Genetic transformation of Ornithogalum via particle bombardment and generation of Pectobacterium carotovorum-resistant plants

摘要

Bacterial soft rot caused by Pectobacterium carotovorum subsp. carotovorum (Pcc) is one of the most devastating diseases of Ornithogalum species. No effective control measures are currently available to use against this pathogen; thus, introduction of resistant genes via genetic transformation into this crop is a promising approach. Tachyplesin I, an antimicrobial peptide, has been shown to effectively control numerous pathogenic bacteria, including Pcc. In this study, liquid-grown cell clusters of Ornithogalum dubium and Ornithogalum thy rsoides were bombarded with a pCAMBIA2301 vector containing a cell leader sequence fused to a gene encoding tachyplesin I, a neomycin phosphotransferase (nptII) gene that served as a selectable marker and a beta-glucuronidase (GUS) gene that served as a reporter. Selection was carried out in the dark in liquid medium containing 80 mg/L kanamycin. Regeneration was executed in the light after 6-14 months depending on the cultivar. Hundreds of transgenic plantlets were produced and their identity was confirmed through GUS activity assays. PCR and RT-PCR were used to confirm the presence of the target, reporter and selection genes in the divergent lines of plantlets. The resistance of the O. dubium plants to Pcc was evaluated in vitro, following infection with a highly virulent isolate from calla lily. Although control plantlets were completely macerated within a week, 87 putative transgenic subclones displayed varying levels of disease resistance. During three growing seasons in the greenhouse, the transgenic O. dubium lines grew poorly, whereas the transgenic O. thy rsoides plants grew similarly to non-transgenic plants. (C) 2014 Elsevier Ireland Ltd. All rights reserved.