The sweet pepper ferredoxin-like protein (pflp) conferred resistance against soft rot disease in Oncidium orchid.

Genetic engineering to date has not been used to introduce disease resistance genes into the orchid gene pool. The ferredoxin-like protein gene originally isolated from sweet pepper is thought to function as a natural defense against infection due to its antimicrobial properties. Hence it was reasoned that introduction of this gene might produce Oncidium plants resistant to Erwinia carotovora, the causal agent for the soft rot disease. An expression vector containing sweet pepper ferredoxin-like protein (pflp) cDNA, hph and gusA coding sequence was successfully transformed into protocorm-like bodies (PLBs) of Oncidium orchid, using Agrobacterium tumefaciens strain EHA105. A total of 17 independent transgenic orchid lines was obtained, out of which six transgenic lines (beta-glucuronidase (GUS) positive) were randomly selected and confirmed by Southern, northern and western blot analyses. A bioassay was conducted on the transgenic lines. Transgenic plants showed enhanced resistance to E. carotovora, even when the entire plant was challenged with the pathogen. Our results suggest that pflp may be an extremely useful gene for genetic engineering strategies in orchids to confer resistance against soft rot disease.

Sweet pepper ferredoxin-like protein ( pflp) gene as a novel selection marker for orchid transformation.

A novel method for selection of transgenic plants utilizing the sweet pepper ( Capsicum annuum L.) ferredoxin-like protein ( pflp) gene as selection marker and Erwinia carotovora as the selection agent has been developed. An expression vector containing a pflp cDNA driven by a cauliflower mosaic virus 35S promoter was successfully transformed into protocorm-like bodies of Oncidium orchid by Agrobacterium tumefaciens and particle bombardment, respectively. Erwinia carotovora was used as a selection agent to screen transformants, thereby obtaining transgenic plants without the use of an antibiotic selection agent. A total of 32 independent transgenic orchid lines were obtained, out of which 9 transgenic lines (beta-glucuronidase positive) were randomly selected and confirmed by Southern and northern blot analyses. The transgenic orchid plants showed enhanced resistance to E. carotovora, even when the entire plant was challenged with the pathogen. Our results suggest the novel use of the pflp gene as a resistance selection marker in plant genetic engineering strategies. In the future, the use of the pflp gene as a selection marker may facilitate the use of smaller gene constructs due to removal of bulky antibiotic selection and reporter genes. These constructs can then be used to incorporate additional genes of choice.