Transgenic Plants: Current Innovations and Future Trends Chapter Abstracts

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Abstract
We have entered the expansion phase of agricultural biotechnology, in which genetically modified (GM) plants are playing an important role in crop germplasm improvement. In the USA, the rate of adoption of GM plants has overshadowed any other introduction of an agricultural technology. The overriding thesis of this book is that while transgenic technologies have come so far in the past 20 years, it is still in its infancy. Some individuals and organized groups have rejected GM crops because they are unable to tolerate change in agriculture especially the rapid technological advances made possible by modern gene transfer techniques. Change is inevitable and will be driven by forces such as climate variation or population shifts that are outside of our control. Perhaps the largest impediment to an even greater rate of global adoption has been questions of biosafety. Ecological and food biosafety concerns are discussed briefly in this text, but the authors have instead focused primarily on technological advances such as plastid transformation, targeted recombination, and the overproduction of pharmaceutical proteins. These breakthroughs will usher in far-greater numbers of commercial and humanitarian opportunities, and will be undeniably attractive to most of the general public; only diehard Luddites will oppose GM technology in 20 years. While the rewards of GM research will be largely reaped by a younger generation this book highlights genuine attempts by scientists to address some of the current concerns on the biosafety of GM crops, to breed new crops that outperform conventional crops and to confer novel properties on crops, such as the ability to cleanse toxic soils, that help to sustain a decent environment.

Abstract
Maturation refers to programmed age-related changes in developmental processes that occur in all organisms. In trees, where maturation occurs over years to decades, numerous morphological and physiological changes associated with this process have been described. However, there has been little progress in elucidating the mechanisms that control maturation, and only limited capability to alter maturation state for horticultural plants and forest trees. The ability to prevent the acquisition of competence to flower during maturation could enable the broad use of genetically engineered trees in plantations with acceptable ecological and social consequence. Conversely, the ability to speed the onset of flowering could allow the use of breeding methods now considered untenable in trees. In addition, the modification of cambial maturation state could allow directed improvements in wood quality, and induced reversion of mature trees to more juvenile maturation states could allow facile clonal propagation of elite, proven genotypes. New genomic information and methods are providing many fresh avenues for probing mechanisms and identifying control points. More so than any other forest tree, Populus possesses the genomics infrastructure, and facile transformation and clonal propagation, that could allow rapid progress in elucidating the regulatory networks that control maturation.

Abstract
Forest trees have great potential to be genetically manipulated, not only for improved fiber production, but also for generation of novel goods and services, by applying the same biotechnological tools that have been used with agronomic crop species. Unlike crop species, however, forest trees are undomesticated and thus present unique problems with regard to genetic manipulation. In vitro propagation, in particular somatic embryogenesis, and gene transfer are two of the tools widely predicted to have substantial impact on forest productivity in the coming decades. Embryogenic culture systems have been developed for most commercially-important forest trees and have already been employed extensively as targets for gene transfer, most commonly via microprojectile-mediated transformation (biolistics). Trees expressing genes for herbicide resistance and modified wood have been produced. In addition, transgenic trees have been proposed for new products and services that have not been previously associated with forestry, such as remediation of contaminated soils and water. Trees engineered with heavy metal detoxification genes are currently being tested for their ability to handle these pollutants. However, in order for these advances in in vitro propagation and genetic manipulation to be translated into economically viable plantations, a number of significant technical bottlenecks must be overcome, with regard to both transformation and somatic seedling production. In addition, recent reports indicate that alternative approaches to embryogenic cultures and biolistics may actually be superior for propagation and gene transfer with some forest trees. Progress is being made in surmounting these problems and novel approaches to improving genetic manipulation of the top commercial forest species are currently under development.

Abstract
Several in planta methods of transformation have been described during the past thirty years. Most of them were not reproducible and the apparent positive results obtained were generally the result of artifacts or the use of methods of investigation leading to ambiguous interpretations Successful Agrobacterium-mediated gene transfer to Arabidopsis thaliana (and some related species) achieved by infiltration (vacuum or surfactant) of adult plants, led to many attempts to replicate the results in other species, which, unfortunately, have remained unsuccesssful. It was recently discovered that this particular transformation process targets the female gametophyte. In the future, a better understanding of the interaction between Agrobacterium and the female germ line of some Brassicaceaes could open possibilities for generalizing applications of this method.

Abstract
Chloroplasts are known to have evolved from free-living microorganisms that were enslaved by plant cells. Chloroplasts are double membrane bound intracellular structures containing their own protein synthetic machinery but function in association with nucleus. Chloroplast transformation in higher plants is an extremely attractive approach for the development of transgenic traits that may be difficult to achieve via nuclear transformation. The chloroplast genome of higher plants is an ideal target for genetic engineering, since foreign proteins in transgenic chloroplasts accumulate to high levels due to the polyploid nature of chloroplast genome. Multiple genes may be expressed as polycistronic units in transgenic chloroplasts. Lack of pollen transmission in most cultivated crops results in natural gene containment, thus minimizing out-cross of transgenes to related weeds or crops and reducing the potential toxicity of transgenic pollen to non-target insects. Chloroplast transformation utilizes two targeting sequences that flank the foreign genes and insert them through homologous recombination at a precise, predetermined location in the chloroplast genome. This results in uniform transgene expression among transgenic lines and eliminates the "position effect" often observed in nuclear transgenic plants. Gene silencing, frequently observed in nuclear transgenic plants, has not been observed in transgenic chloroplasts. The ability to express foreign proteins at high levels in chloroplasts and chromoplasts, and to engineer foreign genes without the use of antibiotic resistant genes make this compartment ideal for development of edible vaccines or oral delivery of biopharmaceuticals. Moreover, the ability of chloroplasts to form disulfide bonds and to fold human proteins has opened the door to high-level production of biopharmaceuticals in plants. Furthermore, several foreign proteins or molecules observed to be toxic in the cytosol are non-toxic when compartmentalized within transgenic chloroplasts. This review highlights these and other recent accomplishments.

Abstract
Plant transformation technologies use antibiotic resistance genes as markers to identify the small fraction of transgenic cells that have taken up trait genes. In addition to plant selectable marker genes, vector-localised genes such as the ampicillin resistance bla(TEM1) gene, can also integrate into the chromosomes of transgenic plants. Integration of vector sequences is particularly problematic when whole plasmids integrate into plant nuclear DNA following their transfer into cells by artificial DNA delivery methods such as particle bombardment. Microbial resistance to antibiotics threatens the success of infectious disease treatment and prevention in the 21st century. While the risk of horizontal transfer of antibiotic resistance genes is minuscule, their elimination from genetically manipulated crops provides a simple solution for ending the continuing debate over the likelihood of pathogen acquisition of plant-derived antibiotic resistance genes. To avoid the presence of antibiotic resistance genes in transgenic crops, they can be removed once they have served their purpose or they can be replaced with alternative marker genes. These two approaches are not mutually exclusive and can be combined where needed to avoid safety evaluations on each new marker gene.

This chapter reviews technologies for removing antibiotic resistance genes from transgenic plants and describes an expanding list of alternative marker genes that do not require antibiotic selection. Plastid engineering illustrates the ease with which both antibiotic resistance genes and vector sequences can be removed from plants using homologous recombination. Efficient marker gene excision technologies and alternative marker genes combine for a better toolkit for the next generation of transgenic crops. This toolkit will facilitate multiple rounds of transformation with the best marker for a particular crop and allow the removal of all excess foreign DNA from a crop. As a consequence the focus of attention will shift from the marker genes to the all important trait genes that are responsible for the added value of genetically manipulated crops.

Abstract
Genetic transformation of the world's major crops has become routine, the result of significant technical advances over the past 10 years. Standard plant transformation methods do not provide for post-insertion manipulation of transgene sequences except through conventional breeding. Progress has been made in the development of new technologies, tools and methodologies to facilitate targeted approaches to gene integration or modification. Sequence-specific recombinases are being increasingly used to introduce or manipulate transgenes and as general tools for genetic manipulations. We focus in this chapter on the adaptation and application of naturally occurring site-specific recombination systems for use in plants, including relevant work from various species that has provided the basis for these new applications. In addition, we discuss some future directions in plants for these and other technologies.

Abstract
The advances in biotechnology have enhanced new opportunities for crop improvement other than conventional plant breeding, which is limited by sexual barriers across species. Transgene-mediated resistance becomes a method of choice for plant disease resistance development. Different sources of transgenes have been proposed; these include plant resistance genes, pathogen-derived resistance genes, and antimicrobial proteins of plant and non-plant origins. Numerous disease resistance transgenes have been inserted into plant genomes and have been successful in protecting the plants from several diseases including nematodes. This chapter discusses the principles of plant-pathogen interaction, mechanisms of defense responses, signals involved in defense responses and the current status of transgenic plants that confer resistance to different kind of pathogens. Promising approaches for the production of transgenic plants with broad-spectrum resistance have also been discussed.

Abstract
The age of genetically engineered foods is here. The promise of plant biotechnology for agriculture and nutrition science is significant. Benefits in the field are being realized already, from reduced pesticide use to increased yields, and biotechnology food products designed to alleviate nutritional deficiencies, protect from cancer, or reduce heart disease will shortly reach the markets. However, there has been public confusion and concern over the use of transgenic plants for crop and food improvement. This chapter will seek to review food safety concerns in order to better understand the risks involved and the benefits to food biotechnology. Food safety evaluations of genetically modified (GM) products are needed to provide continued security of the food supply as well as to maintain consumer confidence in the products they purchase. Safety can be determined by establishment of substantial equivalence of GM products to conventional food varieties, evaluation of any potential toxicity, and identification of products that may be allergenic. Combined, these methodologies will allow for the effective and safe introduction of this technology into conventional food production.

Abstract
The increasing number of new incurable infectious diseases and the re-emergence of antibiotic resistant pathogens over the past several decades, in combination with the expected increase in world population from six to ten billion by the middle of this century, underscores the urgent need for new methods of vaccination which are more effective, easier to administer and less costly to produce. The advent of recombinant DNA technology and plant transformation methods coupled with the ability to regenerate plants from single cells has made possible the synthesis of antigens and autoantigens in edible plants. In this review, we describe recent developments in plant biotechnology where production of mucosal vaccines in edible plants for effective and inexpensive protection against infectious and autoimmune diseases will soon be feasible.

Abstract
Plant transformation technology is an established toolbox for the study of plant genomics; particularly so for model species such as Arabidopsis thaliana and rice. Progress in transformation methods for these species has allowed the use of several strategies for gene identification and characterization. T-DNA and transposons have been used for random insertional mutagenesis with gene constructs designed to create gene knockouts, promoter traps, enhancer traps, and activation tags. RNA silencing is widely used to create down-regulated mutants. The efficiency gained with a better understanding of the mechanisms of post-transcriptional gene silencing allows the use of the method for large-scale approach. Targeted gene inactivation by homologous recombination is an emerging method for plant transformation. These genomics methods already available to plant molecular biologists or under development are described.

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