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Genetically Modified Food Crops: Current Concerns and Solutions for Next Generation Crops
... In most angiosperm plant species, plastid genes are inherited in a uniparental and strictly maternal fashion (Birky, 1995;Mogensen, 1996;Zhang, Liu, and Sodmergen, 2003;Hagemann, 2004). Although plants exhibiting maternal plastid inheritance contain pollen with metabolically active plastids, the plastid DNA itself is lost during the process of pollen maturation and is therefore not transmitted to the next generation (Nagata et al., 1999;Daniell, 1999Daniell, , 2000. The lack of plastid DNA in pollen thereby minimizes the possibility of leaking transgenes to related weeds or crops (Daniell et al., 1998;Scott and Wilkenson, 1999;Daniell, 2002) and reduces the potential toxicity of transgenic pollen to nontarget insects (De Cosa et al., 2001). ...
... Unfortunately, it cannot distinguish between crops and weeds, requiring the use of genetic engineering in order to confer resistance. Although nuclear transgenic plants engineered for herbicide resistance were quite effective, the possibility of transgenes outcrossing to other related species and weeds poses a significant drawback (Daniell, 1999(Daniell, , 2000. Transgenic plants resistant to glyphosate are typically engineered to overexpress EPSPS. ...
Chloroplast genetic engineering offers a number of unique advantages, including a high-level of transgene expression, multi-gene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects, and undesirable foreign DNA. Thus far, over forty transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer important agronomic traits, as well as express industrially valuable biomaterials and therapeutic proteins. The hyperexpression of recombinant proteins within plastid engineered systems offers a cost effective solution for using plants as bioreactors. Additionally, the presence of chaperones and enzymes within the chloroplast help to assemble complex multi-subunit proteins and correctly fold proteins containing disulfide bonds, thereby drastically reducing the costs of in vitro processing. Oral delivery of vaccine antigens against cholera, tetanus, anthrax, plague, and canine parvovirus are made possible because of the high expression levels and antibiotic-free selection systems available in plastid transformation systems. Plastid genetic engineering also has become a powerful tool for basic research in plastid biogenesis and function. This approach has helped to unveil a wealth of information about plastid DNA replication origins, intron maturases, translation elements and proteolysis, import of proteins and several other processes. Although many successful examples of plastid engineering have set a foundation for various future applications, this technology has not been extended to many of the major crops. Highly efficient plastid transformation has been recently accomplished via somatic embryogenesis using species-specific chloroplast vectors in soybean, carrot, and cotton. Transgenic carrots were able to withstand salt concentrations that only halophytes could tolerate; more than twice the effectiveness of other engineering attempts. Recent advances in plastid engineering provide an efficient platform for the production of therapeutic proteins, vaccines, and biomaterials using an environmentally friendly approach. This review takes an in-depth look into the state of the art in plastid engineering and offers directions for further research and development.
... 7) The toxin gene can be expressed in the chloroplast genome and thus the possibility of gene transfer via pollen (Daniell, 2000) is highly variable (Szekacs et al., 2010). It was reported that pollen from Bt corn is highly toxic to Monarch butterflies in the laboratory (losey et al., 1999), but follow up field studies showed that under real-life conditions Monarch butterfly caterpillars rarely come in contact with pollen from corn (losey et al., 1999; Sears et al., 2001). ...
... For example, by expressing insecticidal proteins in chloroplasts, toxins can be put in the part of a plant where they are most likely to be consumed. The toxin gene can be expressed in the chloroplast genome and thus the possibility of gene transfer via pollen (Daniell, 2000), in which toxin concentration is highly variable (Szekacs et al., 2010). Most caterpillars feed on green tissues that are rich in chloroplasts; therefore, they consume the highest level of insecticidal toxins if the toxins are placed in chloroplasts . ...
One of the best modern agricultural defenses against plant-eating insects is Bacillus thuringiensis (Bt), which either can be applied to the surface of the plant, to provide temporary protection, or can be genetically engineered into the plant to protect it against insects throughout its lifespan. Plants can be genetically engineered to produce their own Bt crystal protein (CP), which is toxic to the pest species of concern. As the insect feeds on the plant, it ingests the CP and suffers the same fate as if the leaf tissue was sprayed with Bt. The use of commercial crops expressing Bt toxins has increased in the recent years due to their advantages in plant protection and lower production costs, however, insects-developed resistance against plant defense mechanisms and the consequent effects of Bt-plants on non target species are hence considered disadvantages. This is still a controversial topic and the question is: Within the next few years, will Bt-plants provide hope for the future of crop protection?
... Currently, QTLs/Cry-protein applied in soybean is expressed in the whole plant (Cregan et al. 1999;Ortega et al. 2016), whereas insects feed mostly on the soybean leaf and pods. It was suggested that Cry-protein should be expressed in the chloroplast genome since it offers the possibility of gene transfer to pollen (Daniell 2000) and high-level toxicity in the leaves. Therefore, transforming chloroplast genome provides high dosage of Cry-protein/mg leaf consumed and offers a more effective tool of controlling the destructive insects. ...
The most important insect pests causing severe economic damages to soybean (Glycine max L.) production worldwide are Chrysodeixis includens (Walker, Noctuidae), Anticarsia gemmatalis (Hübner, Erebidae), Helicoverpa gelotopoeon (Dyar, Noctuidae), Crocidosema aporema (Walsingham; Tortricidae), Spodoptera albula (Walker, Noctuidae), S. cosmiodes (Walker, Noctuidae), S. eridania (Stoll, Noctuidae), S. frugiperda (Smith; Noctuidae), Helicoverpa armigera (Hübner, Noctuidae), H. zea (Boddie; Noctuidae) and Telenomus podisi (Hymenoptera,Platygastidae). Despite the success of biotech Bacillus thuringiensis (Bt)/herbicide tolerance (HT)-soybean in the past decade in terms of output, unforeseen mitigated performances have been observed due to changes in climatic events that favors the emergence of insect resistance. Thus, there is a need to develop hybrids with elaborated gene stacking to avert the upsurge in insect field tolerance to crystal (Cry) toxins in Bt-soybean. This study covers the performance of important commercial transgenic soybean developed to outwit destructive insects. New gene stacking soybean events such as Cry1Ac-, Cry1AF- and PAT-soybean (DAS-81419-2®, Conkesta™ technology), and MON-87751-7 × MON-87701–2 × MON 87708 × MON 89788 (bearing Cry1A.105 [Cry1Ab, Cry1F, Cry1Ac], Cry2Ab, Cry1Ac) are being approved and deployed in fields. Following this deployment trend, we recommend herein that plant-mediated RNA interference into Bt-soybean, and the application of RNA-based pesticides that is complemented by other best agricultural practices such as refuge compliance, and periodic application of low-level insecticides could maximize trait durability in Bt-soybean production in the twenty-first century.
... There have been potential environmental concerns about transgene introgression via pollen to weeds or related crops. Besides, there are concerns due to the low expression levels that could lead to the possibility of development of insect resistance from the plant expressing low levels of insecticidal protein (Bogorad, 2000;Daniell, 2000). All these concerns underscore the need for development of alternate approaches. ...
The chloroplast genetic engineering approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. Thus far, more than 40 transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer several agronomic traits and produce vaccine antigens, industrially valuable enzymes, biomaterials, and amino acids. Functionality of chloroplast-derived vaccine antigens and therapeutic proteins have been demonstrated by in vitro assays and animal studies. Oral delivery of vaccine antigens has been facilitated by hyperexpression in transgenic chloroplasts (leaves) or non-green plastids (carrots) and the availability of antibiotic-free selectable markers or the ability to excise selectable marker genes. Additionally, the presence of chaperones and enzymes within the chloroplast help to assemble complex multi-subunit proteins and correctly fold proteins containing disulfide bonds, thereby drastically reducing the costs of in vitro processing. Despite such significant progress in chloroplast transformation, this technology has not been extended to major crops. This obstacle emphasizes the need for plastid genome sequencing to increase the efficiency of transformation and conduct basic research in plastid biogenesis and function. However, highly efficient soybean, carrot, and cotton plastid transformation has been recently accomplished via somatic embryogenesis using species-specific chloroplast vectors. Recent advancements facilitate our understanding of plastid biochemistry and molecular biology. This review focuses on exciting recent developments in this field and offers directions for further research and development.
... Plastids are inherited in a uniparental, strictly maternal fashion in most angiosperm plant species (Hagemann 2004); therefore, gene containment occurs owing to the lack of transgene transmission by pollen. Although pollen contains plastids, the plastid DNA itself is lost during the process of pollen maturation and therefore is not transmitted to the next generation (Daniell 1999(Daniell , 2000Nagata et al. 1999). ...
Chloroplast engineering (or chloroplast transformation technology, CTT) is a strategy consisting of inserting a transgene into the chloroplast genome of a plant instead of its nuclear genome. CTT brings advantages such as control of the site of gene insertion, high rates of transgene expression and protein accumulation, lack of transmission of the transgene via pollen due to the fact that plastid genes are maternally inherited and an absence of epigenetic effects. Tobacco remains the species most amenable to CTT to date, although chloroplast genetic engineering has also been achieved successfully in crops such as maize, tomato, cotton, potato, rice and sugar beets. Improving agricultural traits such as herbicide and pathogen resistance, resistance to drought, salt tolerance and phytoremediation potential are all promising applications. Molecular pharming is another area of chloroplast engineering with high potential; the production of a wide range of products such as vaccine antigens, pharmaceutical proteins (human somatotropin, human serum albumin, human interferon, monoclonal antibodies) and industrial proteins (avidin, beta casein, liquid crystal polymers, xylanase, anthranilate synthase) is economically beneficial in comparison with bacterial cultivation or animal cell cultures. This review summarises the current status of CCT and its potential economic impact from the viewpoint of high levels of transgene expression and high accumulation of foreign proteins.
The sections in this article are
Introduction
Historical Aspects
Unique Features of Chloroplast Genetic Engineering
Maternal Inheritance and Gene Containment
Crop Species Stably Transformed via the Plastid Genome
Agronomic Traits Conferred via the Plastid Genome
Transgenic Plastids as Bioreactors
Biomaterials, Enzymes, and Amino Acids
Conclusions
Acknowledgements
The life-supporting metabolic process of photosynthesis is carried out in endosymbiotic organelles, the chloroplasts. Scientific endeavors over the last decade have enabled us to successfully engineer the chloroplast genome. This singular development has opened new avenues for agricultural biotechnology and has contributed immensely to our understanding of the basic genetic mechanisms operative within the organelle. The chloroplast genome has been engineered to express agronomically important traits such as disease-resistance, drought-tolerance, herbicide-resistance, insect resistance and production of antibodies, biopharmaceuticals and edible vaccines. The ability to hyper-express prokaryotic and eukaryotic proteins without the drawbacks of gene silencing or position effects, combined with the advantage of gene containment due to plastid maternal inheritance, make chloroplast transformation technology both useful and environmentally-friendly. The recent use of a plant-derived selectable marker for chloroplast transformation is an important development that should help allay public fears about GM foods. In addition, the extension of chloroplast transformation technology to edible plant species such as potato and tomato, coupled with the hyper-expression of antigens, opens up the possibility of oral delivery of vaccines and other biopharmaceuticals. This review focuses on some of these recent accomplishments and their impact.
Molecular biotechnology entails deoxyribonucleic acid manipulations of living organisms, including microscopic creatures, plants, and animals by employing various customary and emerging approaches such as omics, molecular biology, molecular genetics, molecular pathology, microbiology, nanotechnology, forensic botany, molecular virology, and their biotechnological applications in the area of agriculture, health, and industry. Of these, genetic manipulation of plants is generally accomplished through a toolbox that allows engineering genes and by relocating the engineered genes into other organisms to develop processes and/or produce useful products. Hence, gene/s can be transferred from one organism to another through bypassing sexual means of reproductionacross kingdoms; contributing significantly toward improvement of agronomic, horticultural, and forest plants. But, when talking about developing transgenic plants, containment of transgenes is one of the main concerns of the consumers; hence, chloroplast transformation comes in place since chloroplasts are predominantly maternally inherited to progenies. This chapter summarizes applications of plant molecular biotechnology in the multidisciplinary perspective.
Transient expression of β-glucuronidase (GUS) in different cellular compartments following biolistic delivery of chloroplast or nuclear expression vectors into wheat leaves or calli, derived from anther culture or immature embryos, is reported here. When pB1121, the nuclear GUS vector, was used to bombard wheat cells, the β-glucuronidase product, an insoluble indigo dye, was observed evenly throughout the cytosol. But, when the chloroplast expression vector pHD203-GUS was used for bombardments, the indigo dye (GUS product) was subcellularly localized within the chloroplasts of wheat cells. The observation of GUS expression in albino plastids, when anther culture derived albino leaves were bombarded with the chloroplast expression vector pHD203-GUS, suggests the presence of a functional protein synthetic machinery in these organelles. GUS expression was also observed in regenerable calli derived from wheat immature embryos bombarded with pHD203-GUS. Leaves or calli bombarded with pUC19, as negative controls, did not show any GUS expression. These results constitute the first demonstration of foreign gene expression in chloroplasts of a monocot and that a dicot chloroplast promoter functions in a monocot chloroplast.
Chimaeric ribonuclease genes that are expressed in the anthers of
transformed tobacco and oilseed rape plants were constructed. Chimaeric
ribonuclease gene expression within the anther selectively destroys the
tapetal cell layer that surrounds the pollen sac, prevents pollen
formation, and leads to male sterility. These nuclear male sterility
genes should facilitate the production of hybrid seed in various crop
plants.
Most common strategies for gene transfer derive from the fact that Agrobacterium tumefaciens contains Ti plasmids which facilitate transfer of T-DNA from the plasmid genome into plant cells with attendant integration of the T-DNA region into the plant nuclear genome (Binns,1990). Alternatively, DNA may be introduced into protoplasts using various chemical agents (Jenes et al, 1993) or by electroporation (Fromm et al., 1987) or microinjection (Neuhans and Spangenberg, 1990). In all these approaches foreign DNA is introduced into the nuclear genome. Sanford and coworkers (1990) have developed a transformation technique that relies on bombardment of recipient cells or tissues with high velocity tungsten microprojectiles coated with foreign DNA. Using this delivery system several groups have demonstrated stable transformation of foreign genes into nuclear genomes of higher plants, mitochondrial genomes of yeast, and chloroplast genomes of Chlamydomonas and higher plants (Sanford, 1990). Microprojectile bombardment is replacing traditional methods of transformation because of the use of simple vectors and unlimited host range. DNA delivery by the Gene Gun into cultured tissues or the scutellum of immature embryos of several cereal crops is generally followed by selection for transformed tissues. Regenerated transgenic plants have been obtained from maize, rice, wheat, oat and numerous dicots (Guerin and Guerin, 1993), demonstrating the broad applicability of the Gene Gun. The Gene Gun is also routinely used in studies on transient foreign gene expression and is a reliable method to deliver foreign DNA into organelles (Daniell, 1993).
Transgenic plants that produce foreign proteins or secondary metabolites with intrinsic industrial or pharmaceutical value represent a cost-effective alternative to fermentation- or organic synthesis-based production systems. There have been a number of recent demonstrations that transgenic plants are capable of producing functional foreign proteins, or that secondary metabolic pathways can be altered for the production of proteins or compounds of known or potential pharmaceutical importance. These examples portend many new and exciting possibilities in the ancient science of plant medicinal chemistry. Here we discuss current progress in the production of phamacologically important proteins and secondary metabolites in transgenic plants.
Despite the great potential and increasing importance of other weed control options (Turner et al. 1992) and unwanted environmental side effects of some herbicides, herbicides constitute a very important means of weed control. The escape of herbicide resistance genes to wild, weedy plants could cause more severe weed problems, and presents a very real threat to the efficacy of herbicides as a weed control option. Therefore, management strategies that prevent, or reduce the likelihood and frequency of HRG escape through containment methods are advisable, as are mitigation plans in the event of HRG escape to wild plants.
We describe here the development of a reproducible plastid transformation system for potato and regeneration of plants with uniformly transformed plastids. Two distinct tobacco-specific plastid vectors, pZS197 (Prrn/aadA/TpsbA) and pMON30125 (Prrn/GFP/ Trps16::PpsbA/aadA/TpsbA), designed for integration into the large single copy and inverted repeat regions of the plastid genome, respectively, were bombarded into leaf explants of potato line FL1607. A total of three transgenic lines were selected out of 46 plates bombarded with pZS197 and three transgenic lines out of 104 plates were obtained with pMON30125. Development of a high frequency leaf-based regeneration system, a stringent selection scheme and optimization of biolistic transformation protocol were critical for recovery of plastid transformants. Plastidexpressed green fluorescent protein was used as a visual marker for identification of plastid transformants at the early stage of selection and shoot regeneration. The establishment of a plastid transformation system in potato, which has several advantages over routinely used nuclear transformation, offers new possibilities for genetic improvement of this crop.
Seattle--In the United States, government officials are approving more and more transgenic crops for widespread field testing.
But based on new data on the frequency of gene transfer, some ecologists fear that advantageous genes, such as those that
confer virus resistance, could escape from the crops into wild plants and create a hardier race of weeds.