Enhanced frequency of transposition of the maize transposable element Activator following excision from T-DNA in Petunia hybrida.

Many of the systems currently employed for heterologous transposon tagging in plants rely on an excision assay to monitor transposon activity. We have used the streptomycin phosphotransferase (SPT) reporter system to assay Ac activity in Petunia hybrida. In other species, such as tobacco or Arabidopsis, excision of Ac from the SPT gene in sporogenous tissue gives rise to streptomycin-resistant seedlings in the following generation. The frequency of fully streptomycin-resistant seedlings in petunia was low (0.4%) but molecular analysis of these indicated that the actual excision frequency may be as low as 0.05%. This indicates that the SPT assay is not a reliable selection criterion for germinal excision in petunia. Extensive molecular screening for reinsertion of Ac was consistent with a low primary transposition frequency (0%-0.6%). In contrast to these findings, the progeny of confirmed germinal transpositions for three independent transformants showed frequent transposition to new sites (9.5%-17.0%). This suggests a high frequency of secondary transposition compared with primary transposition from the T-DNA. Segregation analysis indicates that the high transposition activity is closely associated with transposed copies of Ac. No evidence was found for an altered methylation state for Ac following transposition. The implications of these results for heterologous transposon tagging in petunia are discussed in the context of the reliability of excision reporter systems in general.

Modification of flower color in florist's chrysanthemum: production of a white-flowering variety through molecular genetics.

Chimeric chalcone synthase (CHS) constructs were prepared in both anti-sense and sense orientations, and introduced into the chrysanthemum cultivar Moneymaker, along with a T-DNA vector lacking a CHS construct. For both the anti-sense and sense constructs, the majority of the plants produced pink flowers typical of Moneymaker itself. Of 133 sense and 83 anti-sense transgenic individuals 3 of each set produced fully white or very pale pink flowers. No white-flowering transgenic plants were obtained in control transformations. The white flowers were found to accumulate higher levels of chalcone synthase precursors and to have reduced levels of chalcone synthase message. A small-scale field trial was performed to evaluate the stability of the phenotype throughout a series of vegetative propagation steps and during plant growth. The white-flowering trait was maintained well through vegetative propagation; however, during growth of individual white-flowering plants, some pink color was found in some flowers. At one site 2% of the white-flowering plants produced a few pink flowers; at two other sites, as many as 10-12% of the plants produced pale pink flowers.

Tagging and Cloning of a Petunia Flower Color Gene with the Maize Transposable Element Activator.

We report here the use of the maize transposable element Activator (Ac) to isolate a dicot gene. Ac was introduced into petunia, where it transposed into Ph6, one of several genes that modify anthocyanin pigmentation in flowers by affecting the pH of the corolla. Like other Ac-mutable alleles, the new mutation is unstable and reverts to a functional form in somatic and germinal tissues. The mutant gene was cloned using Ac as a probe, demonstrating the feasibility of heterologous transposon tagging in higher plants. Confirmation that the cloned DNA fragment corresponded to the mutated gene was obtained from an analysis of revertants. In every case examined, reversion to the wild-type phenotype was correlated with restoration of a wild-type-sized DNA fragment. New transposed Acs were detected in many of the revertants. As in maize, the frequency of somatic and germinal excision of Ac from the mutable allele appears to be dependent on genetic background.