Agrobacterium-mediated transformation using embryogenic calli in Satsuma mandarin (Citrus unshiu Marc.) cv. Miyagawa wase

Abstract

Agrobacterium-mediated transformation in Satsuma mandarin (Citrus unshiu Marc.) cv. Miyagawa wase was achieved with reasonable transformation efficiency of about 22%, which was the percentage of transgenic plantlets confirmed by genomic PCR (37 plantlets/168 hygromycin-resistant calli). Embryogenic calli of Miyagawa wase were infected with Agrobacterium tumefaciens strain EHA105 harboring binary vector pCAMBIA1300 that contained miraculin gene (a taste-modifying protein) and hygromycin as a selection marker. After 5 days of co-culture in the medium containing 100 μM acetosyringone, calli were transferred to the liquid half embryogenic cell culture medium (half concentration of Murashige and Tucker’s (MT) medium modified with the addition of 500 mg·L− malt extract, 50 g·L− sucrose and 1.55 g·L− glutamine) with 15 mg·L− hygromycin and 250 mg·L− cefotaxime, and were cultured for two weeks. Then, the calli were grown on the solid selection medium with 20 mg·L− hygromycin for four weeks and 25 mg·L− hygromycin for another four weeks. The resistant embryos were selected and transferred to the embryo maturation medium. After 3 weeks of culture, the heart shaped embryos were transferred to the MT medium containing 1.0 mg ·L− GA3, 20.0 mL·L− coconut water, 0.02 mg·L− NAA and 0.0146 mg·L− coumarin for embryo germination. Finally the germinated embryos were cultured on MT medium containing 3.0% sucrose and 0.8% agar for growing to the normal plant. Stable integration of the transgene in the plant genome was confirmed by PCR and Southern blot analysis.