Agrobacterium-mediated transformation of spinach (Spinacia oleracea) with Bacillus thuringiensis cry1Ac gene for resistance against two common vegetable pests

Abstract

Agrobacterium-mediated genetic transformation was applied to produce transgenic plants of spinach (Spinacia oleracea) resistant to 2 pest species, Trichoplusia ni and Autographa nigrisigna. Leaf segments from in vitro spinach plants of cultivar 'Glory' were co-cultivated with A. tumefaciens strain EHA105, which harbored the plasmid pBE2111FMB containing a synthetic cry1Ac gene encoding an insecticidal crystal protein of Bacillus thuringiensis, and neomycin phosphotransferase II and bialaphos resistance genes as selectable marker genes. Kanamycin (Km)-resistant calluses were obtained from co-cultivated leaf segments 1 month after selection on 2.5 g l � 1 gellan gum-solidified MS medium containing 3 0gl � 1 sucrose, 0.5 mg l � 1 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg l � 1 kinetin, 100 mg l � 1 Km and 20 mg l � 1 meropenem trihydrate. Regeneration of adventitious shoots from Km-resistant calluses was performed 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, 100 mg l � 1 Km and 10 mg l � 1 meropenem trihydrate at 14°C. PCR and Southern blot analyses of genomic DNA isolated from T1 plants confirmed the successful integration of T-DNA into the plant genome. Expression of Cry1Ac protein was confirmed in leaves of transgenic plants by Western blot analysis. Insect bioassays against T. ni and A. nigrisigna performed with T1 plants showed more than 93.3% insect mortality within 1 week. These results suggest that the cry1Ac gene was effectively expressed in spinach.