High-efficiency Agrobacterium-mediated transformation of Phalaenopsis using meropenem, a novel antibiotic to eliminate Agrobacterium

Abstract

SummaryThe type and concentration of antibiotic used to eliminate Agrobacterium affected the efficiency of bacterial elimination from, and phytotoxicity to Phalaenopsis cells. To eliminate Agrobacterium, high concentrations (500 mg l–1) of cefotaxime and carbenicillin were required, which also caused necrosis of cells when added to the culture medium for 2 weeks. In contrast, meropenem successfully suppressed growth of the bacteria at low concentrations (5 mg l–1) and had no phytotoxic effect. A reproducible genetic transformation method for Phalaenopsis was established by co-cultivating cell suspension cultures of Phalaenopsis Wataboushi ‘#6.13’ with A. tumefaciens strain ‘EHA101’ (pIG121Hm), harbouring genes for -glucuronidase (GUS) and hygromycin resistance (hpt). The highest transformation efficiency was obtained when 5 d-old cells were infected for 2 h after sub-culture with Agrobacterium, as assessed by transient as well as stable GUS expression. Successful GUS gene expression in putative transgenic plantlets was confirmed by histochemical GUS assays on calli, protocorm-like bodies (PLBs), leaves and roots of plantlets selected on New Dogashima medium (NDM) containing 25 mg l–1 hygromycin. PCR and Southern blot analysis of genomic DNA confirmed successful incorporation and transformation of the GUS gene in regenerated plantlets. Agrobacterium was completely eliminated using 10 mg l–1 meropenem during a 10 min wash after 3 d co-cultivation followed by 5 mg l–1 for the first 3 months of culture in Gelrite-solidified medium. Ninety-two percent of transgenic PLBs obtained after 6 months, and 97% of transgenic plantlets after 12 months showed almost complete elimination of Agrobacterium.