Statement 
On the in vivo transfer of the Ti-plasmid from A . tumef aciens 
to E.coli 
It was observed in the laboratory for Genetics, State Uni- 
versity, Gent, that the Ti-plasmids from A. tumef aciens can be natural- 
ly transferred to E . coli . 
Two different mechanisms can promote these DNA transfers : 
1°) Mobilisation by the wide host-range P-type plasmid RP4 . 
It is a well documented fact that RP4 is very readily transferred 
and replicates well in various Agrobacterium strains. It was 
furthermore observed that co-integrated plasmids consisting of 
complete RP4 DNA and complete Ti-DNA parts, are formed spontaneously 
at frequencies of 10~5 to 10“3 (i.e. one out of 10 ^ to 10^ cells 
harbouring both plasmid types in fact contain the Ti::RP4 co- 
integrated plasmid) . 
Some of these co-integrated plasmids still convey oncogenicity when 
present in an Agrobacterium host. These plasmids appear to have 
the wide host-range of the RP4 plasmid since they have been shown 
to transfer at a high frequency (>^10”2) to E . coli K12 r“m hosts. 
In E . coli the co-integrated Ti::RP4 plasmid replicates well but can 
be shown to dissociate frequently (1-10%) into RP4 and Ti plasmids 
where upon the Ti-plasmid appears to be lost. 
The oncogenicity of E . coli (Ti::RP4) strains has been extensively 
tested on pea-seedling-sunflower hypocotyls and tobacco leaves and 
stems. At least ten different cultures were tested on each of these 
plants. In no case was there any evidence of crown-gall formation 
or any other form of pathogenicity. The tested E . coli (Ti::RP4) 
strains were subsequently shown to still harbour the complete Ti:: 
RP4 co-integrate by crossing them with plasmid-free Agrobacterium 
C58-C1. The Agrobacterium transcon jugants were shown to be fully 
oncogenic . 
2°) Transfer constitutive mutants of the Ti-plasmid have been isolated 
and the Tnl transposon (conferring resistance to Ampicilline) has 
been introduced in this Ti tra c plasmid yielding Titra c ::Tnl plasmids. 
Some of these plasmids have been shown to be normally oncogenic in 
Agrobacterium . When crossed with E . coli Kl 2 r“m“ it can be shown 
that conjugation takes place since the Tnl marker is transferred 
from Agrobacterium to E . coli with a frequency of 10“^ to 10”^. 
However , the transconjugant cells, behave as "abortive transconju- 
gants" since the ampicilline resistant E.coli colonies observed 
have been shown to contain only about 5-10 Ampicilline resistant 
cells. This indicates that the Ti-plasmid replicates very, very 
poorly in E . coli . Oncogenicity tests on pea-seedlings, sunflower 
hypocotyls and potato discs were invariably negative. 
[Appendix A — 155] 
