Sporophyte Culture – Cloning

Propagate marginal fronds buds from greenhouse plants by either direct cloning to a soil mix or by axenic cloning

C-Fern produces numerous buds on the margins of fronds. Each of these buds can be readily cultured to maintain specific strains for spore production or for use as experimental tissue.

Greenhouse Cloning


  • C-Fern buds
  • Greenhouse mist bench
  • Seed starter propagation tray
  • ProMix®
  • 2×2″ cell well propagation tray
  • 4″ diameter pot
  • 6″ diameter pot 


HARVEST BUDS TO SEED STARTER TRAY Larger buds (1- 2 cm) that may or may not have roots can be pinched directly from the mother plant using fingers or forceps. Transfer pinched buds to appropriate containers filled with ProMix® such as wells in a seed starter tray. Maintain transplanted buds on a greenhouse mist bench for about 30 days until buds produce sufficient roots that grow out the bottom of the seed starter wells. Fertilize at 3 weeks if growth is slow.

TRANSPLANT TO CELL WELL TRAY Transplant developing sporophytes to 2×2″ cell well tray or equivalent filled with ProMix®. Initiate and maintain weekly fertilization. Perform subsequent transplants when roots begin to emerge from the bottom of the container. (see Sporophyte Culture – Transplantation for the potting sequence of greenhouse cultured sporophytes)

TRANSPLANT TO 4″ POT Transplant developing sporophytes to 4″ diameter pot or equivalent filled with ProMix®.

TRANSPLANT TO 6″ POT Transplant developing sporophytes to 6″ diameter pot or equivalent filled with ProMix®.

Axenic Cloning

Axenic or sterile cultures of sporophyte buds can be obtained from a simple surface sterilization procedure.


  • Sterile 50 mL plastic centrifuge tubes
  • Sterile Pasteur pipets
  • Sterile distilled water
  • Sterile pipet bulbs
  • Sterile forceps
  • Clorox or equivalent 5.25% sodium hypochlorite
  • Waste container
  • Styrofoam cup with plastic sandwich bag cover
  • Culture dishes with nutrient agar


COLLECT AND PREPARE BUDS Collect smaller marginal frond buds from greenhouse plants. Buds should be no larger than about 1 cm and most of the leaf tissue from the mother plant should be trimmed into the shape of a small triangle. In addition, leaf tissue from the bud should be removed. As buds are collected, float them in water in a Styrofoam cup covered with a plastic sandwich bag.

STERILIZE AND RINSE BUDS Prepare sterilization solution (1 part Clorox (5.25% sodium hypochlorite) to 4 parts distilled water). Carefully transfer buds to the bottom of a sterile 50 ml centrifuge tube, i.e. do not touch sides of tubes. Do not sterilize more than about 50 buds at one time. With a sterile Pasteur pipet add sufficient sterilizing solution to completely cover buds. Shake tube for 1 minute to thoroughly wet buds with the solution. Carefully decant sterilizing solution to waste container; use the sterile cap of the centrifuge tube to retain buds. Rinse and decant buds three times, each time with about 25 ml of sterile distilled water.

CULTURE BUDS Wash buds into a petri dish using sterile distilled water. Transfer buds (plant) to nutrient agar with sterile forceps. Push the root end of the bud slightly into the agar to establish good contact. If small buds are cloned be sure that the triangle of mother sporophyte tissue is parallel to the agar surface. Once young sporophytes have a well-developed root system and 10 leaves, they can be transplanted to the greenhouse.