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

Abstract

As part of a publicly funded initiative to develop genetically engineered Brassicas (cabbage, cauliflower, and canola) expressing Bacillus thuringiensis Cry-encoded insecticidal (Bt) toxin for Indian and Australian farmers, we designed several constructs that drive high-level expression of modified Cry1B and Cry1C genes (referred to as Cry1BM and Cry1CM). 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 expressing CryM genes, and to remove or alter sequences that might affect gene activity in plants. To assess the insecticidal efficacy of the Cry1BM/Cry1CM genes, constructs were introduced into the model Brassica Arabidopsis thaliana in which Cry1BM/Cry1CM expression was directed from either single (S4/S7) or double (S4S4/S7S7) Subterranean Clover Stunt Virus promoters. The resulting transgenic plants displayed a high-level of Cry1BM/Cry1CM expression. Protein accumulation for Cry1CM ranged from 0.81 to 17.69 g 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 of Cry1BM/Cry1CM genes, with a similar range of Cry1CM transcript abundance and protein content observed from both constructs. First instar Diamondback moth (Plutella xylostella) larvae fed on transgenic Arabidopsis leaves expressing the Cry1BM/Cry1CM genes 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 of Cry1BM and Cry1CM was sufficient to cause insect mortality, suggesting that these modified CryM genes are suitable for the development of insect resistant GM crops. Except for the Cry1B/Cry1C genes themselves, which remain under patent until 2027 and the PAT gene 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 these Cry1M genes in GM crop plants in the future.