|Authors: ||M. Ghislain, J. Tovar, K. Prentice, M. Ormachea, C. Rivera, S. Manrique, J. Kreuze, R. Rukarwa, A. Sefasi, S. Mukasa, G. Ssemakula, L. Wamalwa, J. Machuka|
|Keywords: ||Ipomoea batatas, Cylas sp., Cry proteins, genetic engineering, Bt crops|
African sweetpotato weevils (SPW), Cylas puncticollis and C. brunneus, pose a major threat to sweetpotato, which plays a vital role in food security and income generation for both the urban and rural poor in Sub-Saharan Africa (SSA). SPW can limit sweetpotato production causing total crop loss.
Control methods such as integrated pest management and breeding have not succeeded satisfactorily in curbing out these pests thus opening the door for using biotechnology and genetic engineering to make transgenic sweetpotato that are resistant to weevil infestation.
At least three protein samples from Bacillus thuringiensis (Bt) have been found to be toxic to both SPW species at less than 1 ppm (Cry7Aa1, CryET33/CryET34, and Cry3Ca1). Corresponding gene constructs were developed using sporamin and β amylase regulatory sequences to express and accumulate high Cry protein levels in the storage root.
Approximately 100 transformed events from sweetpotato cultivars (including one African cultivar), were produced by Agrobacterium tumefaciens transformation of petioles and somatic embryos.
Gene expression from leaf tissues using qRT-PCR revealed up to 20X difference among events.
Protein accumulation using DAS-ELISA and storage roots exhibited even larger variation between events.
However, so far only 18 of the 90 events have produced storage roots which could be bio-assayed.
Most events accumulated Cry proteins below the LC50 level, two events accumulated Cry protein at the LC50 level and only one event accumulated Cry protein above the LC50 level (3 times the LC50 level). Bioassays using transgenic tissues infested with SPW larvae are on-going but preliminary results reflect low toxicity as expected based on Cry protein content in the storage roots.
Future steps include the screening of additional events, characterization of competitive binding of these Cry proteins and confined field trials of SPW-resistant events.
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