Heather A. Kochinsky and Peggy G. Lemaux
While using biotechnology for crop improvement used to be a topic for the future, there are now many examples of genetically engineered products in the field and marketplace. These include Bollgard cotton, Roundup Ready Soybean, European corn borer-resistant maize and more. Such genetically engineered crops represented 50% of the cotton acreage, 30% of the soybean and 20% of the maize acreage in the U.S. in 1998, some 60 million acres.
Biotechnology, coupled with classical methods of crop manipulation, holds promise for alfalfa by providing new opportunities as well as some new approaches for old challenges. In the last few years, much progress has been made in such efforts, which include developing strategies for engineering pest resistance and herbicide tolerance and for understanding the process of nitrogen fixation and the symbiotic relationship. In addition work has focused on the characterization of regulatory signals that can be used for targeting the expression of introduced traits in engineered alfalfa. In addition much work in Canada has been focused on stress tolerance.
In some cases the modifications being attempted with biotechnology have been the object of traditional breeding methods but, because these classical methods are sometimes inadequate or the desired trait is unavailable in closely related germplasm, biotechnological methods are attempted. The power of biotechnology is the ability to make fine-tuned changes. This is exemplified in two approaches that were used to move a sugar-degrading enzyme from a wild tomato species into a commercial variety (Bennett et al. 1994). With classical breeding, the researchers achieved a sweeter tomato after many backcrosses to the commercial line. In addition to the desirable sweetness trait, they also kept some additional information from the wild tomato species; in that information was lowered fertility. By contrast, molecular breeders were able to identify a gene in the commercial variety for an enzyme that breaks down sugar; they were able to use genetic engineering technologies to remove the degradative enzyme in a very specific manner. This time they achieved a sweeter tomato and there were no unexpected consequences of the change. That is because the amount of changed genetic material was small with this approach and its precise nature was known. And, although in this case the information was taken from tomato itself, the possibility existed to take the information from any other useful source. This gives the molecular breeder a large toolbox from which to take the information needed for crop improvement.
One of the cornerstones of this technology is the ability to culture plant cells in vitro in a relatively undifferentiated state, to introduce new genetic information into one of those cells, to identify that cell and coax it to divide to give rise ultimately to a plant, each cell of which contains the new genetic information. The most common method of introducing DNA into alfalfa is through the use of Agrobacterium, a naturally occurring, soil-borne microorganism that can inject DNA into plant cells. This DNA can then become a heritable part of the plant's genetic material. Because only a small number of cells actually incorporate the new genetic information, such cells must be identified by selection using one of the introduced genes, normally for antibiotic or herbicide resistance.
Recently considerable effort has focused on optimizing DNA introduction procedures. In general, these methods are well worked out and transformation and regeneration in some varieties is now routine. A new study looked at nine distinct sets of alfalfa germplasm used to develop modern varieties. Correlations were established between the ability to infect with Agrobacterium and the traits for fall dormancy and nodulation. The latter suggests that the genetic loci required for successful symbiosis are also involved in the ability of Agrobacterium to infect. The implication is that it will be necessary to match a precise Agrobacterium strain with specific alfalfa germplasm in order to achieve successful transformation in alfalfa (Samac, 1995).
Other methods of obtaining transformed alfalfa have been reported, such as the direct introduction of DNA into pollen, which is then used to pollinate flowers. Although engineered plants were generated by this method, there were difficulties in maintaining the introduced genetic material in the transformed plants over multiple vegetative generations (Ramaiah and Skinner, 1997).
In addition to developing efficient methods for transformation, it is also necessary to identify regulatory elements that direct the synthesis of desired gene products in certain tissues at appropriate times. Progress in this area for alfalfa has also been made. A promoter from pea has been found to direct expression of genes to developing surface layers or epidermis of vegetative and floral shoot growing points or apices in transgenic alfalfa. This promoter might be particularly suitable for the genetic engineering of defense genes that are effective against insects and diseases that attack the growing points of the plants (Mandaci and Dobres, 1997). It is also possible that by directing the synthesis of insecticidal proteins to these regions, it might be possible to protect the shoot apex from insect damage, causing insects to devour older less agronomically important tissues without drastically increasing selection for resistant insects. Also a promoter from alfalfa (rubisco) that directs high level expression in most tissues has been identified and characterized (Khoudi et al, 1997); the accumulation of products from linked genes appears to be light-dependent. This promoter might be useful for directing expression of traits like pest or herbicide resistance.
There are a number of different objectives being pursued using genetic engineering and some of these introductions have led to field trials. The USDA APHIS reported 17 applications for field trials of alfalfa up to 1997. These represented plants containing 8 different genes, including those for insect, fungal, viral and herbicide resistance, as well as novel traits. There were no APHIS-approved field tests in 1997. In 1998 there were four trials. The Noble Foundation conducted a field trial for a variety in which resveratrol synthase was altered to increase the levels of a secondary metabolite shown to be inhibitory to certain types of fungal pathogens. WL Research tested a variety in which levels of caffeate methyltransferase were altered to affect the synthesis of lignin, a complex polymer located in the cell wall. Monsanto produced a glyphosate-tolerant line, which was tested in Iowa, Idaho, Indiana, and Wisconsin. The University of Wisconsin tested a variety that was engineered for altered cell wall properties.
In Canada in 1998 there were 61 alfalfa release permits for confined field trials, representing 32 different genetically engineered alfalfa varieties. Field trials were focused on evaluating several genes relating to improvements in yield, winter survival (persistence) and feeding quality. More specifically four trials tested plants for abiotic stress tolerance, three for nutritional change, eleven for stress tolerance, two for cold tolerance, two for photosynthetic efficiency, one for herbicide tolerance and one for viral resistance (S. Charlton, Canadian Food Inspection Agency). Seven trials had no information about the introduced genes except for marker genes for antibiotic resistance or herbicide tolerance.
Insect resistance. A now standard method for engineering insect resistance in crop species is the use of the toxin genes from Bacillus thuringiensis; this is the basis for Bollgard cotton and the commercially available maize that is European corn borer-resistant. These toxins are activated by cleavage in the midgut of insects; however, making engineered toxins that were effective against Spodoptera armyworms was difficult. By conducting in vitro testing of synthetic toxins, a synthetic delta-endotoxin gene was found, which when expressed in optimum amounts in alfalfa, showed evidence of protection against the Egyptian cotton leafworm (Spodoptera littoralis) and the beet armyworm (Spodoptera exigua) (Strizhov et al., 1996). When insects were feed leaves from these plants in the laboratory, 100% mortality of Egyptian cotton leafworm larvae occurred in the majority of plants. With one construct, the majority of plants also produced 100% mortality of beet armyworm larvae. Field conditions were imitated as closely as possible in greenhouse experiments by putting 15-20 3rd to 4th instar larvae on each plant. After 6 days no viable insect escapes and barely detectable leaf damage were observed in the engineered plants. When engineered and nonengineered alfalfa plants were mixed, control plants were devastated; engineered plants were not damaged. To date, field testing of these varieties has not occurred.
In leguminous plants, such as alfalfa, products of the phenylpropanoid pathway of secondary metabolism are involved in interactions with beneficial microorganisms and in defense against pathogens. In addition, a polymer derivative, lignin, is a major structural component of certain vascular tissues and fibers in higher plants. With the isolation of genes for key enzymes in phenylpropanoid pathways, it might be possible to engineer alfalfa for: (a) improved forage digestibility (b) increased or broader-spectrum disease resistance and (c) enhanced nodulation efficiency. To date work on the isolation of genes encoding key enzymes of various natural plant resistance compounds, e.g. the phenylpropanoids, has led to a better understanding of the synthesis of antimicrobial flavanoid derivatives in alfalfa (Dixon et al., 1998). Work, such as this, can lead to approaches where increased or broader-spectrum disease resistance can result from modifying expression levels or timing of unmodified phytoalexin synthesis in alfalfa or modifying the phytoalexins themselves to create new phytoalexins with novel activities (Dixon et al. 1996).
Forage Genetics and Agri BioTech, Inc. (see Herbicide Tolerance section) agreed to develop, evaluate and release new varieties with resistance to leaf hopper. The precise manner in which this would be accomplished was not disclosed.
Viral resistance. Infection with alfalfa mosaic virus (AMV) is a problem in many parts of the country. A standard method for engineering viral resistance, the use of the coat protein gene, has been tested in tobacco plants engineered with the AMV coat protein gene under the control of a promoter that drives expression throughout the plant. These plants showed a significant delay in the onset of symptoms and a reduction in viral accumulation (Jayasena et al., 1997). That this approach works in tobacco makes it more likely that it will also work in alfalfa. It is necessary to develop alternative strategies that complement or replace the coat protein strategy in the future. To do this, it is necessary to understand the architecture of the virus itself since many of these approaches involve the use of viral genes. Much work has been done on characterizing alfalfa mosaic virus (van Rossum et al., 1997). The parts of the virus required for its replication and infectivity have been identified. This information will help in developing new generation approaches for the control of AMV infection.
Fungal resistance. Phytophthora root rot is one of the most significant diseases of alfalfa. Methods for protecting against fungal pathogens involve the identification of natural plant protection compounds that, when expressed in higher amounts in appropriate tissues of the host plant, can lessen fungal damage. Two such genes, an alfalfa acidic glucanase (Aglu1) and a rice basic chitinase (RCH10), have been introduced into alfalfa plants; these enzymes are believed to be involved in the degradation of fungal cell walls. Analysis of the resulting engineered plants indicates that symptom reduction occurred only in plants overexpressing Aglu1 when challenged with Phytophthora megasperma. This resistance was crossed from the transformable cultivar into a commercial alfalfa cultivar, Apollo, and resistance was correlated with the presence of the Aglu1 gene (Masoud et al., 1996). This protection is possibly due to indirect effects of the glucanase gene on phenylpropanoid metabolism (Dixon et al., 1996).
A group at the Nobel Foundation has engineered transgenic alfalfa carrying a peanut resveratrol synthase gene. These plant accumulate a range of levels of resveratrol glucoside, a novel antifungal compound. In laboratory tests the lines accumulating the highest levels of resveratrol show increased resistance to the alfalfa fungal pathogen, Phoma medicaginis. In the current field test, to be completed in October 1999, researchers will look for any negative effects of resveratrol accumulation on forage production. At this time, it is not known if increased resveratrol levels will translate to increased resistance in the field (N. Paiva, Nobel Foundation, personnel communication).
Nematode resistance. The mRNA levels for enzymes in the phenylpropanoid pathway were examined in nematode-resistant (Pratylenchus penetrans) and nematode-sensitive alfalfa (Baldridge et al, 1998). Before infection, levels were higher in resistant plants. After infection levels generally declined in resistant plants, but increased and then declined in sensitive plants; there was no effect on isoflavanoid levels. The roots of the two most resistant plants had the highest constitutive levels of the phytoalexin, medicarpin, which inhibits nematode mobility in vitro.
Although herbicides are generally not used with alfalfa, transgenic plants tolerant to herbicides and antibiotics have been generated. In some cases this is because these genes are used as the selectable markers for identifying transformed lines. In Belgium, Plant Genetic Systems developed Basta- or glufosinate (Liberty)-resistant alfalfa, which was field- tested and found to be unaffected by the herbicide.
Roundup tolerance in alfalfa is being developed by Monsanto with Forage Genetics (West Salem WI) as a partner. This tolerance is based on the expression in the plant of a modified target enzyme, EPSP synthase, that is insensitive to the herbicide. To date, such herbicide tolerance has been engineered into a wide variety of crop species. Tolerance to the herbicide in transgenic alfalfa appears to be excellent in both greenhouse and 1998 field tests. Expanded testing is planned for 1999 (M. McCaslin, Forage Genetics, personnel communication). Forage Genetics is an independent breeding company, a wholly owned subsidiary of Research Seeds (St. Joseph MO). Over the past few years Research Seeds has supplied varieties for many marketers of alfalfa, including Northrup King, Novartis, as well as smaller companies. Monsanto with Forage Genetics will develop lines with the Roundup Ready gene and make these lines available on a contract and non-exclusive basis. By this means, other key alfalfa breeding companies can cross the Roundup Ready gene into their own elite lines suitable for their customers' needs and geographic locations. Monsanto expects the Roundup Ready lines to be available in 2000 (J. Zarndt, Monsanto, personnel communication). On January 15, it was announced that Agri BioTech has agreed to market Forage Genetics, Inc.'s Roundup Ready alfalfa products, which are currently in development.
In Australia in 1997, an advisory committee, which regulates the release of transgenic organisms, decided that the herbicide-resistance traits are undesirable in Australia because of the potential for outcrossing with other pasture species.
Nitrogen Fixation. To manipulate complex pathways, such as those involved in nitrogen fixation, it is necessary to have a detailed understanding of the pathway and the process of nodulation by the symbiotic microorganism. The many genes involved in early nodulation in alfalfa are being identified and characterized; understanding this process will provide the tools necessary for its manipulation.
Regulatory elements that control some of the early nodulation genes have been fused to reporter genes. One of the regulatory regions when linked gene to a colored marker gene leads to expression almost exclusively under symbiotic conditions (Fang and Hirsch, 1998). This regulatory region might be useful for directing expression of genes involved in nitrogen fixation. Another promoter only functions in root nodules that have a growing point or meristem; this promoter may provide a molecular tool for analyzing the important step of establishing these growing points during nodule development (Bauer et al., 1997).
A polygalacturonase gene, isolated from alfalfa, is believed to facilitate cell wall rearrangements, penetration of bacteria through the root hair wall or infection thread formation and release of bacteria into the plant cell, which occurs during the early steps of the Rhizobium-legume interaction (Munoz et al., 1998). Information such as this will be important to the development of long-term strategies for manipulating the plant-symbiont relationship. The isolation of key enzymes in the phenylpropanoid pathway may provide a means for enhanced nodulation efficiency by engineering over-production of flavonoid compounds that induce nodulation genes (Dixon et al. 1996).
Basic questions relating to the biochemistry of nitrogen fixation are also being addressed in alfalfa using molecular approaches. Glutamine synthetase (GS) catalyzes the condensation of ammonia with glutamate to produce glutamine and this gene has been isolated (Vance et al., 1995) and used to reduce the levels of this enzyme in alfalfa and, although the plants had only 20% the levels of mRNA level of control plants, there was no reduction in GS activity (Temple et al., 1998). This means that modification of this pathway will not be a simple matter. Regulatory regions have been identified that control expression of genes in the infected zone of nodules (Pathirana et al., 1997) and in nodules and the nodule symbiotic zone (Shi et al., 1997). A promoter for a soybean gene that is expressed in root and root nodules was studied in engineered alfalfa and found to lead to expression of genes in anthers, theca and pollen at a late stage of flower development and in roots (Carrayol et al., 1997).
Tolerance to Environmental Stress. In northern climates alfalfa is very sensitive to winter injury. Winter-hardiness is a composite of tolerances to freezing, desiccation, ice-encasement, flooding and disease, all of which result in the formation of activated forms of oxygen that ultimately result in various types of injuries to membranes. These similarities led to the hypothesis that winter injury might be reduced in crop plants if their tolerance to oxidative stress were increased. Towards that objective, alfalfa plants were engineered to overexpress either both the Mn- and Fe-forms of superoxide dismutase, enzymes that eliminate oxidative radicals. In two sets of field trials of plants overexpressing these enzymes, the plants on average had a 25% higher survival rate over non-engineered controls after one winter (McKersie et al., 1997). Alfalfa plants overexpressing the Mn-superoxide dismutase also had reduced injury from water-deficit stress. A 3-year field trial with these plants indicated that yield and survival of transgenic plants were significantly increased (McKersie et al., 1996). Analysis of the results of field trials of these new engineered alfalfa varieties with enhanced stress tolerance showed that the engineered plants had a 20% yield advantage over control, nonengineered varieties (D. McKersie, University of Guelph, Annual Meeting, Ontario Forage Council, December 1998) . In the experiments assessing cold tolerance, the tests were performed using variety RA3, which is not a good agronomically performing alfalfa variety. This was the only genotype in which striking differences could be seen between control and engineered varieties; the levels of protection were not as great as some commercial varieties already have. Further experimentation is needed to determine whether the engineered trait can augment natural tolerance.
Salt tolerance in alfalfa is being investigated through several avenues. One such avenue includes single-step selection of salt-tolerant cells in culture, followed by regeneration of salt-tolerant plants and identification of genes important in conferring salt tolerance (Winicov and Bastola, 1997). These genes might be a subset that encode functions that are particularly vulnerable under conditions of salt-stress. One such gene, Alfin1 is expressed predominantly in the roots; the promoter driving this gene leads to expression that is root-specific and salt-inducible (Bastola et al. 1998). It appears to have some specificity for certain DNA sequences and might be important in gene regulation in roots in response to salt. This gene may also be an important marker for breeders that will allow them to manipulate salt tolerance by classical breeding .
Aside from their potential use for engineering disease resistance, the isolation of key enzymes in the phenylpropanoid pathway may also provide means for improved forage digestibility through modification of lignin composition and/or content (Dixon et al. 1996).
It has been shown that rumen supplements of sulfur-rich proteins can increase wool growth rates in sheep. At the CSIRO research facility in Australia, a gene encoding a sulfur-rich protein from sunflower was introduced into alfalfa to improve its quality (Tabe et al., 1995). Although the amount of the sunflower protein that accumulated was below that believed to be needed to increase wool growth rate in sheep, this work is the first effort attempted to achieve this important practical objective.
Value-added Products. Alfalfa, engineered with a fungal gene encoding the enzyme phytase, was designed to eliminate the need for phosphorus supplements in swine and poultry food (McGraw, LC, 1998). This inclusion could result in reduced excretion of phytate from animals. This is important because phosphorus-rich manure is becoming an increasingly significant environmental problem; it promotes the growth of algae in lakes and streams that use up the oxygen needed for fish populations. Transgenic lines performed well in the field with no yield reductions. In poultry feeding trials better results were obtained using the engineered plant materials than feed supplemented with commercially produced phytase. If this is reproduced in further testing, phosphorus supplements could be eliminated from poultry feed and this could reduce costs and mitigate the problem of phosphorus pollution.
"Pharming". The potential also exists to use alfalfa as a bioreactor or "pharming" crop to make products of high value through the introduction of novel genes (Austin and Bingham, 1996). The characteristics which make alfalfa attractive for this purpose, are the fact that it is a perennial, fixes nitrogen, conserves soil, has relatively few pests and produces large quantities of biomass. Also technologies exist for the collection of protein juices from alfalfa, thereby facilitating purification of value-added products, and for the marketing the byproducts as feed and food supplements (D. Putnam, personal communication). Calculations based on the worth of both the value-added product and the by-products suggest that this type of alfalfa would be at least eight times more valuable than alfalfa hay.
Numerous novel proteins have been introduced into alfalfa, such as the human b-interferon gene (Smolenskaja et al., 1994), the light and heavy chains of human antibodies, a-amylase to be used in starch-processing and lignin peroxidase to be used in biopulping and bioleaching. Despite this promise, yields to date have been low and there were possible deleterious effects of the transgenic products on the host plants. These hurdles must be overcome before such an approach can be considered practical.
A collaborative project involving DOE, USDA-ARS, the University of Minnesota and the Minnesota Valley Alfalfa Producers is aimed at using alfalfa biomass to generate electricity, coupled with the use of alfalfa as a bioreactor. Stems will be used to produce electricity, leaf meal as a high-quality animal feed, protein juice for production of novel products, and growth of alfalfa for refurbishing the soil. For economic profitability, alfalfa grown for electricity production would be harvested only twice a year and alfalfa grown for these protracted periods tends to be tall, have fewer lower leaves and have decreased feed-value due to a decrease in crude protein content after flowering. To minimize these problems, a gene that causes reduced apical dominance (isopentenyl transferase), which causes more branching, later flowering and longer leaf-hold, was introduced into alfalfa. Characteristics of these engineered alfalfa lines are currently being determined (D. Samac and C. Vance, personal communication).
Using plants to remove contaminants from soil and water, a process, termed phytoremediation, is currently being pursued as an environmental strategy. Researchers are looking at engineered alfalfa for the potential to phytoremediate or remove polynuclear aromatic hydrocarbons (PAHs) from the soil at former manufactured gas plants (Pradhan et al., 1998). Using nonengineered alfalfa and switch grass (Panicum virgatum) for the primary treatment of these contaminated soils, a 57% reduction in the total PAH concentration was observed after 6 months of treatment. Final polishing of that soil using alfalfa further reduced the total PAH level by another 15%. After an understanding of this process is developed, engineering of alfalfa should improve the efficiency and economics of this process.
Work has been also initiated to engineer alfalfa to degrade atrazine, a contaminant from agricultural run-off frequently found in groundwater in the United States. A bacterial gene responsible for atrazine degradation is being introduced into alfalfa with the hope that its expression in alfalfa roots will lead to detoxification of atrazine in the groundwater (C. Vance, personal communication).
Technologies exist to introduce genes into alfalfa via new genetic engineering technologies, although all commercially important varieties cannot be engineered. Significant progress has been made in the areas of pest and herbicide resistance, the changing of agronomic properties and the use of alfalfa as a source for value-added products. There are many alfalfa traits that can be considered for possible improvement through genetic engineering technologies, including other disease resistance traits and stress tolerances and altered forage quality.
The ability to make the precise changes through biotechnology requires a detailed knowledge of the organisms and the biochemical pathways involved in the traits of interest. Therefore, basic research is being conducted that will form the basis for the use of engineering strategies to address some specific problems, such as those involving nitrogen fixation.
One potential obstacle to the successful application of the new genetic technologies to alfalfa stems from the fact that, unlike corn, potato or soybean, it is not possible to retain high-value proprietary seed and therefore commercial investment will be limited. Development of improved lines by this sector will command higher prices for the value-added trait; public sector efforts will be hampered by their lack of freedom to operate in this technology arena. Another large obstacle to alfalfa improvement via genetic engineering lies in its inherent genetic properties. Alfalfa is an autotetraploid, which makes it difficult to construct lines that are missing a gene or which are homozygous for a gene (C. Vance, personal communication); introgressing genes from one line into another is also problematic.
In summary, the promise of genetic engineering technologies are apparent, but there are some significant challenges that must be overcome before it can reach its potential for making significant contributions to the improvement of alfalfa.
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