In vitro regeneration and genetic transformation of the berberine-producing plant, Thalictrum flavum ssp. glaucum.

Abstract

Protocols have been developed for the in vitro regeneration and Agrobacterium-mediated genetic transformation of meadow rue, Thalictrum flavum ssp. glaucum. Ten-day-old seedlings were bisected along the embryonic axis and the cotyledons were co-cultured with various Agrobacterium tumefaciens strains for 3 days. The cotyledons were cultured on a shoot induction medium (B5 salts and vitamins, 30 g l-1 sucrose, 2 mg l-1 kinetin, and 3 g l-1 Gelrite) containing 25 mg l-1 hygromycin B as the selection agent and 250 mg l-1 timentin to facilitate the elimination of Agrobacterium. Only the oncogenic A. tumefaciens strains A281 and C58 produced transgenic T. flavum callus tissues. A281 was the most effective strain producing hygromycin-resistant callus on 85% of the explants. Transgenic callus was subcultured on the shoot induction medium every 2 weeks. After 12 weeks, hygromycin-resistant shoots that formed on explants exposed to strain A281 were transferred to a root induction medium (B5 salts and vitamins, 25 mg l-1 hygromycin B, 250 mg l-1 timentin, and 3 g l-1 Gelrite). Detection of the beta-glucuronidase (GUS) gene using a polymerase chain reaction assay, the high levels of GUS mRNA and enzyme activity, and the cytohistochemical localization of GUS activity confirmed the genetic transformation of callus cultures and regenerated plants. The transformation process did not alter the normal content of berberine in transgenic roots or cell cultures; thus, the reported protocol is valuable to study the molecular and metabolic regulation of protoberberine alkaloid biosynthesis.