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Insertional mutagenesis, whether by transposable elements or T-DNAs fromAgrobacterium tumefaciens, provides a powerful experimental strategy to investigate the genetic basis of plant growth, metabolism and development. The linkage of an insertion element with a mutant phenotype of interest greatly facilitates the isolation of the wild-type gene. A further refinement of this strategy is the incorporation of promoter traps or enhancer traps into the insertion elements. These can act as functional tags of regulatory elements associated with genes in the host genome, potentially can improve further the efficiency of screening for target mutant phenotypes, and may provide valuable markers of specific cell types for developmental analysis. We discuss the use of these techniques to study the molecular genetics of plant development.

Transgenic plants of tobacco and Arabidopsis that produce genetically marked pollen, expressing the reporter gene uidA (gusA), were generated to determine whether pollen proteins can be expressed and stable in honey, a potential route by which foreign proteins might enter the wider environment. Hydrated tobacco pollen was found to lose viability rapidly in honey, while pollen in the natural dehydrated form remained viable for at least several days and in some cases several weeks, as determined by FDA staining activity and germinability. Dehydrated pollen was found to be capable of transient foreign gene expression, following microprojectile bombardment, after incubation in honey for at least 120 h. PCR amplification of transgene sequences in pollen of transgenic plants revealed that pollen DNA can remain relatively intact after 7 weeks in honey. GUS enzyme activity analysis and SDS-PAGE of pollen proteins revealed that foreign and native pollen proteins are stable in pollen incubated in honey for at least 6 weeks. We conclude that pollen may represent an ecologically important vector for transgenic protein products.

As part of a gene tagging strategy to study the developmental regulation of patterns of plant gene expression, a promoterless uidA (gusA) gene, encoding the beta-glucuronidase (GUS) reporter, was introduced into populations of tobacco, Arabidopsis and potato by Agrobacterium-mediated gene transfer. The objective was to generate random functional fusions following integration of the gusA gene downstream of native gene promoters. We describe here a detailed analysis of levels and patterns of gusA activation in diverse organs and cell types in those populations. gusA activation occurred at high frequency in all three species, and unique patterns of fusion gene expression were found in each transgenic line. The frequency of gusA activation was differentially biased in different organs in the three species. Fusion gene activity was identified in a wide range of cell types in all organs studied, and expression patterns were stably transmissible to the T2 and T3 progeny. Developmentally-regulated and environmentally-inducible expression of gusA is described for one transgenic line. Phenotypic variants were detected in the transgenic population. These results demonstrate the potential of T-DNA insertion as a means of creating functional tags of genes expressed in a wide spectrum of cell types, and the value of the approach as a complement to standard T-DNA insertional mutagenesis and transposon tagging for developmental studies is discussed.