Recovery of transgenic calli through Agrobacterium-mediated transformation in Quillaja lancifolia

Abstract

Quillaja lancifolia D. Don (current correct taxonomic designation of the synonym Q. brasiliensis (A. St.-Hil. & Tul.) Mart.) is a promising source of triterpenoid saponins of high biotechnological value, including QS-21, a potent immunological adjuvant used in human vaccine formulations, producible in cell suspension cultures. Herein, an efficient Agrobacterium tumefaciens –mediated genetic transformation protocol for generating transgenic calli of Q. lancifolia was established. Transformation was performed using A. tumefaciens EHA105 harboring the binary vector pH7WG2D-GUS containing the β-glucuronidase (GUS), the green fluorescent protein (GFP), and hygromycin phosphotransferase ( hpt ) genes. Under the established conditions (OD₆₀₀ = 0.6, 30 min infection, 72 h co-cultivation), hygromycin-resistant callus sectors expressing reporter genes were consistently obtained on hygromycin-containing culture medium. Transgene presence was confirmed by PCR amplification of the hpt gene from genomic DNA. Histochemical GUS staining and GFP fluorescence were shown after confirmation of Agrobacterium elimination by successive subcultures on meropenem-containing medium, followed by cultivation on antibiotic-free conditions. The overall transformation efficiency reached 41.6 ± 4.6%, calculated as the percentage of original explants producing GFP-positive hygromycin-resistant callus. The protocol herein described constitutes a useful tool for the establishment of transgenic callus, enabling future metabolic engineering studies in Q. lancifolia cell cultures.

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