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Suberin in roots acts as a physical barrier preventing water/mineral losses. In Arabidopsis, root suberization is regulated by abscisic acid (ABA) and ethylene in response to nutrient stresses. ABA also mediates coordination between microbiota and root endodermis in mineral nutrient homeostasis. However, it is not known whether this regulatory system is common to plants in general, and whether there are other key molecule(s) involved. We show that serotonin acts downstream of ABA in regulating suberization in rice and Arabidopsis and negatively regulates suberization in rice roots in response to salinity. We show that ABA represses transcription of the key gene (OsT5H) in serotonin biosynthesis, thus promoting root suberization in rice. Conversely, overexpression of OsT5H or supplementation with exogenous serotonin represses suberization and reduces tolerance to salt stress. These results identify an ABA-serotonin regulatory module controlling root suberization in rice and Arabidopsis, which is likely to represent a general mechanism as ABA and serotonin are ubiquitous in plants. These findings are of significant importance to breeding novel crop varieties that are resilient to abiotic stresses and developing strategies for production of suberin-rich roots to sequestrate more COsub2/sub, helping to mitigate the effects of climate change.

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or 'vitreous' phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Starch is the primary form of reserve carbohydrate storage in plants. Rice (

Proteomics can map extracellular vesicles (EVs), including exosomes, across disease states between organisms and cell types. Due to the diverse origin and cargo of EVs, tailoring methodological and analytical techniques can support the reproducibility of results. Proteomics scans are sensitive to in-sample contaminants, which can be retained during EV isolation procedures. Contaminants can also arise from the biological origin of exosomes, such as the lipid-rich environment in human milk. Human milk (HM) EVs and exosomes are emerging as a research interest in health and disease, though the experimental characterization and functional assays remain varied. Past studies of HM EV proteomes have used data-dependent acquisition methods for protein detection, however, improvements in data independent acquisition could allow for previously undetected EV proteins to be identified by mass spectrometry. Depending on the research question, only a specific population of proteins can be compared and measured using isotope and other labelling techniques. In this review, we summarize published HM EV proteomics protocols and suggest a methodological workflow with the end-goal of effective and reproducible analysis of human milk EV proteomes.

The interaction of wheat puroindolines (pin) with starch granules from a pin-null cereal crop, rice, was investigated using an in vitro immunofluorescent method. Rice seed cross-sections were incubated with soft (Augusta) and hard (Langdon) durum wheat extracts. The in vitro localization of puroindolines on rice seed cross sections were compared to the in vivo localization of puroindolines from transgenic Pin+ rice seeds using the Durotest anti-Pin monoclonal antibody. Wheat puroindolines were observed to localize with high affinity for rice starch granule surfaces both in vitro and in vivo. The strong interactions of puroindolines with starch granules plays important roles in both plant defense and grain texture. The obtained results confirmed that puroindolines interact with starch granule surfaces in rice, a pin-null cereal, and that an in vitro localization method can be used to predict the in vivo targeting and deposition of puroindoline proteins.

Biodiesel is a low-carbon-intensity renewable fuel with up to 99% lower greenhouse gas emissions than petroleum-based diesel. The use of oil crops for biodiesel is under critical examination. It is expensive and suffers from the food versus fuel risk/benefit problem. Consequently, many countries (e.g. Malaysia and countries in the EU) are scaling back the use of oil crops as feedstock for biofuel production. The limitations of these traditional crops are leading the renewable fuels industry to consider innovative, sustainable, and profitable biomass-based platforms. Plant genetic engineering and other new breeding technologies are essential for developing such biomass-based platforms because they enhance plant tolerance to abiotic and biotic stresses, resulting in higher feedstock yields, greater net energy gain, and the generation of high-value co-products. We review and summarize the recent improvements of oil crops through plant genetic engineering that may increase widespread and cost-effective production of biodiesel and value-added co-products for green chemistry applications. © 2017 Society of Chemical Industry and John Wiley & Sons, Ltd

Gibberella ear rot is a disease of maize associated with low yields and the production of harmful mycotoxins therein. The disease is caused by the infection of hostZea mayswith fungal pathogenFusarium graminearum. Resistant (CO441) and susceptible (CL30) inbred maize line kernels were inoculated with conidial suspensions ofF. graminearumor water (controls). Ears of maize (cobs) from each line were harvested upon maturation and proteins were extracted from the embryo tissue of the kernels to study tissue-specific response of the host. Embryo proteins from both CO441 and CL30 lines were sequenced using mass spectrometry (LC-MS/MS) and quantified using Label Free Quantification (LFQ). Following filtering, 509 proteins were identified. These proteins were grouped into nine functional categories:Fusarium-derived, late embryogenesis abundant, oil-body, metabolism, stress, cellular, protein storage, metabolism, and defense. Defense proteins were up-regulated in response to infection in both CO441 and CL30 lines. Furthermore,F. graminearumderived proteins were only found in CL30 infected kernels suggesting that resistance may be attributed in part to the inability ofFusariumto establish itself in the embryo. To our knowledge this is the first successful application of LFQ mass spectrometry to the study of host-pathogen response toF. graminearum.Biological significanceFungal pathogenFusarium graminearumis responsible for billion dollar losses in crops and contamination of global grains with harmful mycotoxins. By studying host-pathogen interactions ofFusariumand maize on a proteomic level with resistant and susceptible genotypes, the biological interactions occurring during infection of the maturing seed were characterized. Mature kernels of theF. graminearumsusceptible maize line CL30 and resistant CO441 line were dissected to permit a proteomic survey of the new sporophytic generation, the embryo. Detailed knowledge of this Host-pathogen interactome will assist development of new cereal lines resistant to the rot diseases caused byFusarium graminearum.HighlightsSusceptible (CL30) and Resistant (CO441) lines were injected with water mock or F. graminearum LC-MS/MS of maize embryo protein extracts followed by Label Free Quantification (LFQ) permitted identification, quantification and comparison of proteomes between maize genotypes and treatments Fusarium-derived proteins were abundant only in the susceptible infected embryo Defense proteomes were up-regulated in both lines following infection nsLTP and Protease Inhibitor were significantly over-expressed in the Susceptible line after infection; chitinase and WIP1 were significantly over-expressed in the Resistant line after infection

The ‘triple-blue’ cultivar of blue spruce (Picea pungens Hoopsii) is notably recalcitrant towards the realm of traditional vegetative propagation methods. Its ability to naturally proliferate is limited by ovule and embryo abortion during the growing season, leading to low viable seed yield. In this study, we established a protocol using somatic embryogenesis (SE) as a means of propagating this popular ornamental cultivar. We collected cones from Hoopsii trees at seven different timepoints throughout the growing season (mid-June to late July in Ottawa (Plant Hardiness Zone 5A)). Female megagametophytes were harvested following each collection and immature zygotic embryos were plated onto induction media. Early somatic embryos began developing from the embryonic tissue (ET) three to five weeks following induction. The highest ET initiation frequency occurred from embryos collected June 20–July 10, suggesting that developmental stage of the embryo was a significant factor in SE induction. The conversion of mature somatic embryos into plantlets (emblings) was completed in eight–ten weeks at a rate of 92.8%. In this study, we demonstrate that in vitro somatic embryogenesis using our optimized protocol is a fast and prolific method for the mass propagation of Hoopsii blue spruce. This is the first report on the production of somatic Hoopsii emblings.

Wan, S., Greenham, T., Goto, K., Mottiar, Y., Johnson, A. M., Staebler, J. M., Zaidi, M. A., Shu, Q. and Altosaar, I. 2014. A novel nitrous oxide mitigation strategy: expressing nitrous oxide reductase from Pseudomonas stutzeri in transgenic plants. Can. J. Plant Sci. 94: 1013–1025. As a stable greenhouse gas, nitrous oxide (N2O) plays a significant role in stratospheric ozone destruction. The primary anthropogenic N2O source is the use of nitrogen in agriculture. Currently, the annual N2O emissions from this soil–plant–microbial system is more than 2.6 Tg (1 Tg=1 million metric tonnes) of N2O-N globally. So it is important to explore some innovative and effective biology-based strategies for N2O mitigation. If shown to be effective in field trails as well as laboratory-scale experiments, such GMO plants could help guide international policies on adaptation to climate change. The bacterial enzyme nitrous oxide reductase (N2OR) is the only known enzyme capable of catalyzing the final step of the denitrification pathway, conversion of N2O to N2. To “scrub” the N2O emissions, bacterial N2OR was heterologously expressed in plants. Structurally, the enzyme N2OR is encoded by nosZ, but its biosynthesis and assembly in prokaryotes require the products of several nos genes, including a putative ABC-type transporter encoded by nosDFY, and the copper chaperone NosL for biogenesis of the metal centre. We have generated transgenic tobacco plants expressing the nosZ gene, as well as tobacco plants in which the other nos genes were co-expressed under the control of a root-specific promoter (rolD) and a constitutive promoter (d35S). The nosZ gene from Pseudomonas stutzeri heterologously expressed in tobacco produced active recombinant N2OR. The positive results in the preliminary proof-of-principle experiments indicated that plants heterologously expressing N2OR could mitigate emissions at the source before N2O reaches the stratosphere or troposphere.

Soluble CD14 (sCD14) is a pattern recognition receptor and Toll-like co-receptor observed in human milk (5–26μg/mL) and other bodily fluids such as blood (3μg/mL). The most well defined role of sCD14 is to recognize lipopolysaccharide of Gram-negative bacteria and signal an immune response through Toll-like receptor 4 (TLR4). Previous research has shown ingested sCD14 to transfer from the gastrointestinal tract and into the blood stream in neonatal rats. The contribution of human milk sCD14 to circulating levels in the infant and the functionality of the protein, however, remained unknown. Using CD14−/− mouse pups fostered to wild type (WT) mothers expressing sCD14 in their milk, we show herein that ingestion of sCD14 resulted in blood sCD14 levels up 0.16±0.09μg/mL. This represents almost one-third (26.7%) of the circulating sCD14 observed in WT pups fostered to WT mothers (0.60±0.14μg/mL). We also demonstrate that ingested-sCD14 transferred to the blood remains functional in its ability to recognize lipopolysaccharide as demonstrated by a significant increase in immune response (IL-6 and TNF-α) in CD14−/− pups fostered to WT mothers in comparison to control animals (P=0.002 and P=0.007, respectively). Using human intestinal cells (Caco-2), we also observed a significant decrease in sCD14 transcytosis when TLR4 was knocked down (P

Insect pests are the major constraints of grain legumes production and storage. Improvement through conventional breeding strategies has been limited by the lack of resistance traits within the gene pool for most of the economically important pests. This gap can be closed by transgenic approaches using resistance genes from different sources. In this study, we report the development of insect resistant transgenic peas expressing a plant codon optimized cry1Ac gene from Bacillus thuringiensis. The transgenic nature of regenerated in vitro plants and their segregating progenies has been confirmed through molecular analyses (PCR, Southern blot, RT-PCR and immunostrip assay). The introduced transgene was inherited up to the T4 generation. Insect bioassay using larvae of tobacco budworm indicated total larval mortality and significantly reduced feeding damage on the developed transgenic pea plants as compared to 85% larval survival and heavy feeding damage on non-transgenic control plants. The developed transgenic lines can be used for further studies such as gene stacking and field trials.

Plant expression of the entomopathogenic bacteria Bacillus thuringiensis cry gene has reduced the damage created by insect pests in several economically important cultures. For this study, we have conducted genetic transformation of the indica rice ‘IRGA 424,’ via Agrobacterium tumefaciens, using the B. thuringiensis cry1Aa and cry1B genes, with the objective of obtaining rice plants resistant to the insect pests from this culture. The gene constructions harbor the promoters maize proteinase inhibitor and ubiquitin. The results showed that high concentration of the hormone 2,4-dichlorophenoxyacetic acid and agarose as the gelling agent helped the production of embryogenic calli for the analyzed cultivar. More than 80% of the obtained transformed plants revealed the integration, using polymerase chain reaction, of the cry1Aa and cry1B genes. Analysis of the expression of the heterologous protein by Western blotting revealed the expression of the Cry1B δelta-endotoxin in IRGA 424 plants transformed...

Background Milk contains immunological constituents that comprise an edible immune system conveyed from mother to newborn. Soluble Cluster of Differentiation 14 (sCD14) is a protein found in significant quantities in human milk (~8–29 μg/ml). At a tenfold lower concentration in the blood (~3 μg/ml), the most notable role of sCD14 is to sequester lipopolysaccharide of Gram-negative bacteria from immune cells. Methods To explore the pharmacodynamics of this milk protein and its biological fate, the biodistribution of radiolabeled sCD14 (14C, 125I) was monitored in 10 d old rat pups. Results Up to 3.4 ± 2.2% of the radiolabeled-sCD14 administered was observed, intact, in the pup blood for up to 8 h post-ingestion. Additionally, 30.3 ± 13.0% of the radiolabeled-sCD14 administered was observed degraded in the stomach at 8 h post-ingestion. A reservoir of intact, administered sCD14 (3.2 ± 0.3%), however, remained in the stomach at 8 h post-ingestion. Intact sCD14 was observed in the small intestine at 5.5 ± 1.6% of the dose fed at 8h post-ingestion. Conclusions The presence of intact sCD14 in the blood and gastrointestinal tract of newborns post-ingestion has implications in the development of allergies, obesity and other inflammation-related pathogeneses later in life.

Applications of gene technology in agriculture, the environment and human health fields are reviewed. This case study of the intricate historical details of the development of Bt crops like cotton and rice unveils essential elements of productive funding schemes and effective multinational collaborations. Gene technology applied to pest resistance traits in global cotton is analyzed using nation-specific data from India to demonstrate ‘ricochet’ results: Regulatory approval for one crop catalyzes an ‘Enhancer Effect’ for promoting more research funding and more competitive results for other crops-in-waiting, namely rice. Just as cotton commerce promoted philanthropy in unpredictable situations like the Kreenholm dynasty of Ludwig Knoop, research budgets for pesticide and biocide technology have yielded intended effects, but several surprising unintended effects as well. Finally, the case is made for greater control of gene flow and identity preserve issues in plant biotechnology research by invoking Appellation d’Origine Controlee for Bt genes.

Nitrous oxide (N 2 O), the third most abundant greenhouse gas (GHG), is highly stable and plays a significant role in stratospheric ozone destruction. The primary anthropogenic source of N 2 O stems from use of nitrogen fertilizers in soil. The bacterial enzyme nitrous oxide reductase (N 2 OR), naturally found in some soils, is the only known enzyme capable of catalyzing the final step of the denitrification pathway, conversion of N 2 O to N 2 . In this opinion, we discuss potential biology-based strategies to reduce N 2 O by amplifying the amount of available enzyme catalyst in agri-system environments during crop growth and in post-harvest detritus. N 2 OR from Pseudomonas stutzeri has been tested in transgenic plants with promising results. Such seed-borne phytoremediation systems targeted towards GHGs merit field testing.

We hypothesize that amylose is an important in planta sink for iodine. Moreover, starch with greater amylose content could be capable of sequestering higher levels of this important micronutrient. Diets low in iodine can result in goitre, cretinism and a host of other serious medical conditions. Since nearly two billion people worldwide still receive insufficient dietary iodine despite salt iodisation programmes, iodine deficiency disorders (IDD) constitute a serious global health issue in need of innovative solutions strengthened by interdisciplinary systems approaches ( Bouis and Welch, 2010 , World Health Organisation, 2007 ). Iodine can be sequestered within the helical V-amylose component of starch in the form of polyiodide chains ( Yu, Houtman, & Atalla, 1996 ). We argue that stable iodine complexation in high-amylose staple crops could contribute an invaluable and critical biofortification tactic to prevent IDD.

Nitrous oxide (N2O) is a potent greenhouse gas (GHG). Although it comprises only 0.03% of total GHGs produced, N2O makes a marked contribution to global warming. Much of the N2O in the atmosphere issues from incomplete bacterial denitrification processes acting on high levels of nitrogen (N) in the soil due to fertilizer usage. Using less fertilizer is the obvious solution for denitrification mitigation, but there is a significant drawback (especially where not enough N is available for the crop via N deposition, irrigation water, mineral soil N, or mineralization of organic matter): some crops require high-N fertilizer to produce the yields necessary to help feed the world’s increasing population. Alternatives for denitrification have considerable caveats. The long-standing promise of genetic modification for N fixation may be expanded now to enhance dissimilatory denitrification via genetic engineering. Biotechnology may solve what is thought to be a pivotal environmental challenge of the 21st century, reducing GHGs. Current approaches towards N2O mitigation are examined here, revealing an innovative solution for producing staple crops that can ‘crack’ N2O. The transfer of the bacterial nitrous oxide reductase gene (nosZ) into plants may herald the development of plants that express the nitrous oxide reductase enzyme (N2OR). This tactic would parallel the precedents of using the molecular toolkit innately offered by the soil microflora to reduce the environmental footprint of agriculture.

The starch granule surface is a frontline of microbial attack and defence, operating in the background of normal starch granule metabolism. Puroindoline, a wheat protein which binds starch granule surfaces, contains a unique tryptophan-rich domain likely responsible for this property, though direct evidence is lacking. To test puroindoline’s tight association, prime starch granule extracts were water-washed 8 or 20 times and residual puroindoline removed using a solution of 50% isopropanol/50 mM NaCl. We found that this solvent was consistent in the amount of protein extracted from wheat flour and washed starch, regardless of initial protein content. Relative quantification of puroindoline following water-washing was performed using dot blot. Washing more than 8 times did not further reduce puroindoline content of starch granules suggesting a strong association with the starch granule surface. To identify the tryptophan-rich domain tightly associated with the starch granule surface, a combination of in situ tryptic digestion and mass spectrometry was used. Following digestion and water-washing, 50% isopropanol/50 mM NaCl was used to remove tightly-associated peptides for identification by mass spectrometry. Using this method, we identified the tryptophan-rich domain of puroindoline directly bound to the starch granule surface of wheat.

Endosperm texture in wheat is controlled by the Pina and Pinb genes that comprise the Hardness (Ha) locus. Studies have shown that soft and hard varieties differ in the amount of starch bound polar lipids but have not addressed whether PINs are directly involved and whether the presence of one particular PIN affects seed polar lipid levels and cellular localization. Here, we determined the effect of overexpressing PINA or PINB on seed bound polar lipids and PIN localization. F3 recombinants homozygous for either a Pina or Pinb null Ha locus with or lacking a transgenically added Pina or Pinb were analyzed for grain hardness, PIN abundance, and seed bound polar lipid levels. Overexpressed PINs resulted in reduced hardness, increased starch bound PINs, and increased seed bound polar lipids. Addition of PINA to the PINA nulls or PINB to the PINB nulls resulted in higher bound polar lipid levels than the addition of the alternative PIN. Both PINs localized to the starch surface in the presence or absence of the other protein. Our results indicate that PIN overexpression results in reduced endosperm texture and increased seed bound polar lipids and that PINs independently localize to the surface of starch granules.

Porcine trypsin was glycated with glucose and covalently immobilized through its carboxyl groups onto aminated glass beads to produce porcine immobilized glycated-trypsin (IGT). On incubation at 60 °C and pH 8, IGT retained its full activity for 8 h and 50% of its activity after 24 h. In comparison, under the same conditions porcine native trypsin lost 80% of its activity in 2 h and was completely inactivated in less than 4 h. The rate of autolysis of porcine glycated-trypsin at 37 °C was 40% that of native trypsin and with IGT there was no significant autolysis, even at elevated temperatures as high as 60 °C. Glycation significantly increased the stability of trypsin and immobilization also significantly increased the stability of trypsin. The remarkable thermostability of IGT is attributed to a synergistic effect when these two modifications are combined. Tryptic fragmentation of denatured proteins with IGT can be performed at 60 °C for shorter digestion times and with smaller amounts of enzyme than normally employed to achieve complete digestion with soluble forms of trypsin. Prior denaturation of proteins for tryptic digestion is not required with IGT as in situ denaturation and digestion can be achieved simultaneously at 60 °C with an enzyme:protein mass ratio as low as 1:1000.

Expression of cry1Ac gene from Bacillus thuringiensis (Bt) was evaluated under the control of a wound-inducible AoPR1 promoter from Asparagus officinalis in transgenic tobacco plants. The leaves of transgenic plants were mechanically wounded to evaluate the activity of the AoPR1 promoter in driving the expression of Cry1Ac protein at the wound site. Our results indicate that mechanical wounding of transgenic plants was effective in inducing the expression of Cry1Ac protein. As a result of this induction, the accumulated levels of Cry1Ac protein increased during 6–72 h post-wounding period. The leaves of transgenic tobacco plants were evaluated for resistance against Heliothis virescens and Manduca sexta in insect bioassays in two different ways. The detached tobacco leaves were either fed directly to the insect larvae or they were first mechanically wounded followed by a 72 h post-wounding feeding period. Complete protection of mechanically wounded leaves of transgenic plants was observed within 24 h of the bioassay. The leaves of transgenic plants fed directly (without pre-wounding) to the larvae achieved the same level of protection between 24 and 72 h of the bioassay.

A novel method for cloning of genes coding for cytotoxic molecules based on a cell viability assay is described. The working hypothesis is that expression of DNA sequences coding for cytotoxic molecules in bacterial cells will lead to cell death or impairment, and the isolation of the impaired or dead cells could lead to identification of DNA sequences responsible for debilitating the host cells. We verified this concept by isolating the well known antimicrobial Puroindoline b gene in Escherichia coli cells. We further demonstrated the feasibility to use this approach for isolating DNA encoding for antimicrobials from cDNA expression libraries. Sequence analysis and bioassay indicated that the isolated clones encoded previously characterized antimicrobial proteins (AMPs), proteins not previously characterized as AMPs, as well as novel antimicrobial peptides. In addition, clones harboring ribosomal protein encoding cDNA were also identified. Therefore, this method could also be used to identify host genes important in maintaining bacterial cell viability.

A high throughput method for screening cDNA libraries has been developed to identify putative antimicrobial peptides (AMPs). It is based on a rapid dye inclusion assay for assessing antagonism of bacterial viability. Colonies are grown on a membrane on a permissive medium until full colony size is reached. The membrane, supporting the array of colonies, is transferred onto an inductive medium containing a vital dye. Upon expression of any antagonizing peptides, the cell membrane becomes compromised allowing dye infusion to permit visual identification of deleterious peptides. Our approach was validated by screening a synthetic oligonucleotide library expressed in Escherichia coli. A random oligonucleotide library, containing inserts of up to 75 nucleotides in length was constructed and expressed in E. coli. From a potential pool of 100000 peptides, in a single round of screening, three were found to be antimicrobial: L1, L3, and L8. Peptide L1 was shown to have a concentration-dependent bactericidal effect against Gram-negative E. coli and moderate biostatic activity against the Gram-positive bacteria Listeria monocytogenes. L8 was found to have bacteriostatic, and possibly bactericidal effect against E. coli, Pseudomonas aeruginosa and Salmonella typhimurium. These results validated this high throughput AMP identification assay based on filter bound colony array libraries and vital dye inclusion.

Transgenic Korean rice plants containing the cry1Ab gene were developed for resistance against yellow stem borer (Scirpophaga incertulas, YSB). More than 100 independent transgenic lines from three Korean varieties (P-I, P-II and P-III) were generated. The amount of Cry1Ab in transgenic T0 plants was as high as 2.88% of total soluble proteins. These levels were sufficient to cause 100% mortality of YSB larvae. The majority of T1 transgenic lines originated from the varieties P-I and P-II followed a Mendelian fashion of segregation. Deviation from the expected segregation ratio was observed in a small number of the transgenic lines of P-I and P-II origins. However, this deviation was primarily observed in the P-III originated lines. Segregation analysis of the T1 generation indicated that 1–3 copies of the cry1Ab gene were integrated into the genome of the majority of the transgenic lines originating from varieties P-I and P-II. Stunted and semi-fertile mutants were observed in some transgenic lines. These aberrations were either independent or closely linked to the introduced cry1Ab gene loci in different transgenic lines. Reduction in GUS expression levels and loss of toxicity against YSB larvae were found in some transgenic lines. The transgenic T3 and T4 lines causing 100% mortality of third instar YSB larvae in the lab were completely protected in the field. Analysis of important yield components on nine selected transgenic lines indicated that stem length, panicle length, grain number per panicle, and seed setting rates were reduced in transgenic plants compared to those in non-transgenic parental rice lines. Number of panicles per cluster, however, was significantly higher in transgenic plants. The numerical value of the average yield was in general greater in the controls than in all the transgenic lines, indicating some ‘yield drag’. Since some selected lines were highly resistant to the YSB with good yielding potential, they offer effective potential for use in insect resistance management programs.

Somatic embryos of the commercial soybean (Glycine max) cultivar IAS5 were co-transformed using particle bombardment with a synthetic form of the Bacillus thuringiensis delta-endotoxin crystal protein gene cry1Ac, the beta-glucuronidase reporter gene gusA and the hygromycin resistance gene hpt. Hygromycin-resistant tissues were proliferated individually to give rise to nine sets of clones corresponding to independent transformation events. The co-bombardment resulted in a co-transformation efficiency of 44%. Many histodifferentiated embryos and 30 well-developed plants were obtained. Twenty of these plants flowered and fourteen set seeds. The integration and expression of the cry1Ac, gusA and hpt transgenes into the genomes of a sample of transformed embryos and all T0, T1, T2 and T3 plants were confirmed by Gus activity, PCR, Southern and western blot, and ELISA techniques. Two T0 plants out of the seven co-transformed plants produced seeds and were analyzed for patterns of integration and inheritance until the T3 generation. Bioassays indicated that the transgenic plants were highly toxic to the velvetbean caterpillar Anticarsia gemmatalis, thus offering a potential for effective insect resistance in soybean.

Transgenic rice containing insecticidal protein(s) from Bt (Bacillus thuringiensis) effectively control lepidopteran pests under laboratory and field conditions. However, Bt rice has not been widely commercialized. A major concern regarding the deployment of Bt rice is its potential impact on non-target arthropods, including herbivorous insect pests and biological control agents (predators and parasitoids) in the rice ecosystem. A 2-year field study was conducted at 3 sites in Zhejiang Province, China to assess the impacts of a homozygous transgenic japonica rice line KMD1 (Bt rice) containing a synthetic cry1Ab gene from Bacillus thuringiensis Berliner. The impacts on three planthoppers (Homoptera: Delphacidae), Sogatella furcifera (Horvath), Nilaparvata lugens (Stal) and Laodelphax striatellus (Fallen), and the natural enemy, Cyrtorhinus lividipennis (Reuter) (Hemiptera: Miridae), were evaluated using two sampling methods: vacuum-suction and Malaise trap. Population densities of planthoppers and C. lividipennis were not significantly affected by rice type (Bt/non-Bt) over the season at any of the 3 sites in each year, regardless of sampling methods. Both in Bt rice and non-Bt rice plots, S. furcifera was the predominant species of planthoppers as determined by either of the two sampling methods, and comprised >50% of the planthoppers at each site. No consistent effects of Bt rice and Bt rice × sampling date interaction on population dynamics of the predominant planthopper species, S. furcifera, and the predator, C. lividipennis, were observed throughout the sampling period. Overall, this 2-year field study indicates that, in comparison with non-Bt rice, Bt rice did not lead to higher planthopper populations and did not negatively affect the predator C. lividipennis. These results suggest that use of Bt rice may provide control of the key lepidopteran pests while promoting the key biological control agent of planthopppers, C. lividipennis.

Tryptophanins (TRPs) are low molecular weight, tryptophan-rich, basic proteins found in oat ( Avena sativa L.) seeds. Like their counterpart puroindolines (PINs) from wheat ( Triticum aestivum L.), TRPs are thought to be involved in flour softness as well as disease resistance against phytopathogenic fungi. PINs are known to be the major components of ‘friabilin’ associated with the surface of water washed starch grains and possess lipid binding properties. Two polyclonal antisera against puroindoline-a (PIN-a), and puroindoline-b (PIN-b) respectively; and a monoclonal antiserum raised against ‘friabilin’ were used as primary antibodies in immunoblotting experiments. All antisera detected immunoreactive polypeptides, with approximate relative masses of 15–16 kDa, in oat, wheat, and barley ( Hordeum vulgare L.) seed extracts but not in rice ( Oryza sativa L.), maize ( Zea mays L.), bean ( Phaseolus vulgaris L.), pea ( Pisum sativum L.) and lentil ( Lens culinaris Medic.) seed extracts. Immunoreactive polypeptides were detected in aqueous ethanol [52% (v/v) ethanol] seed extracts. Both anti-‘friabilin’ monoclonal and anti-PIN-b polyclonal antisera recognized 15 as well as 16 kDa tryptophanins in oat seeds from different cultivars. On the other hand, anti-PIN-a polyclonal antiserum strongly cross-reacted with 16 kDa TRP and weakly with 15 kDa TRP. Tryptophanins were found to be associated with the surface of starch grains in oat endosperm tissue using both fluorescence and confocal laser scanning microscopies with anti-‘friabilin’ monoclonal antiserum. SDS-PAGE and immunoblotting assays revealed a gradual synthesis of TRPs as early as milk stage in developing oat seeds. On the other hand, TRPs tend to undergo degradation during seed germination.

An animal model for safety assessment of genetically modified foods was tested as part of the SAFOTEST project. In a 90-day feeding study on Wistar rats, the transgenic KMD1 rice expressing Cry1Ab protein was compared to its non-transgenic parental wild type, Xiushui 11. The KMD1 rice contained 15mg Bt toxin/kg and based on the average feed consumption the daily intake was 0.54mg Bt toxin/kg body weight. No adverse effects on animal behaviour or weight gain were observed during the study. Blood samples collected one week prior to sacrifice were analyzed and compared for standard haematological and biochemical parameters. A few parameters were significantly different, but all within the normal reference intervals for rats of this breed and age and not in relation to any other findings, thus not considered treatment related. Upon sacrifice a large number of organs were weighed, macroscopic and histopathological examinations were performed with only minor changes to report. The aim of the study was to use a known animal model in performance of safety assessment of a GM crop, in this case KMD1 rice. The results show no adverse or toxic effects of KMD1 rice when tested in the design used in this 90-day study. Nevertheless the experiences from this study lead to the overall conclusion that safety assessment for unintended effects of a GM crop cannot be done without additional test group(s).

Transgenic potato, Solanum tuberosum L., plants containing a synthetic cry1Ac gene coding for the Bacillus thuringiensis (Bt) crystalline insecticidal protein were produced and evaluated for resistance to Tecia solanivora Povolny (Lepidoptera: Gelechiidae), the larvae of which attack potato tubers. In total, 43 transgenic lines of commercial Andean potato varieties Diacol Capiro, Pardo Pastusa, and Pandeazucar were obtained. These transgenic lines were found to have one to four copies of cry1Ac per genome and expression levels of Cry1Ac protein varying from 0.02 to 17 microg/g fresh tuber tissue. Bioassays of T. solanivora larvae on these transgenic potato tubers showed 83.7-100% mortality, whereas the mortality levels on nontransgenic lines were 0-2.67%. Our data indicate the capability of Bt transgenic technology to control the T. solanivora while reducing the use of chemical insecticides. Further studies under controlled field conditions will be helpful in exploring the potential of CrylAc potatoes in the insect pest management strategies.

An in vitro regeneration system was developed in cowpea [Vigna unguiculata (L.) Walp.] Blackeye. Among several explants studied, shoot initiation response was observed from shoot apices of 3–5-day-old seedlings. The optimal medium for maximum shoot initiation comprised MS salts, B5 vitamins, 8.88 µM N6-benzylaminopurine, 1 gl-1 casein hydrolysate, 342 μM L-glutamine, 3% sucrose, 0.3% phytagel, adjusted to pH 5.8. A shift in pH from 5.8 to 7.0 had no effect on shoot initiation and on number of shoots per explant. The highest shoot initiation frequency (77%) was obtained using this preferred medium, reaching a maximum of eight shoots per explant. For shoot elongation, 14 µM gibberellic acid was supplemented in the shoot initiation medium. Presence of indolebutyric acid in the rooting medium had no effect on root induction. The regenerated plants were fertile and developed normally.

Insecticidal toxins produced by Bacillus thuringiensis (Bt toxins) have been widely used in plant protection in two different ways: spraying of bacterial Bt toxin formulations over plants and production of the toxins in planta by expressing the genes encoding for the proteins. In the second strategy, only the transgenic plants themselves are protected. We have now broadened the application of insect-resistant transgenics in a third scenario: protection of other crops that theoretically could be recalcitrant to DNA transformation. Using cabbage as a model crop well known to be susceptible to many pests, the Bt toxin Cry1Ab was first produced in transgenic rice (Oryza sativa) plants and then was applied to the leaves of cabbage (Brassica juncea) to test its toxicity against larvae of Pieris rapae. Seventy-eight to 100% larval mortality was observed for different instar larvae. Furthermore, no-choice feeding of the larvae on the Bt toxin-treated cabbage leaves had adverse effects on the oviposition rates and egg hatchability of surviving adult moths. Bt toxin-treated larvae had 30% and 46% lower moth emergence rates than their control counterparts. Female moths from the Bt toxin-treated larvae had significantly reduced egg production, 15.1 eggs per female versus the control 326.3 eggs, and lower hatching rates, 3.1% versus 33.8% in the control. Since long term stability is a key factor contributing to the potential of ex situ application of plant-made biocides, Cry1Ab protein stability was assessed in the detached rice leaf tissue. The toxin was found to be stable over a 5-month storage period, declining 31.7% and 20% from the initial content when stored at 4 and 37 °C, respectively.

Rice variety Basmati 370 was transformed with a co-integrate vector pSM6 carrying cry1Ac and cry2A genes. The hygromycin resistance gene on a separate vector, pROB5 was used as a selectable marker. One to two week old scutellum derived calli were transformed with a biolistic gun. Transgenic status of different transformed lines was evaluated by molecular analysis (PCR, Dot blot, Southern blot, Western blot and ELISA) and insect feeding assays with rice leaf folder (RLF) and yellow stem borer (YSB). An average transformation efficiency of 1.98% was observed in a set of experiments. Plants from five different transgenic lines exhibited 100% mortality against rice leaf folder and segregated in Mendelian fashion. Insect bioassays revealed that there is no synergistic or antagonistic effect of cry1Ac and cry2A genes on the development of resistance against RFL and YSB.

Human CD14 plays an important role in innate immunity by being the key receptor of lipopolysaccharide found on Gram-negative bacteria. The recently discovered widespread localization of CD14 in secretions and mucosal surfaces reveals its extensive anti-microbial properties and numerous potential medical applications. To produce active recombinant human CD14 (rhCD14) for massive distribution, transgenic tobacco plants were successfully generated to express rhCD14 in the seed endosperm under the control of two versions (1.8 kb and 5.1 kb) of the rice glutelin Gt-1 promoter. Plant-made rhCD14 proteins reached a concentration of 16 microg/g of seeds and showed stability, proteolytic resistance to pepsin digestion and ability to induce the release of pro-inflammatory IL-6 and IL-8 cytokines in presence of LPS. The expression of plant rhCD14 in tobacco seeds constitutes a promising low-cost and abundant supply of this immune protein to further investigate its roles in, impacts on and potential medical applications for the innate immune system.

Human breast milk contains several proteins that supplement the newborn mucosal defense system and prevent gastrointestinal illnesses. One of these recently identified breast milk proteins is soluble CD14 (sCD14). By being an important component of the lipopolysaccharide (LPS) receptor complex, it has been suggested that breast milk sCD14 could stimulate the newborn immune system and help reduce gastrointestinal Gram-negative infections. However, to deliver its potential immune benefits to the neonate, sCD14 would have to survive the passage through the gastrointestinal tract and retain its biologic activity. We analyzed the presence of breast milk sCD14 in the neonatal digestive system and found breast milk sCD14 to be absent from the stools of breast-fed infants. In vitro digestion analysis with simulated gastric and pancreatic fluids revealed that sCD14 is likely to survive the pepsin digestion but is more prone to been nicked and digested by pancreatin. These findings suggest that the presence of intact breast milk sCD14 in the upper digestive system could promote innate immunity in this low bacteria density lumen. The low concentration of sCD14 in the LPS-rich environment of the distal gastrointestinal tract (i.e. commensal microflora) could prevent excessive inflammation.

Expression of the Cry2Aa2 protein was targeted specifically to the green tissues of transgenic tobacco Nicotiana tabacum cv. Xanthi plants. This deployment was achieved by using the promoter region of the gene encoding the Solanum tuberosum leaf and stem specific (ST-LS1) protein. The accumulated levels of toxin in the leaves were found to be effective in achieving 100% mortality of Heliothis virescens larvae. The levels of Cry2Aa2 expression in the leaves of these transgenic plants were up to 0.21% of the total soluble proteins. Bioassays with R1 transgenic plants indicated the inheritance of cry2Aa2 in the progeny plants. Tissue-specific expression of the Bt toxin in transgenic plants may help in controlling the potential occurrence of insect resistance by limiting the amount of toxin to only predated tissues. The results reported here validate the use of the ST-LS1 gene promoter for a targeted expression of Bt toxins in green tissues of plants.

The use of transgenic plants in the production of recombinant proteins for human therapy, including subunit vaccines, is being investigated to evaluate the efficacy and safety of these emerging biopharmaceutical products. We have previously shown that synthesis of recombinant glycoprotein B (gB) of human cytomegalovirus can be targeted to seeds of transgenic tobacco when directed by the rice glutelin 3 promoter, with gB retaining critical features of immunological reactivity (E.S. Tackaberry et al. 1999. Vaccine, 17: 3020–3029). Here, we report development of second generation transgenic plant lines (T1) homozygous for the transgene. Twenty progeny plants from two lines (A23T1-2 and A24T1-3) were grown underground in an environmentally contained mine shaft. Based on yields of gB in their seeds, the A23T1-2 line was then selected for scale-up in the same facility. Analyses of mature seeds by ELISA showed that gB specific activity in A23T1-2 seeds was over 30-fold greater than the best T0plants from the same transformation series, representing 1.07% total seed protein. These data demonstrate stable inheritance, an absence of transgene inactivation, and enhanced levels of gB expression in a homozygous second generation plant line. They also provide evidence for the suitability of using this environmentally secure facility to grow transgenic plants producing therapeutic biopharmaceuticals.Key words: transgenic tobacco seeds, homozygous second generation, glycoprotein B, human cytomegalovirus, vaccine, underground mine.

Two transgenic rice (Oryza satica L.) lines, KMD1 and KMD2, containing a synthetic cry1Ab gene from Bacillus thuringiensis Berliner, were evaluated for resistance to the rice leaffolder (RLF), Cnaphalocrocis medinalis (Guenee) under field conditions for 3 years in Zhejiang Province, China. Both KMD1 and KMD2 exhibited high and stable resistance against natural infestations by the RLF, and showed no symptoms of damaged leaves throughout the rice-growing season. In contrast, the untransformed parental control line (Xiushui 11) showed serious RLF damage symptoms not only in untreated plots, but also in plots treated once with chemical insecticides. These results demonstrate that both KMD1 and KMD2 have potential for protecting rice from the RLF damage.