SUMMARY
The refined dating technique based on both pollen density and radio‐carbon dating proposed by Middeldorp (1982) was applied to five curves of a pollen‐ and macro‐remain diagram from a peat section from the Engbertsdijksveen (Vriezeveen, The Netherlands). Percentages curves of Alnus, Quercus, Corylus, alkaline soluble humic acids and coarse fraction of peat were then subjected to time series analysis by means of Periodic Regression (Briskin et al. 1980). In this way periodicities of approximately 1450, 1000, 800, 600, 500, 350, 200, 145, 85, 40 and possible 22 years could be established with reasonable confidence. Several of these periods seem to correspond to known sunspot cycles (22, 40, 80, 150, 200 and possibly 1000 years).
A simple strategy to identify and isolate new promoters suitable for driving the expression of selectable marker genes is described. By employing a Brassica napus hypocotyl transformation protocol and a promoterless gus::nptII tagging construct, a series of 20 kanamycin-resistant tagged lines was produced. Most of the regenerated plants showed hardly any GUS activity in leaf, stem and root tissues. However, expression was readily restored in callus tissue induced on in vitro leaf segments. Genomic sequences upstream of the gus::nptII insertions were isolated via plasmid rescue. Three clones originating from single copy T-DNA lines were selected for further evaluation. The rescued plasmids were cloned as linear fragments in binary vectors and re-transformed to Brassica napus hypocotyl and Solanum tuberosum stem segments. The new sequences maintained their promoter activity, demonstrated by transient and stable GUS activity after transformation. Furthermore, the promoters provided sufficient expression of the nptII gene to yield transgenic plants when using kanamycin as selective agent. Database searching (BLASTN) revealed that the promoters have significant homology with three Arabidopsis BAC clones, one Arabidopsis cDNA and one Brassica napus cDNA. The results presented in this paper illustrate the strength of combined methods for identification, isolation and testing of new plant promoters.
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