Vol. 81 No: 3

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

Authors:
RINALDI SJAHRIL and MASAHIRO MII

pp: 458-464

Abstract:
The 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 β-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.

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