Development and Evaluation of Potatoes Transgenic for a cry1Ac9 Gene Conferring Resistance to Potato Tuber Moth

Abstract

<jats:p>Transgenic potato (<jats:italic>Solanum tuberosum</jats:italic> L.) lines of two cultivars, Ilam Hardy and Iwa, were developed using <jats:italic>Agrobacterium</jats:italic>-mediated transformation to transfer a <jats:italic>cry</jats:italic>1Ac9 gene under the transcriptional control of the CaMV 35S promoter. PCR confirmed the presence of the <jats:italic>npt</jats:italic>II selectable marker gene in all recovered lines. All ten lines of Ilam Hardy and 14 of 15 Iwa lines were PCR-positive for the <jats:italic>cry</jats:italic> gene. In greenhouse trials, all Ilam Hardy transgenic lines produced phenotypically normal plants and significantly inhibited larval growth of potato tuber moth (<jats:italic>Phthorimaea operculella</jats:italic> Zeller). In contrast, only 60% of the Iwa transgenic lines produced phenotypically normal plants, but all lines positive for the <jats:italic>cry</jats:italic> gene significantly inhibited larval growth. All transgenic lines with a greenhouse appearance equivalent to the nontransgenic controls and improved resistance to potato tuber moth larvae were planted in the field. Three of the ten Ilam Hardy lines and two of the eight Iwa lines retained phenotypically normal appearance in the field and produced tuber yields equivalent to the nontransgenic controls. All five of these transgenic lines significantly inhibited larval growth of potato tuber moth on excised field-grown leaves. A high correlation was established between larval growth indices from the greenhouse and the field. A transgenic line from each cultivar inhibited larval growth by over 40%, and the line derived from Ilam Hardy prevented pupation of all larvae. Southern analysis on these five elite lines revealed that they contained either one or two copies of the <jats:italic>cry</jats:italic>1Ac9 gene. The amount of Cry protein in all transgenic lines tested was less than 60 ng·g<jats:sup>-1</jats:sup> of fresh leaf tissue. A transgenic line from each cultivar was identified with comparable phenotypic appearance and yield to their parent cultivars coupled with high resistance to PTM.</jats:p>

Expression of cry1Ac9 and cry9Aa2 genes under a potato light-inducible Lhca3 promoter in transgenic potatoes for tuber moth resistance

“…The insects used in the bioassays were obtained from a laboratory colony maintained as previously described (Davidson et al, 2002). The petioles of excised leaves were inserted through a polystyrene stopper (2 cm diameter × 1.5 cm long with a full length radial slit) into each 25 ml glass vial full of water.…”

“…Lanes 14-24 represent a multiplex reaction with cry9Aa2 primers producing an expected 826 bp product, the 612 bp nptII product and the 1069 bp actin product. Lanes 1-8, lines transformed with pART27cab1Ac9 (#102, #107, #110, #116, #121, #122, #123 and #130 respectively); lanes 9 and 22, no DNA template control; lanes 10 and 23, non-transgenic 'Iwa' control; lane 11, 'Iwa' line I52 known to be transgenic for nptII and cry1Ac9 (positive control, ex Davidson et al, 2002); lanes 12 and 13, 100 bp molecular ruler 10380-012 and 1 kb plus molecular ruler 10787-018 (Invitrogen, Carlsbad, California) size markers respectively; lanes 14-21, lines transformed with pART27cab9Aa2 (#7, #12, #17, #18, #22, #27, #28 and #29 respectively); lane 24, 'Iwa' line DG4c known to be transgenic for nptII and cry9Aa2 (positive control, ex Meiyalaghan et al, 2004). reactions to be conveniently distinguished from a nontransgenic line. PCR products from representative lines are illustrated in Figure 2.…”

“…Various management strategies such as cultural practices, biological and chemical control are used to manage PTM (Goldson & Emberson, 1985;Hanafi, 1999). To contribute an additional component to integrated pest management, the development of transgenic potatoes that express the insecticidal toxins produced by different strains of the soil bacterium, Bacillus thuringiensis (Bt), has been investigated (Jansens et al, 1995;Douches et al, 1998;Rico et al, 1998;Cañedo et al, 1999;Li et al, 1999;Chakrabarti et al, 2000;Davidson et al, 2002Davidson et al, , 2004.…”

“…The gene encoding this protein was codon-modified to increase its expression in plants and confer resistance to PTM larvae in tobacco (Beuning et al, 2001). The subsequent transfer of this modified cry1Ac9 gene under the regulatory control of the CaMV 35S promoter to potato confirmed efficacy against PTM larvae in the main crop in which this pest causes economic damage (Davidson et al, 2002. Gleave et al (1992b) cloned and characterized a cry gene, encoding a 129 kDa protein from the Bt strain, DSIR517, serotyped as var.…”

“…The 35S promoter used for transcriptional control of the cry genes in previous investigations in potato Rico et al, 1998;Cañedo et al, 1999;Li et al, 1999;Chakrabarti et al, 2000;Davidson et al, 2002Davidson et al, , 2004 is well known to be constitutively expressed throughout plants (Odell et al, 1985), including potato (Gatehouse et al, 1997). However, it is often desirable to couple high-level expression of the transgene in potato foliage with no expression in tubers.…”