AIR3-CT94-1941 Applications of Biotechnology for Reduced Inputs in Disease Protection and Nitrogen Fertilisation of Rapeseed Cultivation

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AIR Cluster X - Inputs for Non-Food Crops : Biotechnology : Crops for Bulk Chemicals : Integrated Crop Protection & Biological Control : Plant Genetics : Vegetable Oil/Fat

	Proposal No:	AIR3-CT94-1941
	Date Prepared:	September 1999
	Source:	Final Summary June 1998

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Final Summary June 1998

Summary

This Concerted Action assembled five partners from three European states in which rapeseed is an important crop, for close co-operation leading to the application of biotechnology for reducing inputs in disease protection and nitrogen fertilization of rapeseed cultivation. The aim of the present project was to contribute to the improvement of ecological compatibility and to the reduction of production costs of rapeseed cultivation by increasing the nitrogen-uptake and nitrogen-use efficiency, as well as by improving the genetic mechanisms of disease resistance. Within the project, national research activities were performed in each of the participating countries and co-ordinated on a European level. Five different, important research areas of biotechnological applications in rapeseed were identified. From these, two major themes emerged, namely disease resistance (mainly against Phoma lingam) and nitrogen efficiency.

Objectives

The objectives of the project were:

• to increase the ecological compatibility and to reduce the production costs of rapeseed (Brassica napus) by improving nitrogen efficiency and genetic mechanisms of disease resistance
• to co-ordinate the research activities at a European level and to exchange experiences, materials and results obtained in order to guarantee fast progress
• to avoid duplication of work and to use resources efficiently
• to create a research network between partnering institutes in different European countries, working in leading positions on the biotechnological improvement of the rapeseed crop, that should last beyond the duration of the present project
• to identify bottlenecks in current research approaches and to outline future research priorities
• to establish and consolidate co-operation with private plant breeding companies, to guarantee immediate transfer of the results into practical applications

Background

During the last two decades rapeseed has become one of the most important crops in Europe. The rapid development of this crop originated in part from the dramatic breeding progress that took place in the 1970s, mainly in Germany, France and Sweden. The subsequent production by private breeding companies of cultivars low in erucic acid and glucosinolates opened the way for high-value uses of the oil for nutritional purposes and of the meal for animal feeds. During the last few years these three countries - in which rapeseed is extensively grown - have carried out further breeding, such that biotechnological research results may now be readily transferred into economic applications. These are expected to contribute to lowering inputs in rapeseed production for nutritional and non-food uses, as well as for economical and ecological benefits.

In Europe a major concern of rapeseed cultivation is the various diseases of rapeseed that make cost-intensive fungicide applications necessary. Among these, the biggest losses are caused by Phoma lingam, Verticillum dahliae and Altenaria brassicola. These diseases are difficult to control by fungicide-treatment and traditional breeding, but there are good chances to improve plant resistance with the use of biotechnological methods. For Phoma - the most severe fungal rapeseed disease - approaches were started within the present project to find closely-linked molecular markers and ultimately to isolate plant-resistance genes from related cruciferous species. Another major concern are the alleged severe environmental disadvantages rapeseed is said ti have over other crops. High amounts of nitrogen fertilizer are necessary to obtain high grain-yields. Since about only half of the applied N-fertiliser is removed with the harvested seed, the rest of the nitrogen incorporated in the leaves, straw and root matter remain in the field. From this, nitrate is produced rapidly by nitrification processes and may leach out into the groundwater during late fall. Several genes of the nitrate-assimilation pathway have been cloned from model species but also from rapeseed. Transgenic plants over- or under-expressing distinct enzyme activities may help elucidate nitrogen assimilation in rapeseed and support the breeding of cultivars with an improved nitrogen-uptake and nitrogen-use efficiency. However, breeding for improved N-efficiency has not yet received sufficient attention by plant breeding companies and public authorities.

Several gene constructs are available with genes of potential agronomical importance to rapeseed. But, compared to species such as Arabidopsis and Nicotiana, transformation in rapeseed is still rather inefficient. The development of more-efficient transformation protocols and the use of haploid cells could speed up the process for obtaining homozygous transgenic plants that can be propagated via seeds without segregation. Furthermore, considering the increasing public awareness of ecological risks, transformation methods are increasingly sought that allow an integration of "genes of interest" at different positions than the "marker gene" - required for technical means of selection only - in order to facilitate the subsequent elimination of the marker in the segregating offspring. The aim of the present project was to contribute to the above-mentioned areas, by co-ordinating research work that has been started independently in the laboratories of the partners and by discussing different research strategies.

Project work

Within the project, five different, important research areas of biotechnological applications in rapeseed were identified. From these, two major groups have emerged:

• disease resistance (mainly against Phoma lingam)
• nitrogen efficiency

Blackleg, due to Leptosphaeria maculans, is the most severe fungal disease in rapeseed. The aim of project work was to introduce the Brassica B genome-resistance into oilseed rape and to develop closely-linked molecular markers for easy selection in segregating breeding material. Further project work was directed to the development of an efficient co-transformation protocol for rapeseed using Agrobacterium rhizogenes for the generation of transgenic plants free of selection markers. Breeding for increased nitrogen efficiency in winter rapeseed is gaining increased attention for several reasons. The work of the present project was directed towards getting new information about the metabolic control points of N-metabolism in rapeseed, by performing transformation with genes of critical enzymes in the nitrate-uptake and nitrate-assimilation pathway.

Results

For blackleg, only specific or partial resistance is described in oilseed rape, whereas the related mustards Brassica juncea, B. carinata and B. nigra possess a hypersensitive type of resistance efficient throughout the life of the plant. These Phoma-resistance genes have been introduced into rapeseed. The introduced resistance genes confer high Phoma-resistance to different developmental stages of the rapeseed plant. The chromosomal location of the resistance genes has been mapped and several RFLP markers linked closely to the resistance have been identified. Attempts are being made to convert the RFLP markers into PCR markers, and to search for new closely-linked PCR markers. Work is in progress to finally isolate the gene(s) in order to determine their DNA sequence and biological function. In France and Germany Phoma-resistant rapeseed material is now introduced into private plant-breeding programmes. The durability of the Brassica B genome-derived Phoma resistance is studied under field conditions. Sources of resistance to Phoma have also been found in A. thaliana and B. tournefortii; the material is under investigation to support the cloning of the resistance genes and to have new resistance sources for future breeding.

Breeding for increased nitrogen efficiency in winter rapeseed is gaining increased attention for several reasons. In the frame of the present project principal genes involved in the uptake and assimilation of nitrate have been cloned from either Nicotiana, Arabidopsis or Brassica napus, and transgenic plants over- or under-expressing distinct enzyme activities were generated in these species. Transgenic Arabidopsis and Nicotiana plants proved to be extremely useful as model plants, because transgenic lines can be obtained in a much shorter period and at a higher frequency. Transcriptional and translational regulation can effectively be studied in those plants, whereas quantitative effects of the transformed genes on the phenotype can only be analysed in the rapeseed crop. The results from the transgenic Nicotiana, Arabidopsis and first Brassica napus plants indicate that nitrate-assimilation is tightly-regulated in these plants. The expression of principal genes involved so far in N-assimilation - in sense or antisense - did not lead to drastic changes in the plant phenotype or N-metabolism. However, four comprehensive pot experiments, with plants grown under different N-regimes - and perhaps field experiments too - must be awaited before final conclusions can be drawn. It has been discussed among the partners that the principal genes involved in the uptake and assimilation of nitrate cover only some aspects of N-efficiency in rapeseed. Genetic variation for N-efficiency is multi-factorial and to date there have been only a few investigations on genetic variation for N-efficiency in rapeseed.

The Brassica B genome Phoma-resistance genes have been introduced into rapeseed. The introduced resistance-genes confer high Phoma-resistance at different developmental stages of the rapeseed plant. The chromosomal location of the resistance genes has been mapped and several RFLP markers closely-linked to the resistance have been identified. Attempts are being made to convert the RFLP markers into PCR markers and to search for new closely-linked PCR markers. Work is in progress to finally isolate the gene(s) in order to determine their DNA sequence and biological function. In France and Germany Phoma-resistant rapeseed material has been developed and is now introduced into private plant-breeding programmes. The durability of the Brassica B genome-derived Phoma-resistance is studied under field conditions. Sources of resistance to Phoma have also been found in A. thaliana and B. tournefortii; the material is under investigation to support the cloning of the resistance genes and to have new resistance sources for future breeding.

The Agrobacterium rhizogenes-mediated transformation of spring and winter rapeseed lines has been shown to yield up to 100%-transformed hairy roots. A wide range of genotypes and plants can be regenerated from hairy-root cultures. with the method. Co- transformation can be successfully performed using A. rhizogenes, carrying a binary vector in addition to its own Ri-plasmid. Although some improvements can be made in the regeneration of plantlets from hairy-root cultures - and in the early detection of genotypes in which the integrated gene of interest is not linked to the hairy-root locus - the protocol is ready for application in oilseed-rape transformation programmes aimed at the generation of transgenic plants free of selection markers.

Exploitation plans

The Phoma-resistant rapeseed material developed within the present project has now been introduced into private plant-breeding programmes in France and Germany for further evaluation and exploitation. RFLP markers closely linked to Phoma resistance are converted into PCR markers that can be used more effectively in large breeding programmes. The exploitation is supported further by the EC-funded shared-cost project New oilseed rape resistant to diseases through interspecific crosses (NORDIC), which started on January 1, 1998. The developed protocol for the Agrobacterium rhizogenes-mediated co-transformation of rapeseed is ready to be applied to oilseed-rape transformation programmes aimed at the generation of transgenic plants free of selection markers. The exploitation depends on the regulative environment that enforces the generation of selection marker-free transgenic crop plants. Further work is required for the understanding and genetic improvement of N-uptake and N-use efficiency of rapeseed, before results and plant material can be exploited.

Conclusions

The present Concerted Action Project has allowed a faster progress in the different research areas by frequent exchange of results and materials and by the joint planning of experiments and research activities. The following can be concluded.

• The work performed in the group Disease Resistance has greatly increased in effectiveness. Working plans were co-ordinated to avoid doubling work; plant materials were exchanged; experimental data obtained in the different laboratories were immediately disseminated and thus verified more quickly. Furthermore, other research institutes with expertise in Phoma research (e.g. IPK Gatersleben, Germany) were included, and private plant-breeding companies became interested in transferring the results thus far obtained into their practical breeding programmes.

• Through the concerted activity of the partners, together with other research institutes and private plant-breeding companies, a research proposal (NORDIC) was successfully submitted to the EU FAIR programme. This NORDIC project will help to transfer the scientific results obtained so far into practical breeding programmes.

• The research work of the Nitrogen Efficiency group has also been very much stimulated by the support of the Concerted Action Project. Although the problem of possible nitrate-leaching from crop residues following rapeseed cultivation is known, very little experimental work had been done in this field before. N-efficiency is a quantitatively-inherited trait and thus it was of considerable advantage that the experiments of the involved project partners were directed at quite different aspects of the phenomenon.

• Because of the complexity of the N-assimilation pathway and its strong regulation, it is important to perform fundamental research in Nicotiana and Arabidopsis: these experimental plants are easier to handle than rapeseed. The function and regulation of specific genes can be studied sufficiently-well in these species, whereas the importance of the genes for the expression of N-related traits can only be analysed in the rapeseed crop itself.

• Several genes involved in the uptake and assimilation of nitrate have successfully been cloned during the last few years. Experiments are well underway to identify their significance in improving N-efficiency in rapeseed.

• Through an international workshop on N-efficiency in rapeseed organised in Gottingen in October 1996, new international co-operation in the field of N-efficiency in rapeseed was generated with partners beyond the Concerted Action Project, which will last beyond the end of the present project.

• The performance of multi-locational field experiments with different rapeseed cultivars to identify genotypic differences in N-efficiency add important aspects and results to the understanding of N-efficiency. The knowledge of genetic variability among different rapeseed cultivars is also important to evaluate the success of transgenic methods.

• Rapid progress in the improvement of N-efficiency can only be obtained by a combination of conventional breeding and biotechnological approaches. For example, several traits related to N-efficiency, such as protein-content in the seeds or lodging-resistance, can be very much improved by classical breeding approaches. However, the current situation (e.g. price of N-fertiliser, environmental regulations) does not encourage the breeding of N-efficient rapeseed cultivars.
• Private plant-breeding companies became interested and involved in the research work of the project. These companies were:
  • Svalof Weibull AB, Sweden
  • SERASEM, France
  • Norddeutsche Pflanzenzucht, Germany
  • Deutsche Saatveredelung, Germany
  • AgrEvo, Germany

The long-term aim of the present project is to contribute to the improvement of ecological compatibility and to the reduction of production costs of rapeseed cultivation. However, in order to maintain the competitiveness of the rapeseed crop under a new Common Agricultural Policy, considerable efforts are still necessary. The introduction into the market of the new hybrid rapeseed cultivars has led to a substantial increase in the average yield under equal input levels. Thusthose hybrid cultivars show an improved N-efficiency and help to maintain profit margins in rapeseed cultivation. Through agricultural research, further reductions of pesticide- and fertiliser-input can be expected through improvements in crop management systems. Additional biotechnological approaches to modify and improve the quality of the rapeseed oil and meal - and to find new applications in the food, feed and industrial sectors - support the efforts to maintain and increase the competitiveness of the rapeseed crop.