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Mapping of forage grasses using molecular markers is occurring for several different species; however, mapping of forage grasses lags behind that of many other crop plants. It is important that mapping occur for forage grasses as it is expected to provide insights into the organization and evolution of plant genomes. This is important for forage grasses because most o the species are highly polyploid. Molecular maps can provide information on assaying genetic variation within a species. In addition, important genes for desirable traits can be localize by linkage analysis, cloned on the basis of a linkage map, and transformed to produce improved cultivars. It is also hoped that well developed linkage maps can identify useful quantitative trait loci that could be used in marker-assisted selection for forage grass improvement.
Alfalfa (Medicago sativa L.) is an allogamous tetraploid with polysomic inheritance, and not the simplest system for applying molecular marker analyses; however, many advances have been made recently that provide insight into the genetics and breeding of alfalfa. Complete linkage maps of molecular markers have been developed for diploid populations, thereby avoiding the complications of tetraploid inheritance. The high levels of segregation distortion observed for inbred mapping populations can be greatly reduced by using non-inbred F1 populations. Mapping data for tetraploids also has been obtained recently, and this will be important for understanding the genetic control and improving the selection of quantitative traits in populations used for cultivar development. Analysis of meiotic mutants with molecular markers has permitted mapping of centromeres and determining the mode of 2n gamete formation. Molecular markers also have provided information on inbreeding behavior and heterosis in alfalfa, and they may even prove useful as a tool for selecting genotypes that combine well in synthetic cultivars derived from small numbers of parents.
Lack of winterhardiness reduces the dependability of alfalfa (Medicago sativa L.) in cold climates. Cold tolerance is the most important factor that determines winterhardiness potential in alfalfa. A better understanding of the physiological and molecular bases of low temperature adaptation will lead to the development of new breeding strategies for the significant improvement of cold tolerance in alfalfa germplasms of high agronomic value. Changes occurring in gene expression and carbohydrate composition in crowns of cold-acclimated plants have been investigated in search of determinant traits. Electrophoretic analysis of in vitro translation products revealed extensive changes in gene expression that are very similar among cultivars of contrasting winterhardiness; however, a small number of these changes appears to be closely related to hardiness potential in alfalfa. Genes that are cold-regulated (COR) in alfalfa have been isolated and characterized. Some of these genes show a marked differential expression between cultivars of contrasting winterhardiness. Levels of galactose-containing oligosaccharides, stachyose and raffinose, were found to be better related to freezing tolerance than those of the known cryoprotectant sucrose. These results support the hypothesis that even though cold acclimation of alfalfa is a complex trait under the control of a large number of genes, differences in cold tolerance between cultivars of contrasting hardiness might be related to the differential expression of a more limited number of these genes. The potential use of COR genes and soluble sugars as molecular markers for the improvement of cold tolerance in alfalfa is currently being tested by divergent selections based on the expression of these traits within populations.
Genetic transformation has now been reported in numerous crops and bioengineered products are coming into the marketplace. Although this technology has not progressed as rapidly for forage and turfgrasses as it has for certain major cash crops, transgenic plants have now been obtained in several species. These include orchardgrass (Dactylis glomerata L.), tall fescue (Festuca arundinacea Schreb.), red fescue (Festuca rubra L.), meadow fescue (Festuca pratensis Huds.) perennial ryegrass (Lolium perenne L.), creeping bentgrass (Agrostis palustris Huds.), and redtop (Agrostis alba L.). Successful gene transfer has been accomplished by direct uptake of DNA by protoplasts and by bombardment of cells or tissues with DNA coated microprojectiles. In both cases, the transfer is followed by whole plant regeneration. Much of the work has focused on developing and improving protocols for the transformation and have used the reporter gene uidA coding for -glucuronidase (GUS) and the selectable marker bar that confers tolerance to phosphinothricin-based herbicides. Proof of the transformation has been provided by polymerase chain reaction (PCR) techniques, northern hybridization analysis of transcribed RNA, western blot analysis of soluble protein (gene product), and southern blot hybridization of total genomic DNA. Because forage grasses, in general, are not highly domesticated, possess weedy characteristics, and are highly outcrossing, special difficulties may be encountered in the ultimate release of transgenic plants.
Our long-term goal is to improve persistence and yield of alfalfa (Medicago sativa L.) and other forage legumes by identifying and manipulating genes that affect these traits. Future improvements by genetic manipulation depend, however, on new insights into basic physiological and biochemical plant processes. Currently we lack knowledge of discrete traits controlling agronomic performance that can serve as targets for manipulation using modern genetic techniques. Our work, and recent work of others, has failed to show a positive association between root total nonstructural carbohydrate (TNC) levels and genetic variation in regrowth and winterhardiness of forage legumes. We are exploring alternatives to the conventional thinking that root TNC reserves control alfalfa regrowth and persistence. Recent results indicate that root N declines during herbage regrowth after defoliation, and again in spring when shoot growth resumes. Labeling studies have proven that much of the N found in shoots during early regrowth is derived from root N pools. In alfalfa, certain root N pools, especially root vegetative storage proteins (VSPs) are preferentially used as N reserves during the early stages of shoot regrowth. The VSPs represent 25% of the root protein pool. They are unique to alfalfa roots, and their synthesis is developmentally regulated. Work is underway to isolate and characterize the cDNAs for the VSPs to learn more about regulation of VSP synthesis and degradation in alfalfa roots.
Medical research has elucidated a detailed understanding of immunology to where scientific applications are possible in plant sciences. Plant immunology is a relatively recent research discipline, but it has potential to provide powerful tools for diagnostics purposes as well as opportunities for direct improvement of plants via molecular techniques. The purpose of this chapter is to provide a rudimentary understanding of immune function, provide examples of uses of plant immunology in basic and applied research, and describe how plant immunology might be used for forage improvement. Plant immunoassays are based upon polyclonal or monoclonal antibodies, each having defined uses depending upon requirements for specificity of the test being conducted. Monoclonal antibodies are more specific because they are products of individual cell lines with antibody specificity to one epitope of the antigen. Hence, if low molecular weight compounds, such as toxins, are conjugated to immunogenic proteins, it is possible to isolate monoclonal cell lines that produce antibodies to the compound of interest. Monoclonal antibodies can be generated to antigens of specific disease organisms, and the genes responsible for antibody production isolated and inserted into plants to develop novel sources of disease resistance. Similar technology may be useful for providing oral immunizations for livestock grazing forages, thus creating value added forages.
Endophytic fungi of Neotyphodium sp. occur naturally in Festuca sp. and Lolium sp., which are grasses widely managed for forage and turf uses. The endophytes typically produce alkaloids that reduce growth and reproduction in grazing livestock while conferring resistance in the grass hosts to insects, nematodes, and drought stress. Novel endophyte technology involves selecting strains of fungus that do not produce livestock toxins and then introducing them into agronomically desirable host populations to form new compatible associations. The steps involved are to (i) acquire Festuca and Lolium germplasm from diverse sources and screen for endophyte-containing plants, (ii) select endophyte-infected plants via chomatographic analysis that lack ergot-related alkaloids (livestock toxins), but which contain the insect-deterring alkaloids peramine and lolines, (iii) screen those plants for insect resistance using bioassays, (iv) isolate the most promising endophyte strains from the endemic hosts and transfer to improved cultivars via seedling-stab inoculation, (v) grow out the newly infected plants in the field for seed production and select those strains which show a high compatibility and seed transfer with the cultivar, and (vi) increase seed containing the most desirable strains in large enough amounts to establishing livestock grazing trials. Essentially 100% seed transmission of endophyte is a key criterion for full compatibility and to ensure that novel endophytes have commercial viability. A regenerated cultivar containing the new endophyte must be retested for the desirable alkaloid profile. Animal performance and plant persistence trials are critical to demonstrating the success of the novel endophyte in improving a cultivar.
Somatic embryogenesis is fundamental to the genetic improvement of forage legumes for plant transformation and for propagation by synthetic seeds. The trait is sexually inherited in alfalfa as two independent, complementary, dominant nuclear genes. Populations that have different genetic backgrounds, that contain a high proportion of embryogenic individuals and that have good field performance have been created. To evaluate the potential of transformation technology to improve winter survival in alfalfa, we created transgenic alfalfa plants expressing a cDNA for a Mn-superoxide dismutase (SOD). In replicated field plots, all SOD transgenic plants had much higher survival after two winters than the non-transgenic genotype. Releasing these or other transgenic alfalfa plants as commercial varieties requires special consideration because of autotetraploid inheritance and in-breeding depression during seed production. Several alternative seed production methods are described that might produce transgenic varieties from a few transgenic parents. One model is to backcross the transgenic plant into different populations and then identify new parents for the synthetic variety from those populations. Synthetic seeds might be used to produce a double-cross hybrid or a reduced generation synthetic. Alternatively, one or two transgenic plants might be included as parents in conventional synthetic varieties and herbicide resistance used to select those progeny containing the desired transgenes. This last strategy requires the least laboratory labor and over 80% of the forage production plants probably might contain the transgene.