Rayner.—Obligate Symbiosis in Calluna vulgaris. 105 
and to demonstrate infection of the seeds by this Fungus while still enclosed 
in the fruit (p. 98). 
3 . To investigate the possibility of replacing the stimulus resulting 
from fungal infection of the seedling by small amounts of various organic 
substances supplied to sterile seedlings (grown under aseptic conditions), in 
addition to the requisite mineral salts. 
3. To determine the source of infection of the ovary tissues (p. 98). 
4. To isolate the mycorrhizal Fungus and establish its identity by 
successful inoculation of sterile seedlings. 
5. To investigate the life-history of the Fungus when grown in pure 
culture as an independent organism outside the plant. 
1. Dependence of root formation on infection. The conclusions already 
reached with regard to inability of sterile seedlings to form a root-system 
have been confirmed. 
If due precautions are observed, seeds can be sterilized without injury 
to the embryo by washing in 1 per cent, corrosive sublimate solution. Com¬ 
plete sterilization is not easy to effect, and the margin of safety is a narrow 
one, owing no doubt to the delicacy of the testa and the fact that infection 
of the cells of the seed-coat is more extensive and deep-seated than is the 
case with air-infected seeds. 
If seeds were left a few seconds too long in the sterilizing solution, 
the embryo was killed outright, germination was delayed, or the seedlings 
which germinated showed complete chlorosis. 
After sterilization by this method and repeated washings in distilled 
water, seeds were sown by means of a sterile pipette on agar plates, and 
kept under dust-free conditions in a closed germinator. 
The agar medium on which they were sown contained dextrose and 
peptone in addition to mineral salts, in order to facilitate the growth of 
micro-organisms if present. By this means a number of plates of seed¬ 
lings were obtained which remained entirely free from infection by micro¬ 
organisms. 
As an additional test of sterility, seedlings were transferred singly 
to tubes of glucose broth and other media, and kept under observation for 
three weeks. All the tubes remained sterile. 
The evidence is conclusive therefore that the embryo and endosperm of 
Calluna seeds are free from infection, and that, by adequate sterilization, 
seedlings can be obtained free from infection by micro-organisms. Sterile 
seedlings obtained in this way were planted out in sand and in agar, 
in a series of cultures extending over several years. 
The tubes originally used for sterile sand cultures are described 
and figured elsewhere ( 3 ). 
In order to eliminate imperfect aeration as a possible factor in the non¬ 
production of roots, a special apparatus was designed for use in subsequent 
