Dr. Mary Clutter 
Page 2 
The fragments of the Ti plasmid to be tested must first be 
recloned in a vehicle that can replicate in Agrobacterium. 
The easiest choice would be to use RP4, which has a single 
Hind III site in the gene conferring resistance to kanamycin. 
The clones would be isolated by transforming into E. coli . 
and then transferred to Agrobacterium by conjugation. Con- 
jugation of RP4 from E. coli to Agrobacterium occurs with 
high frequency (lO - ''). The Agrobacterium strain would then 
be tested for oncogenicity on carrot discs and sunflower 
stemlets in vitro , and on tobacco and sunflower and kalanchoe 
plants in a high containment greenhouse. 
If the small fragments of T-DNA presently available do not 
confer oncogenicity under the above conditions, we propose 
to clone larger sectors of the Ti plasmid by using partial 
digests. Using the colony-screening procedure in combination 
with Grunstein's colony hybridization technique, v/e can pick 
clones carrying segments of the Ti plasmid of any desired 
molecular weight that include the T-DNA. These recombinant 
DNAs would then similarly be moved to Agrobacterium for 
oncogenicity testing. 
It seems virtually certain that a shrunken Ti plasmid can be 
created in this manner. The oncogenicity testing will reveal 
whether such a plasmid is more effective or less effective, 
whether its host range is altered, and whether its susceptibility 
to biological control by A. tumefaciens strain K84 is altered. 
In addition, the experiment would demonstrate the boundaries 
of the essential part of the Ti plasmid relating to tumor 
induction. This information would be useful to our studies 
of the mechanism of the process. 
Experiment 2. Genetic Engineering with a shrunken Ti plasmid . 
Because the T-DNA is maintained in plant cells, it can in 
principle be used as a vector to carry in nutritionally de- 
sirable traits. The system of choice for this experiment 
is Ti-T37, a plasmid that induces teratomas in tobacco. Dr. 
Armin Braun has demonstrated that cloned teratoma tissue 
can be induced to revert to a healthy plant of normal appear- 
ance. The plant nevertheless maintains copies of the foreign 
DNA, and expresses one tumorous trait, production of the 
amino acid derivative nopaline. Such a teratoma-plant would 
be a suitable test system for expression of foreign nutritional 
genes in the plant. Initial experiments could be done in to- 
bacco, for which the methods are already known. Potato, a 
closely related solanaceous plant, should be amenable to the 
same kinds of manipulations. 
The first step in the experiment is to create, by the methods 
outlined in Experiment 1 above, a shrunken Ti-T37 derivative. 
[Appendix A — 304] 
