POLYEMBRYONY IN CERTAIN FERNS 239 



CULTURE METHODS IN THE PRODUCTION OF POLY- 

 EMBRYONY IN CERTAIN FERNS (POLYPODIACEAE). 



David M. Mottier, Indiana University. 



To Secure the Stock Culture. — In both stock and transplanted cul- 

 tures the i^rothallia are grown upon rich soil, preferably woods earth. 

 Unglazed earthen ware vessels, flower pot saucers, are used as con- 

 tainers. 



The thoroughly moistened (not wet) soil is passed through a sieve 

 with one-fourth inch mesh. First a thin layer of coarser earth is placed 

 in the saucer, which is then filled three-fourths full with the sifted 

 earth. Upon this is evenly spread a thin layer of earth which has been 

 passed through a finer sieve such as may be made from fly screen wire. 

 The surface is then gently firmed with the hand, so that an even and 

 sufficiently compact surface is provided. A moat is now made around 

 the edge by pressing the soil back from the side of the saucer with a 

 stick or finger. 



The vessel with the soil thus pre^iared is sterilized in a steam 

 sterilizer two hours on each of two successive or alternate days. If the 

 soil be too wet before sterilization, the result will be a mud ]}ie instead 

 of a suitable substratum. 



The soil is now evenly coated with the spores, and the culture cov- 

 ered with a bell.jar. It is well to prop up the belljar on one side to a 

 height of about one-fourth inch to insure ventilation and to facilitate 

 evaporation should the culture be made too wet in watering. The soil 

 is kept moist by sub-irrigation, the water being gently poured into the 

 moat. This method is preferred to that of allowing the saucer to stand 

 temporarily in shallow water. Cultures should never be watered by 

 spraying, and care should be taken not to keep the soil wet. 



Good difl'used light provides the best illumination. However, direct 

 sunlight for an hour in early morning is beneficial. 



Transplanting and Cultivation. — The soil is prepared as for the stock 

 culture, and the conditions are to remain the same. 



When the prothallia are from one to three millimeters in breadth, 

 or larger, they are lifted from the stock culture with needle or forceps, 

 along with sufficient earth to preserve the majority of the rhizoids, and 

 set out in the new dish about an inch apart. Care should be taken to 

 remove all smaller adhering i^rothallia. The soil should be quite moist 

 at the time of transplanting so that the earth about the rhizoids may 

 be pushed easily into it. The under free surface of the prothallium 

 should not come into direct contact with the soil. If after a time weed- 

 prothallia, mosses or other plants appear, in the cultures, these 

 should be removed. If the surface of the soil should become caked or 

 grown over with blue-green or other algae, as may happen in long- 

 standing cultures, the earth -should be allowed to dry somewhat and the 

 caked or matted surface removed and fresh sterilized soil added. Some- 

 times it is desirable to transplant the prothallia to newly prepared cul- 

 ture dishes. They should be lifted with enough earth to preserve the 



"Pioc. Ind. Acad. Sci., vol. 33, 1923 (1924)." 



