An efficient protocol for regeneration and transformation of Symphyotrichum novi-belgii

Abstract

A protocol for adventitious shoot formation in Symphyotrichum novi-belgii was developed after investigating the effects of cultivar and hormone combinations. A Murashige and Skoog medium with 1.0 mg l -1 6-benzyladenine induced adventitious shoot formation in 15 out of 19 cultivars. Addition of 0.1 mg l -1 indole-3-acetic acid or naphthaleneacetic acid increased the total number of shoots per explant, but not the number of shoots longer than 1 cm. Addition of dichlorophenoxyacetic acid (2,4-D) promoted callus formation, but inhibited shoot elongation. A transformation system for the two cultivars Victoria Fanny and Victoria Jane was developed by co-cultivation of leaf explants with Agrobacterium tumefaciens. Three bacterial strains (LBA 4404, A281 and C58) all carrying the binary vector, p35S-GUS-INT, and harbouring the uidA gene coding for b-glucuronidase (GUS) were used. Regeneration of transgenic plants after co-cultivation with A281 was independent of cultivar, and all explants produced callus followed by indirect shoot formation. In 'Victoria Fanny' shoots were formed faster and without a callus phase after co-cultivation with LBA 4404 or C58. The highest number of potentially transformed shoots was regenerated after co-cultivation of 'Victoria Fanny' leaf explants with LBA 4404. Integration of the transgenes in the plant genome was confirmed using PCR and Southern blot hybridisation. To verify that the transgenes could be transferred to offspring, crosses were conducted between three transgenic lines of 'Victoria Fanny' and two wild type pollen donors. It was demonstrated that viable seeds were produced and that the uidA gene was inherited.