, Volume 13, Issue 2, pp 135-142

Prototype Demonstration of Transgenic Resistance to the Nematode Radopholus similis Conferred on Banana by a Cystatin

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Abstract

Cavendish banana was transformed using Agrobacterium tumefaciens to express a protein engineered rice cystatin (OcIΔD86) of value for control of plant parasitic nematodes. Expression for each line was under control of a constitutive promoter from the maize ubiquitin gene (UBI-1), a constitutive, chimeric promoter from the octopine and mannopine synthase genes of A. tumefaciens or a promoter from a root-preferentially expressed tubulin gene Arabidopsis (TUB-1). Lines were selected as of potential interest after 8 weeks challenge in containment if their mean R. similis/25 g roots for three sibling plants were more than 1 standard normal variate below the grand mean for all plants in c7-15 lines challenged concurrently. A total of 16 lines were selected on this basis as putative positives. Western blots confirmed that eight of these lines expressed cystatin with a mean of 0.08 ± 0.04% tsp. All but two of 19 negatively selected lines from bioassays did not express cystatin. The mean resistance level of the confirmed positive lines was 69 ± 17%. ELISA established the positive lines under control of UBI provided significantly higher expression levels of OcIΔD86 than recorded for the other two promoters. Lines of interest were confirmed as producing a transcript for OcIΔD86 by RT-PCR. Eight plants of one UBI promoter line expressing only 0.1 ± 0.004% tsp as cystatin were re-challenged with R. similis and achieved a resistance of 70 ± 10%. Subsequent repeat western blotting confirmed that this line still produced the cystatin after the trial. This is the first report of transgenic resistance against a major pest or disease of banana.