In the Lab
We study the full life cycle of showy lady’s slipper at the STEM Labs of New Hampshire Academy of Science and its associated educational organizations. It starts with understanding how to best germinate seed to produce seedlings efficiently; how to vernalize seedlings, which is to mimic their dormancy in the winter; and to how to get the best survival when transplanting to soil, and how to make sure seedlings mature to a flowering state in the wild or in sanctuaries.
Seedpod & Seed
We collect seedpods in mid-September, just as the pods are about to turn brown and before they split open (dehisce). We take the seedpods back to our lab. We allow the pods to dry out for a week or so, and then scrape out the seeds.
Embryo of a seed protected by its inner and outer seed coats
Outer seed coat taken at 70x with SEM
Inner seed coat taken at 400x with SEM
Lady’s slipper seeds have an inner and outer seed coat. In the wild, a mycorrhizal fungus breaks down the inner and outer seed coats and provides the seed with nutrients. Because we grow the seeds in a sterile medium without a fungus present, we pretreat the seeds with bleach to weaken the seed coats so that the nutrients in the medium can reach the embryo. The bleach also sterilizes the outer surfaces of the seeds.
After the seeds have been sterilized, we inoculate the seeds onto a gelatin medium in a sterile hood.
Weakening seed coats in a solution of bleach
Step 1: Sterilize the Inoculation Loop
Step 2: Scrape up Some Sterile Seeds
Step 3: Open the Culture Tube
Step 4: Insert the Seeds
Step 5: Close and Seal the Tube
We monitor the germination and growth of the seeds every week. To help track the growth of the seeds, we use the 5-stage system published by Dr. Faletra and his students in Axenic Seed Culture and Micropropagation of Cypripedium reginae in 1998.
NHAS students monitor the germination and growth of the seeds every week
Stage-1: untreated seed
Stage-2: seed that has been treated with bleach
Stage-3: seed has begun to germinate into a seedling
Stage-4: seedling that has broken through the seed coat
Stage-5: seedling that has developed roots and/or shoots
Seedling
Once a seed has germinated, it is considered a seedling. Although all the seeds in a culture tube are inoculated at the same time, they grow at different rates. A seedling has a rhizome which is an underground stem located where the root meets the shoot. The rhizome stores starch to provide energy to put up the next year’s new shoots, which will grow new leaves. With each successive year, the plant will produce more shoots from the rhizome. The seedling also has a coleoptile or shoot-covering. The coleoptile is a protective sheath found in grasses and orchids that surrounds the developing shoot.
Showy lady's slippers, as with all orchids of New England, must go through a cold dormancy (winter) to put up new healthy growth in the following spring. This cold dormancy process is called vernalization. When the seedlings develop prominent roots and shoots, they must be vernalized. We vernalize seedlings by storing them in a 5º C refrigerator to mimic their natural cold dormancy.
We tested two common methods of vernalization: 1) storing bare-root seedlings in containers with small amounts of water, and 2) storing seedlings layered in a mixture of peat moss and compost. Less than 25% of the seedlings survived when layered in peat moss and compost. The bare-root soilless method increased survival rate to about 60%. We discovered that vernalizing seedlings in their culture tubes is the most efficient and successful method. We simply move the sterile cultures from their storage at room temperature to the refrigerator without transferring the seedlings to any other medium. After three months in the fridge, all of the seedlings were healthy. This method yielded a 100% success rate.
Storing bare-root seedlings in containers with small amounts of water.
Storing seedlings layered in a mixture of peat moss and compost
Storing seedlings in their original sterile culture
Planting & Hydroponic Fen
We often suffer over 50% loss of seedlings upon transplantation to soil (a homemade fen). Sprouting of seedling in soil takes about 3-5 weeks with well-formed leaves not appearing until about 1-2 months. We tried creating an artificial indoor fen environment with sphagnum moss. All of the seedlings died. We then tried transplanting into sphagnum moss and fine pebbles with about 25% survival. We have recently experimented with initially planting seedlings in a highly controlled laboratory setting in a soilless medium (hydroponic) to improve both the percent of seedlings that survive and the speed with which they sprout. The image here shows a very encouraging experiment where we observed sprouting of seedlings and well developed leaves in three weeks. The seedlings in this soilless medium grew much faster and larger than in soil with almost 100% survival.
Sphagnum moss fen; all seedlings died
Sphagnum moss and fine pebbles fen; 25% seedling survival rate.
Soil-less aquaculture medium fen
in 3 weeks, seedlings rocketed to over 2.5 inches in height with100% survival
We intend on using this approach prior to moving seedling to soil in our homemade fens. We are refining our design for building a hydroponic fen.