Agrobacterium-mediated transformation of potato cv. Mnandi for resistance to the potato tuber moth (Phthorimaea operculella)

Potato tuber moth (Phthorimaea operculella) is a major insect pest of potatoes during field cultivation and post harvest storage, causing high annual losses in potato production areas. The Bacillus thuringiensis gene (cryllal) was introduced into the South African potato cultivar Mnandi with Agrobacterium-mediated transformation. The Agrobacterium tumefaciens strain LBA4404 harbouring the binary vector pSPUD5 carrying the cryllal gene under the transcriptional control of the (ocs)(3)mas promoter and the nptll gene (as selectable marker) was used to transform in vitro potato explants. In vitro potato explants were infected with Agrobacterium LBA4404, cultured on the pre-culture medium with 50 mu M acetosyringone, co-cultivated on pre-culture media for two days, and then regenerated on medium containing MS basal medium, 0.27 mu M NAA, 0.58 mu M GA(3), 9.12 mu M zeatin and 50 mg 1(-1) kanamycin. The regeneration frequency of 15% obtained with the potato cultivar Mnandi was an indication that this cultivar is sensitive to transformation with Agrobacterium. Verification of stable integration of the cryllal gene was confirmed by PCR and Southern blot hybridization procedures. The level of resistance to he potato tuber moth was investigated using leaf and tuber bio-assays which involved feeding studies of potato tuber moth larvae on the leaves and tubers of putatively transformed plants. Seven transformed lines expressed potato tuber moth resistance during the leaf and tuber assays. The Southern blot hybridization analysis verified that one to three copies of the cryllal gene was integrated into the genome of five transformed lines. (C) 2012 SAAB. Published by Elsevier B.V. All rights reserved.