Tab A - Page 16 
the surfaces of, above-ground plant parts. It has also been shown that plants 
grown aseptically can tolerate temperatures of several degrees Celsius below 
zero without apparent damage and they are rendered sensitive to such 
temperatures by spraying with suspensions of INA + bacteria prior to the low 
temperature exposure. (I) 
Frost damage to plants can be decreased by reducing the natural population 
of INA' 1 ’ bacteria present on the plant surfaces. Especially suited for this 
purpose are IMA" mutants of the naturally occurring INA + strains. These INA" 
mutants, being basically adapted for epiphytic growth and survival, would 
presumably prevent their wild-type counterparts from occupying the same physical 
spaces on the plant surface and from using the same nutrients. (I) 
INA" deletion mutants of selected strains of P_. syrinqae pv. syrinoae and 
_E. herbicola will be constructed by Drs. Lindow and Pancpoulos in their 
laboratories in Rooms 210, 216, 225 and 227 Hilgard Hall on the Berkeley 
campus. A biosafety cabinet is located in Room 210. INA" deletions will be 
obtained by removing one or more ONA fragments covering all or part of the INA 
region. Recombinant plasmid ONA will be digested with restriction 
endonuclease(s) which cleave the cloned inserts at suitable sites and the DNAs 
will be religated and transformed into £. col 1 . INA" transformants will be 
verified as having specific fragment deletions by plasmid reisolation, 
restriction endonuclease re-cutting and/or blot hybridization. 
To facilitate the monitoring of the mutant IMA bacteria, various types of 
"flagged" deletions, having specific ONA sequences or segments, are proposed to 
be constructed. Elements or segments encoding for resistance to antibiotics 
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