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Thomas R. Warne and Leslie G. Hickok. All rights reserved. |
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C-Fern
Web Manual - Sporophytes
Return to Table of Contents Cloning
of Sporophytes
Greenhouse Cloning
Procedure
1. 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. 2. Transplant developing sporophytes to 2x2" cell well tray or equivalent filled with ProMix®. Initate and maintain weekly fertilization. Perform subsequent transplants when roots begin to emerge form bottom of container. (see Sporophyte Culture - Transplantation for potting sequence) 3. Transplant developing sporophytes to 4" diameter pot or equivalent filled with ProMix® 4. Transplant developing sporophytes to 6" diameter pot or equivalent filled with ProMix®
Axenic Cloning Procedure
Materials:
Procedures:
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 trimmed into the shape
of a small triangle. In addition, leaf tissue from the bud should 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 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 with about 25 ml of sterile distilled water.
CULTURE BUDS Wash buds into a petri dish using sterile distilled
water. Plant buds to nutrient agar with forceps. Push the root end of
the bud slightly into the agar to establish good contact. If small buds
are cloned be sure that triangle of mother sporophyte tissue is parallel
to agar surface. Once young sporophytes have a well developed root system
and 10 leaves, they can be transplanted to the greenhouse.
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