Note: Constant moist conditions will result in mostly vegetative growth. If fertile leaves with spores are desired, a moderate reduction in watering, accompanied by an increased level of water stress will typically assist with the developmental shift from vegetative to reproductive leaf production. This can be accomplished by allowing the soil to dry substantially between waterings. Place the terrarium under the same lighting conditions used for the gametophyte cultures. Warm temperatures and constant 24-hour illumination work best; cold window sills are not recommended. Cooler temperatures and less light will slow development but still allow growth. Other than watering, as described in step 6, little care is needed.
