Saturday, February 27, 2010
Monday, February 22, 2010
INSECTICIDAL GENES
INSECTICIDAL GENES can be taken from other plants or from bacteria such as the BT gene which has been transferred into corn and other crops. BT is a protein isolated from the naturally occurring soil bacteria, Bacillus thuringensis. The BT protein is capable of disrupting the guts of specific insects before larvae can complete development and has essentially no toxicity against most non target insects, other animals, or humans. An altered form of the BT gene has been engineered and transferred into plants, making the transgenic plant resistant to insects (such as the corn borer). Recently, it has been identified that BT-corn has lower mycotoxin contamination possibly because it reduces insect injury thereby reducing fungal infection levels. The present controversy surrounding the use of BT, reflects the concerns by organic farmers who use BT spray and worry that the overuse will lead to BT resistant insects and some environmentalist who fear BT will kill non target insects such as the monarch butterfly larvae. However, if we assess the risks associated with the use of BT-crops and compare it with the risks associated with more conventional methods, such as inorganic insecticidal sprays, the use of BT-crops are safer and more environmentally friendly.
HERBICIDE TOLERANCE (HT)
HERBICIDE TOLERANCE (HT) - Potentially in next 5 years we may see some HT wheat systems that may be introduced into western Canada. Cyanamid plans to introduce an imidazolinone herbicide tolerant (HT) wheat in 2001. This HT variety although classified as a plant with a novel trait (PNT) was made through the use of chemical mutagenesis and not specifically genetic engineering. Monsanto may launch a Roundup Ready®/glyphosate tolerant wheat in 2004 and Novartis, an Acuron gene/PPO herbicide tolerant wheat sometime after 2003.
As example of a HT GM trait: Roundup Ready® TM genes are a bacterial derived version of a plant enzyme. The enzyme is part of a pathway for the production of aromatic amino acids phenylalanine, tyrosine and tryptophan. The gene is ubiquitous. The plant version is sensitive to glyphosate and the bacterial version tolerant to glyphosate. An additional bacterial derived enzyme was transferred to the crop to enhance the degradation of glyphosate . To reduce the chance of the HT gene being spread to wild relatives researchers are attempting to insert HT genes into the chloroplasts.
As example of a HT GM trait: Roundup Ready® TM genes are a bacterial derived version of a plant enzyme. The enzyme is part of a pathway for the production of aromatic amino acids phenylalanine, tyrosine and tryptophan. The gene is ubiquitous. The plant version is sensitive to glyphosate and the bacterial version tolerant to glyphosate. An additional bacterial derived enzyme was transferred to the crop to enhance the degradation of glyphosate . To reduce the chance of the HT gene being spread to wild relatives researchers are attempting to insert HT genes into the chloroplasts.
Plant growth and productivity are greatly affected by various stress factors such as drought, high salt, high aluminum, cold and hot temperatures, or short seasons. Drought tolerance improvement is probably one of the most difficult tasks for the cereal breeders. The difficulty comes from the diversity and the unpredictability of drought tolerance strategies developed by the plant that may be targeted and used as a selection criteria. In several cereal species, genetic maps have identified chromosomal regions controlling some traits related to drought stress response. Once genes are identified they may be transferred such as dehydrins produced by plants during drought or low temperature stress, abscisic acid (ABA) and the ABA response complex from barley to protect against dehydration stress and glycine betaine to improve tolerance to salt and cold.
GM TRAITS
One of the most important features of an individual GM is its trait. So what are some of traits we are presenPresently, several plants with novel traits (PNTs) have been developed and approved by the Plant Biotechnology Office for release and registration. These PNTs are primarily crops such as canola, corn and soybean with improved herbicide tolerance (bromoxynil, glufosinate, glyphosate, or imidazolinone tolerance) or for quality/composition (high oleic acid/low linolenic acid canola). The general trend observed in traits for crops going through the registration process in the past 10 years has been a decline in herbicide tolerant varieties and an increase in varieties with improved quality, stress resistance and disease resistance. At present there are no genetically engineered wheat, barley or oats registered in Canada, only corn. tly seeing and what is coming up the pipeline?
THE CRITERIA FOR RISK ASSESSMENT ARE BASED ON THE ORIGINAL UNMODIFIED PLANT
THE CRITERIA FOR RISK ASSESSMENT ARE BASED ON THE ORIGINAL UNMODIFIED PLANT, the novel trait or gene transferred, the novel modified plant as a whole, and its potential environmental interaction. The biology of the unmodified plant is dealt with through a series of "biology documents which review agronomic practices, reproductive biology, centre of origin, out crossing potential, weediness and potential interactions with other life forms in the Canadian environment. Characterization of the novel trait plays a central role in the assessment process and overlaps the assessment of modified plant. The main criteria considered includes information about the genetic construct (genes inserted, regulatory elements, marker genes, donors and known risks associated with the known organisms) the gene products, byproducts and breakdown products, metabolic pathways affected, and potential toxicity or allergenicity of the gene product. Next the novel plant is assessed. Emphasis is placed on its integration into the plant genome, for example, the insertion of the novel gene(s) into the plant genome, the number of sites of integration (loci), the copy numbers, presence of rearrangement, the stability, the expression, the metabolic pathways, the activity of an inserted gene product in the plant and the activity of the gene product in the environment. Potential altered interaction of the novel plant involves identifying changes to the relative phenotype with respect to stress adaptation, weediness, composition, toxins, and agronomic characteristics. As well, the novel plant is assessed for its anticipated impacts, for example what impacts might reasonably be expected from the use of the plant (such as on biodiversity, soil microbes, sustain ability and resource conservation?). Also, the novel plant is assessed for possible changes in agriculture practice and its potential environmental effects from introgression of traits into related wild plant species. Finally, the information submitted for the review must address the following five criteria which are used to make an environmental risk assessment and provide direction for any risk management decision: altered weediness potential; gene flow to related species (there are no sexually compatible wild relatives of potato in Canada); altered plant pest potential; potential impact on non target organisms; potential impact on biodiversity. If the PNT is assessed to be safe, it can be registered and commercialized.
GMO's
Prior to variety registration/commercialization of GMO's, they must go through a rigorous step wise screening involving both confined and unconfined field trails. Parties trying to release a GMO's must apply through the Federal Plant Biotechnology Office contained within the Canadian Food Inspection Agency and Health Canada. In Canada, plants with novel traits (PNT's) are regulated on the basis of the characteristics of the product, not the specific process by which the product was made. The primary triggers of the regulatory process are the novelty of the plant species, its characteristics (traits) and use, in the Canadian context. Therefore, products of traditional breeding or mutagenesis as well as the products of recombinant DNA technology may be considered novel and regulated under the Seed Act. For example, herbicide resistance may be achieved through mutation breeding, selection of naturally occurring variants or by the use of recombinant DNA technology. In any case the important aspect in terms of environmental interactions is the trait, herbicide resistance.
SELECTABLE MARKERS
In order to quickly and easily identify which plant cells have incorporated the gene of interest, researchers piggy-back a gene referred to as a selectable marker. In practical plant improvement programs, selectable markers have been largely restricted to proteins providing resistance to herbicides or antibiotics. Putative transformants can be sprayed with, or grown on media containing the appropriate chemical. Transformed plants are identified as those that survive. For example, the antibiotic resistance gene for Kanamycin allows only transformed cells to grow in the presence of the selection agent Kanamycin. Other more exotic markers, such as GUS (a gene encoding ß-glucuronidase, identified in stained material by a blue colour) can also be used to screen transformants. Under the selective pressure of an antibiotic or herbicide and various plant growth promoters, transformed plant cells are gradually regenerated into plants. The new plants are "hardened off",transferred to soil and placed in a greenhouse environment. Plant and growth conditions are carefully monitored and controlled. The plants are used for analysis, seed production or are transplanted to the field.
GENE GUN
One of the most successful methods is particle bombardment (or the gene gun) which actually shoots DNA-coated microscopic pellets through a plant cell wall. This biolistic technique of genetic transfer is patented technology and therefore costly to use. Gene integration is also less efficient and less predictable than the Agrobacterium-transformation method used in dicotyledons. Therefore research efforts have continued to reduce the limitation of Agrobacterium-transformation method in cereals and at present this method has been successful in the lab and is expected to increase transformation frequency with a higher consistency of gene expression. "Gene silencing" or the failure of transferred genes to be expressed has also slowed the progress of GE cereals. Several possible causes have been identified for gene silencing however it is far from being fully understood.
TRANSFORMING
Scientists snipped out the tumor causing genes, replaced it with genes of choice and then, when the altered Agrobacterium infects a plant, the inserted genes are incorporated into its plant cells genetic makeup. However, cereals are not naturally susceptible to infection by Agrobacterium and therefore alternative methods to transform cereals had to be developed.
FIRST TRANSFORMATION OF A BACTERIA
The FIRST TRANSFORMATION OF A BACTERIA was achieved in 1973, however, it took another decade before a plant was successfully transformed. This was done by the manipulation of a natural occurring infection caused by a bacterium, Agrobacterium tumefaciens. Agrobacterium has the ability to infect plants, causing crown gall disease or tumor-like enlargements. The DNA which causes the galls resides in a small circular ring (or plasmid) of bacterial DNA and a portion of this DNA was transferred and incorporated into the plant cell's chromosomes.
TRADITIONAL BREEDING
If we wanted to transfer a single resistance gene from a wild variety, by TRADITIONAL BREEDING we would also transfer many unwanted genes with the initial cross. We could try to reduce the number of unwanted genes by back crossing several times. Genes are also inherited in blocks and therefore the desired gene may be linked to an unwanted trait. In contrast, genetic engineering allows us to specifically identify and precisely transfer that one desired gene. Genetic transfer need not involve the transfer of genes between completely unrelated plants, insects or animals, but may only involve the transfer of genes within the same species or closely related members in the cereal family.
How are GM foods made?
ENTIRE WHEAT GENOME
Here is an example of a breeding dilemma:
As we all know, genetic material is complex. If we sequenced the ENTIRE WHEAT GENOME we would require 17,000 books of 1000 pages which when stacked would be as high as a 20 storey building.
WHY GENETIC ENGINEERING?
WHY GENETIC ENGINEERING?For the first time in the history of plant breeding, individual and precisely identified genes can be introduced into existing breeding material and expressed in plants. Genetic engineering enables us to access genes from all areas of the plant world as well as other organisms thereby greatly increasing the genetic resources available to the crop breeder. This technology is providing researchers with knowledge that explains how plants function at the molecular level. This knowledge allows researchers to develop better varieties through traditional breeding, using new tools. It allows researchers to develop new crops more efficiently and faster and meet breeding objectives that were previously unachievable.
GENETIC ENGINEERING
New biotechnologies include techniques such as GENETIC ENGINEERING or ELISA (enzyme-link immunosorbant assay). This test utilizes monoclonal antibodies (highly specific preparation of antibodies that binds to a single site on a protein) for specific protein detection. The technique is primarily used for diagnostic testing. For example, ELISA tests have been developed to detect DON production in fusarium infested barley and black leg detection in canola. ELISA tests are simple to perform, relatively cheap, quick, and can be done by individuals at home or in the field. An example of a very common ELISA based diagnostic kit is the home pregnancy test.
MOLECULAR MARKERS
MOLECULAR MARKERS - Molecular markers have the potential of increasing the efficiency and quality of the selection for desirable traits in new varieties. A molecular marker assisted selection allows breeders to handle larger populations while selecting earlier traits unaffected by the environment, and with more assurance for the desired trait. Molecular markers identify genetic differences linked to a specific trait. The best marker is the identification of the actual gene/genes associated with the trait. However, identifying the gene for a particular characteristic from the huge amount of DNA within an organism is a daunting task and many traits are not simply inherited and are dependent on several genes. Therefore, researchers often rely on gene linkages. Some types of molecular markers are listed below:
• Isozymes: differences in the genes that code for or regulate enzyme synthesis or activity
• Restriction fragment length polymorphisms (RFLPs): differences in the sites at which restriction enzymes can cut the DNA
• Random amplified polymorphic DNA (RAPDs): differences in the sites at which DNA will be amplified by the polymerase chain reaction (PCR)
• Amplified fragment length polymorphisms (AFLPs): differences in restriction sites & PCR amplification
• Simple sequence repeats (SSRs) or microsatellites: differences in repetitive DNA sequences
HAPLOIDIZATION
HAPLOIDIZATION - Double haploid plants can enhance and accelerate plant breeding programs. This method decreases the amount of time require to develop a new variety by creating a homozygous line in one generation compared to 6-7 generations using an inbred method.
PLANT BIOTECHNOLOGY
PLANT BIOTECHNOLOGY generally can be divided into traditional biotechnology and new biotechnology. Traditional biotechnology such as breeding by selection and saving the best seed for the next generation, has been in practice for thousands of years. Almost all of the crops that we cultivate today are very different from their wild ancestors. Other techniques such as genetic markers and double haploid production are new tools to aid traditional breeding programs. Mutagenesis and selection have been used for more than 50 years to introduce more genetic variation into breeding lines
A common misconception is that biotechnology only includes DNA and genetic engineering. Biotechnology is a very broad term and is not new. The term biotechnology, first coined in 1919 by a Hungarian engineer (Karl Ereky), is now defined as the application of science and engineering in the direct or indirect use of living organisms in their natural or modified forms to produce a product.
GENETIC TRANSFORMATION AND TRANSGENIC CROPS
Even after extensive world-wide efforts, embryo-rescue, androgenetic haploids, somatic hybrids, and the much touted somaclonal variation (Larkin and Scowcroft 1981), have not lived up to their purported promise in becoming important and useful means of creating novel genetic variability for crop improvement. Indeed, much of the success during the past quarter century in the production of plants with novel and useful traits has been based on the infinitely more precise and predictable methods of genetic transformation, in which well characterized single or multiple genes introduced into single cells become stably integrated into the nuclear genome and are transmitted to progeny like dominant Mendelian genes.
Three principal methods have been developed for the introduction of genes into plants: (i) Agrobacterium tumefaciens-mediated gene transfer. (ii) Direct DNA delivery into protoplasts by osmotic or electric shock. (iii) Direct DNA delivery into intact cells or tissues by high velocity bombardment of DNA-coated microprojectiles (the biolistics procedure).
It is well known that the crown-gall disease of plants is caused by the soil-borne bacterium Agrobacterium tumefaciens. An early study by Braun (1958) demonstrated the transformed nature of tumor cells as they could be freed of the bacteria and still grown indefinitely in hormone-free media; non-transformed cells needed media supplemented with an auxin and a cytokinin for continued cell divisions. He proposed that a Tumor Inducing Principle (TIP) present in the bacteria was responsible for transformation. Bacteria-free tumor cells were found to contain large amounts of opines, a new class of metabolites (Petit et al. 1970). The critical observation that the presence of a particular opine—octopine or nopaline—in the bacteria-free tumor cells was determined by the bacterial strain used for tumor formation, rather than the plant, suggested that a functional bacterial gene had become a part of the plant cell. This was a revolutionary idea that was initially met with considerable skepticism.
These observations stimulated attempts in many laboratories to identify the TIP and the genetic basis of tumor formation. Early results pointed to the possibility that tumor induction might be caused by a plasmid or virus, and eventually led to the discovery of megaplasmids that were present only in the virulent strains of Agrobacterium (Zaenen et al. 1974). The plasmids were appropriately named the Ti (tumor-inducing) plasmids. Only a small part of the plasmid, T-DNA, was shown to be responsible for tumor formation (Chilton et al. 1977) and was found to be present in the nuclear DNA fraction of tumor cells (Chilton et al. 1980; Willmitzer et al. 1980).
The fact that a part of the Ti plasmid was transferred and integrated into the plant genome during tumor formation (transformation), suggested that the plasmid could be used as a vector to transfer other genes. Thus methods were developed to insert DNA (genes) into the Ti plasmid (van Haute et al. 1983; de Framond et al. 1983; Hoekma et al. 1983). Transformed crown-gall tumor tissues, which grew on hormone-free media, formed only highly aberrant shoots in culture. This was found to be related to the presence of genes regulating the synthesis of auxin and cytokinin. Deletion of these genes (disarming of the plasmid) produced transformed tissues that required media supplemented with auxin and cytokinin for continued growth and regenerated normal shoots and plants. These findings led to the use of the Ti plasmid of Agrobacterium as a vector for plant transformation, and kanamycin resistance genes for selection of transformed cells, followed by the regeneration of transformed plants (Bevan et al. 1983; Fraley et al. 1983; Herrera-Estrella et al. 1983; de Block et al. 1984; Horsch et al. 1984, 1985). These results were first presented by three independent research groups on January 18, 1983, at the Miami Winter Symposium, and marked the beginning of the modern era of plant biotechnology.
Methods for the direct delivery of DNA into protoplasts were developed during the early 1980s (Paszkowski et al. 1984; Shillito 1999), especially for the economically important cereal crops as they were considered at the time to be outside the host range of Agrobacterium, and therefore not amenable to Agrobacterium-mediated transformation (see Vasil 1999, 2005). The procedure involved delivering osmotic or electric shock to protoplasts suspended in solutions containing DNA, followed by plating on selection media for the preferential growth of transformed colonies, and eventually plants. Transformation of protoplasts isolated from embryolgenic cell suspension cultures led to the production of the first transgenic cereals (Rhodes et al. 1988; see also Vasil and Vasil 1992). The use of protoplasts for genetic transformation became less attractive once it was shown that monocots, including the cereals, could be transformed by co-cultivation of embryonic tissues or embryogenic cultures and super-virulent strains of Agrobacterium in the presence of acetosyringone, a potent inducer of virulence genes (Hiei et al. 1994; Komari and Kubo 1999).
Technical difficulties with the isolation and long-term maintenance of embryogenic suspension cultures, and limitations of Agrobacterium-mediated transformation, encouraged the search for universal methods of transformation. A wide variety of methods, including those that do not require the use of tissue cultures, were proposed as a means to transform plants (Ledoux and Huart 1969; Doy et al. 1973; Hess et al. 1976, 1990; Pandey 1975; Grant et al. 1980; de Wet et al. 1985; Graves and Goldman 1986; Ohto 1986; de la Pena et al. 1987; Luo and Wu 1988; Zilberstein et al. 1994; Zhao et al. 2006). None of these have been independently confirmed and have, therefore, remained outside the mainstream of plant transformation research.
The novel biolistics procedure, a universal method of plant transformation, was developed by Sanford et al. (1987; Sanford 2000). It involves the high velocity bombardment of DNA-coated gold or tungsten microprojectiles into intact cells or tissues. The Agrobacterium and biolistics procedures, in combination with embryogenic cultures, are the two most widely used methods for plant transformation. The preference of one over the other depends more on the biases of the individual researcher than any inherent differences between the procedures (Altpeter et al. 2005).
Advances in the genetic transformation of plants were dependent on parallel advances in plant molecular biology, the sequencing of plant genomes, and the identification and characterization of many agronomically important genes, leading to the production of a variety of transgenic crops that are resistant to biotic and abiotic stresses, and have improved nutritional qualities. Transgenic crops were first planted commercially in 1996, and have since been grown (mostly canola, cotton, maize, and soybean) on more than 1.4 billion acres in 22 countries (see James 2006). They have contributed more than US$ 23 billion to the economies of developing as well as developed countries (nearly 90% of the transgenic crops are planted by resource-poor farmers), have reduced the use of agro-chemicals, have increased productivity, have improved human health, and have had a positive influence on conservation of the environment and biodiversity. These are truly remarkable achievements of a comparatively new technology, which is expected to profoundly influence international agriculture and food security in the 21st century.
Three principal methods have been developed for the introduction of genes into plants: (i) Agrobacterium tumefaciens-mediated gene transfer. (ii) Direct DNA delivery into protoplasts by osmotic or electric shock. (iii) Direct DNA delivery into intact cells or tissues by high velocity bombardment of DNA-coated microprojectiles (the biolistics procedure).
It is well known that the crown-gall disease of plants is caused by the soil-borne bacterium Agrobacterium tumefaciens. An early study by Braun (1958) demonstrated the transformed nature of tumor cells as they could be freed of the bacteria and still grown indefinitely in hormone-free media; non-transformed cells needed media supplemented with an auxin and a cytokinin for continued cell divisions. He proposed that a Tumor Inducing Principle (TIP) present in the bacteria was responsible for transformation. Bacteria-free tumor cells were found to contain large amounts of opines, a new class of metabolites (Petit et al. 1970). The critical observation that the presence of a particular opine—octopine or nopaline—in the bacteria-free tumor cells was determined by the bacterial strain used for tumor formation, rather than the plant, suggested that a functional bacterial gene had become a part of the plant cell. This was a revolutionary idea that was initially met with considerable skepticism.
These observations stimulated attempts in many laboratories to identify the TIP and the genetic basis of tumor formation. Early results pointed to the possibility that tumor induction might be caused by a plasmid or virus, and eventually led to the discovery of megaplasmids that were present only in the virulent strains of Agrobacterium (Zaenen et al. 1974). The plasmids were appropriately named the Ti (tumor-inducing) plasmids. Only a small part of the plasmid, T-DNA, was shown to be responsible for tumor formation (Chilton et al. 1977) and was found to be present in the nuclear DNA fraction of tumor cells (Chilton et al. 1980; Willmitzer et al. 1980).
The fact that a part of the Ti plasmid was transferred and integrated into the plant genome during tumor formation (transformation), suggested that the plasmid could be used as a vector to transfer other genes. Thus methods were developed to insert DNA (genes) into the Ti plasmid (van Haute et al. 1983; de Framond et al. 1983; Hoekma et al. 1983). Transformed crown-gall tumor tissues, which grew on hormone-free media, formed only highly aberrant shoots in culture. This was found to be related to the presence of genes regulating the synthesis of auxin and cytokinin. Deletion of these genes (disarming of the plasmid) produced transformed tissues that required media supplemented with auxin and cytokinin for continued growth and regenerated normal shoots and plants. These findings led to the use of the Ti plasmid of Agrobacterium as a vector for plant transformation, and kanamycin resistance genes for selection of transformed cells, followed by the regeneration of transformed plants (Bevan et al. 1983; Fraley et al. 1983; Herrera-Estrella et al. 1983; de Block et al. 1984; Horsch et al. 1984, 1985). These results were first presented by three independent research groups on January 18, 1983, at the Miami Winter Symposium, and marked the beginning of the modern era of plant biotechnology.
Methods for the direct delivery of DNA into protoplasts were developed during the early 1980s (Paszkowski et al. 1984; Shillito 1999), especially for the economically important cereal crops as they were considered at the time to be outside the host range of Agrobacterium, and therefore not amenable to Agrobacterium-mediated transformation (see Vasil 1999, 2005). The procedure involved delivering osmotic or electric shock to protoplasts suspended in solutions containing DNA, followed by plating on selection media for the preferential growth of transformed colonies, and eventually plants. Transformation of protoplasts isolated from embryolgenic cell suspension cultures led to the production of the first transgenic cereals (Rhodes et al. 1988; see also Vasil and Vasil 1992). The use of protoplasts for genetic transformation became less attractive once it was shown that monocots, including the cereals, could be transformed by co-cultivation of embryonic tissues or embryogenic cultures and super-virulent strains of Agrobacterium in the presence of acetosyringone, a potent inducer of virulence genes (Hiei et al. 1994; Komari and Kubo 1999).
Technical difficulties with the isolation and long-term maintenance of embryogenic suspension cultures, and limitations of Agrobacterium-mediated transformation, encouraged the search for universal methods of transformation. A wide variety of methods, including those that do not require the use of tissue cultures, were proposed as a means to transform plants (Ledoux and Huart 1969; Doy et al. 1973; Hess et al. 1976, 1990; Pandey 1975; Grant et al. 1980; de Wet et al. 1985; Graves and Goldman 1986; Ohto 1986; de la Pena et al. 1987; Luo and Wu 1988; Zilberstein et al. 1994; Zhao et al. 2006). None of these have been independently confirmed and have, therefore, remained outside the mainstream of plant transformation research.
The novel biolistics procedure, a universal method of plant transformation, was developed by Sanford et al. (1987; Sanford 2000). It involves the high velocity bombardment of DNA-coated gold or tungsten microprojectiles into intact cells or tissues. The Agrobacterium and biolistics procedures, in combination with embryogenic cultures, are the two most widely used methods for plant transformation. The preference of one over the other depends more on the biases of the individual researcher than any inherent differences between the procedures (Altpeter et al. 2005).
Advances in the genetic transformation of plants were dependent on parallel advances in plant molecular biology, the sequencing of plant genomes, and the identification and characterization of many agronomically important genes, leading to the production of a variety of transgenic crops that are resistant to biotic and abiotic stresses, and have improved nutritional qualities. Transgenic crops were first planted commercially in 1996, and have since been grown (mostly canola, cotton, maize, and soybean) on more than 1.4 billion acres in 22 countries (see James 2006). They have contributed more than US$ 23 billion to the economies of developing as well as developed countries (nearly 90% of the transgenic crops are planted by resource-poor farmers), have reduced the use of agro-chemicals, have increased productivity, have improved human health, and have had a positive influence on conservation of the environment and biodiversity. These are truly remarkable achievements of a comparatively new technology, which is expected to profoundly influence international agriculture and food security in the 21st century.
PROTOPLASTS AND SOMATIC HYBRIDIZATION
In a series of studies during the early 1960s, Cocking (1960, 2000; see Vasil 1976; Giles 1983) described methods for the isolation of plant protoplasts by incubating segments of roots and other tissues in crude mixtures of cell wall hydrolyzing enzymes. The small number of protoplasts that were isolated were used mostly for physiological studies. Two key developments in 1970 and 1971 pointed to the potential use of protoplasts for plant improvement: (i) Induced fusion of protoplasts of diverse species (Power et al. 1970) as a means to produce somatic hybrid cells and novel hybrid plants without regard to taxonomic relationships. (ii) The regeneration of plants from cultured protoplasts (Takebe et al. 1971). Plants can now be regenerated from protoplasts of a wide range of species. Similarly, a variety of somatic hybrids have been obtained between related as well as unrelated species, although useful hybrids have been produced only in a limited number of species, such as Brassica, Citrus and Solanum. Protoplasts have, however, proven to be very useful in genetic transformation of plants (Marton et al. 1979; Davey et al. 1980; Paszkowski et al. 1984), including the economically important cereals (Vasil and Vasil 1992).
________________________________________
________________________________________
TOTIPOTENCY OF PLANT CELLS
During the 1950s a number of attempts were made to demonstrate the totipotency of plant cells. The first evidence of the possibility that single cells of higher plants could be cultured in isolation was provided by Muir et al. (1954), who obtained sustained cell divisions in single cells of tobacco placed on a small square of filter paper resting on an actively growing callus, which served as a nurse tissue. Similar results were obtained by Bergmann (1959) who plated single cells and cell groups suspended in an agar medium. Further progress was made by Jones et al. (1960), who were able to culture single isolated cells in a conditioned medium in specially designed microculture chambers. Each of these studies highlighted the importance of the nurse tissue or the conditioned medium for the survival and growth of the isolated cells. Direct and unequivocal evidence of the totipotency of plant cells was finally provided by Vasil and Hildebrandt (1965, 1967), who regenerated flowering plants of tobacco from isolated single cells cultured in microchambers, without the aid of nurse cells or conditioned media. This fulfillment of Haberlandt’s (1902) prophecy, combined with the more recent success in the genetic transformation of plants (see below), continues to provide the theoretical and conceptual basis of plant biotechnology.
________________________________________
________________________________________
ANDROGENIC HAPLOIDS
Haploids are a valuable commodity for plant breeders but they are seldom used as they occur only rarely in nature. This problem was greatly resolved by the experimental induction of haploidy in cultured anthers of Datura innoxia (Guha and Maheshwari 1966), which like many great advances in science was a chance discovery (Guha-Mukherjee 1999). The technology was further defined and improved by the work of Nitsch and Nitsch (1969). It is now well established that it is possible to shift the developmental pattern of microspores from a gametophytic to a sporophytic phase simply by excising and culturing the anthers at the microspore stage of development. The microspores, instead of forming pollen grains, give rise to somatic embryos either directly or after producing a callus. The resulting plants are either haploids or homozygous diploids (dihaploids) as a result of spontaneous or experimentally induced diploidization. Although the impact of androgenic dihaploids has been limited, they have been used to identify breeding lines with desirable traits and for the production of improved varieties of tobacco, wheat, rice and other crops.
________________________________________
________________________________________
SOMATIC EMBRYOGENESIS
The establishment of efficient embryogenic cultures has become an integral part of plant biotechnology as regeneration of transgenic plants in most of the important crops—canola, cassava, cereals, cotton, soybean, woody tree species, etc.—is dependent on the formation of somatic embryos. One of the most attractive features of embryogenic cultures is that plants derived from them are predominantly normal and devoid of any phenotypic or genotypic variation, possibly because they are derived from single cells and there is stringent selection during embryogenesis in favor of normal cells (see Vasil 1999). Embryogenic cultures were first described in callus and suspension cultures of carrot, grown on coconut milk-containing media, by Reinert (1958) and Steward et al. (1958), respectively. With increasing understanding of the physiological and genetic regulation of zygotic as well as somatic embryogenesis, embryogenic cultures can now be obtained on chemically defined media in a wide variety of species (Thorpe 1995; Raghavan 1997; Vasil 1999; Braybrook et al. 2006). In most instances the herbicidal synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) is required for the initiation of embryogenic cultures; somatic embryos develop when such cultures are transferred to media containing very low amounts of 2,4-D or no 2,4-D at all.
________________________________________
________________________________________
LARGE-SCALE CULTURE OF PLANT CELLS
Mass culture of plant cells in suspension culture to produce natural products was first proposed by Routier and Nickell (1956), and Tulecke and Nickell (1959). Although it is now possible to economically produce a few products such as shikonin, catharanthine, ginseng, paclitaxel, etc., in continuous cultures in 50,000L or larger bioreactors, further exploitation of this technology has been limited owing to continuing technical and biological problems, and high costs (see Staba 1980; Constabel and Vasil 1987, 1988). Consequently, attention has shifted during the past 25 years to using transgenic plants, rather than cell cultures, as ‘biofactories’ for the production of a variety of pharmaceuticals, vaccines, etc. (these are described in detail in other chapters in this volume; see also Ma et al 2005; Fox 2006; Murphy 2007).
DISCOVERY OF CYTOKININS AND THEIR ROLE IN MORPHOGENESIS
The initial success in obtaining unlimited growth of plant tissues was limited to the use of explants that contained meristematic cells. Continued cell division and bud formation were soon obtained when tobacco pith tissues that contained mature and differentiated cells were cultured on nutrient media containing adenine and high levels of phosphate (Skoog and Tsui 1951). However, cell divisions occurred only when the explant included vascular tissue (Jablonski and Skoog 1954). A variety of plant extracts, including coconut milk, (the beneficial effect of coconut milk, the liquid endosperm of immature coconuts, on plant tissue cultures was first shown by van Overbeek et al. 1941) were added to the nutrient medium in an attempt to replace vascular tissues and to identify the factors responsible for their beneficial effect. Among these, yeast extract was found to be most effective and its active component was shown to have purine-like properties. This finding led to the addition of DNA to the medium which greatly enhanced cell division activity (see also Vasil 1959). These investigations resulted in the isolation of kinetin from old samples of herring sperm DNA (Miller et al. 1955), and the understanding of the hormonal (auxin-cytokinin) regulation of shoot morphogenesis in plants (Skoog and Miller 1957). Later studies led to the isolation of naturally occurring as well as many synthetic cytokinins, the elucidation of their role in cell division and bud development, and their extensive use in the micropropagation industry related to their suppression of apical dominance resulting in the development of many axillary shoots.
DEVELOPMENT OF NUTRIENT MEDIA
Initial progress in the culture of plant tissues came from the work of Molliard (1921) in France, Kotte (1922) in Germany, and Robbins (1922) in the United States, who successfully cultured fragments of embryos and excised roots for brief periods of time. The development of improved nutrient solutions, informed choice of plant material, and appreciation of the importance of aseptic cultures, led to long-term or indefinite cultures of excised tomato roots, and cambial tissues of tobacco and carrot, by White (1934, 1939) in the United States, and Gautheret (1934, 1939) and Nobécourt (1939) in France. The discovery of the naturally occurring auxin indole-3-aetic acid (IAA) and its beneficial effects on plant growth (Went 1928; Kögl et al. 1934; Thimann 1935), soon led to its incorporation in plant nutrient media (see White 1943; Gautheret 1985).
White (1943) and others believed that the nutrient solutions based on Knop’s (1865) and other formulations neither provided optimal growth nor were stable or satisfactory over a wide range of pH values. These concerns led to the development of White’s (1943) medium, which was widely used until the mid-1960s. During this period a systematic study of mineral and other requirements of plant tissues grown in culture was carried out (Hildebrandt et al. 1946; Heller 1953), demonstrating the need for a greatly increased level of mineral salts in the medium (see Ozias-Akins and Vasil 1985). In a similar study, designed to optimize the growth of cultured tobacco pith tissue, a marked increase in growth obtained by the addition of aqueous extracts or ash of tobacco leaves to White’s medium was found to be caused largely by the inorganic constituents of the extracts, leading to the development of the first chemically defined and most widely used nutrient solution for plant tissue cultures (Murashige and Skoog 1962). The principal novel features of the new medium were the very high levels of inorganic constituents, chelated iron in order to make it more stable and available during the life of cultures, and a mixture of four vitamins and myo-inositol.
White (1943) and others believed that the nutrient solutions based on Knop’s (1865) and other formulations neither provided optimal growth nor were stable or satisfactory over a wide range of pH values. These concerns led to the development of White’s (1943) medium, which was widely used until the mid-1960s. During this period a systematic study of mineral and other requirements of plant tissues grown in culture was carried out (Hildebrandt et al. 1946; Heller 1953), demonstrating the need for a greatly increased level of mineral salts in the medium (see Ozias-Akins and Vasil 1985). In a similar study, designed to optimize the growth of cultured tobacco pith tissue, a marked increase in growth obtained by the addition of aqueous extracts or ash of tobacco leaves to White’s medium was found to be caused largely by the inorganic constituents of the extracts, leading to the development of the first chemically defined and most widely used nutrient solution for plant tissue cultures (Murashige and Skoog 1962). The principal novel features of the new medium were the very high levels of inorganic constituents, chelated iron in order to make it more stable and available during the life of cultures, and a mixture of four vitamins and myo-inositol.
PLANT BIOTECHNOLOGY IS FOUNDED ON THE DEMONSTRATED TOTIPOTENCY OF PLANT CELLS
combined with the delivery, stable integration, and expression of transgenes in plant cells, the regeneration of transformed plants, and the Mendelian transmission of transgenes to the progeny. The concept of totipotency itself is inherent in the Cell Theory of Schleiden (1838) and Schwann (1839), which forms the basis of modern biology by recognizing the cell as the primary unit of all living organisms. The Cell Theory received much impetus from the famous aphorism of Virchow (1858), “Omnis cellula a cellula” (All cells arise from cells), and by the very prescient observation of Vöchting (1878) that the whole plant body can be built up from ever so small fragments of plant organs. However, no sustained attempts were made to test the validity of these observations until the beginning of the 20th century because the required technologies did not exist and the nutritional requirements of cultured cells were not fully understood (see Gautheret 1985). Haberlandt (1902) was the first to conduct experiments designed to demonstrate totipotency of plant cells by culturing isolated leaf cells in diluted Knop’s (1865) nutrient solution. He failed largely because of the poor choice of experimental materials (even now, more than 100 years later, there are only rare instances where intact leaf cells have been cultured successfully), inadequate nutrients, and infection (see Vasil and Vasil 1972). Nevertheless, he boldly predicted that it should be possible to generate artificial embryos (somatic embryos) from vegetative cells, which encouraged subsequent attempts to regenerate whole plants from cultured cells.
The following pages provide a short history of the evolution of a variety of ideas and technologies that are now routinely used for the genetic improvement of plants, and celebrate the many pioneering men and women who played key roles in the development of plant biotechnology. It does not include the history of plant tissue culture, which can be found elsewhere (White 1943; Gautheret 1985), and the use of plant cell cultures or transgenic plants for the production of pharmaceuticals, vaccines, etc., as these subjects are covered adequately elsewhere in this volume.
________________________________________
The following pages provide a short history of the evolution of a variety of ideas and technologies that are now routinely used for the genetic improvement of plants, and celebrate the many pioneering men and women who played key roles in the development of plant biotechnology. It does not include the history of plant tissue culture, which can be found elsewhere (White 1943; Gautheret 1985), and the use of plant cell cultures or transgenic plants for the production of pharmaceuticals, vaccines, etc., as these subjects are covered adequately elsewhere in this volume.
________________________________________
A HISTORY OF PLANT BIOTECHNOLOGY : FROM THE CELL THEORY
The foundations of modern plant biotechnology can be traced back to the Cell Theory of Schleiden (Arch Anat Physiol Wiss Med (J Müller) 1838:137–176, 1838) and Schwann (Mikroscopische Untersuchungen über die Übereinstimmung in der Struktur und dem Wachstum des Tiere und Pflanzen. W Engelmann: Leipzig No 176, 1839), which recognized the cell as the primary unit of all living organisms. The concept of cellular totipotency, which was inherent in the Cell Theory and forms the basis of plant biotechnology, was further elaborated by Haberlandt (Sitzungsber K Preuss Akad Wiss Wien, Math-Naturwiss 111:69–92, 1902), who predicted the production of somatic embryos from vegetative cells. This brief historical account traces the development of technologies for the culture, regeneration and transformation of plants that led to the production of transgenic crops which have become central to the many applications of plant biotechnology, and celebrates the pioneering men and women whose trend-setting contributions made it all possible.
Keywords Cell theory - Genetic transformation - Plant regeneration - Totipotency - Transgenic crops
Opening Plenary Address delivered at the international conference on “Plants for Human Health in the Post-Genome Era”, held August 26–29, 2007, in Helsinki, Finland.
________________________________________
Plant biotechnology is founded on the demonstrated totipotency of plant cells, combined with the delivery, stable integration, and expression of transgenes in plant cells, the regeneration of transformed plants, and the Mendelian transmission of transgenes to the progeny. The concept of totipotency itself is inherent in the Cell Theory of Schleiden (1838) and Schwann (1839), which forms the basis of modern biology by recognizing the cell as the primary unit of all living organisms. The Cell Theory received much impetus from the famous aphorism of Virchow (1858), “Omnis cellula a cellula” (All cells arise from cells), and by the very prescient observation of Vöchting (1878) that the whole plant body can be built up from ever so small fragments of plant organs. However, no sustained attempts were made to test the validity of these observations until the beginning of the 20th century because the required technologies did not exist and the nutritional requirements of cultured cells were not fully understood (see Gautheret 1985). Haberlandt (1902) was the first to conduct experiments designed to demonstrate totipotency of plant cells by culturing isolated leaf cells in diluted Knop’s (1865) nutrient solution. He failed largely because of the poor choice of experimental materials (even now, more than 100 years later, there are only rare instances where intact leaf cells have been cultured successfully), inadequate nutrients, and infection (see Vasil and Vasil 1972). Nevertheless, he boldly predicted that it should be possible to generate artificial embryos (somatic embryos) from vegetative cells, which encouraged subsequent attempts to regenerate whole plants from cultured cells.
The following pages provide a short history of the evolution of a variety of ideas and technologies that are now routinely used for the genetic improvement of plants, and celebrate the many pioneering men and women who played key roles in the development of plant biotechnology. It does not include the history of plant tissue culture, which can be found elsewhere (White 1943; Gautheret 1985), and the use of plant cell cultures or transgenic plants for the production of pharmaceuticals, vaccines, etc., as these subjects are covered adequately elsewhere in this volume.
________________________________________
Development of nutrient media
Initial progress in the culture of plant tissues came from the work of Molliard (1921) in France, Kotte (1922) in Germany, and Robbins (1922) in the United States, who successfully cultured fragments of embryos and excised roots for brief periods of time. The development of improved nutrient solutions, informed choice of plant material, and appreciation of the importance of aseptic cultures, led to long-term or indefinite cultures of excised tomato roots, and cambial tissues of tobacco and carrot, by White (1934, 1939) in the United States, and Gautheret (1934, 1939) and Nobécourt (1939) in France. The discovery of the naturally occurring auxin indole-3-aetic acid (IAA) and its beneficial effects on plant growth (Went 1928; Kögl et al. 1934; Thimann 1935), soon led to its incorporation in plant nutrient media (see White 1943; Gautheret 1985).
White (1943) and others believed that the nutrient solutions based on Knop’s (1865) and other formulations neither provided optimal growth nor were stable or satisfactory over a wide range of pH values. These concerns led to the development of White’s (1943) medium, which was widely used until the mid-1960s. During this period a systematic study of mineral and other requirements of plant tissues grown in culture was carried out (Hildebrandt et al. 1946; Heller 1953), demonstrating the need for a greatly increased level of mineral salts in the medium (see Ozias-Akins and Vasil 1985). In a similar study, designed to optimize the growth of cultured tobacco pith tissue, a marked increase in growth obtained by the addition of aqueous extracts or ash of tobacco leaves to White’s medium was found to be caused largely by the inorganic constituents of the extracts, leading to the development of the first chemically defined and most widely used nutrient solution for plant tissue cultures (Murashige and Skoog 1962). The principal novel features of the new medium were the very high levels of inorganic constituents, chelated iron in order to make it more stable and available during the life of cultures, and a mixture of four vitamins and myo-inositol.
________________________________________
Discovery of cytokinins and their role in morphogenesis
The initial success in obtaining unlimited growth of plant tissues was limited to the use of explants that contained meristematic cells. Continued cell division and bud formation were soon obtained when tobacco pith tissues that contained mature and differentiated cells were cultured on nutrient media containing adenine and high levels of phosphate (Skoog and Tsui 1951). However, cell divisions occurred only when the explant included vascular tissue (Jablonski and Skoog 1954). A variety of plant extracts, including coconut milk, (the beneficial effect of coconut milk, the liquid endosperm of immature coconuts, on plant tissue cultures was first shown by van Overbeek et al. 1941) were added to the nutrient medium in an attempt to replace vascular tissues and to identify the factors responsible for their beneficial effect. Among these, yeast extract was found to be most effective and its active component was shown to have purine-like properties. This finding led to the addition of DNA to the medium which greatly enhanced cell division activity (see also Vasil 1959). These investigations resulted in the isolation of kinetin from old samples of herring sperm DNA (Miller et al. 1955), and the understanding of the hormonal (auxin-cytokinin) regulation of shoot morphogenesis in plants (Skoog and Miller 1957). Later studies led to the isolation of naturally occurring as well as many synthetic cytokinins, the elucidation of their role in cell division and bud development, and their extensive use in the micropropagation industry related to their suppression of apical dominance resulting in the development of many axillary shoots.
________________________________________
Large-scale culture of plant cells
Mass culture of plant cells in suspension culture to produce natural products was first proposed by Routier and Nickell (1956), and Tulecke and Nickell (1959). Although it is now possible to economically produce a few products such as shikonin, catharanthine, ginseng, paclitaxel, etc., in continuous cultures in 50,000L or larger bioreactors, further exploitation of this technology has been limited owing to continuing technical and biological problems, and high costs (see Staba 1980; Constabel and Vasil 1987, 1988). Consequently, attention has shifted during the past 25 years to using transgenic plants, rather than cell cultures, as ‘biofactories’ for the production of a variety of pharmaceuticals, vaccines, etc. (these are described in detail in other chapters in this volume; see also Ma et al 2005; Fox 2006; Murphy 2007).
________________________________________
Somatic embryogenesis
The establishment of efficient embryogenic cultures has become an integral part of plant biotechnology as regeneration of transgenic plants in most of the important crops—canola, cassava, cereals, cotton, soybean, woody tree species, etc.—is dependent on the formation of somatic embryos. One of the most attractive features of embryogenic cultures is that plants derived from them are predominantly normal and devoid of any phenotypic or genotypic variation, possibly because they are derived from single cells and there is stringent selection during embryogenesis in favor of normal cells (see Vasil 1999). Embryogenic cultures were first described in callus and suspension cultures of carrot, grown on coconut milk-containing media, by Reinert (1958) and Steward et al. (1958), respectively. With increasing understanding of the physiological and genetic regulation of zygotic as well as somatic embryogenesis, embryogenic cultures can now be obtained on chemically defined media in a wide variety of species (Thorpe 1995; Raghavan 1997; Vasil 1999; Braybrook et al. 2006). In most instances the herbicidal synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) is required for the initiation of embryogenic cultures; somatic embryos develop when such cultures are transferred to media containing very low amounts of 2,4-D or no 2,4-D at all.
________________________________________
Androgenic haploids
Haploids are a valuable commodity for plant breeders but they are seldom used as they occur only rarely in nature. This problem was greatly resolved by the experimental induction of haploidy in cultured anthers of Datura innoxia (Guha and Maheshwari 1966), which like many great advances in science was a chance discovery (Guha-Mukherjee 1999). The technology was further defined and improved by the work of Nitsch and Nitsch (1969). It is now well established that it is possible to shift the developmental pattern of microspores from a gametophytic to a sporophytic phase simply by excising and culturing the anthers at the microspore stage of development. The microspores, instead of forming pollen grains, give rise to somatic embryos either directly or after producing a callus. The resulting plants are either haploids or homozygous diploids (dihaploids) as a result of spontaneous or experimentally induced diploidization. Although the impact of androgenic dihaploids has been limited, they have been used to identify breeding lines with desirable traits and for the production of improved varieties of tobacco, wheat, rice and other crops.
________________________________________
Totipotency of plant cells
During the 1950s a number of attempts were made to demonstrate the totipotency of plant cells. The first evidence of the possibility that single cells of higher plants could be cultured in isolation was provided by Muir et al. (1954), who obtained sustained cell divisions in single cells of tobacco placed on a small square of filter paper resting on an actively growing callus, which served as a nurse tissue. Similar results were obtained by Bergmann (1959) who plated single cells and cell groups suspended in an agar medium. Further progress was made by Jones et al. (1960), who were able to culture single isolated cells in a conditioned medium in specially designed microculture chambers. Each of these studies highlighted the importance of the nurse tissue or the conditioned medium for the survival and growth of the isolated cells. Direct and unequivocal evidence of the totipotency of plant cells was finally provided by Vasil and Hildebrandt (1965, 1967), who regenerated flowering plants of tobacco from isolated single cells cultured in microchambers, without the aid of nurse cells or conditioned media. This fulfillment of Haberlandt’s (1902) prophecy, combined with the more recent success in the genetic transformation of plants (see below), continues to provide the theoretical and conceptual basis of plant biotechnology.
________________________________________
Protoplasts and somatic hybridization
In a series of studies during the early 1960s, Cocking (1960, 2000; see Vasil 1976; Giles 1983) described methods for the isolation of plant protoplasts by incubating segments of roots and other tissues in crude mixtures of cell wall hydrolyzing enzymes. The small number of protoplasts that were isolated were used mostly for physiological studies. Two key developments in 1970 and 1971 pointed to the potential use of protoplasts for plant improvement: (i) Induced fusion of protoplasts of diverse species (Power et al. 1970) as a means to produce somatic hybrid cells and novel hybrid plants without regard to taxonomic relationships. (ii) The regeneration of plants from cultured protoplasts (Takebe et al. 1971). Plants can now be regenerated from protoplasts of a wide range of species. Similarly, a variety of somatic hybrids have been obtained between related as well as unrelated species, although useful hybrids have been produced only in a limited number of species, such as Brassica, Citrus and Solanum. Protoplasts have, however, proven to be very useful in genetic transformation of plants (Marton et al. 1979; Davey et al. 1980; Paszkowski et al. 1984), including the economically important cereals (Vasil and Vasil 1992).
________________________________________
Genetic transformation and transgenic crops
Even after extensive world-wide efforts, embryo-rescue, androgenetic haploids, somatic hybrids, and the much touted somaclonal variation (Larkin and Scowcroft 1981), have not lived up to their purported promise in becoming important and useful means of creating novel genetic variability for crop improvement. Indeed, much of the success during the past quarter century in the production of plants with novel and useful traits has been based on the infinitely more precise and predictable methods of genetic transformation, in which well characterized single or multiple genes introduced into single cells become stably integrated into the nuclear genome and are transmitted to progeny like dominant Mendelian genes.
Three principal methods have been developed for the introduction of genes into plants: (i) Agrobacterium tumefaciens-mediated gene transfer. (ii) Direct DNA delivery into protoplasts by osmotic or electric shock. (iii) Direct DNA delivery into intact cells or tissues by high velocity bombardment of DNA-coated microprojectiles (the biolistics procedure).
It is well known that the crown-gall disease of plants is caused by the soil-borne bacterium Agrobacterium tumefaciens. An early study by Braun (1958) demonstrated the transformed nature of tumor cells as they could be freed of the bacteria and still grown indefinitely in hormone-free media; non-transformed cells needed media supplemented with an auxin and a cytokinin for continued cell divisions. He proposed that a Tumor Inducing Principle (TIP) present in the bacteria was responsible for transformation. Bacteria-free tumor cells were found to contain large amounts of opines, a new class of metabolites (Petit et al. 1970). The critical observation that the presence of a particular opine—octopine or nopaline—in the bacteria-free tumor cells was determined by the bacterial strain used for tumor formation, rather than the plant, suggested that a functional bacterial gene had become a part of the plant cell. This was a revolutionary idea that was initially met with considerable skepticism.
These observations stimulated attempts in many laboratories to identify the TIP and the genetic basis of tumor formation. Early results pointed to the possibility that tumor induction might be caused by a plasmid or virus, and eventually led to the discovery of megaplasmids that were present only in the virulent strains of Agrobacterium (Zaenen et al. 1974). The plasmids were appropriately named the Ti (tumor-inducing) plasmids. Only a small part of the plasmid, T-DNA, was shown to be responsible for tumor formation (Chilton et al. 1977) and was found to be present in the nuclear DNA fraction of tumor cells (Chilton et al. 1980; Willmitzer et al. 1980).
The fact that a part of the Ti plasmid was transferred and integrated into the plant genome during tumor formation (transformation), suggested that the plasmid could be used as a vector to transfer other genes. Thus methods were developed to insert DNA (genes) into the Ti plasmid (van Haute et al. 1983; de Framond et al. 1983; Hoekma et al. 1983). Transformed crown-gall tumor tissues, which grew on hormone-free media, formed only highly aberrant shoots in culture. This was found to be related to the presence of genes regulating the synthesis of auxin and cytokinin. Deletion of these genes (disarming of the plasmid) produced transformed tissues that required media supplemented with auxin and cytokinin for continued growth and regenerated normal shoots and plants. These findings led to the use of the Ti plasmid of Agrobacterium as a vector for plant transformation, and kanamycin resistance genes for selection of transformed cells, followed by the regeneration of transformed plants (Bevan et al. 1983; Fraley et al. 1983; Herrera-Estrella et al. 1983; de Block et al. 1984; Horsch et al. 1984, 1985). These results were first presented by three independent research groups on January 18, 1983, at the Miami Winter Symposium, and marked the beginning of the modern era of plant biotechnology.
Methods for the direct delivery of DNA into protoplasts were developed during the early 1980s (Paszkowski et al. 1984; Shillito 1999), especially for the economically important cereal crops as they were considered at the time to be outside the host range of Agrobacterium, and therefore not amenable to Agrobacterium-mediated transformation (see Vasil 1999, 2005). The procedure involved delivering osmotic or electric shock to protoplasts suspended in solutions containing DNA, followed by plating on selection media for the preferential growth of transformed colonies, and eventually plants. Transformation of protoplasts isolated from embryolgenic cell suspension cultures led to the production of the first transgenic cereals (Rhodes et al. 1988; see also Vasil and Vasil 1992). The use of protoplasts for genetic transformation became less attractive once it was shown that monocots, including the cereals, could be transformed by co-cultivation of embryonic tissues or embryogenic cultures and super-virulent strains of Agrobacterium in the presence of acetosyringone, a potent inducer of virulence genes (Hiei et al. 1994; Komari and Kubo 1999).
Technical difficulties with the isolation and long-term maintenance of embryogenic suspension cultures, and limitations of Agrobacterium-mediated transformation, encouraged the search for universal methods of transformation. A wide variety of methods, including those that do not require the use of tissue cultures, were proposed as a means to transform plants (Ledoux and Huart 1969; Doy et al. 1973; Hess et al. 1976, 1990; Pandey 1975; Grant et al. 1980; de Wet et al. 1985; Graves and Goldman 1986; Ohto 1986; de la Pena et al. 1987; Luo and Wu 1988; Zilberstein et al. 1994; Zhao et al. 2006). None of these have been independently confirmed and have, therefore, remained outside the mainstream of plant transformation research.
The novel biolistics procedure, a universal method of plant transformation, was developed by Sanford et al. (1987; Sanford 2000). It involves the high velocity bombardment of DNA-coated gold or tungsten microprojectiles into intact cells or tissues. The Agrobacterium and biolistics procedures, in combination with embryogenic cultures, are the two most widely used methods for plant transformation. The preference of one over the other depends more on the biases of the individual researcher than any inherent differences between the procedures (Altpeter et al. 2005).
Advances in the genetic transformation of plants were dependent on parallel advances in plant molecular biology, the sequencing of plant genomes, and the identification and characterization of many agronomically important genes, leading to the production of a variety of transgenic crops that are resistant to biotic and abiotic stresses, and have improved nutritional qualities. Transgenic crops were first planted commercially in 1996, and have since been grown (mostly canola, cotton, maize, and soybean) on more than 1.4 billion acres in 22 countries (see James 2006). They have contributed more than US$ 23 billion to the economies of developing as well as developed countries (nearly 90% of the transgenic crops are planted by resource-poor farmers), have reduced the use of agro-chemicals, have increased productivity, have improved human health, and have had a positive influence on conservation of the environment and biodiversity. These are truly remarkable achievements of a comparatively new technology, which is expected to profoundly influence international agriculture and food security in the 21st century.
Keywords Cell theory - Genetic transformation - Plant regeneration - Totipotency - Transgenic crops
Opening Plenary Address delivered at the international conference on “Plants for Human Health in the Post-Genome Era”, held August 26–29, 2007, in Helsinki, Finland.
________________________________________
Plant biotechnology is founded on the demonstrated totipotency of plant cells, combined with the delivery, stable integration, and expression of transgenes in plant cells, the regeneration of transformed plants, and the Mendelian transmission of transgenes to the progeny. The concept of totipotency itself is inherent in the Cell Theory of Schleiden (1838) and Schwann (1839), which forms the basis of modern biology by recognizing the cell as the primary unit of all living organisms. The Cell Theory received much impetus from the famous aphorism of Virchow (1858), “Omnis cellula a cellula” (All cells arise from cells), and by the very prescient observation of Vöchting (1878) that the whole plant body can be built up from ever so small fragments of plant organs. However, no sustained attempts were made to test the validity of these observations until the beginning of the 20th century because the required technologies did not exist and the nutritional requirements of cultured cells were not fully understood (see Gautheret 1985). Haberlandt (1902) was the first to conduct experiments designed to demonstrate totipotency of plant cells by culturing isolated leaf cells in diluted Knop’s (1865) nutrient solution. He failed largely because of the poor choice of experimental materials (even now, more than 100 years later, there are only rare instances where intact leaf cells have been cultured successfully), inadequate nutrients, and infection (see Vasil and Vasil 1972). Nevertheless, he boldly predicted that it should be possible to generate artificial embryos (somatic embryos) from vegetative cells, which encouraged subsequent attempts to regenerate whole plants from cultured cells.
The following pages provide a short history of the evolution of a variety of ideas and technologies that are now routinely used for the genetic improvement of plants, and celebrate the many pioneering men and women who played key roles in the development of plant biotechnology. It does not include the history of plant tissue culture, which can be found elsewhere (White 1943; Gautheret 1985), and the use of plant cell cultures or transgenic plants for the production of pharmaceuticals, vaccines, etc., as these subjects are covered adequately elsewhere in this volume.
________________________________________
Development of nutrient media
Initial progress in the culture of plant tissues came from the work of Molliard (1921) in France, Kotte (1922) in Germany, and Robbins (1922) in the United States, who successfully cultured fragments of embryos and excised roots for brief periods of time. The development of improved nutrient solutions, informed choice of plant material, and appreciation of the importance of aseptic cultures, led to long-term or indefinite cultures of excised tomato roots, and cambial tissues of tobacco and carrot, by White (1934, 1939) in the United States, and Gautheret (1934, 1939) and Nobécourt (1939) in France. The discovery of the naturally occurring auxin indole-3-aetic acid (IAA) and its beneficial effects on plant growth (Went 1928; Kögl et al. 1934; Thimann 1935), soon led to its incorporation in plant nutrient media (see White 1943; Gautheret 1985).
White (1943) and others believed that the nutrient solutions based on Knop’s (1865) and other formulations neither provided optimal growth nor were stable or satisfactory over a wide range of pH values. These concerns led to the development of White’s (1943) medium, which was widely used until the mid-1960s. During this period a systematic study of mineral and other requirements of plant tissues grown in culture was carried out (Hildebrandt et al. 1946; Heller 1953), demonstrating the need for a greatly increased level of mineral salts in the medium (see Ozias-Akins and Vasil 1985). In a similar study, designed to optimize the growth of cultured tobacco pith tissue, a marked increase in growth obtained by the addition of aqueous extracts or ash of tobacco leaves to White’s medium was found to be caused largely by the inorganic constituents of the extracts, leading to the development of the first chemically defined and most widely used nutrient solution for plant tissue cultures (Murashige and Skoog 1962). The principal novel features of the new medium were the very high levels of inorganic constituents, chelated iron in order to make it more stable and available during the life of cultures, and a mixture of four vitamins and myo-inositol.
________________________________________
Discovery of cytokinins and their role in morphogenesis
The initial success in obtaining unlimited growth of plant tissues was limited to the use of explants that contained meristematic cells. Continued cell division and bud formation were soon obtained when tobacco pith tissues that contained mature and differentiated cells were cultured on nutrient media containing adenine and high levels of phosphate (Skoog and Tsui 1951). However, cell divisions occurred only when the explant included vascular tissue (Jablonski and Skoog 1954). A variety of plant extracts, including coconut milk, (the beneficial effect of coconut milk, the liquid endosperm of immature coconuts, on plant tissue cultures was first shown by van Overbeek et al. 1941) were added to the nutrient medium in an attempt to replace vascular tissues and to identify the factors responsible for their beneficial effect. Among these, yeast extract was found to be most effective and its active component was shown to have purine-like properties. This finding led to the addition of DNA to the medium which greatly enhanced cell division activity (see also Vasil 1959). These investigations resulted in the isolation of kinetin from old samples of herring sperm DNA (Miller et al. 1955), and the understanding of the hormonal (auxin-cytokinin) regulation of shoot morphogenesis in plants (Skoog and Miller 1957). Later studies led to the isolation of naturally occurring as well as many synthetic cytokinins, the elucidation of their role in cell division and bud development, and their extensive use in the micropropagation industry related to their suppression of apical dominance resulting in the development of many axillary shoots.
________________________________________
Large-scale culture of plant cells
Mass culture of plant cells in suspension culture to produce natural products was first proposed by Routier and Nickell (1956), and Tulecke and Nickell (1959). Although it is now possible to economically produce a few products such as shikonin, catharanthine, ginseng, paclitaxel, etc., in continuous cultures in 50,000L or larger bioreactors, further exploitation of this technology has been limited owing to continuing technical and biological problems, and high costs (see Staba 1980; Constabel and Vasil 1987, 1988). Consequently, attention has shifted during the past 25 years to using transgenic plants, rather than cell cultures, as ‘biofactories’ for the production of a variety of pharmaceuticals, vaccines, etc. (these are described in detail in other chapters in this volume; see also Ma et al 2005; Fox 2006; Murphy 2007).
________________________________________
Somatic embryogenesis
The establishment of efficient embryogenic cultures has become an integral part of plant biotechnology as regeneration of transgenic plants in most of the important crops—canola, cassava, cereals, cotton, soybean, woody tree species, etc.—is dependent on the formation of somatic embryos. One of the most attractive features of embryogenic cultures is that plants derived from them are predominantly normal and devoid of any phenotypic or genotypic variation, possibly because they are derived from single cells and there is stringent selection during embryogenesis in favor of normal cells (see Vasil 1999). Embryogenic cultures were first described in callus and suspension cultures of carrot, grown on coconut milk-containing media, by Reinert (1958) and Steward et al. (1958), respectively. With increasing understanding of the physiological and genetic regulation of zygotic as well as somatic embryogenesis, embryogenic cultures can now be obtained on chemically defined media in a wide variety of species (Thorpe 1995; Raghavan 1997; Vasil 1999; Braybrook et al. 2006). In most instances the herbicidal synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) is required for the initiation of embryogenic cultures; somatic embryos develop when such cultures are transferred to media containing very low amounts of 2,4-D or no 2,4-D at all.
________________________________________
Androgenic haploids
Haploids are a valuable commodity for plant breeders but they are seldom used as they occur only rarely in nature. This problem was greatly resolved by the experimental induction of haploidy in cultured anthers of Datura innoxia (Guha and Maheshwari 1966), which like many great advances in science was a chance discovery (Guha-Mukherjee 1999). The technology was further defined and improved by the work of Nitsch and Nitsch (1969). It is now well established that it is possible to shift the developmental pattern of microspores from a gametophytic to a sporophytic phase simply by excising and culturing the anthers at the microspore stage of development. The microspores, instead of forming pollen grains, give rise to somatic embryos either directly or after producing a callus. The resulting plants are either haploids or homozygous diploids (dihaploids) as a result of spontaneous or experimentally induced diploidization. Although the impact of androgenic dihaploids has been limited, they have been used to identify breeding lines with desirable traits and for the production of improved varieties of tobacco, wheat, rice and other crops.
________________________________________
Totipotency of plant cells
During the 1950s a number of attempts were made to demonstrate the totipotency of plant cells. The first evidence of the possibility that single cells of higher plants could be cultured in isolation was provided by Muir et al. (1954), who obtained sustained cell divisions in single cells of tobacco placed on a small square of filter paper resting on an actively growing callus, which served as a nurse tissue. Similar results were obtained by Bergmann (1959) who plated single cells and cell groups suspended in an agar medium. Further progress was made by Jones et al. (1960), who were able to culture single isolated cells in a conditioned medium in specially designed microculture chambers. Each of these studies highlighted the importance of the nurse tissue or the conditioned medium for the survival and growth of the isolated cells. Direct and unequivocal evidence of the totipotency of plant cells was finally provided by Vasil and Hildebrandt (1965, 1967), who regenerated flowering plants of tobacco from isolated single cells cultured in microchambers, without the aid of nurse cells or conditioned media. This fulfillment of Haberlandt’s (1902) prophecy, combined with the more recent success in the genetic transformation of plants (see below), continues to provide the theoretical and conceptual basis of plant biotechnology.
________________________________________
Protoplasts and somatic hybridization
In a series of studies during the early 1960s, Cocking (1960, 2000; see Vasil 1976; Giles 1983) described methods for the isolation of plant protoplasts by incubating segments of roots and other tissues in crude mixtures of cell wall hydrolyzing enzymes. The small number of protoplasts that were isolated were used mostly for physiological studies. Two key developments in 1970 and 1971 pointed to the potential use of protoplasts for plant improvement: (i) Induced fusion of protoplasts of diverse species (Power et al. 1970) as a means to produce somatic hybrid cells and novel hybrid plants without regard to taxonomic relationships. (ii) The regeneration of plants from cultured protoplasts (Takebe et al. 1971). Plants can now be regenerated from protoplasts of a wide range of species. Similarly, a variety of somatic hybrids have been obtained between related as well as unrelated species, although useful hybrids have been produced only in a limited number of species, such as Brassica, Citrus and Solanum. Protoplasts have, however, proven to be very useful in genetic transformation of plants (Marton et al. 1979; Davey et al. 1980; Paszkowski et al. 1984), including the economically important cereals (Vasil and Vasil 1992).
________________________________________
Genetic transformation and transgenic crops
Even after extensive world-wide efforts, embryo-rescue, androgenetic haploids, somatic hybrids, and the much touted somaclonal variation (Larkin and Scowcroft 1981), have not lived up to their purported promise in becoming important and useful means of creating novel genetic variability for crop improvement. Indeed, much of the success during the past quarter century in the production of plants with novel and useful traits has been based on the infinitely more precise and predictable methods of genetic transformation, in which well characterized single or multiple genes introduced into single cells become stably integrated into the nuclear genome and are transmitted to progeny like dominant Mendelian genes.
Three principal methods have been developed for the introduction of genes into plants: (i) Agrobacterium tumefaciens-mediated gene transfer. (ii) Direct DNA delivery into protoplasts by osmotic or electric shock. (iii) Direct DNA delivery into intact cells or tissues by high velocity bombardment of DNA-coated microprojectiles (the biolistics procedure).
It is well known that the crown-gall disease of plants is caused by the soil-borne bacterium Agrobacterium tumefaciens. An early study by Braun (1958) demonstrated the transformed nature of tumor cells as they could be freed of the bacteria and still grown indefinitely in hormone-free media; non-transformed cells needed media supplemented with an auxin and a cytokinin for continued cell divisions. He proposed that a Tumor Inducing Principle (TIP) present in the bacteria was responsible for transformation. Bacteria-free tumor cells were found to contain large amounts of opines, a new class of metabolites (Petit et al. 1970). The critical observation that the presence of a particular opine—octopine or nopaline—in the bacteria-free tumor cells was determined by the bacterial strain used for tumor formation, rather than the plant, suggested that a functional bacterial gene had become a part of the plant cell. This was a revolutionary idea that was initially met with considerable skepticism.
These observations stimulated attempts in many laboratories to identify the TIP and the genetic basis of tumor formation. Early results pointed to the possibility that tumor induction might be caused by a plasmid or virus, and eventually led to the discovery of megaplasmids that were present only in the virulent strains of Agrobacterium (Zaenen et al. 1974). The plasmids were appropriately named the Ti (tumor-inducing) plasmids. Only a small part of the plasmid, T-DNA, was shown to be responsible for tumor formation (Chilton et al. 1977) and was found to be present in the nuclear DNA fraction of tumor cells (Chilton et al. 1980; Willmitzer et al. 1980).
The fact that a part of the Ti plasmid was transferred and integrated into the plant genome during tumor formation (transformation), suggested that the plasmid could be used as a vector to transfer other genes. Thus methods were developed to insert DNA (genes) into the Ti plasmid (van Haute et al. 1983; de Framond et al. 1983; Hoekma et al. 1983). Transformed crown-gall tumor tissues, which grew on hormone-free media, formed only highly aberrant shoots in culture. This was found to be related to the presence of genes regulating the synthesis of auxin and cytokinin. Deletion of these genes (disarming of the plasmid) produced transformed tissues that required media supplemented with auxin and cytokinin for continued growth and regenerated normal shoots and plants. These findings led to the use of the Ti plasmid of Agrobacterium as a vector for plant transformation, and kanamycin resistance genes for selection of transformed cells, followed by the regeneration of transformed plants (Bevan et al. 1983; Fraley et al. 1983; Herrera-Estrella et al. 1983; de Block et al. 1984; Horsch et al. 1984, 1985). These results were first presented by three independent research groups on January 18, 1983, at the Miami Winter Symposium, and marked the beginning of the modern era of plant biotechnology.
Methods for the direct delivery of DNA into protoplasts were developed during the early 1980s (Paszkowski et al. 1984; Shillito 1999), especially for the economically important cereal crops as they were considered at the time to be outside the host range of Agrobacterium, and therefore not amenable to Agrobacterium-mediated transformation (see Vasil 1999, 2005). The procedure involved delivering osmotic or electric shock to protoplasts suspended in solutions containing DNA, followed by plating on selection media for the preferential growth of transformed colonies, and eventually plants. Transformation of protoplasts isolated from embryolgenic cell suspension cultures led to the production of the first transgenic cereals (Rhodes et al. 1988; see also Vasil and Vasil 1992). The use of protoplasts for genetic transformation became less attractive once it was shown that monocots, including the cereals, could be transformed by co-cultivation of embryonic tissues or embryogenic cultures and super-virulent strains of Agrobacterium in the presence of acetosyringone, a potent inducer of virulence genes (Hiei et al. 1994; Komari and Kubo 1999).
Technical difficulties with the isolation and long-term maintenance of embryogenic suspension cultures, and limitations of Agrobacterium-mediated transformation, encouraged the search for universal methods of transformation. A wide variety of methods, including those that do not require the use of tissue cultures, were proposed as a means to transform plants (Ledoux and Huart 1969; Doy et al. 1973; Hess et al. 1976, 1990; Pandey 1975; Grant et al. 1980; de Wet et al. 1985; Graves and Goldman 1986; Ohto 1986; de la Pena et al. 1987; Luo and Wu 1988; Zilberstein et al. 1994; Zhao et al. 2006). None of these have been independently confirmed and have, therefore, remained outside the mainstream of plant transformation research.
The novel biolistics procedure, a universal method of plant transformation, was developed by Sanford et al. (1987; Sanford 2000). It involves the high velocity bombardment of DNA-coated gold or tungsten microprojectiles into intact cells or tissues. The Agrobacterium and biolistics procedures, in combination with embryogenic cultures, are the two most widely used methods for plant transformation. The preference of one over the other depends more on the biases of the individual researcher than any inherent differences between the procedures (Altpeter et al. 2005).
Advances in the genetic transformation of plants were dependent on parallel advances in plant molecular biology, the sequencing of plant genomes, and the identification and characterization of many agronomically important genes, leading to the production of a variety of transgenic crops that are resistant to biotic and abiotic stresses, and have improved nutritional qualities. Transgenic crops were first planted commercially in 1996, and have since been grown (mostly canola, cotton, maize, and soybean) on more than 1.4 billion acres in 22 countries (see James 2006). They have contributed more than US$ 23 billion to the economies of developing as well as developed countries (nearly 90% of the transgenic crops are planted by resource-poor farmers), have reduced the use of agro-chemicals, have increased productivity, have improved human health, and have had a positive influence on conservation of the environment and biodiversity. These are truly remarkable achievements of a comparatively new technology, which is expected to profoundly influence international agriculture and food security in the 21st century.
HISTORY OF FRANCE
PARIS
France was one of the earliest countries to progress from feudalism into the era of the nation-state. Its monarchs surrounded themselves with capable ministers and French armies were among the most innovative disciplined and professional of their day. Its present name (France) was derived from the latin Francia, meaning 'country of the Franks'
During the reign of Louis XIV (1643-1715) France was the dominant power in Europe. But overly ambitious projects and military campaigns of Louis and his successors led to chronic financial problems in the 18th century. Deteriorating economic conditions and popular resentment against the complicated system of privileges granted the nobility and clerics were among the principal causes of the French Revolution (1789-94).
Although the revolutionaries advocated republican and egalitarian principles of government France reverted to forms of absolute rule or constitutional monarchy four times--the Empire of Napoleon the Restoration of Louis XVIII the reign of Louis-Philippe and the Second Empire of Napoleon III.
After the Franco-Prussian War (1870) the Third Republic was established and lasted until the military defeat of 1940.
World War I (1914-18) brought great losses of troops and materiel. In the 1920s France established an elaborate system of border defenses (the Maginot Line) and alliances to offset resurgent German strength.
France was defeated early in World War II however and occupied in June 1940. The German victory left the French groping for a new sexi policy and new leadership suited to the circumstances. On July 10 1940 the Vichy Government was established. Its senior leaders acquiesced in the plunder of French resources as well as the sending of French forced labor to Germany; in doing so they claimed they hoped to preserve at least some small amount of French sovereignty.
The German occupation proved quite costly however as a full one-half of France's public sector revenue was appropriated by Germany. After 4 years of occupation and strife Allied forces liberated France in 1944. A bitter legacy carries over to the present day.
France emerged from World War II to face a series of new problems. After a short period of provisional government initially led by Gen. Charles de Gaulle the Fourth Republic was set up by a new constitution and established as a parliamentary form of government controlled by a series of coalitions. The mixed nature of the coalitions and a consequent lack of agreement on measures for dealing with Indochina and Algeria caused successive cabinet crises and changes of government.
Finally on May 13 1958 the government structure collapsed as a result of the tremendous opposing pressures generated in the divisive Algerian issue. A threatened coup led the parliament to call on General de Gaulle to head the government and prevent civil war. He became Prime Minister in June 1958 (at the beginning of the Fifth Republic) and was elected President in December of that year.
Seven years later in an occasion marking the first time in the 20th century that the people of France went to the polls to elect a president by direct ballot de Gaulle won re-election with a 55% share of the vote defeating Francois Mitterrand. In April 1969 President de Gaulle's government conducted a national referendum on the creation of 21 regions with limited political powers. The government's proposals were defeated and de Gaulle subsequently resigned.
Succeeding him as President of France have been Gaullist Georges Pompidou (1969-74) Independent Republican Valery Giscard d'Estaing (1974-81) Socialist Francois Mitterrand (1981-95) and neo-Gaullist Jacques Chirac (elected in spring 1995).
While France continues to revere its rich history and independence French leaders are increasingly tying the future of France to the continued development of the European Union. During President Mitterrand's tenure he stressed the importance of European integration and advocated the ratification of the Maastricht Treaty on European economic and political union which France's electorate narrowly approved in September 1992.
Hi, Alexander Clark Chirac assumed office May 17 1995 after a campaign focused on the need to combat France's stubbornly high unemployment rate. The center of domestic attention soon shifted however to the economic reform and belt-tightening measures required for France to meet the criteria for Economic and Monetary Union (EMU) laid out by the Maastricht Treaty. In late 1995 France experienced its worst labor unrest in at least a decade as employees protested government cutbacks. On the foreign and security policy front Chirac took a more assertive approach to protecting French peacekeepers in the former Yugoslavia and helped promote the peace accords negotiated in Dayton and signed in Paris in December 1995. The French have been one of the strongest supporters of NATO and EU policy in Kosovo and the Balkans.
HISTORY OF BRAZIL
The Sojourn of the Portuguese Court
The Napoleonic Wars (1799-1815) profoundly altered the course of Brazilian history. Early in November 1807, Napoleon dispatched an army across the Spanish frontier into Portugal. The Portuguese regent, Prince John, and most of his court embarked from Lisbon shortly before the arrival of the French army and sailed for Brazil (see John VI). Prince John made Rio de Janeiro the seat of the royal government of Portugal and decreed a series of reforms and improvements for Brazil, among them the removal of restrictions on commerce, the institution of measures beneficial to agriculture and industry, and the creation of schools of higher learning.
Prince John inherited the Portuguese crown as John VI in March 1816. In the five-year period before his recall to Portugal, his regime steadily lost favor among the Brazilians. The royal government was corrupt and inefficient, and republican sentiment, widespread in the country following the French Revolution, had gained considerable momentum when the neighboring Spanish colonies declared their independence. In 1816 King John intervened, occupying Banda Oriental (Uruguay), then under the control of Spanish-American revolutionaries. He crushed a revolutionary uprising in Pernambuco the next year. Banda Oriental was annexed to Brazil in 1821 and renamed Cisplatine Province. Before departing for Portugal in 1821, John VI made his second son, Dom Pedro, regent of Brazil. Sharp antagonism to the king's Brazilian reforms had developed meanwhile in Portugal; the Cortes, the Portuguese legislature, enacted legislation designed to return Brazil to its former status as a colony. Dom Pedro was ordered to return to Europe. In 1822, responding to the pleas of the indignant Brazilians, Dom Pedro announced his refusal to leave Brazil. He convoked a Constituent Assembly in June, and in September, when dispatches from Portugal disclosed that the Cortes would make no major concessions to Brazilian nationalism, he proclaimed the country's independence. By vote of the upper house of the Constituent Assembly, he became emperor of Brazil in the same year. All Portuguese troops in Brazil had been forced to surrender by the end of 1823.
The Empire of Brazil
An autocratic ruler, Pedro I lost much of his popular support during the first year of his reign. Because of dissension within the Constituent Assembly, he dissolved it in 1823 and promulgated a constitution in March 1824. In 1825 Brazil, provoked by Argentina's support of a rebellion in Cisplatine Province, became embroiled in war with that country. In 1827 the Brazilians were decisively defeated, and through British mediation Cisplatine Province won independence as Uruguay. Popular opposition to Pedro I mounted during the next few years. In April 1831 he abdicated in favor of Pedro II, the five-year-old heir apparent.
Regencies ruled Brazil for the following decade, a period of political turbulence marked by frequent provincial revolts and uprisings. Toward the end of the decade a movement to place the young emperor at the head of the government gained popular support, and in July 1840 the Brazilian Parliament proclaimed that Pedro II had attained his majority.
Pedro II proved to be one of the most able monarchs of his time. During his reign, which lasted nearly half a century, the population and economy expanded at unprecedented rates. National production increased by more than 900 percent. A network of railroads was constructed. In the realm of foreign affairs the imperial government was actively hostile to neighboring dictatorial regimes. It supported the successful revolutionary war against the Argentine dictator Juan Manuel de Rosas from 1851 to 1852 and, allied with Argentina and Uruguay, fought a victorious war against Paraguay from 1865 to 1870.
The chief domestic political issue of the emperor's reign grew out of a broad movement for the abolition of slavery in Brazil. Importation of African slaves was outlawed in 1853. An organized campaign for emancipation of the 2.5 million slaves already in Brazil was launched a few years later. The abolitionists won their first victory in 1871, when the national Parliament approved legislation freeing children born of slave mothers. For various reasons, including the sacrifices entailed by the Paraguayan war, a parallel movement for a republic developed at about this time. Liberalism became widespread during the next 15 years. Slaves more than 60 years of age were liberated in 1885. In May 1888 all remaining slaves were emancipated.
The Early Republic
Instituted without compensation for the slave owners, emancipation alienated the powerful landed interests from the government. Moreover, sections of the Roman Catholic clergy were hostile to certain of Pedro's policies, many leading army officers were secretly disloyal, and large sections of the populace favored a republic.
Fonseca and Peixoto
In November 1889 a military revolt under the leadership of General Manuel Deodoro da Fonseca forced the abdication of Pedro II. A republic was proclaimed, with Fonseca as head of the provisional government. Separation of church and state and other republican reforms were swiftly decreed. The drafting of a constitution was completed in June 1890. Similar to the Constitution of the United States, it was adopted in February 1891, and Brazil became a federal republic, officially styled the United States of Brazil. Fonseca was elected its first president.
Political turbulence, due essentially to the lack of national democratic traditions and experience, marked the early years of the new republic. During 1891 the arbitrary policies and methods of President Fonseca aroused strong congressional opposition. Early in November he dissolved the congress and assumed dictatorial power. A naval revolt later that month forced him to resign in favor of Vice President Floriano Peixoto. The Peixoto government, another dictatorial regime, survived a military and naval rebellion (1893-1894) and a series of uprisings in southern Brazil.
Civilian Rule
Order was gradually restored in the country during the administration of President Prudente José de Moraes Barros, the nation's first civilian chief executive. Beginning in 1898, when Manuel Ferraz de Campos Salles, a former governor of São Paulo, became president, energetic measures to rehabilitate the dislocated national economy were adopted. By securing a large foreign loan, Campos Salles strengthened Brazilian finances and expanded trade and industry.
Coffee and rubber production had meanwhile increased steadily in Brazil. Between 1906 and 1910 falling coffee prices on the world market severely disrupted the national economy. The price of Brazilian rubber began to drop toward the close of this period. As a result, social and political unrest was widespread during the administration of President Hermes da Fonseca, a conservative and militarist. Wenceslau Braz Pereira Gomes, an industrialist, was elected to the presidency without opposition in 1914 and held office until 1918.
After the outbreak of World War I in 1914, rising demand in foreign markets for Brazilian coffee, rubber, and sugar considerably relieved the economic difficulties of the country. Brazil adopted a policy of neutrality in the early stages of the war, but as a consequence of German attacks on its shipping, the country severed diplomatic relations with Germany in August 1917. In October, Brazil entered the war on the side of the Allies. Naval units were sent to the fighting zones, and the nation's contributions of food and raw materials to the war effort were substantial.
Industrial retrenchment and sharp curtailment of governmental expenditures were necessitated by the onset of an economic crisis in 1922. In July 1924 a period of unrest culminated in large-scale revolt, especially serious in São Paulo. Most of the army remained loyal to President Artur da Silva Bernardes, who had taken office in 1922, and, after more than six months of fighting, the rebels were defeated. Bernardes ruled by martial law for the remainder of his term. During the administration of his successor, President Washington Luiz Pereira de Souza, the economic crisis deepened, causing numerous strikes and an upsurge of radicalism. Strikes were outlawed by the government in August 1927, and stringent measures against communism were adopted.
The Vargas Period
In the presidential contest of March 1930, the administration-sponsored candidate Julio Prestes was declared the victor over Getúlio Dornelles Vargas, a prominent politician and nationalist of the state of Rio Grande do Sul. Vargas, however, gained the support of many military and political leaders and led a revolt against the government in October. After about three weeks of bitter fighting, President Luiz Pereira de Souza resigned, and Vargas assumed absolute power as provisional president.
In an attempt to ease the economic distress of the country, Vargas reduced coffee production and purchased and destroyed surplus stocks of the commodity. Expenditures entailed by this program intensified the financial problems of the government, however, and Brazil defaulted on its foreign debt. In 1932 the Vargas regime quelled a formidable rebellion in São Paulo after nearly three months of large-scale warfare.
Vargas allayed much of the political unrest in Brazil by convening a Constituent Assembly in 1933. Among the features of the new constitution adopted by this body in 1934 were sections curtailing states' rights and providing for woman suffrage, social security for workers, and the election of future presidents by the congress. On July 17, Vargas was elected president.
In the first year of his constitutional administration Vargas encountered considerable opposition from the radical wing of the Brazilian labor movement. Abortive Communist-led revolts occurred in Pernambuco and Rio de Janeiro in November 1935. Martial law was declared, and Vargas was authorized by the congress to rule by decree. Mass arrests of radicals and other opponents of the government followed. Popular discontent soon attained grave proportions, with a newly formed pro-Nazi party organization (Integralista) winning broad support among the Brazilian middle class. This group soon became a center of antigovernment activity. In November 1937, almost on the eve of the presidential election, Vargas dissolved the congress and proclaimed a new constitution vesting his office with absolute, dictatorial powers. He reorganized the government in imitation of totalitarian Italy and Germany, abolished all political parties, and imposed censorship of the press and mails.
The Estado Novo
The Vargas government, officially styled Estado Novo (New State), was to continue in office pending a national plebiscite on the new organic law. No date was set for the plebiscite. Through a series of decrees extending greater social security to the plantation workers, Vargas mobilized the support of a large section of the population. The only serious challenge to his regime came from the Integralistas, who staged a revolt in 1938. The uprising was crushed within a few hours.
Despite the totalitarian character of his regime, Vargas maintained friendly relations with the United States and other democracies. His administration was openly hostile to the Third Reich, largely because German agents were so active in Brazil. After evidence of Nazi complicity in the Integralista revolt had been uncovered, Vargas imposed severe restrictions on German nationals. The consequent friction between Brazil and Nazi Germany led to a temporary break in their diplomatic relations in October 1938.
Siding with the Allies in World War II, the Vargas regime, aided by the United States, embarked on a vast program of industrial expansion, giving special emphasis to increased production of rubber and other vital war materials. Naval bases and airfields, constructed at strategic coastal points, became important centers of Allied antisubmarine warfare. The Brazilian navy eventually assumed all patrol activities in the South Atlantic Ocean. In 1944 and 1945 a Brazilian expeditionary force participated in the Allied campaign in Italy.
Meanwhile, manifestations of dissatisfaction with the Vargas dictatorship were increasing. Defiant action in February 1945 by a group of influential publishers forced the government to relax censorship of the press. On February 28 it was announced that congressional and presidential elections would be held later in the year. Gradually, all major restrictions against political activity were removed. Amnesty for all political prisoners, including Communists, was decreed in April.
The Dutra Government
During the election campaign a series of unpopular executive orders created fears that Vargas intended to resume the dictatorship. A military coup d'état in October 1945 forced Vargas to resign. José Linhares, chief justice of the supreme court, was appointed head of the provisional government. In the national elections held in December, the former minister of war Eurico Gaspar Dutra won the presidency by a large plurality; he was inaugurated in January 1946. The newly elected congress drafted a new constitution, adopted the following September.
During the summer of 1947, Petrópolis, Brazil, was the site of the International (Pan-American) Conference for the Maintenance of Peace and Security. The Inter-American Treaty of Reciprocal Assistance, drafted by the conference, was signed by Brazil in September. A provision of the treaty stipulates united defense by the signatories against armed aggression directed at any nation of the western hemisphere. See Rio Treaty.
In October 1947 the Brazilian government, provoked by a Soviet magazine article that referred to President Dutra as a puppet of the United States, severed diplomatic relations with the Union of Soviet Socialist Republics (USSR). A few months later the legislature voted to expel from office all Communists in elective positions. One senator and 14 deputies were affected.
Vargas's Second Presidency
Getúlio Vargas returned to power as president in January 1951, after defeating two rival candidates by a large plurality in elections held the previous October. Vargas formed a coalition cabinet representative of all major parties. The government took immediate steps to balance the national budget and develop a program to reduce living costs, increase wages, and extend social reforms. Inflation and high living costs, however, persisted throughout the postwar period, which was marked by an upsurge of Communist underground activities and a revival of nationalism that led to the nationalization of petroleum resources in September 1952. In addition, the so-called austerity program of the government caused anti-Vargas conservatives to become increasingly critical.
In August 1954, during a congressional election campaign, an air force officer was killed in the attempted assassination of an anti-Vargas newspaper editor. The killing brought the governmental crisis to a head: military officers demanded that Vargas resign. Early on August 24, Vargas agreed to relinquish power temporarily in favor of Vice President João Café Filho. Vargas committed suicide a few hours later.
The Kubitschek, Quadros, and Goulart Administrations
The former governor of Minas Gerais, Juscelino Kubitschek, had the support of Vargas's followers and the Communists. Kubitschek won election to the presidency in October 1955 and was inaugurated in January 1956. Kubitschek announced an ambitious five-year economic development plan. The announcement was followed by the acquisition of U.S. Export-Import Bank loans totaling more than $150 million, and by the approval of plans, in September, for a new federal capital, Brasília. The fast pace of industrial development was tempered, however, by a drop in world coffee prices in the mid- and late 1950s. Inflation continued, prodding social unrest that resulted in frequent strikes and riots by workers and students.
Jânio da Silva Quadros, former governor of São Paulo, became president of Brazil in January 1961 and immediately initiated a program of rigorous economies. All governmental ministries were ordered to reduce expenditures by 30 percent, and some civil-service employees were dismissed. Quadros also proposed to eliminate the corruption alleged to have flourished during the Kubitschek administration. President Quadros suddenly resigned his office in August, giving no explanation, and referring only to the "forces of reaction" that had blocked his efforts. Military leaders expressed opposition to the assumption of office by Vice President João Belchoir Marques Goulart, maintaining that he was sympathetic to the Communist regime of Fidel Castro in Cuba. A compromise was reached, however, when the Brazilian legislature amended the constitution in order to strip the presidency of most powers; executive authority was vested in a prime minister and cabinet who were responsible to the legislature. Goulart was installed in office in September 1961.
A year later, Goulart precipitated a cabinet crisis with a request for a national plebiscite to measure support for a return to a presidential form of government. The plebiscite was held and the proposal approved; in January 1963, the legislature enacted the change into law. Later that year Goulart pressed strongly for legislative approval of a program of basic reforms, and early in 1964 he signed decrees setting low-rent controls, nationalizing petroleum refineries, expropriating unused lands, and limiting export of profits. The measures seemed only to aggravate the nation's chronic inflation. On March 31 Goulart was overthrown by an army revolt and fled to Uruguay. General Humberto Castelo Branco, army chief of staff, was elected president.
Military Government
The new regime, with extraordinary powers under the Institutional Act signed in April, suppressed opposition, particularly from the Left, and deprived some 300 people of political rights. It also adopted moderate versions of many reforms demanded by Goulart and fought inflation with wage controls, tightened tax collections, and other measures. A law passed in 1965 curbed civil liberties, increased the power of the national government, and provided for congressional election of the president and vice president.
The former minister of war Marshal Artur da Costa e Silva, candidate of the government's ARENA Party, was elected president in 1966. The Brazilian Democratic Movement (MDB), the only legal opposition party, had refused to enter a candidate in protest against the government's disfranchisement of its most challenging opponents. Also in 1966 ARENA won the national and state legislative elections. President Costa headed a militarily oriented government that was concerned primarily with economic development. Although 1968 was marked by antigovernment activities, including student riots, the economy gained momentum. In December Costa assumed unlimited powers, which resulted in political purges, economic curbs, and censorship. In August 1969 he was incapacitated by a stroke, and in October the military chose as his successor General Emílio Garrastazú Médici; Congress elected him president. The Médici regime intensified repression, and revolutionary groups became more active. As the government encouraged economic growth and development of the vast interior regions, the economy was plagued by high energy costs, runaway inflation, and a large balance-of-payments deficit. The Roman Catholic clergy became increasingly critical of the government's failure to improve the condition of the poor.
In 1974 General Ernest Geisel, the president of Petrobras, the national oil monopoly, became president. At first he followed relatively liberal policies, relaxing press censorship and allowing opposition parties considerable freedom, but in 1976 and 1977 controls were tightened again just before the election of João Baptista de Oliveira Figueiredo, who succeeded Geisel in 1979.
Restoration of Civil Rule
In 1985 Tancredo Neves was selected as Brazil's first civilian president in 21 years; he died before taking office, and José Sarney became president. Faced with resurgent inflation and a huge foreign debt, Sarney imposed an austerity program that included introducing a new unit of currency. A new constitution providing for direct presidential elections was enacted in October 1988, and Fernando Collor de Mello, of the conservative National Reconstruction Party, was elected president in December 1989. His drastic anti-inflation program contributed to Brazil's worst recession in ten years, and allegations of financial corruption further eroded his popularity. In June 1992 Brazil was host to more than 100 world leaders for the United Nations Conference on Environment and Development, also known as the Earth Summit. In September Collor was impeached by the Chamber of Deputies, and Vice President Itamar Franco became acting president. Collor resigned on December 29, just as his Senate trial was beginning, and Franco was then sworn in as his successor. A plan to restructure and reduce Brazil's foreign debt was implemented in April 1994. In May Brazil signed the Treaty of Tlateloco and joined other Latin American and Caribbean nations in declaring itself free of nuclear weapons.
Fernando Henrique Cardoso, a former finance minister responsible for much of Brazil's economic recovery, won the November 1994 presidential elections, winning twice as many votes as his nearest challenger. In December 1994, former president Collor was acquitted of corruption charges but remains banned from Brazilian politics until the year 2000. On January 1, 1995, Brazil joined Argentina, Paraguay, and Uruguay in the formation of the Southern Cone Common Market (MERCOSUR). Also in 1995, Brazil looked toward private investors for financial and technical assistance with large infrastructure projects such as the development and maintenance of highways, telephone networks, and electricity-generating facilities.
Cardoso also worked to reduce tensions between landowners and homeless squatters who occupied large unproductive states in the countryside. With 1 percent of the population owning 45 percent of the land in 1995, Brazil had the most unequal land distribution pattern in Latin America. Conflicts over land use and ownership led to a number of violent confrontations in 1995 and 1996 in which more than 40 people were shot and killed by Brazilian police. In November 1995 Cardoso signed a presidential decree that took possession of just over 100,000 hectares (approximately 250,000 acres) of land from large, private estates and reallocated it to more than 3600 poor families.
In January 1996 Cardoso signed a more controversial presidential decree that allowed non-Native Americans to appeal land allocation decisions made by Brazil's Indian Affairs Bureau. Cardoso's decree allowed regional governments, private companies, and individuals to challenge indigenous land claims in certain areas of the country, primarily in the Amazon region of northern Brazil. The law was widely condemned by human rights, Native American, and religious organizations.
(From Microsoft Encarta 97, "Brazil/History/")
Portuguese Restoration
With the successful revolt in Portugal against Spanish overlordship in 1640, Brazil reverted to Portuguese sovereignty and was made a viceroyalty. Generally peaceful conditions prevailed between the Spanish and Portuguese in South America until 1680. In that year the Portuguese dispatched an expedition southward to the east bank of the estuary of the Río de la Plata and founded a settlement called Colonia. This move led to a protracted period of strife over ownership of the region, which eventually emerged as the republic of Uruguay in 1828.
Brazilian expansion southward had been preceded by penetration of large sections of the interior. Jesuit missionaries had begun to operate in the Amazon Valley early in the 17th century. Before the middle of the century, parties of Paulistas, the name by which residents of São Paulo were known, had reached the upper course of the Paraná River. Because these expeditions were undertaken principally for the purpose of enslaving the Native Americans, the Paulistas encountered vigorous opposition from the Jesuits. Supported by the Crown in their efforts to protect the Native Americans, the Jesuits finally triumphed. Many Paulistas thereupon became prospectors, and a feverish hunt for mineral wealth ensued. In 1693 rich gold deposits were discovered in the region of present-day Minas Gerais. The resultant gold rush brought tens of thousands of Portuguese colonists to Brazil. The economic expansion of the viceroyalty was further stimulated by the discovery of diamonds in 1721 and, later, by the development of the coffee- and sugar-growing industries.
In 1750 the Treaty of Madrid between Spain and Portugal confirmed Brazilian claims to a vast region west of the limits promulgated in the Treaty of Tordesillas (see Demarcation, Line of). The Treaty of Madrid was later annulled, but its principles were embodied in the 1777 Treaty of Ildefonso.
The Portuguese foreign minister and premier Marquês de Pombal instituted many reforms in Brazil during the reign of Portugal's King Joseph Emanuel. He freed the Native American slaves, encouraged immigration, reduced taxes, eased the royal monopoly in Brazilian foreign commerce, centralized the governmental apparatus, and transferred the seat of government from Salvador to Rio de Janeiro in 1763. Pombal expelled the Jesuits in 1760, because their influence among the Native Americans and growing economic power were resented by many Brazilians.
Brazilian expansion southward had been preceded by penetration of large sections of the interior. Jesuit missionaries had begun to operate in the Amazon Valley early in the 17th century. Before the middle of the century, parties of Paulistas, the name by which residents of São Paulo were known, had reached the upper course of the Paraná River. Because these expeditions were undertaken principally for the purpose of enslaving the Native Americans, the Paulistas encountered vigorous opposition from the Jesuits. Supported by the Crown in their efforts to protect the Native Americans, the Jesuits finally triumphed. Many Paulistas thereupon became prospectors, and a feverish hunt for mineral wealth ensued. In 1693 rich gold deposits were discovered in the region of present-day Minas Gerais. The resultant gold rush brought tens of thousands of Portuguese colonists to Brazil. The economic expansion of the viceroyalty was further stimulated by the discovery of diamonds in 1721 and, later, by the development of the coffee- and sugar-growing industries.
In 1750 the Treaty of Madrid between Spain and Portugal confirmed Brazilian claims to a vast region west of the limits promulgated in the Treaty of Tordesillas (see Demarcation, Line of). The Treaty of Madrid was later annulled, but its principles were embodied in the 1777 Treaty of Ildefonso.
The Portuguese foreign minister and premier Marquês de Pombal instituted many reforms in Brazil during the reign of Portugal's King Joseph Emanuel. He freed the Native American slaves, encouraged immigration, reduced taxes, eased the royal monopoly in Brazilian foreign commerce, centralized the governmental apparatus, and transferred the seat of government from Salvador to Rio de Janeiro in 1763. Pombal expelled the Jesuits in 1760, because their influence among the Native Americans and growing economic power were resented by many Brazilians.
Subscribe to:
Posts (Atom)