Transgenic spinach plants produced by Agrobacterium-mediated method based on the low temperature-dependent high plant regeneration ability of leaf explants

Abstract

Agrobacterium strain EHA101 harboring plasmid pIG121-Hm containing the genes for β-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase (nptII) was co-cultivated with leaf explants from in vitro grown spinach plants. Hygromycin-resistant calli were obtained 1 month after selection of transformed cells on 2.5 g l−1 gellan gum-solidified MS medium containing 30 g l−1 sucrose, 0.5 mg l−1 2,4-D, 2 mg l−1 kinetin, 20 mg l−1 hygromycin and 20 mg l−1 meropenem trihydrate. Regeneration of adventitious shoots was affected by culture temperature, and the highest frequency of shoot formation was 40% when the calli were cultured at 14°C during regeneration process on 2.5 g l−1 gellan gum-solidified MS medium containing 30 g l−1 sucrose, 0.01 mg l−1 2,4-D, 1 mg l−1 kinetin, 1 mg l−1 gibberellic acid (GA3), 20 mg l−1 hygromycin and 10 mg l−1 meropenem trihydrate. Stable expression of gus gene was indicated by histochemical GUS assay in the leaves and roots of putative transgenic plants. Southern blot analysis of genomic DNA isolated from T0 plants confirmed the successful integration of T-DNA into the plant genome. Segregation of the gus gene in transgenic T1 progeny was confirmed by PCR analysis. These results show that Agrobacterium-mediated transformation combined with plant regeneration at a low temperature, 14°C, can be efficiently used for producing transgenic spinach plants with useful genes.