T-DNA TAGGING OF GENES INFLUENCING POLYAMINE METABOLISM - ISOLATION OF MUTANT PLANT LINES AND RESCUE OF DNA PROMOTING GROWTH IN THE PRESENCE OF A POLYAMINE BIOSYNTHETIC INHIBITOR

Activation T-DNA tagging has been used to generate cell lines characterized by their ability to grow and form callus in vitro under selective concentrations of the polyamine biosynthetic inhibitor methylglyoxal-bis(guanylhydrazone) (MGBG). These have been regenerated into plants of which the majority have been shown by Southern analysis to contain single T-DNA inserts. In the mutant lines the T-DNA is contained on differing restriction fragments of genomic DNA, suggesting that they represent different insertion events. Regenerated plants display characteristic phenotypic changes when compared with untransformed plants including leaf and floral malformation as well as parthenocarpy. In two of the plant lines studied in detail, the activity of S-adenosylmethionine decarboxylase, the target of MGBG, was increased in callus tissue and in one line this is coupled with elevated levels of putrescine and spermidine. Plant genomic DNA flanking the T-DNA insert has been rescued from one mutant plant line and Northern blot analysis indicates that transcripts corresponding to this DNA accumulate to higher levels in the T-DNA tagged line compared with untransformed tobacco. Rescued genomic DNA flanking the T-DNA, once re-introduced into untransformed tobacco protoplasts by PEG-mediated uptake, confers the ability to grow under selective levels of MGBG. This confirms that sequences responsible for the selected phenotype have been isolated. It is anticipated that the analysis of such mutants and the further characterization of the tagged genomic sequences may shed light on the action of polyamines in plant growth and development.