SummaryNicotiana plurnbaginifolia haploid protoplasts were co-transformed with two plasmids, one with a NPT-II/Ds element and one with a gene encoding an amino-terminal truncated Ac transposese. It is shown that Ds can efficiently transpose from extrachromosomal DNA to IV. plurnbaginifolia chromosomes when the Ac transposese gene is present in trans. Ds has been shown to have transposed into the plant genome in a limited number of copies (1.9 copies per genome), for 21/32 trensgenic lines tested. The flanking sequences present in the original plasmid are missing in these 21 plants. In only two of 21 plants was pert of the transposese construct integrated. By segregation analysis of tranegenic progeny, Ds was shown to be present in the heterozygous state in 10 lines even though haploid protoplaste had been originally transformed. This observation could indicate that integration occurred after or during DNA replication that leads to protoplast diploidization.

A seedling lethal mutant of Nicotiana plumbaginifolia (sdl-1) was isolated by transposon tagging using a maize Dissociation (Ds) element. The insertion mutation was produced by direct co-transformation of protoplasts with two plasmids: one containing Ds and a second with an Ac transposase gene. sdl-1 seedlings exhibit several phenotypes: swollen organs, short hypocotyls in light and dark conditions, and enlarged and multinucleated cells, that altogether suggest cell growth defects. Mutant cells are able to proliferate under in vitro culture conditions. Genomic DNA sequences bordering the transposon were used to recover cDNA from the normal allele. Complementation of the mutant phenotype with the cDNA confirmed that the transposon had caused the mutation. The Ds element was inserted into the first exon of the open reading frame and the homozygous mutant lacked detectable transcript. Phenocopies of the mutant were obtained by an antisense approach. SDL-1 encodes a novel protein found in several plant genomes but apparently missingfrom animal and fungal genomes; the protein is highly conserved and has a potential plastid targeting motif.

We have recently shown that a plasmid-borne Dissociation (Ds) element can excise from extrachromosomal plasmid DNA and integrate into a plant genome in the presence of the Activator (Ac) transposase. Ds and Ac-carrying plasmids were used to co-transform Nicotiana plumbaginifolia protoplasts. Transgenic plants were regenerated and analyzed. Here we describe further characterization of the system and discuss its efficiency in terms of DNA transformation and transposon tagging.