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AIR1-CT92-0349
Inter-Disciplinary Research for Poplar Improvement (IRPI) |
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Proposal No: | AIR1-CT92-0349 |
| Date Prepared: | September 1999 | |
| Source: | Final summary report 1996 |
Introduction
The project aimed at the genetic improvement of fast growing poplars, having particular regard to resistance to pests and diseases. Several approaches were considered in five tasks.
Task I: Genetic transformation The objective was to introduce foreign DNA into commercially desirable poplar stock by means of Agrobacterium tumefaciens as gene vector, a process that had already proved viable for several poplar species. Genes from Bacillus thuringiensis and proteinase inhibitors would confer resistance to insect pests; sequences derived from Poplar Mosaic Virus would slow down viral replication in transformed poplars.
Task 2: Genetics of resistance and virulence Stable resistance or tolerance to diseases in long-living crops is supposed to be achieved by multiple genes with distinct mechanisms of action. The genetic components of resistance to economically important pathogens were analized, as well as the reliability of in vivo and in vitro tests. Molecular and biochemical markers were studied in relation to resistance to leaf and bark diseases and insect pests as tools for clonal selection and breeding work.
Task 3: Improvement of breeding tools Poplar breeding is largely based on inter-specific hybridization, which is often limited by sexual incompatibility; the project addressed both pre- fertilization barriers and post-zygotic causes of embryo abortion: mentor pollen, in vitro pollination, and embryo rescue were investigated. The task included also the development of techniques for long-term storage of germplasm, cryo-preservation of pollen and seed; maintenance of in vitro collections with minimal requirements for periodical subculturing.
Task 4: Pathogen variability and evolution Variability in pathogenicity is known to exist for bacterial canker and rusts and suspected for other relevant pathogens. The project explored the genetic variability of pathogens as a necessary step for understanding their potential evolution ability and for developing breeding strategies for stable resistance. Isolates from different geographic areas, and in some cases, strains obtained by recombination in confined environment were studied: differences of aggressivity towards common sets of poplar genotypes were investigated,with the use of molecular techniques.
Task 5: Clone and environment interactions A network of clonal trials already available in quite distinct environmental conditions in four countries helped determine the main limiting environmental factors, as well as the clonal response to them. Data concerning annual growth and phytosanitary status vis a vis of climatic and pedological factors was fed into a GIS-compatible database and will orientate the future choice of clones and sites for commercial poplar culture.
Discussion of Results
Genetic transformation and evaluation of transgenic material In the few poplars clones that were investigated so far, there were a handful which contain protease inhibitors or other chemicals with potential value against insects. However, there are as yet too few data to justify serious enthusiasm about any of the current anti-insect strategies in commercially useful poplar backgrounds. In parallel, the genetic modification of poplar metabolism has been assessed and flavonoid biosynthesis was one focus - at least in part because of the possible spin-off in terms of pathogen resistance.
The same approaches are being addressed in other crops but the reduction of lignin biosynthesis (linked to the flavonoid pathway) potentially has special benefits for the environment. It is hoped that diminishing lignin may tend to lessen pollution from pulping plants based on poplar (which is in any event a relatively white fibre crop that does not require much chemical bleaching). The principle objective was expected to possibly have unexpected side effects in terms of pathogenesis. This component of the work has hardly been addressed in the time available within the project.
A substantial effort was devoted to transformation of hybrid poplars of current or potential economic value in member states. Despite the multinational effort, the most promising results have been obtained with only one commercial cultivar of Populus nigra of limited economic value. Although the use of such transgenic genotypes is hoped to provide benefits (in terms of diminished pesticide usage) there are a number of substantial and unresolved issues concerning the unanticipated impact on components of the natural environment. In the context of trees, there are uncertainties about the duration of benefit and potential problems of interaction which are reflections of the durability of transgenic product expression.
Having regard to the imperatives of human population growth, there is no consensus about the most desirable environment, the consequential needs for adequate food production or the balance with aesthetics/recreation, safety and "quality of life". No clear durable benefit from laboratory-based genetic manipulation of any plant has yet been demonstrated and, despite publicity which implies a contrary view, the benefits of biotechnology in field crops remain unproven. There is a less obvious but nevertheless important need to recognise and respond to the concerns which are expressed, particularly by non-governmental organisations, in some parts of the European Union. Against this background, it may be prudent to proceed very slowly and with adequate consultation.
Genetics of resistance and virulence: molecular, biochemical and conventional approaches The close linkage between a RAPD marker OPM03/04.480 and the locus of qualitative resistance to Melampsora larici-populina in Populus deltoides can now be used to screen, potential P. deltoides parents for breeding purposes. This fragement has been cloned and sequenced and can now be used as a hybridization probe. Genome mapping can be and will be employed as the basis for the location of genes related to other qualitative as well as quantitative traits and will constitute the reference for marker-oriented selection for breeding institutions in Europe. Results on molecular systematics have demonstrated among other things, that P. nigra is not correctly classified with P. deltoides, P. fremontii and P.wislizeni in the Aigeiros section. This information can be used to orientate intersectional hybridization programmes which are among the most successful of breeding activities. Moreover the markers used revealed intraspecific polymprphism that could be used in breeding programmes based on Marker Aided Selection.
The efficiency of a breeding strategy for resistance to diseases can be improved, in terms of durability, by using a suitable methodology for the breakdown of resistance into its components, which offers the possibility to characterize the quality of the resistance which is selected for. During this project, a breakdown of resistance to rust was proposed, and discussed in terms of genetic efficiency as well as of practical feasibility. This new approach to the problem, based on old techniques but clearly prone to dramatic synergies with molecular techniques, should help the breeders in facing the rapid evolution of poplar parasites.
The main immedite application will be the adaptation of current breeding strategies (breeding populations and selection procedures) for the selection of durable resistance. Significant progress was also achieved in the techniques for the study of the genetics of pathogenicity genes in the fungus The inoculation of alternate hosts in the laboratory in order to obtain new rust genotypes in a confined environment is now available, thus enabling the pathologists to test new virulences in the laboratory before they are expressed as the result of natural genetic recombination in the field.
The work done under the contract has permitted the isolation, the-identification and the quantification of phenolic glycosides in poplar leaves. Some of them have been shown to positively affect the chrysomelid beetle C. tremulae and others to exert a negative influence. Results can be applied by breeders to select clones rich in antifeedant phenolic glycosides and poor in phagostimulant phenolic glycosides. The relationship between the resistance behaviour of Populus nigra to Phloemyzus passerinii and the amount of two phenolic compounds (4-hydroxybenzoic acid and 4-hydroxy-3-methoxybenzoic acid) in the bark paves the way for the setup of a susceptibility test based on HPLC analysis of bark contents.
As regard the fungus Discosporium populeum, although no relationship was established between the susceptibility to this organism and the composition of the phenolic compounds within P. nigra a and high variability was found for D. populeum and Melampsora susceptibility, no genetic correlation between the two susceptibilities was observed. However, it was proved that rust is a predisposing factor that enhances the damages due to D. populeum. This shows that breeders will have to separately select for the resistance to both parasites, in absence of one another.
Development of new tools for the improvement of breeding procedures One objective of this study was the improvement of breeding procedures in order to remove the incompatibility barriers in interspecific crosses. Considering the large economic interest of Euro-american clones in Europe, particular attention was devoted to the incompatible cross between P.nigra females and P.deltoides males. Embryo culture has been optimized in various intra and interspecific combinations, Crosses P.nigra females X P.deltoides have been tested through the combination of mentor techniques and embryo culture. Numerous seedlings have been obtained but they proved not to be hybrids although P. nigra pollen has participated in the fertilization process, confirmed by the observation of the growth of pollen tubes in the female tissues by a fluorescent dye. The alternative procedure (removal of the stylar barriers and in vitro fertilization) seems to be more promising but are still in need of refinements.
The results of the tests for the conservation of germplasm showed that long-stored pollen and seed give good results in vivo fertilisation if stored with a low moisture content. From a practical point of view this represents an advantage in planning the crosses in a breeding programme and also for the conservation of genetic resources. Seed and pollen storage represents the most convenient way to maintain a tremendous genetic diversity thanks to the possibility of keeping a large number of different genotypes in a very small space (a freezer).
Results of experiments on the long-term in vitro preservation of shoots indicate that it is possible to maintain poplar shoots in vitro at low temperature for about 18 months without damage. In this way it is possible to reduce the frequency of subculturing and to maintain in culture a larger number of genotypes of particular interest (specific genotypes for genetic transformation, germplasm conservation of a limited number of genotypes and so on).
Pathogen variability and evolution Generic and specific markers were reqired to rapidly name new xanthomonads isolated from poplar lesions. PCR-RFLP methods were found efficient to delineate related bacterial species. At specific level, a clear discrimination of X. campestris pv populi and X. populi was obtained with X. populi appearing as a particular lineage clearly separated from other xanthomonads. However, X. campestris pv populi could not be distinguished from other xanthomonads, because X. campestris pv populi belongs to a complex genomic species that includes several other pathovars.
Intraspecific markers were required for epidemiological purposes in defining whether xanthomonads isolated from different foci of the disease belong to the same clone (or clonal lineage) or not, and thus to provide essential information on disease spreading. Multiple neutral loci were investigated, including serological, biochemical and molecular (AFLP) markers. Results showed that X. campestris is very contagious, whereas X. populi is not. Phylogenetic studies within the species X. populi suggest the involvement of mobile element of the genome such as plasmids or insertion sequences, or instability of a genetic character known to be monogenic in numerous instances. From an epidemiological point of view, the data obtained in this study are largely unforeseen, and give a new conception of the epidemiology of poplar diseases caused by xanthomonads.
The aggressiveness of ten German isolates of X. populi was compared to selected Belgian, French, and British isolates. The tested clones exhibited significant differences in susceptibility to the German isolates, confirming host-pathogen interactions. Several German isolates were found to be at least as aggressive as the race-4 isolates from the UK and Beigium. In order to control bacterial canker on poplars in Europe and other countries, future breeding programmes for disease resistance need to incorporate X populi isolates classified as race-4 isolates. Moreover, a continuous survey of the aggressiveness of the natural X popiili population is also necessary. Improved efficiency in screening a large number of poplar clones for high resistance to X. populi can be obtained by using plantlets which are in vitro propagated and in vitro artificially infected with the bacterium. Since in vitro inoculated plantlets are more sensitive to bacterial canker than greenhouse plants, trials can be evaluated in a shorter period of time. The analysis of pathogenicity genes on the molecular level promises to yield new insights into the various phases of disease development. Therefore, the aim for future research should focus on the identification and isolation of virulence genes from X populi. The role of the gene products in the plant-bacterium interaction should be elucidated in further studies.
A method was developed to extract and amplify (PCR) DNA from two rust species: M. allii-poptilina and M. larici-populina, which produced a clear distinction for RFLP patterns between the two species. No difference was found between physiological races of a given species. It has also been proved that race populations of M. larici-populina in differently located nurseries are very distinct and that some recent, but dangerous virulences, may be present at very low frequencies. As a consequence, field tests for resistance may declare some clones resistant in one location while they may -be badly infected in another one. This knowledge will lead in the future to a more accurate strategy for testing resistance of poplar clones in the EU.
While looking for the new race E4 in different.locations, it has been possible to develop a new laboratory method which allows an easy detection even when such race is infrequent. Consequently, it, becomes possible to detect very early unknown vi'ruiences, well before they induce evident field infection. Those new virulences show clearly that there is an urgent need to increase knowledge on the available cultivars in Europe and that breeders must select now for long-term resistance.
Interaction of poplar clones with site and climatic factors The database of 97 existing poplar trials, built up with ORACLE and structured as a space-related database in a GIS under ArcInfo, was shared by all the interested partners with the intention to enrich it with new information as soon as this becomes available. In due course it can be progressively extended and form the basis of an expert system guiding the choice of appropriate sites for poplar production, the choice of clones to be cultivated and decisions about cultural models. The analysis carried out using multiple regression tools showed that, at least for the most represented clones that are widely in commercial stands, water supply appears to be the most important factor affecting growth over a wide range of site characteristics (climate and soil). Such study proved that a multisite analysis of growth performance in a wide range of ecological conditions can help:
Local tests on a clone by clone basis may still be necessary before the commercial use of a new clone in a new site, but at least broad classes of potential performance of poplars can now be established. The 34 experimental trials established in different zones of Great Britain, Belgium, France, Luxembourg and Italy with common clones and experimental protocols will produce important information on clones that have recently entered or are about to enter the European market. The partners have agreed to continue cooperation after the contract ended in order to share and to publish the results at a later date. Farmers and Agricultural advisors will have at their disposal first hand information on newly released clones at the time of their appearance for sale.
Conclusions
Gene transfer and molecular markers appear to be among the most promising tools for geneticists and breeders,and potential applications are being conceived and discovered continuously as scientific insight is obtained and new techniques are developed. Marker-assisted selection in its broadest sense can boost poplar breeding at an unprecedented level. Therefore all the research studies leading to the drawing or saturation of molecular maps, their extension and testing from pedigrees to outcrossiiig populations, the location of qualitative and quantitative traits of agronomic interest are all activities that deserve the highest attention. However, as all these methods have a reason to exist in support of poplar breeding programmes, a simultaneous approach of traditional breeding and genetic analysis (e.g. breakdown of resistance into components) and modern DNA technologies (for a genetic dissection) will be the best strategy for breeding in a clonal forestry system as used for poplars.
Less "glamorous" activities, such as conventional breeding, based on genetically diverse populations and on long-term plans will survive and must be provided with the resources to survive. The undesirable consequence of over-emphasizing the role of gene technologies and molecular approaches at the expense of breeding activities would be that advanced technologies, developed as supporting tools for breeding, would have no breeding programme to effectively support.
© Copyright 2006 Policy Statements
Updated
by CPL Press:
03/07/2007
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