Transformation of potato(Solanum tuberosum)cultivars with acry1Ac9 gene confers resistance to potato tuber moth(Phthorimaea operculella)

Abstract

Eight potato (Solanum tuberosum) cultivars were transformed with a modified cry 1AC9 gene under the transcriptional control of the CaMV 35S promoter using Agrobacterium-mediated transformation with a nptII gene conferring kanamycin resistance as a selectable marker. Initially the transformation efficiency of two Agrobacterium strains (LBA4404 and AGL1) were compared using the potato cultivars 'Iwa' and 'Ilam Hardy'. Both strains resulted in similar numbers of regenerated shoots, but there was a higher proportion of off-types among plants transformed using the AGL1 strain. Subsequently, the cultivars 'Karaka', 'Pacific', 'Red Rascal', 'Rua', 'Russet Burbank', and 'White Delight' were transformed with strain LBA4404. Putatively transformed lines with kanamycin resistance were assayed using multiplex polymerase chain reaction (PCR) with an endogenous potato actin as an internal control. From a total of 116 lines, 105 were confirmed as being PCR-positive for the nptII gene, of which 93 were also PCR-positive for the presence of the cry1Ac9 gene. all transgenic lines tested was less than 60 ng/g fresh leaf tissue. The growth of potato tuber moth (Phthorimaea operculella) larvae was inhibited by foliage from greenhouse-grown plants of 77 of the transgenic lines (P < 0.05). Fifty-five of these resistant transgenic lines were described as having a phenotypic appearance comparable to the corresponding parental cultivars, when grown in the greenhouse. Southern analysis on four highly resistant lines revealed they contained two to five copies of the cry1Ac9 gene. Several transgenic lines severely reduced larval growth while exhibiting a phenotype similar to their parent cultivar. These lines warrant further investigation in the field.

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

“…The bioassay method used was based on the assumption that the level of Cry protein in excised leaves was similar to that in the foliage of intact plants. The same assumption has been made in similar studies involving PTM bioassays (Ebora et al, 1994;Jansens et al, 1995;Westedt et al, 1998;Beuning et al, 2001;Douches et al, 1998Douches et al, , 2002Li et al, 1999;Davidson et al, 2002Davidson et al, , 2004, and has been validated by the observed high correlation between excised leaves and intact plants for growth of PTM larvae in response to cry gene expression (Davidson et al, 2002).…”

“…In potato, it is often desirable to couple high-level expression of the transgene in foliage with no expression in tubers. Constitutive promoters have been used to drive cry gene expression in all previous studies targeting PTM resistance in transgenic potatoes (Douches et al, , 2002Jansens et al, 1995;Cañedo et al, 1999;Li et al, 1999;Chakrabarti et al, 2000;Mohammed et al, 2000;Davidson et al, 2002Davidson et al, , 2004. Since PTM generally infests foliage of potato crops prior to the tubers (Foot, 1979), preventing foliar infestation may be sufficient to avoid tuber infestation.…”

“…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 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.…”

Potato Transformation with Modified Nucleotide Sequences of the cry9Aa2 Gene Improves Resistance to Potato Tuber Moth

“…To contribute an additional component to integrated pest management, the development of transgenic potatoes that express genes encoding insecticidal compounds has been investigated (Meiyalaghan et al 2005). The use of cry genes from the soil bacterium, Bacillus thuringiensis, has been especially successful (e.g., Jansens et al 1995;Douches et al 1998;Li et al 1999;Davidson et al 2002Davidson et al , 2004Meiyalaghan et al 2006). However, the transfer of native cry genes to plants has often resulted in inadequate gene expression.…”