The maize transposable element Ac exhibits variable activity in transgenic tomato.

The identification and cloning of genes in tomato will be facilitated if transposable elements suitable for transposon tagging experiments in tomato can be identified and their behavior characterized. In a previous report (Tomato Genetics Cooperative 39:33) we described a transposable element/reporter gene combination based on the Activator (Ac) transposable element of maize and the beta-glucuronidase (GUS) reporter gene (Jefferson et al. 1987). The Ac element was inserted between the GUS coding region and the strong, (somewhat) constitutive 35S promoter from Cauliflower Mosaic Virus (Finnegan et al. 1989). The GUS gene was not expressed while the Ac element was in place; however excision of Ac resulted in the appearance of sectors of tissue expressing the GUS gene. Sectors as small as a single cell can be readily detected in cotyledons and petiole sections with the histochemical stain (X-gluc), which stains GUS-expressing cells blue, or by a quantitative fluorometric assay (Jefferson et al, 1987). The GUS gene thus serves as a very sensitive indicator of Ac excision.

We have studied the behavior of the Ac element in two transgenic tomato lines. In one line, designated pBT375-53, four plants were regenerated from a single transformed callus. The Ac transposition frequency, as measured by both the appearance of GUS expressing sectors and by fluorometric assay of GUS activity in excised leaf disks, varied dramatically between the four regenerants. In one plant, pBT375-53B, activity levels were very high. Most, but not all, of the vascular bundle in petiole sections of this plant stained blue with X-gluc (GUS activity driven by the 35S promoter is expressed preferentially in the vascular bundle region), while in pBT375-53C and D sectors were small and very infrequent (less than 0.1 per section). The levels of GUS activity in the leaves of these plants were determined using the fluorometric GUS assay and are shown in Figure 1A. (pBT375-53A did not exhibit detectable sectoring with the X-gluc stain, and was not assayed). Transposition was also very infrequent in the other transgenic tomato line studied, pBT375-54 (Figure 1B).

Southern analysis of the pBT375-53 and pBT375-54 plants using Ac DNA as probe showed that each plant carried a single copy of the Ac-GUS gene. pBT375-53A and C had identical patterns; pBT375-53B and pBT375-53D varied in that two bands present in A and C were missing and D had an extra band not found in the others. The nature of these variable bands and their relationship, if any, to the observed Ac activity levels has not been determined.

The effect of germline transmission on the activity of each of these alleles was determined by comparing activity levels in parent lines and progeny obtained by selfing. A general increase in the level of GUS activity (and hence Ac activity) was observed in the progeny of the pBT375-54 transformant (Figure 1B), whereas the levels of GUS activity in the pBT375-53D progeny were the same as the parent plant (not shown). We are currently investigating the possibility that changes in the level of DNA methylation can occur between generations and either stimulate or repress the level of Ac activity.