Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants.

A series of chimeric promoters for higher-level expression of foreign genes in plants was constructed as fusions of a gene for beta-glucuronidase (GUS) with the terminator of a gene for nopaline synthase (nos) or of the cauliflower mosaic virus (CaMV) 35S transcript, and the strength of these promoters was assayed in transient and stable expression systems in tobacco and rice. As parts of these promoters, the CaMV 35S core promoter, three different 5'-upstream sequences of the 35S promoter, the first intron of a gene for phaseolin, and a 5'-untranslated sequence (omega sequence) of tobacco mosaic virus were used in various combinations. In tobacco and rice protoplasts, all three fragments of the 35S promoter (-419 to -90, -390 to -90 and -290 to -90, relative to the site of initiation of transcription), the intron, and the omega sequence effectively enhanced GUS activity. Some chimeric promoters allowed levels of GUS activity that were 20- to 70-fold higher than those obtained with the 35S promoter in pBI221. In tobacco protoplasts, the two longer fragments of the 35S promoter were more effective than the shortest fragment. In rice cells, by contrast, the shortest fragment was as effective as the two longer ones. The terminator of the 35S transcript was more effective than that of the nos gene for gene expression. In transgenic tobacco plants, a representative powerful promoter, as compared to the 35S promoter, allowed 10- and 50-fold higher levels of expression on average and at most, respectively, with no clear qualitative differences in tissue- and organ-specific patterns of expression. When the representative promoter was introduced into tobacco with a gene for luciferase, the autofluorescence of detached leaves after a supply of luciferin to petioles was great and was easily detectable by the naked eye in a dark room.

Efficient regeneration of plants independent of exogeneous growth regulators in bell pepper (Capsicum annumm L.).

Organogenesis of shoots of bell pepper (Capsicum annuum L.) was achieved in fourteen cultivars on Murashige and Skoog's medium (MS medium) supplemented only with 0.4% (w/v) Gellan gum (pH 5.8). Mature seeds of cv. Shinsakigake-2 were sown on filter paper that had been wetted with sterilized water and precultured for zero to five days in under 16 hr of light per day at 25 °C. Explants, consisting of the proximal part of the hypocotyl and the radicle, were excised from the seeds and formed adventitious buds around the cut surfaces of elongated hypocotyls after four weeks of culture. When explants were subcultured on MS medium, 57% of the explants that had produced adventitious buds extended shoots after an additional three weeks of culture. Shoots were rooted on MS medium after two further weeks of culture. Chromosome numbers of all 30 regenerated plants that weexamined were normal (2n=24). The morphology of the mature plants was also normal and they set normally shaped fruits with mature seeds. Regenerated whole plants were also obtained in the case of 13 other cultivars by applying this simple procedure.