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Journal articles on the topic 'Plant transgenesis'

Author: Grafiati
Published: 4 June 2021
Last updated: 14 February 2022

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1

Skarzyńska, Agnieszka, Magdalena Pawełkowicz, and Wojciech Pląder. "Influence of transgenesis on genome variability in cucumber lines with a thaumatin II gene." Physiology and Molecular Biology of Plants 27, no. 5 (April 28, 2021): 985–96. http://dx.doi.org/10.1007/s12298-021-00990-8.

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AbstractThe development of new plant varieties by genetic modification aims at improving their features or introducing new qualities. However, concerns about the unintended effects of transgenes and negative environmental impact of genetically modified plants are an obstacle for the use of these plants in crops. To analyze the impact of transgenesis on plant genomes, we analyze three cucumber transgenic lines with an introduced thaumatin II gene. After genomes sequencing, we analyzed the transgene insertion site and performed variant prediction. As a result, we obtained similar number of variants for all analyzed lines (average of 4307 polymorphisms), with high abundance in one region of chromosome 4. According to SnpEff analysis, the presence of genomic variants generally does not influence the genome functionality, as less than 2% of polymorphisms have high impact. Moreover, analysis indicates that these changes were more likely induced by in vitro culture than by the transgenesis itself. The insertion site analysis shows that the region of transgene integration could cause changes in gene expression, by gene disruption or loss of promoter region continuity.
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2

McHughen, Alan. "Genetically modified organisms-transgenesis in plants." In Vitro Cellular & Developmental Biology - Plant 39, no. 6 (November 2003): 669. http://dx.doi.org/10.1079/ivp2003452.

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3

Смирнова, О. Г., and O. G. Smirnova. "TGP – the Database on Promoters for Plant Transgenesis." Mathematical Biology and Bioinformatics 7, no. 2 (July 30, 2012): 444–60. http://dx.doi.org/10.17537/2012.7.444.

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4

Smirnova, Olga G., Salmaz S. Ibragimova, and Alex V. Kochetov. "Simple database to select promoters for plant transgenesis." Transgenic Research 21, no. 2 (August 3, 2011): 429–37. http://dx.doi.org/10.1007/s11248-011-9538-2.

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5

JONES, H. D. "Tourte Y. Genetically modified organisms: transgenesis in plants." Annals of Botany 93, no. 1 (January 1, 2004): 115. http://dx.doi.org/10.1093/aob/mch001.

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6

Ambrosini, Veronica, Mohammad Issawi, Stéphanie Leroy-Lhez, and Catherine Riou. "How protoporphyrinogen IX oxidase inhibitors and transgenesis contribute to elucidate plant tetrapyrrole pathway." Journal of Porphyrins and Phthalocyanines 23, no. 04n05 (April 2019): 419–26. http://dx.doi.org/10.1142/s1088424619300076.

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Several families of herbicides, especially diphenyl ether (DPE) and pyrimidinedione, target the plant tetrapyrrole biosynthesis pathways and in particular one key enzyme, protoporphyrinogen IX oxidase (PPO). When plants are treated with DPE or pyrimidinedione, an accumulation of protoporphyrin IX, the first photosensitizer of this pathway, is observed in cytosol where it becomes very deleterious under light. Indeed these herbicides trigger plant death in two distinct ways: (i) inhibition of chlorophylls and heme syntheses and (ii) a huge accumulation of protoporphyrin IX in cytosol. Recently, a strategy based on plant transgenesis that induces deregulation of the tetrapyrrole pathway by up- or down-regulation of genes encoding enzymes, such as glutamyl-[Formula: see text]RNA reductase, porphobilinogen deaminase and PPO, has been developed. Against all expectations, only transgenic crops overexpressing PPO showed resistance to DPE and pyrimidinedione. This herbicide resistance of transgenic crops leads to the hypothesis that the overall consumption of herbicides will be reduced as previously reported for glyphosate-resistant transgenic crops. In this review, after a rapid presentation of plant tetrapyrrole biosynthesis, we show how only PPO enzyme can be the target of DPE and how transgenic crops can be further resistant not only to herbicide but also to abiotic stress such as drought or chilling. Keeping in mind that this approach is mostly prohibited in Europe, we attempt to discuss it to interest the scientific community, from plant physiologists to chemists, who work on the interface of photosensitizer optimization and agriculture.
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7

Lampropoulos, Athanasios, Zoran Sutikovic, Christian Wenzl, Ira Maegele, Jan U. Lohmann, and Joachim Forner. "GreenGate - A Novel, Versatile, and Efficient Cloning System for Plant Transgenesis." PLoS ONE 8, no. 12 (December 20, 2013): e83043. http://dx.doi.org/10.1371/journal.pone.0083043.

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8

Tretiakova, Polina Ya, and Aleksandr A. Soloviev. "Application of small RNAs for plant protection." Ecological genetics 18, no. 4 (December 12, 2020): 467–82. http://dx.doi.org/10.17816/ecogen35203.

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Double-stranded small RNAs (dsRNA) perform various regulatory functions via RNA-interference. Additionally, they can be transported between various plant species and their pathogens and pests via extracellular vesicles, protecting RNA from nucleases. Plants secrete short dsRNA molecules to defend themselves against pathogens. The latter also use small RNAs when infecting crops. Some dsRNAs of pathogens are known as ribonucleic effectors. Host-induced gene silencing (HIGS) was shown to be effective when breeding resistant varieties and analyzing plant-pathogen interactions. However, complexity of transgenesis and society fear of genetically modified products make HIGS application difficult. The appearance of a new strategy based on plant spraying with dsRNA gave a new perspective of plant protection. Currently such a strategy requires accurate studying as well as the development of efficient systems stably producing high-quality dsRNA.
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9

Herrera‐Estrella, Luis. "My journey into the birth of plant transgenesis and its impact on modern plant biology." Plant Biotechnology Journal 18, no. 7 (March 18, 2020): 1487–91. http://dx.doi.org/10.1111/pbi.13319.

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10

Wang, Shasha, Yaling Song, Taihe Xiang, Pan Wu, Ting Zhang, Daze Wu, Shuyu Zhou, and Yafei Li. "Transgenesis of Agrobacterium rhizogenes K599 orf3 into plant alters plant phenotype to dwarf and branch." Plant Cell, Tissue and Organ Culture (PCTOC) 127, no. 1 (July 11, 2016): 207–15. http://dx.doi.org/10.1007/s11240-016-1043-0.

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11

Kaitlyn, Hull, Hodgson Dee, Clark Bob, W. Hickok Timothy, N. Petitte James, and E. Mozdziak Paul. "Lentiviral transgenesis of the leopard gecko, Eublepharis macularius." African Journal of Microbiology Research 8, no. 10 (March 5, 2014): 1070–79. http://dx.doi.org/10.5897/ajmr2013.6532.

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12

Giraldo, Patricia, and Lluís Montoliu. "Artificial Chromosome Transgenesis in Pigmentary Research." Pigment Cell Research 15, no. 4 (July 3, 2002): 258–64. http://dx.doi.org/10.1034/j.1600-0749.2002.02030.x.

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13

Delporte, Fabienne, Jean-Marie Jacquemin, Patrick Masson, and Bernard Watillon. "Insights into the regenerative property of plant cells and their receptivity to transgenesis." Plant Signaling & Behavior 7, no. 12 (December 2012): 1608–20. http://dx.doi.org/10.4161/psb.22424.

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14

Kaitlyn, Hull, Hodgson Dee, N. Petitte James, Clark Bob, Hickok, Tim, Fee Lanette, and E. Mozdziak Paul. "Restriction enzyme mediated integration and FIV lentiviral transgenesis applied to amphibians." African Journal of Microbiology Research 8, no. 1 (January 1, 2014): 1–11. http://dx.doi.org/10.5897/ajmr2013.5657.

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15

Boudet, Alain-M., and Matthieu Chabannes. "Gains achieved by molecular approaches in the area of lignification." Pure and Applied Chemistry 73, no. 3 (January 1, 2001): 561–66. http://dx.doi.org/10.1351/pac200173030561.

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In this article we highlight the contribution of molecular biology and lignin genetic engineering toward a better understanding of lignin biosynthesis and spatio-temporal deposition of lignin. Specific examples from the literature and from our laboratory will serve to underline the chemical flexibility of lignins, the complexity of the regulatory circuits involved in their synthesis, and the specific behavior of different cell types within the xylem. We will also focus on strategies aiming to reduce the lignin content or to modify the lignin composition of plants and present their impact on plant development. We will show that the ectopic expression of a specific transgene may have a different impact, depending on the genetic background, and that plants with a severe reduction in lignin content may undergo normal development. Lignification is currently benefiting enormously from recent developments in molecular biology and transgenesis, and the progress made opens the way for future developments to study how the walls of lignified plant cells are built and organized.
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16

Kowalczyk, Tomasz, Joanna Wieczfinska, Ewa Skała, Tomasz Śliwiński, and Przemysław Sitarek. "Transgenesis as a Tool for the Efficient Production of Selected Secondary Metabolites from Plant in Vitro Cultures." Plants 9, no. 2 (January 21, 2020): 132. http://dx.doi.org/10.3390/plants9020132.

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The plant kingdom abounds in countless species with potential medical uses. Many of them contain valuable secondary metabolites belonging to different classes and demonstrating anticancer, anti-inflammatory, antioxidant, antimicrobial or antidiabetic properties. Many of these metabolites, e.g., paclitaxel, vinblastine, betulinic acid, chlorogenic acid or ferrulic acid, have potential applications in medicine. Additionally, these compounds have many therapeutic and health-promoting properties. The growing demand for these plant secondary metabolites forces the use of new green biotechnology tools to create new, more productive in vitro transgenic plant cultures. These procedures have yielded many promising results, and transgenic cultures have been found to be safe, efficient and cost-effective sources of valuable secondary metabolites for medicine and industry. This review focuses on the use of various in vitro plant culture systems for the production of secondary metabolites.
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17

Edwards, Robert, Daniele Del Buono, Michael Fordham, Mark Skipsey, Melissa Brazier, David P. Dixon, and Ian Cummins. "Differential Induction of Glutathione Transferases and Glucosyltransferases in Wheat, Maize and Arabidopsis thaliana by Herbicide Safeners." Zeitschrift für Naturforschung C 60, no. 3-4 (April 1, 2005): 307–16. http://dx.doi.org/10.1515/znc-2005-3-416.

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Abstract By learning lessons from weed science we have adopted three approaches to make plants more effective in phytoremediation: 1. The application of functional genomics to identify key components involved in the detoxification of, or tolerance to, xenobiotics for use in subsequent genetic engineering/breeding programmes. 2. The rational metabolic engineering of plants through the use of forced evolution of protective enzymes, or alternatively transgenesis of detoxification pathways. 3. The use of chemical treatments which protect plants from herbicide injury. In this paper we examine the regulation of the xenome by herbicide safeners, which are chemicals widely used in crop protection due to their ability to enhance herbicide selectivity in cereals. We demonstrate that these chemicals act to enhance two major groups of phase 2 detoxification enzymes, notably the glutathione transferases and glucosyltransferases, in both cereals and the model plant Arabidopsis thaliana, with the safeners acting in a chemical- and species-specific manner. Our results demonstrate that by choosing the right combination of safener and plant it should be possible to enhance the tolerance of diverse plants to a wide range of xenobiotics including pollutants.
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18

Lacoux, J., L. Gutierrez, F. Dantin, B. Beaudoin, D. Roger, and E. Lain�. "Antisense transgenesis of tobacco with a flax pectin methylesterase affects pollen ornamentation." Protoplasma 222, no. 3-4 (December 1, 2003): 205–9. http://dx.doi.org/10.1007/s00709-003-0019-1.

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19

Nasti, Ryan A., and Daniel F. Voytas. "Attaining the promise of plant gene editing at scale." Proceedings of the National Academy of Sciences 118, no. 22 (April 30, 2021): e2004846117. http://dx.doi.org/10.1073/pnas.2004846117.

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Crop improvement relies heavily on genetic variation that arises spontaneously through mutation. Modern breeding methods are very adept at combining this genetic variation in ways that achieve remarkable improvements in plant performance. Novel traits have also been created through mutation breeding and transgenesis. The advent of gene editing, however, marks a turning point: With gene editing, synthetic variation will increasingly supplement and, in some cases, supplant the genetic variation that occurs naturally. We are still in the very early stages of realizing the opportunity provided by plant gene editing. At present, typically only one or a few genes are targeted for mutation at a time, and most mutations result in loss of gene function. New technological developments, however, promise to make it possible to perform gene editing at scale. RNA virus vectors, for example, can deliver gene-editing reagents to the germ line through infection and create hundreds to thousands of diverse mutations in the progeny of infected plants. With developmental regulators, edited somatic cells can be induced to form meristems that yield seed-producing shoots, thereby increasing throughput and shrinking timescales for creating edited plants. As these approaches are refined and others developed, they will allow for accelerated breeding, the domestication of orphan crops and the reengineering of metabolism in a more directed manner than has ever previously been possible.
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20

Nishizawa‐Yokoi, Ayako, and Seiichi Toki. "A piggyBac ‐mediated transgenesis system for the temporary expression of CRISPR/Cas9 in rice." Plant Biotechnology Journal 19, no. 7 (February 23, 2021): 1386–95. http://dx.doi.org/10.1111/pbi.13559.

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21

Sharma, Rakesh, Manan Azad, Vandana Nunia, S. L. Kothari, and Sumita Kachhwaha. "Differential and developmental stage specific abundance of Zmdreb2a mRNA transcripts under drought stress and root development in Zea mays (L.)." Plant Omics, no. 12(02):2019 (September 20, 2019): 78–86. http://dx.doi.org/10.21475/poj.12.02.19.p1763.

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Environmental stress constraints like temperature, drought and salinity affect plant growth, development and productivity negatively. In maize (Zea mays L.) water-deficiency affects flowering, pollination and embryo development. Transgenic plants having drought tolerance is a priority target in maize breeding programs worldwide. Various transcription factors play a key role in plant development and stress management. DRE-Binding Protein 2A (DREB2A) has been shown to play a central role in drought tolerance in Arabidopsis thaliana. The homologue of dreb2a in Zea mays is Zmdreb2a, a well-known transcription factor regulating genes induced under stress conditions. Studies indicated that Zmdreb2a transgenic plants show not only stress tolerance but also growth retardation. It is a major targeted gene for transgenesis to produce drought-tolerant plants. However, to make construct for transgenic plants, there is need to consider functional transcripts and regulating mechanism of Zmdreb2a mRNAs under drought condition. We have analyzed publicly available RNASeq data of leaf meristem, ovary and developing root and used a host of FastQC, Tophat, Cufflinks, Cuffmerge, Cuffdiff and ‘R’ packages to functionally characterize the differentially expressed genes (DEGs). In addition, prediction of protein structure, nuclear localization signals and transactivation domain were performed using cNLS mapper and 9aaTAD prediction tools. Alternative splicing of Zmdreb2a pre-mRNA was shown to be strongly associated with developmental stage and isoforms show fluctuating expression under stress. This study provides putative functional transcripts of Zmdreb2a, and NMD as a regulating mechanism to control their abundance.
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22

Khakhar, Arjun, Cecily Wang, Ryan Swanson, Sydney Stokke, Furva Rizvi, Surbhi Sarup, John Hobbs, and Daniel F. Voytas. "VipariNama: RNA viral vectors to rapidly elucidate the relationship between gene expression and phenotype." Plant Physiology 186, no. 4 (May 2, 2021): 2222–38. http://dx.doi.org/10.1093/plphys/kiab197.

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Abstract Synthetic transcription factors have great promise as tools to help elucidate relationships between gene expression and phenotype by allowing tunable alterations of gene expression without genomic alterations of the loci being studied. However, the years-long timescales, high cost, and technical skill associated with plant transformation have limited their use. In this work, we developed a technology called VipariNama (ViN) in which vectors based on the tobacco rattle virus are used to rapidly deploy Cas9-based synthetic transcription factors and reprogram gene expression in planta. We demonstrate that ViN vectors can implement activation or repression of multiple genes systemically and persistently over several weeks in Nicotiana benthamiana, Arabidopsis (Arabidopsis thaliana), and tomato (Solanum lycopersicum). By exploring strategies including RNA scaffolding, viral vector ensembles, and viral engineering, we describe how the flexibility and efficacy of regulation can be improved. We also show how this transcriptional reprogramming can create predictable changes to metabolic phenotypes, such as gibberellin biosynthesis in N. benthamiana and anthocyanin accumulation in Arabidopsis, as well as developmental phenotypes, such as plant size in N. benthamiana, Arabidopsis, and tomato. These results demonstrate how ViN vector-based reprogramming of different aspects of gibberellin signaling can be used to engineer plant size in a range of plant species in a matter of weeks. In summary, ViN accelerates the timeline for generating phenotypes from over a year to just a few weeks, providing an attractive alternative to transgenesis for synthetic transcription factor-enabled hypothesis testing and crop engineering.
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23

Lambirth, Kevin C., Adam M. Whaley, Jessica A. Schlueter, Kenneth J. Piller, and Kenneth L. Bost. "Transcript Polymorphism Rates in Soybean Seed Tissue Are Increased in a Single Transformant of Glycine max." International Journal of Plant Genomics 2016 (November 29, 2016): 1–12. http://dx.doi.org/10.1155/2016/1562041.

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Transgenic crops have been utilized for decades to enhance agriculture and more recently have been applied as bioreactors for manufacturing pharmaceuticals. Recently, we investigated the gene expression profiles of several in-house transgenic soybean events, finding one transformant group to be consistently different from our controls. In the present study, we examined polymorphisms and sequence variations in the exomes of the same transgenic soybean events. We found that the previously dissimilar soybean line also exhibited markedly increased levels of polymorphisms within mRNA transcripts from seed tissue, many of which are classified as gene expression modifiers. The results from this work will direct future investigations to examine novel SNPs controlling traits of great interest for breeding and improving transgenic soybean crops. Further, this study marks the first work to investigate SNP rates in transgenic soybean seed tissues and demonstrates that while transgenesis may induce abundant unanticipated changes in gene expression and nucleotide variation, phenotypes and overall health of the plants examined remained unaltered.
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24

Iwakawa, Hidekazu, Benjamin C. Carter, Brett C. Bishop, Joe Ogas, and Stanton B. Gelvin. "Perturbation of H3K27me3-Associated Epigenetic Processes Increases Agrobacterium-Mediated Transformation." Molecular Plant-Microbe Interactions® 30, no. 1 (January 2017): 35–44. http://dx.doi.org/10.1094/mpmi-12-16-0250-r.

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Agrobacterium-mediated transformation is a core technology for basic plant science and agricultural biotechnology. Improving transformation frequency is a major goal for plant transgenesis. We previously showed that T-DNA insertions in some histone genes decreased transformation susceptibility, whereas overexpression of several Arabidopsis H2A and H4 isoforms increased transformation. Overexpression of several histone H2B and H3 isoforms had little effect on transformation frequency. However, overexpression of histone H3-11 (HTR11) enhanced transformation. HTR11 is a unique H3 variant that lacks lysine at positions 9 and 27. The modification status of these lysine residues in canonical H3 proteins plays a critical role in epigenetic determination of gene expression. We mutated histone H3-4 (HTR4), a canonical H3.3 protein that does not increase transformation when overexpressed, by replacing either or both K9 and K27 with the amino acids in HTR11 (either K9I, K27Q, or both). Overexpression of HTR4 with the K27Q but not the K9I substitution enhanced transformation. HTR4K27Q was incorporated into chromatin, and HTR4K27Q overexpression lines exhibited deregulated expression of H3K27me3-enriched genes. These results demonstrate that mutation of K27 in H3.3 is sufficient to perturb H3K27me3-dependent expression in plants as in animals and suggest a distinct epigenetic role for histone HTR11. Further, these observations implicate manipulation of H3K27me3-dependent gene expression as a novel strategy to increase transformation susceptibility.
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25

Hoenicka, Hans, Denise Lehnhardt, Ove Nilsson, Dieter Hanelt, and Matthias Fladung. "Successful crossings with early flowering transgenic poplar: interspecific crossings, but not transgenesis, promoted aberrant phenotypes in offspring." Plant Biotechnology Journal 12, no. 8 (June 29, 2014): 1066–74. http://dx.doi.org/10.1111/pbi.12213.

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26

Khoo, Kelvin H. P., Amanda J. Able, Timothy K. Chataway, and Jason A. Able. "Preliminary characterisation of two early meiotic wheat proteins after identification through 2D gel electrophoresis proteomics." Functional Plant Biology 39, no. 3 (2012): 222. http://dx.doi.org/10.1071/fp11253.

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Various genetic-based approaches including mutant population screens, microarray analyses, cloning and transgenesis have broadened our knowledge of gene function during meiosis in plants. Nonetheless, these genetic tools are not without inherent limitations. One alternative approach to studying plant meiosis, especially in polyploids such as Triticum aestivum L. (bread wheat), is proteomics. However, protein-based approaches using proteomics have seldom been described, with only two attempts at studying early plant meiosis reported. Here, we report the investigation of early bread wheat meiosis using proteomics. Five differentially expressed protein spots were identified using 2D gel electrophoresis (2DGE) on protein extracts from four pooled stages of meiosis and three genotypes (Chinese Spring wild-type, ph1b and ph2a wheat mutant lines). Tandem mass spectrometry (MS/MS) identification of peptides from these protein spots led to the isolation and characterisation of the full-length clones of a wheat Speckle-type POZ protein, an SF21-like protein and HSP70, and a partial coding sequence of a hexose transporter. Significantly, the putative functions of the Speckle-type POZ protein and HSP70 were confirmed using in vitro DNA binding assays. Through the use of a 2DGE proteomics approach, we show that proteomics is a viable alternative to genetic-based approaches when studying meiosis in wheat. More significantly, we report a potential role for a Speckle-type POZ protein and a HSP70 in chromosome pairing during the early stages of meiosis in bread wheat.
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27

Da Lage, Jean-Luc. "The Amylases of Insects." International Journal of Insect Science 10 (January 2018): 117954331880478. http://dx.doi.org/10.1177/1179543318804783.

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Alpha-amylases are major digestive enzymes that act in the first step of maltopolysaccharide digestion. In insects, these enzymes have long been studied for applied as well as purely scientific purposes. In many species, amylases are produced by multiple gene copies. Rare species are devoid of Amy gene. They are predominantly secreted in the midgut but salivary expression is also frequent, with extraoral activity. Enzymological parameters are quite variable among insects, with visible trends according to phylogeny: Coleopteran amylases have acidic optimum activity, whereas dipteran amylases have neutral preference and lepidopteran ones have clear alkaline preference. The enzyme structure shows interesting variations shaped by evolutionary convergences, such as the recurrent loss of a loop involved in substrate handling. Many works have focused on the action of plant amylase inhibitors on pest insect amylases, in the frame of crop protection by transgenesis. It appears that sensitivity or resistance to inhibitors is finely tuned and very specific and that amylases and their inhibitors have coevolved. The multicopy feature of insect amylases appears to allow tissue-specific or stage-specific regulation, but also to broaden enzymological abilities, such as pH range, and to overcome plant inhibitory defenses.
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28

Benavente, Elena, and Estela Giménez. "Modern Approaches for the Genetic Improvement of Rice, Wheat and Maize for Abiotic Constraints-Related Traits: A Comparative Overview." Agronomy 11, no. 2 (February 20, 2021): 376. http://dx.doi.org/10.3390/agronomy11020376.

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After a basic description of the different sets of genetic tools and genomic approaches most relevant for modern crop breeding (e. g., QTL mapping, GWAS and genomic selection; transcriptomics, qPCR and RNA-seq; transgenesis and gene editing), this review paper describes their history and the main achievements in rice, wheat and maize research, with a further focus on crop traits related to the improvement of plant responses to face major abiotic constrains, including nutritional limitations, drought and heat tolerance, and nitrogen-use efficiency (NUE). Remarkable differences have been evidenced regarding the timing and degree of development of some genetic approaches among these major crops. The underlying reasons related to their distinct genome complexity, are also considered. Based on bibliographic records, drought tolerance and related topics (i.e., water-use efficiency) are by far the most abundantly addressed by molecular tools among the breeding objectives considered. Heat tolerance is usually more relevant than NUE in rice and wheat, while the opposite is true for maize.
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Ricroch, Agnès E., Michèle Guillaume-Hofnung, and Marcel Kuntz. "The ethical concerns about transgenic crops." Biochemical Journal 475, no. 4 (February 28, 2018): 803–11. http://dx.doi.org/10.1042/bcj20170794.

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It is generally accepted that transgenesis can improve our knowledge of natural processes, but also leads to agricultural, industrial or socio-economical changes which could affect human society at large and which may, consequently, require regulation. It is often stated that developing countries are most likely to benefit from plant biotechnology and are at the same time most likely to be affected by the deployment of such new technologies. Therefore, ethical questions related to such biotechnology probably also need to be addressed. We first illustrate how consequentialist and nonconsequentialist theories of ethics can be applied to the genetically modified organism debate, namely consequentialism, autonomy/consent ethics (i.e. self-determination of people regarding matters that may have an effect on these people) and virtue ethics (i.e. whether an action is in adequacy with ideal traits). We show that these approaches lead to highly conflicting views. We have then refocused on moral ‘imperatives', such as freedom, justice and truth. Doing so does not resolve all conflicting views, but allows a gain in clarity in the sense that the ethical concerns are shifted from a technology (and its use) to the morality or amorality of various stakeholders of this debate.
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Cazzola, Federico, Carolina Julieta Bermejo, Ileana Gatti, and Enrique Cointry. "Speed breeding in pulses: an opportunity to improve the efficiency of breeding programs." Crop and Pasture Science 72, no. 3 (2021): 165. http://dx.doi.org/10.1071/cp20462.

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Pulses form an important component of the human diet, provide animal feed, and replenish soil fertility through biological nitrogen fixation. However, pulse breeding is a time consuming process. Most of the traditional breeding programs take 10–15 years to release an improved cultivar. In the breeder’s equation, a model of the expected change in a trait in response to selection, cycle time is the most powerful parameter for increasing genetic gain. Shuttle breeding, double haploids and in vitro culture are some of the methodologies that have been developed; however, they have not been able to be implemented efficiently in the breeding programs for pulses. In this context, speed breeding emerges as a technology that allows increased efficiency of the programs, reducing costs and the work required. The technique uses optimal light quality, light intensity, daylength and temperature control to accelerate photosynthesis and flowering, coupled with early seed harvest. It can be integrated with other breeding technologies, does not include transgenesis or gene editing, and is presented as a revolution to increase the efficiency of the programs. We present different advances in pulse breeding programs and propose a speed breeding system for pea (Pisum sativum L.) that includes hybridisations and advancing generations in a growth chamber. This review concludes by highlighting the opportunities and challenges to incorporating speed breeding into pulse breeding programs.
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Zheng, Qingbo, Yakun Chen, Xiaolin Jia, Yi Wang, Ting Wu, Xuefeng Xu, Zhenhai Han, Zhihong Zhang, and Xinzhong Zhang. "MicroRNA156 (miR156) Negatively Impacts Mg-Protoporphyrin IX (Mg-Proto IX) Biosynthesis and Its Plastid-Nucleus Retrograde Signaling in Apple." Plants 9, no. 5 (May 22, 2020): 653. http://dx.doi.org/10.3390/plants9050653.

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Plastid-nucleus retrograde signaling (PNRS) play essential roles in regulating nuclear gene expression during plant growth and development. Excessive reactive oxygen species can trigger PNRS. We previously reported that in apple (Malus domestica Borkh.) seedlings, the expression of microRNA156 (miR156) was significantly low in the adult phase, which was accompanied by high levels of hydrogen peroxide (H2O2) accumulation in chloroplasts. However, it was unclear whether adult-phase-specific chloroplast H2O2 may induce PNRS and affect miR156 expression, or miR156 triggers adult phase PNRS during the ontogenesis. In this paper, we examined the relationship between miR156 levels and six PNRS components in juvenile and adult phase leaves from ‘Zisai Pearl’בRed Fuji’ hybrids. We found that PNRS generated by singlet oxygen (1O2), the photosynthetic redox state, methylerythritol cyclodiphosphate (MEcPP), SAL1-3-phosphoadenosine 5-phosphate (PAP) and WHIRLY1 were not involved. The accumulation of Mg-protoporphyrin IX (Mg-Proto IX), the expression of the synthetic genes MdGUN5 and MdGUN6, and Mg-Proto IX PNRS related nuclear genes increased with ontogenesis. These changes were negatively correlated with miR156 expression. Manipulating Mg-Proto IX synthesis with 5-aminolevulinic acid (ALA) or gabaculine did not affect miR156 expression in vitro shoots. In contrast, modulating miR156 expression via MdGGT1 or MdMIR156a6 transgenesis led to changes in Mg-Proto IX contents and the corresponding gene expressions. It was concluded that the Mg-Proto IX PNRS was regulated downstream of miR156 regardless of adult-phase-specific plastid H2O2 accumulation. The findings may facilitate the understanding of the mechanism of ontogenesis in higher plants.
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Lam, Emily K., Kaitlin N. Allen, Julia María Torres-Velarde, and José Pablo Vázquez-Medina. "Functional Studies with Primary Cells Provide a System for Genome-to-Phenome Investigations in Marine Mammals." Integrative and Comparative Biology 60, no. 2 (June 9, 2020): 348–60. http://dx.doi.org/10.1093/icb/icaa065.

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Synopsis Marine mammals exhibit some of the most dramatic physiological adaptations in their clade and offer unparalleled insights into the mechanisms driving convergent evolution on relatively short time scales. Some of these adaptations, such as extreme tolerance to hypoxia and prolonged food deprivation, are uncommon among most terrestrial mammals and challenge established metabolic principles of supply and demand balance. Non-targeted omics studies are starting to uncover the genetic foundations of such adaptations, but tools for testing functional significance in these animals are currently lacking. Cellular modeling with primary cells represents a powerful approach for elucidating the molecular etiology of physiological adaptation, a critical step in accelerating genome-to-phenome studies in organisms in which transgenesis is impossible (e.g., large-bodied, long-lived, fully aquatic, federally protected species). Gene perturbation studies in primary cells can directly evaluate whether specific mutations, gene loss, or duplication confer functional advantages such as hypoxia or stress tolerance in marine mammals. Here, we summarize how genetic and pharmacological manipulation approaches in primary cells have advanced mechanistic investigations in other non-traditional mammalian species, and highlight the need for such investigations in marine mammals. We also provide key considerations for isolating, culturing, and conducting experiments with marine mammal cells under conditions that mimic in vivo states. We propose that primary cell culture is a critical tool for conducting functional mechanistic studies (e.g., gene knockdown, over-expression, or editing) that can provide the missing link between genome- and organismal-level understanding of physiological adaptations in marine mammals.
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Sant’Ana, Rodrigo Ribeiro Arnt, Clarissa Alves Caprestano, Rubens Onofre Nodari, and Sarah Zanon Agapito-Tenfen. "PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts." Genes 11, no. 9 (September 2, 2020): 1029. http://dx.doi.org/10.3390/genes11091029.

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Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology allows the modification of DNA sequences in vivo at the location of interest. Although CRISPR-Cas9 can produce genomic changes that do not require DNA vector carriers, the use of transgenesis for the stable integration of DNA coding for gene-editing tools into plant genomes is still the most used approach. However, it can generate unintended transgenic integrations, while Cas9 prolonged-expression can increase cleavage at off-target sites. In addition, the selection of genetically modified cells from millions of treated ones, especially plant cells, is still challenging. In a protoplast system, previous studies claimed that such pitfalls would be averted by delivering pre-assembled ribonucleoprotein complexes (RNPs) composed of purified recombinant Cas9 enzyme and in vitro transcribed guide RNA (gRNA) molecules. We, therefore, aimed to develop the first DNA-free protocol for gene-editing in maize and introduced RNPs into their protoplasts with polyethylene glycol (PEG) 4000. We performed an effective transformation of maize protoplasts using different gRNAs sequences targeting the inositol phosphate kinase gene, and by applying two different exposure times to RNPs. Using a low-cost Sanger sequencing protocol, we observed an efficiency rate of 0.85 up to 5.85%, which is equivalent to DNA-free protocols used in other plant species. A positive correlation was displayed between the exposure time and mutation frequency. The mutation frequency was gRNA sequence- and exposure time-dependent. In the present study, we demonstrated that the suitability of RNP transfection was proven as an effective screening platform for gene-editing in maize. This efficient and relatively easy assay method for the selection of gRNA suitable for the editing of the gene of interest will be highly useful for genome editing in maize, since the genome size and GC-content are large and high in the maize genome, respectively. Nevertheless, the large amplitude of mutations at the target site require scrutiny when checking mutations at off-target sites and potential safety concerns.
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Huang, He, Yuting Liu, Ya Pu, Mi Zhang, and Silan Dai. "Transcriptome Analysis of Chrysanthemum lavandulifolium Response to Salt Stress and Overexpression a K+ Transport ClAKT Gene-enhanced Salt Tolerance in Transgenic Arabidopsis." Journal of the American Society for Horticultural Science 144, no. 4 (July 2019): 219–35. http://dx.doi.org/10.21273/jashs04629-18.

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Plant growth and development are significantly affected by salt stress. Chrysanthemum lavandulifolium is a halophyte species and one of the ancestors of chrysanthemum (C. ×morifolium). Understanding how this species tolerates salt stress could provide vital insight for clarifying the salt response systems of higher plants, and chrysanthemum-breeding programs could be improved. In this study, salt tolerance was compared among C. lavandulifolium and three chrysanthemum cultivars by physiological experiments, among which C. lavandulifolium and Jinba displayed better tolerance to salt stress than the other two cultivars, whereas Xueshan was a salt-sensitive cultivar. Using the transcriptome database of C. lavandulifolium as a reference, we used digital gene expression technology to analyze the global gene expression changes in C. lavandulifolium seedlings treated with 200 mm NaCl for 12 hours compared with seedlings cultured in normal conditions. In total, 2254 differentially expressed genes (DEGs), including 1418 up-regulated and 836 down-regulated genes, were identified. These DEGs were significantly enriched in 35 gene ontology terms and 29 Kyoto Encyclopedia of Genes and Genomes pathways. Genes related to signal transduction, ion transport, proline biosynthesis, reactive oxygen species scavenging systems, and flavonoid biosynthesis pathways were relevant to the salt tolerance of C. lavandulifolium. Furthermore, comparative gene expression analysis was conducted using reverse transcription polymerase chain reaction to compare the transcriptional levels of significantly up-regulated DEGs in C. lavandulifolium and the salt-sensitive cultivar Xueshan, and species-specific differences were observed. The analysis of one of the DEGs, ClAKT, an important K+ transport gene, was found to enable transgenic Arabidopsis thaliana to absorb K+ and efflux Na+ under salt stress and to absorb K+ under drought stress. The present study investigated potential genes and pathways involved in salt tolerance in C. lavandulifolium and provided a hereditary resource for the confinement of genes and pathways responsible for salt tolerance in this species. This study provided a valuable source of reference genes for chrysanthemum cultivar transgenesis breeding.
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Mykhalska, S. I., A. G. Komisarenko, V. M. Kurchii, and O. M. Tishchenko. "Agrobacterium-mediated in planta genetic transformation of winter wheat (Triticum aestivum L.)." Faktori eksperimental'noi evolucii organizmiv 22 (September 9, 2018): 293–98. http://dx.doi.org/10.7124/feeo.v22.964.

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Aim. To optimize the agrobacterium-mediated method of winter wheat transformation (Triticum aestivum L.); to select the conditions and period of inoculation to effectively transfer the genes during pollination. Methods. Agrobacterium-mediated in planta genetic transformation of winter wheat (Triticum aestivum L.) during pollination. Results. The conditions for agrobacterium-mediated transformation method of winter wheat during natural (frequency pollination was 1 %) and non-natural (frequency pollination was 4 %) pollination were defined. Conclusions. The possibility of integrating transgenes into the genome of winter wheat plants by the method of Agrobacterium-mediated transformation in planta in the process of forced and natural pollination is demonstrated. It is found that the transformation efficiency to a large extent depends on the plant genotype and the method of carrying out the transformation procedure. The selection of transgenic plants under water deficit conditions allowed to identify the plants with functional transgene. The signs of functioning transgene have been remaining in the next generation of genetically modified winter wheat. Keywords: Triticum aestivum L., Agrobacterium-mediated transformation in planta, transgenic plants, seeds.
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Scott, Alicia, D. R. Woodfield, Anne Allan, Dorothy Maher, and D. W. R. White. "Inheritance and expression of transgenes in white clover." NZGA: Research and Practice Series 6 (January 1, 1996): 131–35. http://dx.doi.org/10.33584/rps.6.1995.3352.

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White clover plants from a range of cultivars can now be routinely transformed with cloned foreign genes. However, before transgenic white clover cultivars can be developed, these inserted genes must be stably inherited and expressed at appropriate levels in progeny. Primary transgenic white clover plants containing a uidA (GUS) reporter transgene were outcrossed and the inheritance and expression of the uidA gene was examined over two generations, in several different cultivar backgrounds. Both Mendelian inheritance and consistent expression in different genetic backgrounds were obtained from strongly expressing primary transgenic plants. However, primary transgenic plants with weak or variable expression gave non-Mendelian inheritance and inconsistent expression of the transgene in progeny plants. Transgenic BC1 plants were also intercrossed to produce a segregating F2 population containing individuals heterozygous or homozygous for the transgene. In these populations heterozygous and homozygous plants had similar levels of uidA gene expression. These results indicate that F2 plants, homozygous for a transgene, might be used to develop a transgenic cultivar. However, both selection of a primary transgenic plant with stable, high level expression of the introduced gene and progeny testing, to determine the influence of genetic background, are prerequisites to such a development. Keywords: gene expression, inheritance, transgene, uidA reporter, white clover
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Bakhsh, Allah, Rao Qayyum, Zeeshan Shamim, and Tayyab Husnain. "A mini review: RuBisCo small subunit as a strong, green tissue-specific promoter." Archives of Biological Sciences 63, no. 2 (2011): 299–307. http://dx.doi.org/10.2298/abs1102299b.

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Plant genetic transformation is a powerful application used to study gene expression in plants. Transcriptomics has the potential to rapidly increase our knowledge of spatial and temporal gene expression and lead to new promoters for research and development. The availability of a broad spectrum of promoters with the ability to regulate the temporal and spatial expression patterns of transgenes can increase the successful application of transgenic technology. A variety of promoters is necessary at all levels of genetic engineering in plants, from basic research, to the development of economically viable crops and plant commodities, it can address legitimate concerns raised about the safety and containment of transgenic plants in the environment. Compared with temporal- or spatial-specific expression of a toxin, constitutive expression of foreign proteins in transgenic plants can cause adverse effects. The constitutive overexpression of transgenes that interferes with normal processes in a plant underscores the need for refinement of transgene expression. The development of tissue-specific promoters to drive transgene expression has helped fulfill that need. Therefore, in certain circumstances it is desirable to use expression-specific promoters which only express the foreign gene in specific plant tissues or organs. This review highlights the uses and benefits reaped by the use of green tissue-specific promoter for the RuBisCo small subunit in different crops and systems and thus establishing a broad range of tissue-specific promoters. Such plant promoters that are activated precisely when and where they are needed would be ideal for genetic engineering strategies.
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Dubrovina, Alexandra, Olga Aleynova, Alexander Kalachev, Andrey Suprun, Zlata Ogneva, and Konstantin Kiselev. "Induction of Transgene Suppression in Plants via External Application of Synthetic dsRNA." International Journal of Molecular Sciences 20, no. 7 (March 29, 2019): 1585. http://dx.doi.org/10.3390/ijms20071585.

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Recent investigations show that exogenously applied small interfering RNAs (siRNA) and long double-stranded RNA (dsRNA) precursors can be taken up and translocated in plants to induce RNA interference (RNAi) in the plant or in its fungal pathogen. The question of whether genes in the plant genome can undergo suppression as a result of exogenous RNA application on plant surface is almost unexplored. This study analyzed whether it is possible to influence transcript levels of transgenes, as more prone sequences to silencing, in Arabidopsis genome by direct exogenous application of target long dsRNAs. The data revealed that in vitro synthesized dsRNAs designed to target the gene coding regions of enhanced green fluorescent protein (EGFP) or neomycin phosphotransferase II (NPTII) suppressed their transcript levels in Arabidopsis. The fact that, simple exogenous application of polynucleotides can affect mRNA levels of plant transgenes, opens new opportunities for the development of new scientific techniques and crop improvement strategies.
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Li, Jiarui, Timothy C. Todd, and Harold N. Trick. "Rapid in planta evaluation of root expressed transgenes in chimeric soybean plants." Plant Cell Reports 29, no. 2 (December 15, 2009): 113–23. http://dx.doi.org/10.1007/s00299-009-0803-2.

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40

Kiselev, Konstantin V., Andrey R. Suprun, Olga A. Aleynova, Zlata V. Ogneva, and Alexandra S. Dubrovina. "Physiological Conditions and dsRNA Application Approaches for Exogenously induced RNA Interference in Arabidopsis thaliana." Plants 10, no. 2 (January 30, 2021): 264. http://dx.doi.org/10.3390/plants10020264.

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Recent studies have revealed that foliar application of double-stranded RNAs (dsRNAs) or small-interfering RNAs (siRNAs) encoding specific genes of plant pathogens triggered RNA interference (RNAi)-mediated silencing of the gene targets. However, a limited number of reports documented silencing of plant endogenes or transgenes after direct foliar RNA application. This study analyzed the importance of physiological conditions (plant age, time of day, soil moisture, high salinity, heat, and cold stresses) and different dsRNA application means (brush spreading, spraying, infiltration, inoculation, needle injection, and pipetting) for suppression of neomycin phosphotransferase II (NPTII) transgene in Arabidopsis thaliana, as transgenes are more prone to silencing. We observed a higher NPTII suppression when dsRNA was applied at late day period, being most efficient at night, which revealed a diurnal variation in dsRNA treatment efficacy. Exogenous NPTII-dsRNA considerably reduced NPTII expression in 4-week-old plants and only limited it in 2- and 6-week-old plants. In addition, a more discernible NPTII downregulation was detected under low soil moisture conditions. Treatment of adaxial and abaxial leaf surfaces by brushes, spraying, and pipetting showed a higher NPTII suppression, while infiltration and inoculation were less efficient. Thus, appropriate plant age, late time of day, low soil moisture, and optimal dsRNA application modes are important for exogenously induced gene silencing.
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41

Morozov, S. Y., A. G. Solovyev, N. O. Kalinina, and M. E. Taliansky. "Double-Stranded RNAs in Plant Protection Against Pathogenic Organisms and Viruses in Agriculture." Acta Naturae 11, no. 4 (December 15, 2019): 13–21. http://dx.doi.org/10.32607/20758251-2019-11-4-13-21.

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Recent studies have shown that plants are able to express the artificial genes responsible for the synthesis of double-stranded RNAs (dsRNAs) and hairpin double-stranded RNAs (hpRNAs), as well as uptake and process exogenous dsRNAs and hpRNAs to suppress the gene expression of plant pathogenic viruses, fungi, or insects. Both endogenous and exogenous dsRNAs are processed into small interfering RNAs (siRNAs) that can spread locally and systemically through the plant, enter pathogenic microorganisms, and induce RNA interference-mediated pathogen resistance in plants. There are numerous examples of the development of new biotechnological approaches to plant protection using transgenic plants and exogenous dsRNAs. This review summarizes new data on the use of transgenes and exogenous dsRNAs for the suppression of fungal and insect virulence genes, as well as viruses to increase the resistance of plants to these pathogens. We also analyzed the current ideas about the mechanisms of dsRNA processing and transport in plants.
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Dafny-Yelin, Mery, and Tzvi Tzfira. "Delivery of Multiple Transgenes to Plant Cells." Plant Physiology 145, no. 4 (December 2007): 1118–28. http://dx.doi.org/10.1104/pp.107.106104.

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43

Lee, Ye-Rin, Chae-Yeon Lim, Sohee Lim, Se Ra Park, Jong-Pil Hong, Jinhee Kim, Hye-Eun Lee, Kisung Ko, and Do-Sun Kim. "Expression of Colorectal Cancer Antigenic Protein Fused to IgM Fc in Chinese Cabbage (Brassica rapa)." Plants 9, no. 11 (October 30, 2020): 1466. http://dx.doi.org/10.3390/plants9111466.

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The epithelial cell adhesion molecule (EpCAM) is a tumor-associated antigen and a potential target for tumor vaccine. The EpCAM is a cell-surface glycoprotein highly expressed in colorectal carcinomas. The objective of the present study is to develop an edible vaccine system through Agrobacterium-mediated transformation in Chinese cabbage (Brassica rapa). For the transformation, two plant expression vectors containing genes encoding for the EpCAM recombinant protein along with the fragment crystallizable (Fc) region of immunoglobulin M (IgM) and Joining (J)-chain tagged with the KDEL endoplasmic reticulum retention motif (J-chain K) were constructed. The vectors were successfully transformed and expressed in the Chinese cabbage individually using Agrobacterium. The transgenic Chinese cabbages were screened using genomic polymerase chain reaction (PCR) in T0 transgenic plant lines generated from both transformants. Similarly, the immunoblot analysis revealed the expression of recombinant proteins in the transformants. Further, the T1 transgenic plants were generated by selfing the transgenic plants (T0) carrying EpCAM–IgM Fc and J-chain K proteins, respectively. Subsequently, the T1 plants generated from EpCAM–IgM Fc and J-chain K transformants were crossed to generate F1 plants carrying both transgenes. The presence of both transgenes was validated using PCR in the F1 plants. In addition, the expression of Chinese cabbage-derived EpCAM–IgM Fc × J-chain K was evaluated using immunoblot and ELISA analyses in the F1 plants. The outcomes of the present study can be utilized for the development of a potential anti-cancer vaccine candidate using Chinese cabbage.
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Scorza, Ralph, Laurene Levy, Vern Damsteegt, Ann Callahan, Kevin Webb, and Michel Ravelonandro. "Transfer of Plum Pox Virus Coat Protein Genes from a Plum Pox-resistant Transgenic Clone of Prunus domestica Plum to Its Progeny through Hybridization." HortScience 33, no. 3 (June 1998): 532a—532. http://dx.doi.org/10.21273/hortsci.33.3.532a.

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Sharka or plum pox virus (PPV) is a major disease of stone fruit and causes severe economic losses in Europe. There is little resistance to PPV in most Prunus species, thus genetic engineering represents a potentially useful approach to obtain resistant germplasm. Transgenic plums containing the PPV coat protein (CP) or the related papaya ringspot virus (PRV)-CP gene were produced through Agrobacterium tumefaciens-mediated transformation. These transgenic plum clones were then evaluated for resistance to PPV infection in the greenhouse by graft or aphid inoculation with PPV. While symptoms of PPV appeared in most transgenic clones, all plants of PPV-CP transgenic clone C5 were symptomless and ELISA and immunocapture-reverse transcriptase PCR negative for over three years following inoculation with two strains of PPV (Ravelonandro et al., Plant Dis. 81:1231-1235, 1997). Clone C5, which contains multiple copies of the PPV-CP gene, was hybridized with PRV-CP transgenic plants or untransformed plum cultivars. Progeny were obtained containing no transgenes, only the PPV-CP, only the PRV-CP, or both the PRV-CP and PPV-CP transgenes. Seedlings were inoculated with PPV. At 5 and 11 months post-inoculation, seedlings containing the PPV-CP genes from C5 were symptomless and ELISA negative. Seedlings containing only PRV-CP transgenes or non-transformed controls showed symptoms of PPV infection and were ELISA positive. These results indicate that the PPV-CP transgenes can be transferred to progeny through hybridization and that these genes can impart resistance to PPV in transgenic seedlings. The inheritance of the multicopy inserts of the PPV-CP and PRV-CP transgenes is being analyzed. The combined effects of both transgenes on resistance to PPV and the stability of PPV resistance in the progeny of the resistant C5 transgenic line are currently under evaluation.
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Stokes, T. "Maize barrier of transgenes." Trends in Plant Science 6, no. 1 (January 2001): 12. http://dx.doi.org/10.1016/s1360-1385(00)01852-5.

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Chen, Weiqiang, Gurminder Kaur, Lili Hou, Ruyu Li, and David W. Ow. "Replacement of stacked transgenes in planta." Plant Biotechnology Journal 17, no. 11 (June 12, 2019): 2029–31. http://dx.doi.org/10.1111/pbi.13172.

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47

Allison, Richard F., William L. Schneider, and Ann E. Greene. "Recombination in plants expressing viral transgenes." Seminars in Virology 7, no. 6 (December 1996): 417–22. http://dx.doi.org/10.1006/smvy.1996.0050.

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48

Matzke, Antonius J. M., and Marjori A. Matzke. "Position effects and epigenetic silencing of plant transgenes." Current Opinion in Plant Biology 1, no. 2 (April 1998): 142–48. http://dx.doi.org/10.1016/s1369-5266(98)80016-2.

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Darbani, Behrooz, Safar Farajnia, Shahin Noeparvar, C. Neal Stewa, Seyed A. Mohammadi, and Saeed Zakerbosta. "Plant Transformation: Needs and Futurity of the Transgenes." Biotechnology(Faisalabad) 7, no. 3 (June 15, 2008): 403–12. http://dx.doi.org/10.3923/biotech.2008.403.412.

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Darbani, Behrooz, Safar Farajnia, Shahin Noeparvar, C. Neal Stewart Jr., Seyed A. Mohammadi, and Saeed Zakerbosta. "Plant Transformation: Needs and Futurity of the Transgenes." Biotechnology(Faisalabad) 10, no. 4 (June 15, 2011): 341–50. http://dx.doi.org/10.3923/biotech.2011.341.350.

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