An Agrobacterium-Mediated Transformation System from Thin Cell Layer Explants of Pyrethrum (Tanacetum cinenariifolium)

Pyrethrum (Tanacetum cinenariifolium, (Trev.) Sch. Bip., syn. Chrysanthemum cinerariaefolium (Trev.) Vis., Pyrethrum cinerariifolium Trev.) is a well known plant cultivated as industrial plant for the production of the pyrethrins insecticide. Pyrethrum has also been reported to be one of the recalcitrant species for shoot regeneration in the aim of the improvement of the molecular breeding by transgenic researches. In this study, an efficient regeneration system was set up based on the transverse thin cell layer (tTCL) method. Shoot buds emerged from the stem tTCL explants after one week on regeneration media. The best regeneration frequency of 2 shoots per explant was obtained on medium containing 2.0 mg L-1 6-Benzylaminopurine (6-BA) and 0.4 mg L-1 Indole-3-butyric acid (IBA). By comparison, longitudinal TCL (lTCL) generated very few shoots (only 1 shoot out of 20 explants) on the same regeneration media series. Kanamycin added in the medium significantly reduced explant survival and shoot regeneration and at 20 mg L-1 kanamycin no green shoots were formed after 3 weeks incubation. Filter paper was found to buffer the phytotoxic effects of kanamycin on the explant survival. Agrobacterium-mediated genetic transformation was carried out using the green fluorescent protein (GFP) gene under control of the ribulose-1, 5-bisphosphate carboxylase small subunit (RbcS) promoter as a visual reporter of transformation. Nine shoots out of 200 explants were obtained which were positive both for green fluorescence of leaf tissue and in a PCR analysis of genomic DNA. Based on this efficient regeneration and transformation system, further transgenic work of pyrethrum can be done for scientific studies and to improve the content of pyrethrins and other agronomically relevant traits.