448 LEVINE: STUDIES ON PLANT CANCERS—I 
It is well known that when Bryophyllum calycinum leaves are 
detached from the mother plant and are put on moist soil the 
marginal notches of the leaves, at which totipotent cells are found, 
develop into leafy shoots and eventually form new plants. For 
this reason, leaves and stems of B. calycinum were used to study 
the effect of the bacterium on the leafy shoot formation. 
METHOD AND MATERIAL 
The leaves of B. calycinum were detached from the plant and 
were placed on moist soil in pots in the greenhouse. The marginal 
notches of one side of the leaf, right or left, upper or lower, were 
inoculated, by pricking the tissue five to ten times with a deli- 
cate needle containing a culture of Bacterium tumefaciens from 
five to forty days old. The uninoculated notches of the opposite 
side of the leaf, served as controls, after they had been pricked 
with a sterile needle. Entire leaves with each notch pricked 
five to ten times with a sterile needle were also used as controls. 
Leaves in all stages of development were used. The veins of 
leaves and growing regions of stems of the Bryophyllum were also 
inoculated with Bacterium tumefaciens. In all over a thousand 
inoculations were made. It may be stated that thin young leaves 
did not lend themselves to these experiments, because they dried 
out too rapidly. It was likewise found that normal embryos 
develop better in the greenhouse than in the open. 
As a rule, two days after inoculation with Bacterium tume- 
faciens both the infected and control notches showed necrotic 
areas in those regions. It appeared, however, that the wounds 
infected showed greater areas of dead tissue, which subsequently 
caused deeper indentations at the margin of the leaf. This was 
also observed by Levin and Levine (3) for a number of plants, 
The uninfected or control notches recovered readily and although 
slight scars were formed, the bud Anlagen in the notches developed 
into normal embryos forty to seventy days after injury. Inocula- 
tions were made: (1) into the notches of Bryophyllum calycinum 
leaves; (2) into the leaf in the vicinity of the notches; (3) into the 
midveins of the leaf; and (4) into the growing regions of the 
young stems of this plant. 
