Designing Bt constructs for Brassicas, with minimal IP issues – A case study

Abstract

AbstractAs part of a publicly funded initiative to develop genetically engineered Brassicas (cabbage, cauliflower, and canola) expressingBacillus thuringiensis Cry-encoded insecticidal (Bt) toxin for Indian and Australian farmers, we designed several constructs that drive high-level expression of modifiedCry1BandCry1Cgenes (referred to asCry1BMandCry1CM). The two main motivations for modifying the DNA sequences of these genes were to minimise any licencing cost associated with the commercial cultivation of transgenic crop plants expressingCryMgenes, and to remove or alter sequences that might affect gene activity in plants. To assess the insecticidal efficacy of theCry1BM/Cry1CMgenes, constructs were introduced into the model BrassicaArabidopsis thalianain whichCry1BM/Cry1CMexpression was directed from either single (S4/S7) or double (S4S4/S7S7) Subterranean Clover Stunt Virus promoters. The resulting transgenic plants displayed a high-level ofCry1BM/Cry1CMexpression. Protein accumulation forCry1CMranged from 0.81 to 17.69 μg Cry1CM/g fresh weight of leaves. Contrary to previous work on stunt promoters, we found no correlation between the use of either single or double stunt promoters and the expression levels ofCry1BM/Cry1CMgenes, with a similar range ofCry1CMtranscript abundance and protein content observed from both constructs. First instar Diamondback moth (Plutella xylostella) larvae fed on transgenic Arabidopsis leaves expressing theCry1BM/Cry1CMgenes showed 100% mortality, with a mean leaf damage score on a scale of zero to five of 0.125 for transgenic leaves and 4.2 for wild-type leaves. Under laboratory conditions, even low-level expression ofCry1BMandCry1CMwas sufficient to cause insect mortality, suggesting that these modifiedCryMgenes are suitable for the development of insect resistant GM crops. Except for theCry1B/Cry1Cgenes themselves, which remain under patent until 2027 and thePATgene in the USA, our assessment of the intellectual property landscape of the constructs described here suggest that they can be used without the need for further licencing. This has the capacity to significantly reduce the cost of developing and using theseCry1Mgenes in GM crop plants in the future.