This presentation is described by the attached abstract

Marc Ghislain^1a, Lydia Wamalwa^1a, Sandra Manrique^1b, Jan Kreuze^1b, Runyararo Rukarwa^1c Robert Mwanga^1c, Maria Soto-Aguilar², Katterinne Prentice³, Olivier Christiaens³, Ine Pertry³, Godelieve Gheysen³, Guy Smagghe³

¹ International Potato center, ^a Nairobi Kenya, ^b Lima Peru. ^C Namulonge Uganda

² Donald Danforth Plant Science Center, USA

³ Ghent University, Belgium.

Weevils, Cylas puncticollis and C. brunneus, are responsible for 28% of crop losses in Uganda according to a farmer survey completed in 2009. There is currently little farmers can do when weevils infest their fields, other than to quickly harvest and salvage what is left of their crop. In addition, our recent study has also highlighted a potential health threat when farmers consume the undamaged parts of infected sweetpotato storage roots due to high accumulation of the plant toxic ipomeamarone compound.

In order to render sweetpotato resistant to weevils, we have introduced synthetic cry genes that produce proteins with activity against the C. puncticollis and C. brunneus weevils into sweetpotato. A total of 117 transgenic events were produced with cry genes that resemble sweetpotato genes. Thirty six of them were tested by oviposition to adult emergence at NaCRRI/NARO, Uganda and found to be susceptible to weevils. However, one of two of these retested in BecA showed significantly less damage than non-transgenic storage roots. Sixty three transgenic events were screened for presence of Cry3Ca protein dosage in storage roots. Four of 27 transgenic events with detectable levels of Cry3Ca presented significantly less damage than the non-transgenic storage roots. New cry gene constructs designed for higher cry gene expression, were used to produce a total of 174 transgenic events at ABL in Peru. Of 38 screened for Cry3Ca protein, 5 and 12 were transferred to CIP and BecA greenhouse, respectively. One out of 4 of them did not have damage. The remaining 136 transgenic events are kept in tissue culture. At DDPSC, 225 transgenic events were produced of which 125 were transferred to the greenhouse in their facilities at St. Louis, USA. Interestingly, 7 transgenic events bear the three cry genes obtained by co-transformation. Storage roots are currently being harvested and will be shipped to BecA for bioassays.

In parallel, we are pursuing an RNA interference strategy to complement the Cry protein expression approach. Nano-injections of double stranded (ds) RNA of 24 essential genes led to larvae mortality. Eleven of them were most effective. Nine of them were tested by ingestion using soaking and for the most effective artificial diet was used. Three of them have given good results for both weevil species and in both soaking and artificial diets: Proteasome 20 kD subunit, ribosomal protein S13e and Snf7. Five hairpin gene constructs were designed based on Prot20kd and Snf7 from C. puncticollis and C. brunneus in single and double combinations. When these are completed, genetic transformation of Jonathan variety will be initiated.

In conclusion, we have for the first time observed 6 transgenic events expressing Cry protein displaying minimum, if any, damage (small tunnels, dead pupae or adult) when infested by weevils from a total of 67 tested. All of them were replanted for a second bioassay in order to ensure these results were not due to experimental errors. More transgenic events will be tested in the next three months.