Baseline Susceptibility of the Field Populations of Ostrinia furnacalis in Indonesia to the Proteins Cry1A.105 and Cry2Ab2 of Bacillus thuringiensis.

Genetically modified MON 89034 corn (Zea mays L.) expressing Bacillus thuringiensis (Bt) insecticidal proteins, viz. Cry1A.105 and Cry2Ab2, is a biotechnological option being considered for the management of the major corn pest in Indonesia, the Asian corn borer (Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae)). As a part of a proactive resistance-management program for MON 89034 corn in Indonesia, we assessed the baseline susceptibility of field-collected populations of O. furnacalis to Cry1A.105 and Cry2Ab2 proteins. Dose-response bioassays using the diet-dipping method indicated that the lethal concentration (LC50) values of Cry1A.105 and Cry2Ab2 in 24 different field populations of O. furnacalis ranged from 0.006 to 0.401 µg/mL and from 0.044 to 4.490 µg/mL, respectively, while the LC95 values ranged from 0.069 to 15.233 µg/mL for Cry1A.105 and from 3.320 to 277.584 µg/mL for Cry2Ab2. The relative resistance ratios comparing the most tolerant field populations and an unselected laboratory population were 6.0 for Cry1A.105 and 2.0 for Cry2Ab2 based on their LC50 values. Some field populations were more susceptible to both proteins than the unselected laboratory population. The LC99 and its 95% fiducial limits across the field populations were calculated and proposed as candidate diagnostic concentrations. These data provide a basis for resistance monitoring in Bt Corn and further support building resistance-management strategies in Indonesia.

Host plant resistance for fall armyworm management in maize: relevance, status and prospects in Africa and Asia.

KEY MESSAGE: Sustainable control of fall armyworm (FAW) requires implementation of effective integrated pest management (IPM) strategies, with host plant resistance as a key component. Significant opportunities exist for developing and deploying elite maize cultivars with native genetic resistance and/or transgenic resistance for FAW control in both Africa and Asia. The fall armyworm [Spodoptera frugiperda (J.E. Smith); FAW] has emerged as a serious pest since 2016 in Africa, and since 2018 in Asia, affecting the food security and livelihoods of millions of smallholder farmers, especially those growing maize. Sustainable control of FAW requires implementation of integrated pest management strategies, in which host plant resistance is one of the key components. Significant strides have been made in breeding elite maize lines and hybrids with native genetic resistance to FAW in Africa, based on the strong foundation of insect-resistant tropical germplasm developed at the International Maize and Wheat Improvement Center, Mexico. These efforts are further intensified to develop and deploy elite maize cultivars with native FAW tolerance/resistance and farmer-preferred traits suitable for diverse agro-ecologies in Africa and Asia. Independently, genetically modified Bt maize with resistance to FAW is already commercialized in South Africa, and in a few countries in Asia (Philippines and Vietnam), while efforts are being made to commercialize Bt maize events in additional countries in both Africa and Asia. In countries where Bt maize is commercialized, it is important to implement a robust insect resistance management strategy. Combinations of native genetic resistance and Bt maize also need to be explored as a path to more effective and sustainable host plant resistance options. We also highlight the critical gaps and priorities for host plant resistance research and development in maize, particularly in the context of sustainable FAW management in Africa and Asia.

Development of marker-free insect resistant transgenic okra (Abelmoschus esculentus L. Moench) expressing the cry1Ac gene and identification of vector backbone-free events.

Agrobacterium-mediated co-transformation method was used to generate marker-free insect resistant transgenic okra plants expressing the cry1Ac gene. The cry1Ac gene was borne on the T-DNA of one plasmid while nptII and uidA (GUS) marker genes were present on the T-DNA of a second plasmid. Putative transgenic plants were screened by histochemical GUS assay for expression of -glucuronidase and 32 transgenic events were positive for GUS in which 21 transgenic events were positive in ELISA for the presence of Cry1Ac protein. Out of 21 Cry1Ac positive T0 events, three events displayed Mendelian inheritance of the transgenes in (9:3:3:1 ratio) T1 generation for Cry1Ac and GUS. Selected events were chosen for further genetic and molecular analysis. The cry1Ac and marker genes were found to segregate independently, of each other in 10 events in T1 generation out of 11 Cry1Ac gene inheriting events analysed indicating that the two T-DNAs insertions were genetically unlinked and identification of marker-free plants were possible in these 10 events. The marker-free nature and vector backbone-free Bt events (clean T-DNA insertions carrying cry1Ac gene) were confirmed by Southern analysis using suitable probes. The plants from selected transgenic events were rigorously screened in whole plant insect bioassays using the larvae of shoot and fruit borer, Earias vittella, an important pest of okra. Insect bioassays indicated 100% larval mortality without any infestation in five of the transgenic events and two events showed 5 to 10 percent infestation establishing the insect resistant nature of the transgenic plants. Finally the events inheriting transgenes in Mendelian fashion were characterized further and marker-free and vector backbone-free events were identified showing complete protection from the target pest Earias vittella in whole-plant insect bioassays. Quantification of Cry1Ac protein levels in the plant parts of selected events (lines) was consistent with the results of bioassays. Further, two lines identified in this study met the criteria for inclusion in commercial breeding programs.

Physico-chemical studies on the interaction of dendrimers with lipid bilayers. 1. Effect of dendrimer generation and liposome surface charge.

Studies on the interaction of different generation poly (amido amine) (PAMAM) dendrimers (2G, 4G and 6G) and liposomes of different compositions were carried out by a combined turbidity, dynamic light scattering and atomic force microscopic measurements. Liposomes comprising soy lecithin (SLC, negative surface charge), 1, 2-palmitoyl-sn-glycero-3-phosphatidylcholine (DPPC, mildly positive surface charge), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol (DPPG, negatively charged) and a biologically simulated mixture of DPPC + DPPG (7:3, M/M, negatively charged) were used as model bilayers. 30 wt% cholesterol was used in each combination as it is known to control the fluidity of membrane bilayers. Silica was used as a negatively charged hard sphere model with an aim to compare the results. Both the turbidity and hydrodynamic diameter values of all the liposomes, except DPPC, passed through maxima upon the progressive addition of PAMAM; the effect was insignificant in case of DPPC. Formation of dendriosome, a complex formed between dendrimer and liposome, resulted in the charge reversal of the negatively charged liposomes. Interaction between PAMAM and liposome was found to be governed by electrostatic as well as hydrogen bonding. Generation dependent PAMAM activity followed the order: 6G >4G>2G in terms of overall dendrimer concentration. However, interestingly, the order was reverse when PAMAM activity was considered in terms of total end group concentrations. AFM studies reveal the rupture of bilayer structure upon addition of dendrimer.

Efficient genetic transformation of okra (Abelmoschus esculentus (L.) Moench) and generation of insect-resistant transgenic plants expressing the cry1Ac gene.

KEY MESSAGE: Agrobacterium -mediated transformation system for okra using embryos was devised and the transgenic Bt plants showed resistance to the target pest, okra shoot, and fruit borer ( Earias vittella ). Okra is an important vegetable crop and progress in genetic improvement via genetic transformation has been impeded by its recalcitrant nature. In this paper, we describe a procedure using embryo explants for Agrobacterium-mediated transformation and tissue culture-based plant regeneration for efficient genetic transformation of okra. Twenty-one transgenic okra lines expressing the Bacillus thuringiensis gene cry1Ac were generated from five transformation experiments. Molecular analysis (PCR and Southern) confirmed the presence of the transgene and double-antibody sandwich ELISA analysis revealed Cry1Ac protein expression in the transgenic plants. All 21 transgenic plants were phenotypically normal and fertile. T1 generation plants from these lines were used in segregation analysis of the transgene. Ten transgenic lines were selected randomly for Southern hybridization and the results confirmed the presence of transgene integration into the genome. Normal Mendelian inheritance (3:1) of cry1Ac gene was observed in 12 lines out of the 21 T0 lines. We selected 11 transgenic lines segregating in a 3:1 ratio for the presence of one transgene for insect bioassays using larvae of fruit and shoot borer (Earias vittella). Fruit from seven transgenic lines caused 100 % larval mortality. We demonstrate an efficient transformation system for okra which will accelerate the development of transgenic okra with novel agronomically useful traits.

Off-pump coronary artery bypass in congenital absence of pericardium.

Off-pump coronary artery bypass involves precise manipulations for proper positioning the heart without causing hemodynamic compromise. Pericardial stay sutures exerting appropriate traction play an important role in off-pump surgery. We report a rare case in which a congenital pericardial defect was discovered after sternotomy in a patient undergoing off-pump coronary artery bypass.

Mechanistic insight into cell growth, internalization, and cytotoxicity of PAMAM dendrimers.

We report on the role of PAMAM dendrimer concentration and generation (G2, G4, G6) on cell growth and cytotoxicity in HEK293T and HeLa cell lines and make comparisons with dendrimer-induced leakage from liposomes to probe the mechanisms in action. Specifically, we observed a striking transition from cell growth enhancement to a reduction in cell viability at a critical PAMAM dendrimer concentration, that is, approximately 500 nM. Confocal microscopy studies show evidence of a transition from cell membrane adhesion to cell internalization and cell nucleus interaction at equivalent dendrimer concentrations. A dendrimer concentration window of 500-700 nM was identified for effective cell internalization without significant cytotoxicity. Though liposome leakage correlated with cytotoxicity, no quantitative agreement was observed, that is, cells are 100 times (based on surface coverage) more resistant to dendrimers than liposomes. These findings have significant implications in the design of effective drug/gene delivery vehicles based on dendrimers.

PAMAM dendrimer interactions with supported lipid bilayers: a kinetic and mechanistic investigation.

The interaction kinetics of polyamidoamine (PAMAM) dendrimers with supported lipid bilayers of 1,2-sn-glycero-dimyristoylphosphocholine prepared by the vesicle deposition has been probed by optical waveguide lightmode spectroscopy and atomic force microscopy (AFM). In particular, the influence of PAMAM dendrimer generation (G2, G4, and G6) and concentration (1 to 100 nM) on the levels of adsorption and lipid bilayer removal have been determined as a function of time; hence interaction kinetics and mechanisms have been further elucidated. Dendrimer interaction kinetics with the lipid bilayer are concentration dependent in a complex manner, with net bilayer removal at 1 and 100 nM and net adsorption at 10 nM; these effects are irrespective of dendrimer generation. The pseudo first order rate constant for bilayer removal (at 1 and 100 nM) follows the order G6 > G4 > G2. In contrast, the pseudo first order rate constant for adsorption at 10 nM follows the order G2 > G4 > G6. AFM has confirmed expansion of lipid bilayer defects, hole formation, and adsorption to the bilayer or bilayer defects, and their concentration and generation dependence. These findings have implications when designing dendrimers for specific biopharmaceutical activities, e.g., as drugs, drug delivery vehicles, transfection agents, or antimicrobials.

Monitoring western corn rootworm (Coleoptera: Chrysomelidae) susceptibility to carbaryl and cucurbitacin baits in the areawide management pilot program.

Areawide pest management involves the uniform application of a pest control strategy over wide geographic areas. Therefore, these programs are likely to impose intense selective pressures, and the risk for resistance development among pest species for which areawide management programs are implemented is likely to be high. Pilot studies for areawide management of western corn rootworm, Diabrotica virgifera virgifera LeConte, were conducted from 1996 to 2002 at four different sites across the Corn Belt. This program used cucurbitacin baits to deliver high doses of a traditional neurotoxic insecticide (carbaryl) to individual insects while reducing the overall rate of insecticide use. Because of the concern and potential for resistance evolution, annual assessments of susceptibility to the active ingredient carbaryl were conducted both within the managed area as well as from untreated control areas. Significantly reduced susceptibility to carbaryl based on survival at a diagnostic concentration was detected in three of the four management sites (Kansas, Iowa, and Illinois/Indiana), whereas susceptibility of beetles collected outside the managed areas remained unchanged. Additionally, significantly reduced responsiveness to cucurbitacin baits was observed in beetles collected from the managed area relative to the control area at the same three sites. These results suggest strongly that areawide management has the potential to select for resistance and that a strategy for managing resistance and reducing selective pressure should be proactively implemented.

Inheritance of methyl-parathion resistance in Nebraska western corn rootworm populations (Coleoptera: Chrysomelidae).

Field populations of western corn rootworm, Diabrotica virgifera virgifera LeConte, were collected from three different sites (York Co., Phelps Co., and Saunders Co.) in Nebraska during 1996. Adult bioassays of these three populations were conducted with different concentrations of methyl-parathion and at a diagnostic concentration (1.0 microg/ml) to determine resistance levels among these populations. Self and reciprocal crosses were made between the two resistant and one susceptible laboratory-reared populations. Dose-responses and dominance ratios calculated for the four reciprocal crosses indicated that resistance was incompletely dominant in both strains, although in one of the strains there was an indication of sex linkage. However, evaluation of native polyacrylamide gels stained for nonspecific esterases and nonspecific esterase activity of parents and F1 progeny of the crosses suggested that esterase inheritance was completely dominant and autosomal. The results of this study were inconclusive with regard to the precise nature of inheritance, because the bioassays and esterase assays could not discriminate between heterozygotes and homozygotes. However, they do provide insight into the potential for developing simple diagnostic assays to assess resistance frequencies. Based on the inheritance studies described in this investigation, we can begin to generate information on specific genetic factors that dictate the evolutionary divergence of discrete resistant populations and facilitate modeling efforts designed to approximate the movement of genes for resistance among populations.

Diagnostic assays based on esterase-mediated resistance mechanisms in western corn rootworms (Coleoptera: Chrysomelidae).

Resistance to methyl-parathion among Nebraska western corn rootworm, Diabrotica virgifera virgifera LeConte, populations is associated with increased hydrolytic metabolism of an organophosphate insecticide substrate. An electrophoretic method to identify resistant individuals based on the staining intensity of esterase isozymes on nondenaturing polyacrylamide gels was developed. Three groups of esterases (I, II, and III) were visible on the gels, but only group II esterase isozymes were intensified in resistant populations. A total of 26 and 31 field populations of western corn rootworms from Nebraska (in 1998 and 1999, respectively) were assessed with nondenaturing polyacrylamide gel electrophoresis (PAGE) assays and diagnostic concentration bioassays. Significant correlations were observed between the two diagnostic assays. Group II esterase isozymes provide a reliable biochemical marker for detection of methyl-parathion resistance in individual western corn rootworms and a tool for monitoring the frequency of resistant individuals in field populations.