Transgenic Maize Lines Research Articles

Overexpression of a novel Cry1Ie gene confers resistance to Cry1Ac-resistant cotton bollworm in transgenic lines of maize

Although transgenic crops expressing either Cry1Ab or Cry1Ac, both derived from Bacillus thuringiensis (Bt), have been used commercially, the evolution of insects resistance to these CRY proteins has become a challenge. Thus, it has been proposed that co-expression of two Bt proteins with different modes of action may delay the development of resistance to Bt. However, few Bt proteins have been identified as having different modes of action from those of Cry1Ab or Cry1Ac. In this study, transgenic lines of maize over-expressing either Cry1Ie or Cry1Ac gene have been developed. Several independent transgenic lines with one copy of the foreign gene were identified by Southern blot analysis. Bioassays in the laboratory showed that the transgenic plants over-expressing Cry1Ie were highly toxic against the wild-type cotton bollworm (Heliothis armigera), producing mortality levels of 50 % after 6 days of exposure. However, the mortality caused by these plants was lower than that caused by the Cry1Ac transgenic plants (80 %) and MON810 plants expressing Cry1Ab (100 %), which both exhibited low toxicity toward the Cry1Ac-resistant cotton bollworm. In contrast, three transgenic maize lines expressing Cry1Ie induced higher mortality against this pest and were also highly toxic to the Asian corn borer (Ostrinia furnacalis) in the field. These results indicate that the Cry1Ie protein has a different mode of action than the Cry1Ab and Cry1Ac proteins. Therefore, the use of transgenic plants expressing Cry1Ie might delay the development of Bt-resistant insects in the field.

Detection of genetically modified maize events in Brazilian maize-derived food products

The Brazilian government has approved many transgenic maize lines for commercialization and has established a threshold of 1% for food labeling, which underscores need for monitoring programs. Thirty four samples including flours and different types of nacho chips were analyzed by conventional and real-time PCR in 2011 and 2012. The events MON810, Bt11, and TC1507 were detected in most of the samples, and NK603 was present only in the samples analyzed in 2012. The authorized lines GA21, T25, and the unauthorized Bt176 were not detected. All positive samples in the qualitative tests collected in 2011 showed a transgenic content higher than 1%, and none of them was correctly labeled. Regarding the samples collected in 2012, all positive samples were quantified higher than the threshold, and 47.0% were not correctly labeled. The overall results indicated that the major genetically modified organisms detected were MON810, TC1507, Bt11, and NK603 events. Some industries that had failed to label their products in 2011 started labeling them in 2012, demonstrating compliance with the current legislation observing the consumer rights. Although these results are encouraging, it has been clearly demonstrated the need for continuous monitoring programs to ensure consumers that food products are labeled properly.

Root Growth of Neighboring Maize and Weeds Studied with Minirhizotrons

Competition between crops and weeds may be stronger at the root than at the shoot level, but belowground competition remains poorly understood, due to the lack of suitable methods for root discrimination. Using a transgenic maize line expressing green fluorescent protein (GFP), we nondestructively discriminated maize roots from weed roots. Interactions between GFP-expressing maize, common lambsquarters, and redroot pigweed were studied in two different experiments with plants arranged in rows at a higher plant density (using boxes with a surface area of 0.09 m2) and in single-plant arrangements (using boxes with a surface area of 0.48 m2). Root density was screened using minirhizotrons. Relative to maize that was grown alone, maize root density was reduced from 41 to 87% when it was grown with redroot pigweed and from 27 to 73% when it was grown with common lambsquarters compared to maize grown alone. The calculated root : shoot ratios as well as the results of shoot dry weight and root density showed that both weed species restricted root growth more than they restricted shoot growth of maize. The effect of maize on the root density of the weeds ranged from a reduction of 25% to an increase of 23% for common lambsquarters and a reduction of 42 to 6% for redroot pigweed. This study constitutes the first direct quantification of root growth and distribution of maize growing together with weeds. Here we demonstrate that the innovative use of transgenic GFP-expressing maize combined with the minirhizotron technique offers new insights on the nature of the response of major crops to belowground competition with weeds.

Fast Quantitative Method for the Analysis of Carotenoids in Transgenic Maize

A fast method was developed to determine carotenoid content in transgenic maize seeds. The analysis was carried out using an ultrahigh-pressure liquid chromatograph coupled to a photodiode array detector and a mass spectrometer (UHPLC-PDA-MS/MS). Sixteen carotenoid pigments were detected and quantified in <13 min. In addition, it was possible to obtain good resolution of both polar xanthophylls and nonpolar carotenes. The method exhibited (a) a high degree of repeatability (%RSD < 13%), (b) linear calibration curves (R² > 0.9952), (c) satisfactory recoveries for most of the pigments (between 82 and 108%), and (d) low detection (from 0.02 to 0.07 μg/mL) and quantification limits (from 0.05 to 0.20 μg/mL) (LOD and LOQ, respectively). The methodology was applied to the analysis of transgenic maize lines TM1, TM2, and TM3, expressing several carotenogenic genes.

Enhanced virus resistance in transgenic maize expressing a dsRNA-specific endoribonuclease gene from E. coli.

Maize rough dwarf disease (MRDD), caused by several Fijiviruses in the family Reoviridae, is a global disease that is responsible for substantial yield losses in maize. Although some maize germplasm have low levels of polygenic resistance to MRDD, highly resistant cultivated varieties are not available for agronomic field production in China. In this work, we have generated transgenic maize lines that constitutively express rnc70, a mutant E. coli dsRNA-specific endoribonuclease gene. Transgenic lines were propagated and screened under field conditions for 12 generations. During three years of evaluations, two transgenic lines and their progeny were challenged with Rice black-streaked dwarf virus (RBSDV), the causal agent of MRDD in China, and these plants exhibited reduced levels of disease severity. In two normal years of MRDD abundance, both lines were more resistant than non-transgenic plants. Even in the most serious MRDD year, six out of seven progeny from one line were resistant, whereas non-transgenic plants were highly susceptible. Molecular approaches in the T12 generation revealed that the rnc70 transgene was integrated and expressed stably in transgenic lines. Under artificial conditions permitting heavy virus inoculation, the T12 progeny of two highly resistant lines had a reduced incidence of MRDD and accumulation of RBSDV in infected plants. In addition, we confirmed that the RNC70 protein could bind directly to RBSDV dsRNA in vitro. Overall, our data show that RNC70-mediated resistance in transgenic maize can provide efficient protection against dsRNA virus infection.

Elucidating the controlling role of the pyruvate phosphate dikinase regulatory protein on C4 photosynthesis

Pyruvate phosphate dikinase (PPDK) is a C4 photosynthetic pathway enzyme that converts Pyr to PEP, the substrate for CO2 fixation in C4 leaves. PPDK activity is regulated in response to light intensity to ensure the PEP synthesis rate is in balance with the amount of light energy incident on the leaf. Light regulation of PPDK occurs by reversible phosphorylation of an active‐site Thr. A bifunctional protein kinase/phosphotransferase, the PPDK regulatory protein (PDRP), catalyzes this reversible phosphorylation. Although the biochemical properties of PDRP are well known, its role in overall C4 cycle regulation in leaves is unclear. Our approach for resolving this question entails creating PDRP‐RNAi transgenic knockdown maize lines for assessing the interdependence of C4 PS and PDRP&gt;;PPDK regulation. The first phase of the study involved isolation of homozygous PDRP knockdown T2 maize lines. This was accomplished using PCR and qPCR based screen. A total of 22/134 T2 plants were found to be homozygous for the PDRP‐RNAi transgene. A subset of these T2 plants was analyzed for leaf PDRP transcript abundance via Taqman based qPCR assay. This revealed a 5–50% suppression of PDRP transcript vs. Wt. Western blot analysis of leaf extracts showed a corresponding decrease in PDRP protein. Lines showing severe PDRP suppression developed chlorotic leaves and failed to reach maturity, suggesting PDRP is required for optimal C4 PS.

Porównanie wybranych elementów bionomii Ostrinia nubilalis Hbn. (Lep., Crambidae) na kukurydzy Bt i non-Bt w południowo-wschodniej Polsce

A study on the dynamics of the oviposition and hatching of Ostrinia nubilalis Hbn. larvae on transgenic maize line MON 810 (DKC 3421YG cultivar) and isogenic maize (DKC 3420 cultivar) was carried out in 2008-2011 in Głuchów (south-eastern Poland). No significant differences were found between the number of egg clusters deposited by O. nubilalis on Bt and non-Bt maize based on the carried out observations. A small, statistically significant difference in the number of egg clusters between the studied cultivars was found only in 2011 and was most likely associated with weather changes. Female O. nubilalis did not significantly prefer any of the cultivars when depositing eggs. In addition, we found no differences in the dynamics of oviposition and larvae hatching between Bt and non-Bt maize. The first egg clusters, their maximum number, and the last egg clusters were found on similar dates for both studied cultivars. Empty egg clusters proving larval hatching were also found on similar dates for Bt and non-Bt maize.

Comparison of some aspects of the bionomy of Ostrinia nubilalis Hbn. (Lep., Crambidae) on Bt and non-Bt maize in south-eastern Poland

A study on the dynamics of the oviposition and hatching of Ostrinia nubilalis Hbn. larvae on transgenic maize line MON 810 (DKC 3421YG cultivar) and isogenic maize (DKC 3420 cultivar) was carried out in 2008-2011 in Gluchow (south-eastern Poland). No significant differences were found between the number of egg clusters deposited by O. nubilalis on Bt and non-Bt maize based on the carried out observations. A small, statistically significant difference in the number of egg clusters between the studied cultivars was found only in 2011 and was most likely associated with weather changes. Female O. nubilalis did not significantly prefer any of the cultivars when depositing eggs. In addition, we found no differences in the dynamics of oviposition and larvae hatching between Bt and non-Bt maize. The first egg clusters, their maximum number, and the last egg clusters were found on similar dates for both studied cultivars. Empty egg clusters proving larval hatching were also found on similar dates for Bt and non-Bt maize.

Maize Histone H2B-mCherry: A New Fluorescent Chromatin Marker for Somatic and Meiotic Chromosome Research

Cytological studies of fluorescent proteins are rapidly yielding insights into chromatin structure and dynamics. Here we describe the production and cytological characterization of new transgenic maize lines expressing a fluorescent histone fusion protein, H2B-mCherry. The transgene is expressed under the control of the maize ubiquitin1 promoter, including its first exon and intron. Polymerase chain reaction-based genotyping and root-tip microscopy showed that most of the lines carrying the transgene also expressed it, producing bright uniform staining of nuclei. Further, plants showing expression in root tips at the seedling stage also showed expression during meiosis, late in the life cycle. Detailed high-resolution three-dimensional imaging of cells and nuclei from various somatic and meiotic cell types showed that H2B-mCherry produced remarkably clear images of chromatin and chromosome fiber morphology, as seen in somatic, male meiotic prophase, and early microgametophyte cells. H2B-mCherry also yielded distinct nucleolus staining and was shown to be compatible with fluorescence in situ hybridization. We found several instances where H2B-mCherry was superior to DAPI as a generalized chromatin stain. Our study establishes these histone H2B-mCherry lines as new biological reagents for visualizing chromatin structure, chromosome morphology, and nuclear dynamics in fixed and living cells in a model plant genetic system.

Genetically modified Bt maize lines containing cry3Bb1, cry1A105 or cry1Ab2 do not affect the structure and functioning of root-associated endophyte communities

Transgenic maize expressing Bacillus thuringiensis (Bt) insecticidal crystal (Cry) proteins may contribute to a decreased need for chemical pesticides, providing benefits for human health and the environment. However, there is public concern regarding the environmental safety of transgenic crops, which relates also to potential adverse effects on plant-microbe interactions by affecting the plant-associated, endophytic microflora. Thus, we compared the endophytic bacterial communities in plants of the three transgenic Bt maize lines MON89034 (cry3Bb1), MON88017 (cry1A.105 and cry2Ab2) and the stacked event MON88017xMON89034 (cry1A.105 and cry2Ab2, cry3Bb1), with those of the respective near-isogenic line, and with those in plants of three additional, conventional maize lines. The maize plants were grown in a containment system on two different soils which were commonly used for maize cultivation in Lower Austria.We obtained 700 bacterial endophytes and characterized them regarding their phylogenetic diversity and specific plant growth promoting functions, including the production of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and indole-3-acetic acid (IAA). Both the soil environment and the plant cultivar had an effect on the phylogenetic diversity of the endophytic communities, but there were no specific effects of the transgenic varieties. Diversity measures of endophytic isolates were not different in Bt- versus non-Bt maize varieties. A variable number of isolates showed ACC deaminase activity irrespective of the soil and plant cultivar. Generally, the number of high IAA producers appeared to be affected by the soil environment but not by the plant cultivar. Cultivation-independent profiling via terminal restriction fragment polymorphism (T-RFLP) analysis of the 16S rRNA gene revealed that maize root endophytes were differentially composed dependent on soil type and plant cultivar, but they were not discriminated with respect to Bt- versus non-Bt varieties. In conclusion, maize endophyte communities studied were highly diverse and showed a high potential for plant growth promotion, emphasizing their importance in modern agricultural systems irrespective of the maize cultivar, including Bt-modifications.

Establishment and characterization of a maize Hi-II endosperm culture

An in vitro continuous endosperm callus culture derived from developing endosperm of transformation-amenable maize Hi-II genotype was obtained. The endosperm callus was composed of cells that differentiated into aleurone-like and starchy endosperm-like cell types. This callus has been maintained for 4 yr. Endosperm callus cells transcribe and produce zein proteins at a level similar to developing endosperm tissue. Starchy endosperm cells of the endosperm callus displayed active starch biosynthetic activity. The dual cell physiology of this culture limited the utility of the cell line for promoter analysis and transient assays of gene expression in the current culture conditions. However, because such cell line can be readily initiated and easily maintained for a long period of time, it provides an alternative tool for analysis of transgene expression in endosperm callus derived from transgenic maize lines in Hi-II background.

Effect of insect-resistant transgenic maize on growth and development, utilization of nutrients and in vivo activity of the detoxification enzymes of the Asian corn borer, Ostrinia furnacalis (Lepidoptera: Pyralidae)

There is little systematic research on the effect of transgenic maize expressing Bacillus thuringiensis (Bt) toxins on insect survival and the response in vivo of their detoxification enzymes. Results of laboratory bioassays indicate that the whorl leaves of transgenic maize lines G03-2396 and G03-2739 significantly affected the survival of neonate and third instar larvae of the Asian corn borer, Ostrinia furnacalis (Guenee). Neonate mortality two days after being fed on G03-2739 was 72.5% and exceeded 92.5% four days later. The percentage mortality of third instar larvae six days after being fed whorl leaves from G03-2396 exceeded 65%. The resistance of whorl leaves to insect attack was greater than that of maize-ears, but less effective against attacks by third instar larvae. Neonate development was more prolonged when they were fed on whorl leaves of both the transgenic maize lines. In contrast, low pupation (

T-DNA integration patterns in transgenic maize lines mediated by Agrobacterium tumefaciens

To explore transfer deoxyribonucleic acid (T-DNA) integration patterns in the maize genome, we improved the protocol of thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR), and amplified the flanking sequences around T-DNA integration sites from 70 independent transgenic maize lines mediated by Agrobacterium tumefaciens . Out of 64 specific amplified fragments, 32 and 9 are homologous to the sequences of the maize genome and the expression plasmid, respectively. For 26 of them, a filler sequence was found flanking the cleavage sites. These results demonstrate that cleavage occurs not only during the T-DNA borders but also inside or outside the borders. The border sequences and some inside sequences can be deleted, and filler sequences can be inserted. Illegitimate recombination is a major pattern of T-DNA integration, while some hot spots and preference are present on maize chromosomes. Key words : Agrobacterium tumefaciens, maize, thermal asymmetric interlaced PCR, transfer DNA,transgenics.

Duplicate Maize Wrinkled1 Transcription Factors Activate Target Genes Involved in Seed Oil Biosynthesis

WRINKLED1 (WRI1), a key regulator of seed oil biosynthesis in Arabidopsis (Arabidopsis thaliana), was duplicated during the genome amplification of the cereal ancestor genome 90 million years ago. Both maize (Zea mays) coorthologs ZmWri1a and ZmWri1b show a strong transcriptional induction during the early filling stage of the embryo and complement the reduced fatty acid content of Arabidopsis wri1-4 seeds, suggesting conservation of molecular function. Overexpression of ZmWri1a not only increases the fatty acid content of the mature maize grain but also the content of certain amino acids, of several compounds involved in amino acid biosynthesis, and of two intermediates of the tricarboxylic acid cycle. Transcriptomic experiments identified 18 putative target genes of this transcription factor, 12 of which contain in their upstream regions an AW box, the cis-element bound by AtWRI1. In addition to functions related to late glycolysis and fatty acid biosynthesis in plastids, the target genes also have functions related to coenzyme A biosynthesis in mitochondria and the production of glycerol backbones for triacylglycerol biosynthesis in the cytoplasm. Interestingly, the higher seed oil content in ZmWri1a overexpression lines is not accompanied by a reduction in starch, thus opening possibilities for the use of the transgenic maize lines in breeding programs.

Improving iron bioavailability with ferritin overexpression in low phytate maize

Iron deficiency anemia (IDA) is a major global nutritional problem may be due to lack of diversity in the diet, with maize as a staple crop. Iron bioavailability from maize is low due to its high phytate content. Iron content can be increased by expressing ferritin but this in itself may not overcome the inhibitory effect of phytate on bioavailability. We hypothesized that iron from low phytate maize (lpa‐1‐1 with 50–60% lower phytate than wild type) is more bioavailable than from wild type maize (A188), and increasing the iron content of lpa‐1‐1 will further increase iron bioavailability. We generated transgenic maize lines with enhanced iron content by over expressing soybean ferritin, and used the Caco‐2 cell culture model to determine iron bioavailability from these transgenic lines. Total iron content in transgenic maize was 2.5‐fold and 1.8‐fold higher in A188 and lpa‐1‐1 maize, respectively. Iron bioavailability from non‐transgenic lpa1‐1 was 50% (p&lt;0.01) higher than A188 maize. Ferritin over expression improved iron bioavailability by 60% in A188 maize. Overall, transgenic lpa1‐1 showed higher bioavailability (16–38%; p&lt;0.02) when compared to A188 transgenic maize. Our results suggest that over expression of ferritin in low phytate maize significantly improves iron bioavailability from maize, and is a potential approach to alleviate IDA. Supported by NWRC, Iowa State University

Quantification of transgene expression in maize (Zea mays L.) throughout the vegetation period

With 3 figures and 1 table Abstract Evaluation of transgenic plants during their development is of major importance for breeders. To facilitate this evaluation, we applied a real-time, reverse transcription polymerase chain reaction method to maize tissues that allows quantitative expression profiling of transgenes. To obtain consistent results, sampling, transport, storage and extraction protocols were standardized for a wide range of samples (leaves, stems, roots, pollen and kernels) and phenological stages (from the 4–6 leaves stage to physiological maturity). RNA integrity was monitored by Agilent 2100 Bioanalyzer to ensure that decomposed RNA samples were excluded from further study. A simple and effective SYBR Green methodology was applied for relative quantification of transgene expression. Several endogenous controls were tested to achieve the best possible results. Quantitave results presented illustrate the application of our protocol to the quantification of cry3Bb1 (encoding corn root worm resistance) expression in MON88017 event derived transgenic maize lines. The methodology described here can be routinely performed for the quantification of gene expression in maize.

Creation of a high-yielding recombinant maize hybrid for the production of a microbicide to prevent HIV-1 transmission

The aim of this study was to use conventional breeding to increase the production in maize of the human monoclonal antibody 2G12, known to have potential therapeutic properties in the prevention of HIV-1 transmission. The recombinant antibody, together with a fluorescent marker, was introduced into two South African high-performing maize elite inbred lines by crossing them with a transgenic maize line that had been transformed with the monoclonal antibody 2G12. The effect of breeding to produce high-expressing recombinant hybrid seed was evaluated by comparing 2G12 production in the different breeding lines with the original maize line. ‘Good production practice standards’ were followed throughout the breeding programme. ‘Conventional drug regulations’ adapted to plant- made pharmaceuticals were also followed, with the seeds being stored in a ‘master seed bank’. The maize hybrid expressed a higher level of the antibody than the recombinant maize elite lines. This plant-derived antibody provides a means of producing a microbicide component that could be used with other HIV-neutralising antibodies as an additional approach to prevent HIV infection.

Comparison of two GM maize varieties with a near-isogenic non-GM variety using transcriptomics, proteomics and metabolomics

The aim of this study was to evaluate the use of four nontargeted analytical methodologies in the detection of unintended effects that could be derived during genetic manipulation of crops. Three profiling technologies were used to compare the transcriptome, proteome and metabolome of two transgenic maize lines with the respective control line. By comparing the profiles of the two transgenic lines grown in the same location over three growing seasons, we could determine the extent of environmental variation, while the comparison with the control maize line allowed the investigation of effects caused by a difference in genotype. The effect of growing conditions as an additional environmental effect was also evaluated by comparing the Bt-maize line with the control line from plants grown in three different locations in one growing season. The environment was shown to play an important effect in the protein, gene expression and metabolite levels of the maize samples tested where 5 proteins, 65 genes and 15 metabolites were found to be differentially expressed. A distinct separation between the three growing seasons was also found for all the samples grown in one location. Together, these environmental factors caused more variation in the different transcript/protein/metabolite profiles than the different genotypes.

Transgenic maize lines with cell‐type specific expression of fluorescent proteins in plastids

Plastid number and morphology vary dramatically between cell types and at different developmental stages. Furthermore, in C4 plants such as maize, chloroplast ultrastructure and biochemical functions are specialized in mesophyll and bundle sheath cells, which differentiate acropetally from the proplastid form in the leaf base. To develop visible markers for maize plastids, we have created a series of stable transgenics expressing fluorescent proteins fused to either the maize ubiquitin promoter, the mesophyll-specific phosphoenolpyruvate carboxylase (PepC) promoter, or the bundle sheath-specific Rubisco small subunit 1 (RbcS) promoter. Multiple independent events were examined and revealed that maize codon-optimized versions of YFP and GFP were particularly well expressed, and that expression was stably inherited. Plants carrying PepC promoter constructs exhibit YFP expression in mesophyll plastids and the RbcS promoter mediated expression in bundle sheath plastids. The PepC and RbcS promoter fusions also proved useful for identifying plastids in organs such as epidermis, silks, roots and trichomes. These tools will inform future plastid-related studies of wild-type and mutant maize plants and provide material from which different plastid types may be isolated.

Improved methodology for the characterisation of transgenic Bt-11 maize cultivars using RP-HPLC profiles of albumin, globulin, prolamin, and glutelin protein fractions and chemometric analysis

A clear differentiation between Bt-11 transgenic and isogenic non-transgenic maize cultivars has successfully achieved analysing the perfusion and monolithic RP-HPLC profiles of the albumin, globulin, prolamin, and glutelin maize fractions together with a discriminant analysis. Significant differences between transgenic and isogenic non-transgenic cultivars were observed in the chromatograms obtained from any of the four protein fractions. The application of linear discriminant analysis to the area percentages corresponding to every peak detected in every protein fraction was successfully employed for the classification of transgenic Bt maize lines obtaining a global percentage of correct classification of 100%. For perfusion RP-HPLC, the variables with more discriminant power and prediction capability were the following peaks: peaks 1 and 3 for albumins, peak 2 for globulins, peaks 3 and 6 for prolamins, and peaks 7 and 8 for glutelins. In the case of monolithic RP-HPLC, the variables were peaks 2 and 3 for albumins, peak 5 for globulins, peaks 5, 6, and 7 of prolamins, and peak 10 for glutelins.