Minimizing IP issues associated with gene constructs encoding the Bt toxin - a case study.

BACKGROUND: As part of a publicly funded initiative to develop genetically engineered Brassicas (cabbage, cauliflower, and canola) expressing Bacillus thuringiensis Crystal (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; with M indicating modified). The two main motivations for modifying the DNA sequences of these genes were to minimise any licensing cost associated with the commercial cultivation of transgenic crop plants expressing CryM genes, and to remove or alter sequences that might adversely affect their activity in plants. RESULTS: 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 (SCSV) promoters. The resulting transgenic plants displayed a high-level of Cry1BM/Cry1CM expression. Protein accumulation for Cry1CM ranged from 5.18 to 176.88 µg Cry1CM/g dry 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. CONCLUSIONS: Our work indicates that the modified Cry1 genes are suitable for the development of insect resistant GM crops. Except for the PAT gene in the USA, our assessment of the intellectual property landscape of components presents within the constructs described here suggest that they can be used without the need for further licensing. This has the capacity to significantly reduce the cost of developing and using these Cry1M genes in GM crop plants in the future.

Estimating the bioaccessibility of flocculants in the presence of sediments in model wastewater.

The cationic degradable polymer poly(lactic acid) choline iodide ester methacrylate, poly(PLA4ChMA), can be used to flocculate particles and dewater sediments from tailings ponds and wastewater. A suitable bioaccessibility method is required to characterize the interactions of this novel flocculant in the human gastrointestinal system. To this end, a physiologically based extraction test (PBET) was modified to evaluate the bioaccessibility of flocculants. Bioaccessibility (bioaccessible fraction) is a measure of the solubility of a contaminant in gastrointestinal fluids and that may be available for systemic absorption. The flocculants poly(PLA4ChMA), SNF C3276, and FLOPAM A3338 were tested at a solid-to-liquid ratio of 1:200 in the absence and presence of kaolin clay, which is used as a model sediment compound. Bioaccessible fractions were characterized by proton nuclear magnetic resonance spectroscopy and estimated by gravimetry. The bioaccessibility of poly(PLA4ChMA) in gastric and intestinal PBET solutions decreases from 78% to 100%, respectively, in the absence of kaolin to approximately 0% with kaolin, indicating that poly(PLA4ChMA) remains adsorbed onto the clay surface throughout the PBET, a result confirmed by thermogravimetric analysis. The bioaccessibility of cationic SNF C3276 and anionic FLOPAM A3338 in gastric solution is approximately 76% and 26%, respectively, and is not affected by the presence of kaolin. However, in intestinal solutions, the bioaccessibility of SNF C3276 and FLOPAM A3338 (60-85% in the absence of kaolin) changes to 0% and 100%, respectively, in the presence of kaolin. These results, interpreted in terms of solution pH and surface charge, demonstrate that interactions with kaolin influence the solubility of flocculants and must be considered in the evaluation of bioaccessibility. In future works, such bioaccessibility methods can be applied to assess the human-health safety of using flocculants in wastewater treatments.

Overexpressing Arabidopsis thaliana ACBP6 in transgenic rapid-cycling Brassica napus confers cold tolerance.

BACKGROUND: Rapid-cycling Brassica napus (B. napus-RC) has potential as a rapid trait testing system for canola (B. napus) because its life cycle is completed within 2 months while canola usually takes 4 months, and it is susceptible to the same range of diseases and abiotic stress as canola. However, a rapid trait testing system for canola requires the development of an efficient transformation and tissue culture system for B. napus-RC. Furthermore, effectiveness of this system needs to be demonstrated by showing that a particular trait can be rapidly introduced into B. napus-RC plants. RESULTS: An in-vitro regeneration protocol was developed for B. napus-RC using 4-day-old cotyledons as the explant. High regeneration percentages, exceeding 70%, were achieved when 1-naphthaleneacetic acid (0.10 mg/L), 6-benzylaminopurine (1.0 mg/L), gibberellic acid (0.01 mg/L) and the ethylene antagonist silver nitrate (5 mg/L) were included in the regeneration medium. An average transformation efficiency of 16.4% was obtained using Agrobacterium-mediated transformation of B. napus-RC cotyledons using Agrobacterium strain GV3101 harbouring a plasmid with an NPTII (kanamycin-selectable) marker gene and the Arabidopsis thaliana cDNA encoding ACYL-COA-BINDING PROTEIN6 (AtACBP6). Transgenic B. napus-RC overexpressing AtACBP6 displayed better tolerance to freezing/frost than the wild type, with enhanced recovery from cellular membrane damage at both vegetative and flowering stages. AtACBP6-overexpressing B. napus-RC plants also exhibited lower electrolyte leakage and improved recovery following frost treatment, resulting in higher yields than the wild type. Ovules from transgenic AtACBP6 lines were better protected from frost than those of the wild type, while the developing embryos of frost-treated AtACBP6-overexpressing plants showed less freezing injury than the wild type. CONCLUSIONS: This study demonstrates that B. napus-RC can be successfully regenerated and transformed from cotyledon explants and has the potential to be an effective trait testing platform for canola. Additionally, AtACBP6 shows potential for enhancing cold tolerance in canola however, larger scale studies will be required to further confirm this outcome.

Quantitative analyses to estimate the bioaccessibility of a hydrolytically degradable cationic flocculant.

Poly(lactic acid) choline iodide ester methacrylate, poly(PLA4ChMA), is a cationic degradable polymer that can flocculate particles and dewater oil sands from tailings ponds. This novel material has yet to be characterized in terms of environmental and human health. If ingested, this substance may become bioaccessible. The bioaccessibility (bioaccessible fraction) of an ingested contaminant is a measure of the portion of an ingested dose that solubilizes and may be available for systemic absorption. In the present study, the partially degraded flocculant and its degradation products, modelled using lactic acid and choline chloride, were subjected to a modified physiologically based extraction test (PBET). Bioaccessible fractions were estimated by proton nuclear magnetic resonance (1H-NMR) spectroscopy and by high-performance liquid chromatography (HPLC). The measured bioaccessibility of lactic acid in gastric solution containing choline chloride is ∼100% but slightly dropped to 94% in intestinal solution at a solid-to-liquid ratio of 1:200. The partially degraded poly(PLA4ChMA) did not degrade further during the PBET and is not solubilized (i.e., 0% bioaccessibility) in the gastric phase but is fully solubilized (i.e., 100% bioaccessibility) in the intestinal phase. At the end of PBET intestinal digestion, the molar ratio of lactic acid to choline chloride in the presence of degraded poly(PLA4ChMA) was 2, approximately the same as in the initial solution. Thus, lactic acid and choline chloride are solubilized to the same extents in both gastric and intestinal solutions. Results suggest that HPLC can be used to directly estimate the bioaccessibility of lactic acid, whereas 1H - NMR may be used to indirectly quantify the bioaccessibility of both lactic acid and choline chloride by determining their molar ratio in PBET extracts. In future works, these findings may be applied to the estimation of risks from exposure to poly(PLA4ChMA) as well as to the remediation of contaminants flocculated by poly(PLA4ChMA) in tailings ponds and in other wastewaters.

Infection of Slugs with Theronts of the Ciliate Protozoan, Tetrahymena rostrata.

Tetrahymena rostrata is a free-living ciliated protozoan and is a facultative parasite of some species of terrestrial mollusks. It is a potential biopesticide of pest slugs, such as the grey field slug, which cause considerable damage to crops. T. rostrata has several developmental forms. Homogeneous preparations of the feeding stage cells (trophonts) and excysted stage cells (theronts) were compared for their ability to infect and kill Deroceras reticulatum slugs. Theronts were more effective and remained viable and infective, even after prolonged starvation.

Extracellular endonucleases in the midgut of Myzus persicae may limit the efficacy of orally delivered RNAi.

Myzus persicae is a major pest of many crops including canola and Brassica vegetables, partly because it vectors plant viruses. Previously it has been reported that double-stranded RNA delivered to aphids by injection, artificial diet or transgenic plants has knocked down target genes and caused phenotypic effects. While these studies suggest that RNA interference (RNAi) might be used to suppress aphid populations, none have shown effects sufficient for field control. The current study analyses the efficacy of dsRNA directed against previously reported gene-targets on Green peach aphid (Myzus persicae) strains. No silencing effect was observed when dsRNA was delivered in artificial diet with or without transfection reagents. dsRNA produced in planta also failed to induce significant RNAi in M. persicae. Transcriptome analyses of the midgut suggested other potential targets including the Ferritin heavy chain transcripts, but they also could not be knocked down with dsRNA. Here we show that dsRNA is rapidly degraded by midgut secretions of Myzus persicae. Analysis of the transcriptome of the M. persicae midgut revealed that an ortholog of RNases from other insects was abundant.

Resistance and Synergism of Novel Insecticides in Field Populations of Cotton Bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) in Pakistan.

The cotton bollworm Helicoverpa armigera (Hübner) is still a serious pest of non-Bt crops in Asia and Africa. It has been a notorious pest in developing resistance to all the insecticide classes applied for its control. Response of field populations of H. armigera to new chemistries having novel modes of action was monitored during 2003-2016 using a leaf-dip bioassay. No or very low level of resistance was found to newer insecticides, such as spinetoram, chlorantraniliprole, and flubendiamide during 2008-2016. There was also no or very low resistance to chlorfenapyr from 2003 to 2016. Resistance to spinosad, abamectin, and emamectin benzoate was none or very low as well during 2003-2013 and then it rose to a low level for spinosad and to a high level for avermectins by the year 2016. Methoxyfenozide resistance was very low during 2003-2010, low during 2011-2013, and moderate during 2014-2016. Resistance to thiocyclam was very low to low during 2009-2011, but it increased to moderate to high levels during 2012-2016. Resistance to indoxacarb was moderate during 2003-2006, which then decreased to low level during 2007-2010, very low level during 2011-2014, and no resistance during 2015 and 2016 corresponding to the reduced use of indoxacarb in the Pakistani agriculture. Piperonyl butoxide and tribufos exhibited a good synergism with indoxacarb and chlorfenapyr but not with spinosad in H. armigera populations. Rotation of new insecticides, having no, very low, and low resistance along with other IPM practices, is recommended to manage resistance to insecticides in H. armigera.

Mitochondrial DNA analysis of field populations of Helicoverpa armigera (Lepidoptera: Noctuidae) and of its relationship to H. zea.

BACKGROUND: Helicoverpa armigera and H. zea are amongst the most significant polyphagous pest lepidopteran species in the Old and New Worlds respectively. Separation of H. armigera and H. zea is difficult and is usually only achieved through morphological differences in the genitalia. They are capable of interbreeding to produce fertile offspring. The single species status of H. armigera has been doubted, due to its wide distribution and plant host range across the Old World. This study explores the global genetic diversity of H. armigera and its evolutionary relationship to H zea. RESULTS: We obtained partial (511 bp) mitochondrial DNA (mtDNA) Cytochrome Oxidase-I (COI) sequences for 249 individuals of H. armigera sampled from Australia, Burkina Faso, Uganda, China, India and Pakistan which were associated with various host plants. Single nucleotide polymorphisms (SNPs) within the partial COI gene differentiated H. armigera populations into 33 mtDNA haplotypes. Shared haplotypes between continents, low F-statistic values and low nucleotide diversity between countries (0.0017-0.0038) suggests high mobility in this pest. Phylogenetic analysis of four major Helicoverpa pest species indicates that H. punctigera is basal to H. assulta, which is in turn basal to H. armigera and H. zea. Samples from North and South America suggest that H. zea is also a single species across its distribution. Our data reveal short genetic distances between H. armigera and H. zea which seem to have been established via a founder event from H. armigera stock at around 1.5 million years ago. CONCLUSION: Our mitochondrial DNA sequence data supports the single species status of H. armigera across Africa, Asia and Australia. The evidence for inter-continental gene flow observed in this study is consistent with published evidence of the capacity of this species to migrate over long distances. The finding of high genetic similarity between Old World H. armigera and New World H. zea emphasises the need to consider work on both pests when building pest management strategies for either.