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INSECTICIDAL GENES

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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)

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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.

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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?

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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

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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

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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

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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

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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

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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

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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.

PROTOPLASTS AND SOMATIC HYBRIDIZATION

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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).
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TOTIPOTENCY OF PLANT CELLS

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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.
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ANDROGENIC HAPLOIDS

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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.
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