Dr. Peter Faletra: After placing second in the world in the Team Projects at the 47th International Science and Engineering Fair and spending eight days in the sunny parts of Arizona, all expenses paid by NYNEX, my students were flying pretty high. They were soon receiving e-mail from around the country (and occasionally from overseas) inquiring about their tissue culture research on lady's slippers. We've had quite a few inquiries on what type of medium they used and many orchid lovers were excited to learn if the students had micropropagated any of the species. The students have recently set up an impressive interactive web page on their experiment where they referred many of these inquiries. Others, who wanted more detailed information, were told that we would soon be publishing the results in a few different articles. I'll leave the rest of the story to my students, April, Tyler, and Katie:

Two years ago we began converting a few small rooms in our public high school into a small tissue culture laboratory. Doctor Faletra had quite a bit of tissue culture equipment from when he was an animal tissue culture biotech researcher. All we had to learn was how to use the equipment and adapt it to plant culture. To test our procedures we performed a pilot study with the African Violet, which is easily cultured. After six months we had had a great deal of success but needed a long-term goal for the lab. We found some information on the culturing of endangered species, prompting the decision to try and grow locally endangered plants.

The Natural Heritage Inventory publishes a list of endangered plants which includes a number of lady's slippers. We knew we could find the pink slipper because it grows in abundance in our area of the White Mountains. The pink slipper is protected by law from collection, but is not technically endangered. Listed as critically endangered in most New England states were the species reginae calceolus and arietenum. We set out to try and find a source of these for our restoration plan. At the time, we had no knowledge of their historic difficulty in propagation. We didn't even know they were orchids! Although C. reginae is not the only critically endangered orchid in New Hampshire, we decided to try culturing it since it had the only seed source we could find. Dr. Faletra's wife, Elaine, found them in a fen on the border of New Hampshire and Vermont. With moving water and a calcarious substrata, the fens of the border area had close to a neutral pH.

Speaking with local naturalists and wildflower lovers, we learned that these spectacular flowers were almost totally gone from the region, making this species an excellent flagship for a restoration project. Dr. Faletra had received a Tapestry grant from the Toyota Company for a pilot attempt at the restoration of locally endangered plants, which would use schools as a work force. The plan was to raise thousands of seedlings in culture, create a kit containing the seedlings, and deliver them to New Hampshire schools along with detailed instructions on how to use the kits. The schools, which ranged from elementary to high school, would eventually plant the grown seedlings in a suitable local habitat and keep track of their survival.

After about three months we moved the cultures into new medium. This caused a growth spurt in rhizome growth which is desirable for future seedling survival. We placed a number of cultures in light and monitored their development carefully. After placement in the light, the cultures greened up and produced a pair (or more) of well-formed leaves. After transplantation to soil, these seedlings grew for a few months and then the leaves died off, leaving healthy rhizomes. They could then be placed into a refrigerator for three or four months or if left in a garden, heavily mulched.

Another researcher's approach is somewhat different: he puts early stage seedlings from culture directly into the refrigerator for three to four months. We are not yet sure which approach works best, but cold dormancy certainly produces good results. This seems logical because dormancy is often required for tissue-cultured plants to be in step with the seasons for eventual out-planting.

Preliminary studies make us optimistic that we can micropropagate. We consider a specimen successful when it generates both shoots and rhizomes on each of the two halves. Five micropropagation experiments have been done with about 25 specimens per experiment. Our experiments indicate seedlings cut longitudinally (rather than transversely) produce positive results. It seems as though tissue committed to the growth of either shoots or rhizomes was separated when transverse cuts were made, leading to one half of the specimen growing shoots and rhizomes and the other half growing only rhizomes. It was apparent that cutting longitudinally increased chances of getting shoot and root tissue on each half of the seedling. We have observed a small green spot about .1 mm in diameter near the apex of the seedling. This meristematic region developed into a rhizome in all the seedlings we monitored.

It seems that the tissues in early stages of development in C. regina are committed to becoming certain tissues. The challenge now is to pinpoint these tissues and manipulate the hormone levels to induce consistent generation of both shoots and rhizomes on each half of the specimen. As is known to generally be the case in development, especially in animals, the more mature the tissue, the more likely it is to be committed to develop into a certain tissue. What seems to be the advantage in plants is that with more simple manipulations of hormones, almost any plant tissue can be convinced to grow into a healthy, normal, well-adjusted adult.

Since the start of the program we have delivered kits, free of charge, to over 55 schools and gardening groups. We intend to continue our research with a variety of orchid species to optimize the efficiency for restoration attempts so everyone can enjoy these beautiful flowers both in the wild and in their gardens. This research was funded in part by grants from the Toyota Company and the Lincoln-Woodstock Rotary. A special thanks to NYNEX for their continued support of science education. u