1. The PIP-driven workshop entitled: "A workshop defining strategies for information exchange and methods to expedite the application of results", funded by the EU Commission DGXII, was held in Enkhuizen, the Netherlands on 10-11 May, 1995. The proceedings of this meeting have been included in this Newsletter, together with an account of the topics which have been discussed, as well as an outline of the initiatives PIP is proposing for the immediate future. The results of this meeting form the starting point for further discussions and activities. One of the first occasions will be during the upcoming workshop, organised by the coordinators of the EU supported Project of Technological Priority, 25-26 July, entitled: "Study on progress todate and opportunities to increase interactions with European industry".
2. Short Term Training Fellowships. The coordinators of the PTP were given by the European Commission the mandate to receive applications from European scientists (also from industry) working in the field of plant molecular biology and allied disciplines, for a number of PTP short term training fellowships. Grants provide for travel, subsistence allowance and a contribution to the research costs of the host institution. Applications are invited at any time and further details have been outlined on page 28.
3. Research proposals. Another 10 researchers have responded to the invitation of the PIP to publish details of their research proposals in the PIP Newsletter. PIP members are encouraged to respond to suggestions for cooperation before applications for funding will be submitted to the EU Commission. The second call for proposals in the Framework-4 programme will be open in September 1995, and this will be the first opportunity to submit full research proposals in the area of plant molecular and cellular biology.
4. Email address. Please note the Email address of the PIP secretariat:
Plant Industrial Platform Workshop Programme:
"A workshop defining strategies for information exchange and methods to expedite the application of results", May 10 - 11, 1995, S&G Seeds, Enkhuizen, the Netherlands.
Wednesday May 10
@TABEL3 = 11.00 Guided tour through S&G facilities (optional).
@TABEL3 = 12.00 Arrival, Coffee, Lunch.
@TABEL3 = 13.00 Welcome by the president of S&G Seeds, prof.dr. E. Veltkamp.
@TABEL3 = Opening by the chairman of the PIP Steering Committee.
@TABEL3 = 13.15 Dr. D. Bartels. PTP theme B1. Isolation and characterisation of genes relevant to abiotic stress.
@TABEL3 = 14.00 Dr. M. Caboche. PTP theme D. Nitrogen utilisation efficiency.
@TABEL3 = 14.45 Coffee / Tea
@TABEL3 = 15.15 Dr. M. Bevan. The Arabidopsis sequencing project (ESSA).
@TABEL3 = 16.00 Dr. A. Karp. Molecular Screening Tools programme.
@TABEL3 = 16.45 Dr. J. Senker. How and why do companies link with university science?
@TABEL3 = 19.00 Dinner and panel discussion
@TABEL3 = - Contribution by Dr. J. Schell.
@TABEL3 = - Dissemination of results (yearly reports, sequences, communication channels)
@TABEL3 = - 'Partnering' of industry and scientific researchers (development, structure)
@TABEL3 = - The competitive advantage principle for EU industry.
@TABEL3 =
@TABEL3 = Thursday May 11
@TABEL3 =
@TABEL3 = 08.30 Dr. S. de Vries. PTP theme A1. Embryo pattern formation, transition of vegetative to reproductive growth, development of seeds and leaves.
@TABEL3 = 09.15 Dr. P. de Wit. Human Capital and Mobility Network: Molecular genetics of fungal plant pathogens.
@TABEL3 = 10.00 Dr. R. Scheffer. Biological Seed Coatings, S&G Seeds.
@TABEL3 = 10.45 Coffee / Tea
@TABEL3 = 11.15 Dr. J. Azc˘n-Bieto. The Agro-Industrial Research Programme
@TABEL3 = 12.00 Prof.dr. A. Hoeveler. Application of results, Industrial Platforms, future EU programmes.
@TABEL3 = 12.45 Final discussion and closing of the workshop
@TABEL3 = 13.00 Lunch
@TABEL3 = 14.00 Guided tour through S&G facilities (optional)
Participants:
Dr. J Azc˘n-Bieto, Comm. of the European Communities, Belgium
Dr. D. Bartels, Max Planck Institut, Germany
Dr. N. Batty, PBI / Unilever PLC, United Kingdom
Dr. A.D. Beadle, John Innes Centre, United Kingdom
Dr. A. Beltran, Min. de Industria, Comercio,Turismo, Spain
Dr. M.W. Bevan, Coordination ESSA project, United Kingdom
Dr. M. Boutry, University of Louvain, Belgium
Dr. A. Bruncri, ENEA / CRE Casaccia, Italy
Prof.dr. M. Caboche, INRA, France
Dr. Ir. L.C. Davidse, Res.St.f.Floricult.&Glasshouse Veg., The Netherlands
Dr.Ir. A.M.M. de Laat, D.J van der Have B.V., The Netherlands
Dr. G.E. de Vries, ProBio Partners, The Netherlands
Dr. S. de Vries, Wageningen Agricultural University, the Netherlands
Prof.dr. P. de Wit, Wageningen Agricultural University, The Netherlands
Dr. O.P.E. Doyle, Nat. Agric. Veter. Biotech. Centre, Ireland
Dr. D.A. Eichholz, Monsanto, USA
Prof.dr. R.B. Flavell, John Innes Centre, United Kingdom
Dr. G. Freyssinet, Rhne Poulenc Agrochemie, France
Dr. M.T. Gauthier, INRA, France
Dr. C. Grand, R.A.G.T. S.A., France
Prof.dr. E. Herberle-Bors, Vienna Biocenter, Austria
Dr. A. Hoeveler, Comm. of the European Communities, Belgium
Dr. D. Inz, Rijksuniversiteit Gent, Belgium
Dr. A. Karp, T-Programme Genetic Screening Tools, United Kingdom
Dr. B. Knust, Projecttrger Biol,Energ,kologie, Germany
Prof.dr. M. Koornneef, Wageningen Agricultural University, The Netherlands
Dr. R. Long, Green Crop Ltd., Ireland
Mr. A. Nilsson, Svalf Weitsbull AB., Sweden
Dr. D. Rawlins, Biotechn & Biol. Sci. Res. Council, United Kingdom
Dr. R. Scheffer, S&G Seeds B.V., The Netherlands
Prof.dr. J. Schell, Max-Planck-Institut, Germany
Dr. A.W. Schram, S&G Seeds B.V., The Netherlands
Dr. J. Senker, University of Sussex, United Kingdom
Dr. W. Spek, SENTER, The Netherlands
Dr. D. Stahl, PLANTA Pflanzengenetik Biotech.GmbH, Germany
Dr. R.D. Thompson, Max Planck Institute, Germany
Dr. C. Tir, Rijksuniversiteit Gent, Belgium
Mr. R. Torgersen, The Research Council of Norway, Norway
Dr. A.S. Tsaftaris, University of Thessaloniki, Greece
Ir. L.G. van den Berkmortel, Bruinsma Seeds B.V., The Netherlands
Dr. L. van Houten, SENTER, The Netherlands
Dr. P. Vos, Keygene N.V., The Netherlands
Dr. D. Vreugdenhil, Agricultural University, The Netherlands
PIP workshop defining strategies for information exchange and methods to expedite the plication of results
Funding provided by the EC-DGXII.
Starting with a workshop dinner on the first day, there was ample time during the conference to discuss how to improve the information transfer between academia and industry in the area of plant biotechnology. Other topics included the initiation of a lasting dialogue on possible improvements of the ways EU funded research programmes are developed, how these are given form and executed. The unique opportunity, bringing together key players in plant biotechnology and industry, resulted in a lively discussion on a range of issues and problems specific to current practice in EU funded projects. The following abstract is by no means a complete list of the discussion points, rather it should be considered as a base for reflection and as a starting point for further discussion between members of the PIP platform (as representatives from industry), academia and the EC.
Prof. Schell introduced the evening debate and made a number of points relating to interactions between the scientific community and industry within European funded research projects. The stereotype difference in objectives for scientific researchers (publish or perish) and industrial researchers (produce commercial results) does not exclude further expansion of existing cooperations. In the area of the plant biotechnology basic science it is of vital importance to feed and support innovation as this may revolutionize breeding practices. Science therefore needs to be of top quality and should be well appreciated and understood by industry. Both academia and industry have however an obligation to ensure that biotechnology actually contributes to, and is being applied in practice. In this context both parties would need to take account of the wider social aspects of activities and direct these towards environmental compatibility, sustainability and profitability. This is especially true within the agricultural sector. The results of excellent science must be taken forward through an academic/industrial partnership and applied in a manner that is both socially acceptable and commercially viable. The Commission's measure of Demonstration Projects is one of the tools to reach these goals.
Industrial perspective by dr. A. Schram:
Using overhead sheets, three key issues were presented, which were felt to form the bottlenecks in realising an increased return from EU funded research efforts.
1. Industrial involvement in the definition of framework programmes. The industrial platforms which have been formed during the Framework-2/3 period have, each to its own degree, developed technology transfer activities. The industrial know how and insight in technology, however, may not be appreciated to their full extend while defining EU framework programmes. Next to the roles of IRDAC and ESTA, the practical industrial input in this process seems diffuse or too general and increased use of the well organised platforms as 'collectives of customers of research results' may be considered.
2. Industrial involvement in EU research programmes and project definitions. The plant biotech industry, as represented by PIP, would favour EU funding of thematic research programmes, rather than individual research projects. This will provide opportunities for the development of coherent strategic research activities and these should be coordinated by a scientific/industrial board. The process of individual networking activities is a too random process to be sufficiently effective. A problem is that small companies, typical in the European plant breeding world, do not have the (human) resources to participate in EU funded programmes at the appropriate level.
3. Industrial involvement in the dissemination of results. As stated before, the apparent contradiction between the academic 'publish or perish' and the industrial 'commercial interest' should be recognised and effectively dealt with. For the EU funding efforts to be maximally effective it is important to facilitate possibilities for the protection of intellectual property. Scientific researchers should gain greater awareness of the required procedures for granting patents and make full use of the expertise present in industries participating or interested in research projects. In practice it may be necessary to devise a system which would ensure a confined flow of information from source to users in the EU funded research programmes. A three stage process could be envisioned, where at the first level participation industries interact directly, industrial platforms are, at the second level, involved in a wider information distribution, while ensuring that the process of scientific publications, the third level, is not hampered. The European precompetitive funding principle should contribute to the development and maintenance of a strong European industrial position. Academia and industry should develop future projects collaboratively within the framework of 'push/pull' interactions.
General discussion topics during the workshop and perspectives.
1. The information flow between industry and academia was considered as yet not efficient. The information material provided by the European Commission was not enough adjusted to the needs of companies. It was suggested to simplify the information flow by producing information material which it quickly distributed, easy readable and allude more to the specific requirements of enterprises. An active role of PIP members and academia in formulating and defining the structure of the information materials required were strongly stipulated.
On a larger time scale the creation of an informatic network for information in plant science was proposed. For instance, PIP members could be included within the PIP electronic information exchange system and have an early sight/input into the scientific progress and position reports. The EC could help to develop this informatic network in establishing a reasonable data bank composed of addresses of industrial companies involved in the plant industrial sector with the basic assumption that any other European company declaring interest late would be equally included in the list. Since industry often does not take the time to read available literature it might be desirable to designate a person in each company in charge of screening and looking at the transmitted data.
2. It seems evident that PIP members should be informed about contractor meetings and every conference related to plant science sponsored by the Commission. Industrial participation in contractor meetings is strongly desirable in particular when participation is understood in the active sense. In particular the informal links between different persons created during the meetings are very helpful for developing mutual confidence and trust which eventually will lead to increased outputs. The Commission should continue to stimulate joint meetings between contractors and industrial companies.
3. There was a perceived view from industry that academics lack sufficient patent awareness, and were primarily concerned with publication. Industry felt that potential business opportunities were being missed, due to insufficient or too localised contacts. Although substantial experience of patent applications did exist within the scientific community, this could well be clustered within specific departments or institutions. Scientific researchers should gain greater awareness of the required procedures for granting patents and make full use of the expertise present in industries participating or interested in research projects.
There is a strong need for education in this field. Therefore it was suggested, that workshops should be organised on patent rights for educational training of contractors and for trainees receiving EU fellowships. On this key issue, assistance from the EC might be very fruitful and contribution of PIP necessary.
4. Matters concerning intellectual property ownership and potential licensing arrangements should be agreed upon within EU supported consortia, prior to the launch of the research projects. These projects should include the maximum of industrial participation. Companies are often not well informed about the possibilities for protection of intellectual properties in an EC contract (see standard contract model). The EC should increase its efforts in explaining the different possibilities on licences and user rights to the industrial side. Furthermore several of these project consortia could form a technology interaction board, whose primary role would be to satisfy the demands of all parties and ensure effective and relevant technology transfer. Industry in turn could take initiatives to organise these consortia. This approach is proposed under Framework IV through thematic networks in the FAIR programme or commercial exploitation committees emanating from some industry platforms in the Biotech programme (Microbial biotechnology).
5. The current political impetus encouraged the direct or indirect involvement of small/medium sized enterprises (SMEs) within project submissions. Again, PIP as well as the EC could help academia to identify relevant SMEs across 15 Member States. However, another difference was highlighted: academia was intrinsically long term in its outlook, whilst SMEs very often needed quick solutions to current problems, which mainly related to a reduction in production times and costs. However long term research was needed, if answers to immediate problems were to be forthcoming. It was vital that industry fully appreciated the value and the need for long term horizons of between 5 and 10 years.
6. In order to transform knowledge generated in EU funded programmes into applications through product development, a huge investment is still necessary and must be made by industry. These funds are obtained from actual sales of products and from shareholders, willing to invest in risky stages of product development. The world wide investment in basic plant science research is currently not balanced by similar funds for development of new applications and for development of new products. In order to close this gap between research and the technical viability of a new technology, the implementation of Demonstration Projects under Framework IV in Biotechnology and Agriculture and Fisheries programmes is considered as an incentive to reduce this imbalance.
Isolation and Characterisation of Genes relevant to Abiotic Stress.
Dr. D. Bartels.
The theme B1 of the Project of Technological priority is divided into the following subgroups:
Osmotic stress including water and salt stress.
Low and high temperature stress.
Nutrient stress.
Interaction of abiotic stress.
The objectives of theme B1 are the isolation of structural genes and promotor elements which positively contribute to stress tolerance and the elucidation of signal transduction pathways. The major research activities can be summarised as follows:
Gene isolation and characterisation: Stress responsive genes have been isolated from different stress situations and from different plant species. A cDNA clone designed AF93 represents a gene inducible under cold condition and drought stress. From maize embryos the Rab17 gene has been isolated and was found to be highly phosphorylated in vivo. This protein contains in the middle part of its sequence a cluster of serines followed by a casein kinase-2 substrate consensus sequence. Biochemical experiments showed that sugar metabolism is greatly effected by different forms of osmotic stress, therefore genes for key enzymes have been isolated. By utilizing yeast as an experimental system three genes have been isolated which improve salt tolerance in yeast. These genes affect either ion transport systems or crucial metabolic pathways sensitive to salt stress and they are likely to have counterparts in plants.
Mutant isolation: Mutants effecting the stress phenotype or the signal transduction pathways were isolated from Arabidopsis. Four non-allelic Arabidopsis mutants which exhibit a programmed cell death phenotype were isolated. Those mutants are termed lesion simulating disease resistance (lsd) mutants. Two classes of mutants were defined: One class controls "initiation" of cell death and the other controls "propagation" of cell death after external initiation. Lesion positive mutants express increased resistance to virulent pathogens. Late flowering and early flowering mutants are being selected under different environmental conditions. These lines are being organised in phenotypic groups that are subjected to complementation analysis, analysis of dominance and backcrossing. During the course of the work the following materials were generated:
1. Molecular probes:
superoxide dismutates, different forms.
catalases.
ascorbate peroxidase.
gluthatione peroxidase.
sucrose synthetase.
sucrose phosphatase.
yeast acetaldehyde dehydrogenase.
2. Molecular probes for transcription factors:
Heat shock factor (HSF-24) from Arabidopsis, myb-2.
3. Specific stress inducible structural genes:
Maize Rab17, Rab28, MI;
Soybean and Arabidopsis small HSP genes, extensine;
Barley cold regulated genes and antibodies for chloroplast cold regulated protein;
Yeast ha1, acetaldehyde dehydrogenase, DDR48.
4. Inducible promotors or promotor constructs:
Embryo-specific Rab gene promotor constructs with GUS or CAT as reporter genes.
ABA and drought stress inducible promotors (CDeT6-19 Craterostigma).
5. Seed stocks:
Arabidopsis lines.
Tobacco and Arabidopsis, transgenic for heatshock promotor-GUS constructs.
Arabidopsis cold acclimation mutants.
Arabidopsis winter habitat mutants.
6. cDNA/genomic banks:
Arabidopsis cDNA bank / genomic banks.
Arabidopsis cDNA bank / cold-acclimated seedlings.
Arabidopsis cDNA bank / in yeast expression vector.
Barley cDNA bank (cold stressed shoots).
Barley genomic bank.
Craterostigma cDNA bank (leaf and callus water-stressed, ABA treated).
Yeast genomic bank.
For further information, please contact the coordinators D. Bartels (or F. Salamini), Max Planck Institute fur Zuchtungsforschung, Carl-von-Linneweg 10, D-5000 Koln 30, Germany, phone: (+49)-221-5062-430, fax: (+49)-221-5062-213.
Molecular Strategies to modify Nitrogen / Carbohydrate Partitioning in Crop Plants
Dr. M. Caboche.
Theme D2 of the Project of Technological priority
Introduction. Crops are depending on the supply of mineral nitrogen, mainly nitrate and ammonium for their growth. Yields are promoted on a wide range of doses by N-containing fertilizers, and this has led to an ever increasing use of such fertilizers for the growth of a number of crops. As a consequence of this, nitrate has been found to leach out and contaminate water resources to unacceptable levels. To a significant extent nitrate pollution can be limited by providing optimal doses of fertilizers at specific steps of plant growth. However there is a need to further increase N utilization efficiency and this may be achieved by appropriate modifications of N metabolism, once the characteristics of this metabolism are properly understood and molecular tools are available to obtain such modifications. The collaborative network "Molecular strategies to modify nitrogen / carbohydrate partitioning in crop plants" has been funded by EC to identify control point of N metabolism and develop news strategies to improve N metabolism.
Status of our knowledge at the start of the programme
The catalytic steps of nitrate assimilation have been identified quite some time ago. Two years ago our understanding of N metabolism was including the molecular identification of the different classes of structural genes coding for these catalytic steps involved in the pathway (nitrate and nitrite reductase (NR and NIR), glutamine synthetases (GS1 chloroplastic and GS2 cytosolic), glutamate oxaloacetate amino transferase (GOGAT) and several features of their regulation. However very little was known about the transport processes involved in the flow of N metabolites to sink tissues, the links between N metabolism and carbon metabolism.
Several observations suggest that there is an effective coupling between N and C metabolism. For instance it is known that up to 20% of the reducing power derived from photosynthesis is used for nitrate reduction and that deficiencies in NR or NIR activity lead to chlorotic phenotypes. Another interesting observations concerns the inducibility of transcription of the NR structural gene by sucrose in the absence of light. It would therefore be interesting to evaluate to which extent this coupling can be loosened, for the benefit of an increased N utilization efficiency and to identify which metabolites or enzymes are involved in the detection and signalling of this balance.
Important milestones of the ongoing programme
Nitrate and aminoacid transport: Three of the contributing laboratories develop molecular approaches to identify nitrate in photosynthetic organisms transporters. The group of E. Fernandez (Cordoba) has identified and cloned three genes essential for nitrate transport in Chlamydomonas. Whereas the exact role of Nar 2 in the transport remains to be elucidated, Nar 3 and Nar 4 are transmembrane proteins sharing strong homology with a gene involved in chlorate transport in Aspergillus. In the group of B. Forde (Rothamsted) and our laboratory similar plant genes have been identified in barley and Nicotiana
respectively. Experiments are underway to test for a possible complementation of nar 3 - nar 4 double mutants deficient for nitrate transport with these genes expressed under appropriate control in Chlamydomonas. T. Miller is setting up an electrophysiology analysis of the specificity of these transporters by expression in Xenopus oocytes. A first series of experiments done with Chl 1, another putative low affinity transporter of nitrate and chlorate identified by Nigel Crawford, led to the observation that this transporter is non specific for chlorate and nitrate transport and can also transport peptides as well as aminoacids provided at high concentrations. Oocytes expressing Nar 2 and Nar 3 proteins are being characterized. Aminoacid transporters have been identified by functional complementation of yeast mutants in the group of W. Frommer. Six genes with different expression patterns are being characterized. The presence of multigene family is unexpected since genetic data suggest that a single locus is involved in neutral and acid aminoacid transport. It remains to be tested if genetic loci correspond to one of the transporters or to genes controlling their expression. These aminoacid transporters most probably contribute to assimilate translocation to sink tissues.
Nitrate reduction and ammonium utilization: Several laboratories involved in this programme study the physiological consequences of deregulation affecting the N assimilatory pathway. In our group and that of J.Wray (St Andrews) the consequences of deregulations affecting NR expression are studied. Transgenic potato plants are produced, in which an antisense construct has been introduced to prevent root or shoot expression of NR. Transformants constitutively expressing a 35 S-driven normal NR sequence, or carrying a deletion in the N-terminal part of this coding sequence have been found to be no longer regulated by light at the post transcriptional level.
Transgenic plant expressing an antisense against GOGAT have been obtained by B. Hirel. These plants have a reduced growth, unless they are provided with high CO2 suggesting a primary defect in the photorespiratory cycle. The groups of Hans Heldt, Christine Foyer and Mark Stitt are characterizing the different types of transgenic plants produced in Versailles. From their studies a number of interesting observations have been made. For instance, the group of C. Foyer has found a significant reduction of the amount of nitrate stored in tissues of 35S-expressing transgenic plants, and other modification affecting the rate of photosynthesis. This appears to be the consequence of an increased reduction and a decreased uptake by roots, as suggested by a collaboration with A. Gojon. The group of H. Heldt has characterized plants defective for nitrite reduction. These plants have perturbated nitrate and ammonium utilization. Growth is limited by nitrogen availability, thus resulting in modified photosynthetic apparatus and nitrogen remobilization. The group of M. Stitt found that different levels of expression of NR lead to different / compensating levels of phosphorylation of the enzyme. They also found that nitrate, apart from being a metabolite is also a signal affecting shoot/root ratio. For instance if plant expressing a very low level of functional NR are compared to the wild type grown with limiting amounts of nitrate under conditions where identical biomass production is achieved, the NR limited plants grown at high nitrate have a reduced root development compared to wildtype plants receiving limiting amounts of nitrate. This suggests that a sensing of nitrate effects root/shoot growth identification of the corresponding genes could provide a tool to modify at will the shoot/root ratios in crop.
Links between N and C metabolism: Three series of potato plants perturbated in C metabolism have been produced in the group of L. Willmitzer. A first class of plants are defective for the expression of chloroplastic fructose-1.6-bisphosphatase and show decreased photosynthesis and growth. A second series are defective for starch biosynthesis leaves, tubers or leaves and tubers.
Isocitrate dehydrogenase has been assumed by P. Gadal to be control point linking N and C metabolisms. The group of L. Willmitzer has devised antisense strategies to inhibit the expression of the cytosolic isoform of IDH. This led to no phenotype, most probably as a consequence of an up regulation of the chloroplastic isoform. In parallel plants have been produced where the Calvin cycle functioning has been reduced by antisense strategy also. These different transgenic plants are being characterized by the group of H. Heldt, to study the consequences of these modifications on N metabolism.
Possible outpout for improvements: During the first two years of the programme significant progresses were made as regards our understanding of N metabolism. Some of the discoveries made have stimulated collaborations with private companies to improve crop quality as regards nitrate storage in the green tissues of tobacco, spinach, lettuce and chicory. The main approach was to modify the regulation of nitrate reductase expression to increase reduction at the expense of storage. Alternative approaches based on antisense strategies may now be envisaged on the nitrate transporters once their identity and function are more precisely identified.
For further information, please contact the coordinator prof.dr. M. Caboche (and dr. B. Forde), INRA, Lab. de Biologie Cellulaire, Route de Saint-Cyr, F-78026 Versailles Cedex, France, phone: (+33)-1-3083-3017, fax: (+33)-1-3083-3111.
EU Arabidopsis Sequencing Project
Dr. M. Bevan
The goal of the EC Arabidopsis sequencing project is to begin the systematic sequencing of the genome of this model species, and to lay foundations for an EU-wide network that can contribute, in later Framework programmes, to the complete sequencing of the 100Mb genome. The Arabidopsis Multinational Steering Committee has indicated a possible target date of 2004 for completing this task, with a roughly equal contribution from US and EU scientists.
The complete genomic sequence of an organism, and in particular that of a model organism which has an extensive history of focused research, will provide an unprecedented opportunity to carry out many types of biological experiments, from taxonomy, evolutionary studies, through to whole plant physiology, in a more comprehensive manner, using the strong foundations genetics can provide. The basis for many of these studies will be the identification of all the 20-25,000 protein-encoding genes in Arabidopsis. It is relevant at this stage to ask how potential genes can be recognised in sequence data, and how the function of potential genes can be determined. Another question particularly relevant to interactions between EU biotechnology and agricultural industries and this sector of plant sciences is how to ensure the most productive communication and exploitation of this data.
The technical challenges of getting a distributed network of laboratories to produce large amounts of sequence to a common standard of accuracy, and organising the central collection, analysis, annotation and distribution of the data have been addressed and to a large extent solved by the pioneering EU yeast sequencing network. The specific challenge which the Arabidopsis sequencing project faces is to scale up the production in the individual sequencing labs in order to complete the far larger task of sequencing Arabidopsis
in a reasonable time. This is being done by introducing automated sequencing operations into all labs, and setting up a number of larger-scale centres which can sequence 100-300kb/year. This scale up is being achieved by shotgun sequencing, automating template preparation and sequence reaction steps, and using high capacity sequencers and data analysis systems. These approaches can be directly adopted from the examples provided by the Human Genome Project. A quick calculation indicates that a group of 10 labs, each sequencing at a rate significantly less that presently achieved by larger groups such as the Sanger Centre, could complete 10 Mb a year. If continued refinements in the techniques used were achieved, this output could doubled in five years. Lower unit costs are associated with this improved efficiency, permitting funding agencies to contemplate funding multi-megabase sequencing projects within a reasonable timescale. It will therefore be very important to reduce the unit costs of network sequencing, presently about 1.5 ECU/bp, to less than 0.5 ECU/bp in the next 3-4 years.
A related challenge, which is perhaps more customary for scientists than the quasi-industrial strategy described above, is to interpret the emerging genomic sequence to the greatest possible extent. This is being addressed in two ways. First, potential protein coding regions in Arabidopsis are being identified in genomic sequence by software originally developed for ORF (open-reading frame) recognition in the human genome. The software "learns" as it is supplied with more information regarding correct matches, and has already proved its value in defining the intron-exon structure of several complex Arabidopsis genes. Once the potential coding regions have been identified, the ORF can be compared to all other ORFs using software such as FASTA and BLAST. Functional domains of proteins (such as phosphorylation sites, nucleotide binding sites etc) can be defined, and if there is sufficient overall similarity to a class of proteins with a known function (eg. membrane transport, enzymatic function etc) then the function of the similar plant gene can be inferred. Based on results from other more advanced genome projects about half of the potential ORFs identified can be ascribed a "function" using these criteria. The other half of the potential ORFs identified in the yeast and C. elegans sequencing programmes have no known counterparts nor have they been identified in earlier genetic studies. One could reasonably expect a similar proportion of unknown ORFs in Arabidopsis, and perhaps even more considering the evolutionary distance between vascular plants and animals. The complete cataloguing of sequences from these three organisms, together with Drosophila, will permit plant-specific genes to be identified, and these may the subject of focused studies in the future. The second strategy in function search is to introduce mutations into ORFs and examine resulting phenotypes. Plants are very well suited for a systematic approach to this subject, using endogenous or specially engineered ransposons. Large pools of plants n be screened en masse for insertions at a variety of sequenced loci in a heterozygous state. Individuals can then be selfed and effects of gene disruption observed. Alleles of potential genes can be generated using transposon excision. This type of work will provide the users of genomic sequence with information regarding actual function and permit phenotypes resulting from multiple mutations, assembled by crossing, to be observed.
A compelling interest of biotech companies and other users of sequence information is in obtaining access to information, and further obtaining proprietary rights to information of potential use to them. If a suitable strategy for this can be devised, then the centralised data collection, uniformity of the data, and systematic organisation of data collection, makes the Arabidopsis sequence project an attractive source of information. Present plans for seeking proprietary rights to sequence lie in the individual laboratories who own the sequence they produce until it is made public.
Exploitation strategies and philosophies differ greatly among participants of course, and cannot be the object of obtaining a uniform strategy. It appears unlikely that a patent based solely on sequence will be a strong one, indicating that a reasonable idea of the function and application of a sequence is necessary before a useful patent can be obtained. Therefore the sequence project must not be the only source of information for the relevant biotech sectors, instead they must seek to ensure that the most effective means of identifying the function of sequenced genes is in place, and that this can work at a scale commensurate with the pace of sequencing, and provide a centralised collection of data of assured quality. This is a medium-term strategy, and for the short-term one could imagine the collective exploitation by PIP members of the "gap" that exists between the exploitation of sequences in the originating labs and the collective publication of large amounts genomic sequence.
For further information, please contact the ESSA coordinator: Dr. M. Bevan, IPSR Cambridge Laboratory, Colney Lane, NR4 7UJ Norwich, UK, phone: (+44)-1603-452-571, fax: (+44)-16-3-505-725.
The Molecular Screening Tools Generic Project
Dr. A. Karp
Introduction : DNA technology has provided a range of powerful techniques which can be used for the screening, characterisation and evaluation of genetic diversity. These techniques have broad-ranging applications from evaluation and conservation of biotechnology, including germplasm and genetic resources, through to ecology and agroecology and plant and animal breeding. Although their potential is easy to recognise, further development is required before they can be most effectively applied to these end-uses. Recognising both the potential and the need for further development of the techniques, the EC included an area on molecular genetic screening techniques for conservation in the Framework-3 BIOTECHNOLOGY programme. Four proposals were selected and after the evaluation and selection process, these independently approved projects were combined together. The resultant generic project has 35 groups from 12 EC countries (see PIP Newsletter #3), of which four are industrial partners (Diagen, Beckman, Zeneca and Nickerson Biocem).
The work in the project is organised into eight coordination programmes (CPs), four of which are technological development and four strategic development:
Technological development:
CP-A DNA extraction procedures
CP-B Data scoring and analyses
CP-C Scaling down, rapid assays, automation
CP-D Universal\broad range tools
Strategic development :
CP-E Assessing diversity on accessions and collections
CP-F Assessing diversity in natural populations
CP-G Assessing classifications of natural diversity
CP-H Screening "useful" variation
This short overview will not attempt to cover all the work in the programmes. Instead, a few activities have been selected which should serve both to create an impression of the research underway and also to illustrate the contributions of the industrial participants.
Technological development: There is quite substantial involvement of all four industrial partners in the four technological coordination programmes. In CP A, Diagen has provided protocols for DNA extraction which should assist those trying to handle difficult species such as woody shrubs and forest trees. Quite recently, for example, it was agreed that a whole set of Rhododendron samples would be sent from participants at the Royal Botanic Garden Edinburgh, Scotland, to the participating group at Diagen in Germany and that a member from a lab in the Rhododendron Network (described later on in CP G) would work with Diagen to extract DNA from the samples. The DNA will then be sent out to all members of the Rhododendron network for applying the different molecular screening techniques. In CP C, four areas are being covered under the general objective of "speeding things up or making screens more efficient": scaling-down procedures, non-radioactive techniques, rapid assays and automation. There is insufficient time to comment on scaled down procedures except to say that many have been developed through the course of the project. Nickerson Biocem provided a full protocol for non-radioactive RFLPs which was published in the first issue of "Molecular Screening News" and gave help and advice to those who tried the methods out. In addition, Nickerson Biocem have designed and constructed a machine to automate the washing steps which are the most time-consuming part of the procedure. They are now in the process of comparing results from manual and automated washing. Rapid methods of detecting DNA sequence differences without having to sequence the fragments (TGGE, DGGE, SSCP, heteroduplex formation) are also being considered in the project. TGGE is being optimised by Prof. Dr Riesner and co-workers with industrial assistance. Automation aspects of the project have been advised upon by Beckman and a BIOMEK 2000 workstation is being loaned to one of the labs in the project to work on aspects of automation.
A lot of effort has been placed in the project on the development of universal tools (CP D). Agricultural companies rarely work on only one plant or animal species and will not want to have to invest in a new set of technologies every time they consider a different species. Similarly, conservationists have to deal with a whole range of organisms, some of which have never been studied previously and universal technologies are therefore of high priority. Three areas are being considered in the project under "Universal Tools" (1) techniques that can be universally applied, eg RAPDs, AFLPs, (2) sequences that are universally conserved in wide range of taxa (and therefore for which primers could be designed that will work on a wide range of organisms) and (3) techniques that result in fast retrieval of the desired sequences from any genome. In the case of (1) it is known already that techniques such as RAPDs can be used for obtaining DNA profiles revealing diversity from any organism, given only a PCR machine and the necessary consumables for a PCR reaction with an arbitrary single primer. The question considered in the project, however, is "how useful is the data generated from these profiles in terms of transferability not only between different labs but over time?". This question was put to test in a network reproducibility experiment in which Zeneca was involved. Each lab provided a "Genetic Screening Package" (GSP) which comprised DNA from their favourite species, details of the enzyme, buffer, mix, thermo-cyling conditions, PCR machine and a photograph of the RAPD profile obtained. GSPs were exchanged among all labs and each then tried to reproduce the results of the others. All 9 labs were only able to reproduce the profiles of at most one or two of the GSPS received, nor was there any pattern to which GSPs gave most problems. In practice it seemed most labs did not adhere strictly to the conditions stipulated preferring, for example, to substitute their own enzyme\buffer rather than purchase the one indicatd.The network experiment was thus taken a step further by one lab sending a single comprehensive GSP, including a thermoprofile from the PCR machine used. This time most labs were able to obtain a similar RAPD profile to the sender lab but differences from the original were still apparent. The conclusions reached were that whilst RAPDs could be used for a diversity study in a single lab they could not confidently be recommended as a universal tool (in our definition) since reproducibility between labs was problematic and, given the improvements in PCR machines and enzymes etc occurring continuously it is questionable whether the same lab would be able to reproduce RAPD profiles after a period of time had elapsed. A similar reproducibility experiment for AFLPs and sequence-tagged SSRs is now being initiated.
With respect to the second class of universal tools, primers for conserved regions are already available for organelle sequences in particular. In animals the mt-DNA genome is highly conserved and primers can be used in a wide range of taxa. In plants the same can be said of the cp genome, although not quite to the same degree. Primers for conserved regions have been identified by Antoine Kremer (INRA, Pierroton) and tested out on a range of species including forest trees and Rhododendron. Aside from the internal transcribed spacer of the ribosomal genes (ITS) being studied by Tautz and colleagues at Munich University, there is a general paucity of suitable nuclear genes to use as universal tools. The problem is not in having sufficient genes but in being able to identify genes which have regions conserved enough that primers would work on a wide enough range of taxa and yet which contain regions divergent enough that diversity can be detected at the below species level. At the Institute of Zoology, London Zoo, Mike Bruford and colleagues are pursuing a strategy for isolation of suitable anonymous nuclear sequences from sheep whilst at the University of East Anglia, UK, Godfrey Hewitt and co-workers have identified anonymous nuclear sequences that can be used to study geographical patterns of diversity in grasshoppers. Similar efforts are now underway in plants. One problem faced with this general approach is that of contamination, whilst another, particularly in plants where polyploidy is more common, is of pseudogenes being picked up by the PCR amplification. The major difficulty, however is finding sequences that are polymorphic at the below species level. Microsatellites are stretches of simple base pair repeats (they are often called SSRS or simple sequence repeats) that are hypervariable and therefore polymorphic at the below species level. When used as a probe they hybridise to many different sites and the resultant multi-locus profiles provide highly discriminatory fingerprints that can be used to idenfy cultivars or study parentage. However, because they are multi-loci probes, they cannot be used for population studies or for mapping. However, if single microsatellites can be retrieved from a library such that sufficient flanking sequence is available on either side, primers can be designed to amplify up only that single SSR. Such sequence-tagged SSRS provide extremely powerful tools for below species level diversity studies. The problem is that such sites are not conserved among a wide range of species and this therefore brings the discussion to the third category of universal tools ie. a tool that is universally accepted but for which fast and effective means of retrieval from any genome are required. Strategies for efficient isolation of large numbers of sequence-tagged SSRS are under development in the project by several groups, particularly Michele Morgante at the University of Udine, Italy, Robbie Waugh and Wayne Powell at SCRI, Scotland and Ben Vosman at CPRO-DLO, the Netherlands.
Strategic development : Alongside the technological development of CPs A-D it was recognised that there should be testing of the tools on appropriate diversity systems. In this programme the word "tool" includes primers, probes etc, and associated techniques for efficient screening. Four classes of test systems were selected which form the four strategic coordination programmes E-H. In the first (CPE), the techniques are being tested on accessions and collections. In Hordeum a network of four lab is collaborating to compare and contrast the different techniques on the same subset of the barley core collection. Ahmed Jahoor and co-workers at Munich have constructed pedigrees of 71 barley cultivars based on RFLPs with 31 probes and 3 enzymes. The pedigrees separate spring and wheat barleys and the two-rowed from the six-rowed cultivars. Similar but not identical pedigrees where obtained using RAPDs by Robbie Waugh at SCRI based on 20 primers and 98 bands. In contrast PCR-sequence data on ITS and a few other nuclear and organellar genes has not yet yielded sufficient polymorphism. Microsatellite and AFLP analyses are under way. Also in this CP, Zeneca have been comparing and contrasting RAPDs and AFLPs in two crops of complex genomes - wheat and sugar beet. Cluster analysis of AFLPs in wheat revealed pedigrees in close agreement with the known origins from the breeders provided that more than two primer combinations were used in the data. AT CPRO-DLO, Ben Vosman and colleagues are comparing the sequence tagged SSR approach with oligonucleotide fingerprinting and RAPDS in cultivars and accessions of tomato and lettuce.
Accessions and collections represent just one side of the below species question - in effect the ex-situ conservation side. To test the tools against in situ conservation aspects they are being used to study diversity in natural populations of a range of different species (CP F). One example is from the work of Peter Arctander and co-workers at Copenhagen University on North Atlantic whales where variation in the mt control region (or d-loop) and polymorphisms in sequence-tagged SSRS are being studied. The results so far have revealed behavioural constraints on gene flow which relate to feeding behaviour and the presence of an isolated population in the Mediterranean where levels of diversity are low. In plants, the population question is mostly being addressed in the project in forest trees, where most of the diversity exists in natural populations. Here RAPDS were attempted, but have largely been abandoned due to problems with reproducibility, and microsatellites that have been identified in CP D are being tested out. In a collaborative effort, for example, Michele Morgante at Udine University in Italy, Giovanni Vendramin at Florence University and Wayne Powell at SCRI Scotland have identified SSRS in the chloroplast genome that are proving to be useful polymorphic markers in populations of Pinus species.
Although the case studies on a populations (CP F) are obviously concerned with natural variation they represent a similar test of the tools as accessions and collections (CP E) in the sense that both CPs are concerned with genotypes that are relatively closely related. In fact, in both CP E and F the major problem is finding tools that are sensitive enough at this level. It was therefore thought pertinent to include, as a contrast, a test case from a botanic garden, which would provide an example of a problem of diversity in nature in which species relatedness is not always clear and in which too much rather then too little diversity is the difficulty faced. The test case chosen, that of Rhododendron, had also been little studied at the DNA level and therefore provided an additional test of how readily the different techniques could be applied from scratch. A network approach was undertaken which included the industrial partner Zeneca. The Royal Botanic Garden Edinburgh, Scotland, UK where one of the worlds largest collections of Rhododendron is maintained and where the classification of the genus is being studied, initially sent out three samples (A,B,C) to the members of the network, with instructions that one sample may be different, although they were in fact identical. They sent out 12 species, one of which was the A,B,C species and finally six samples from within two species. The whole range of screening techniques is being covered by the network: RAPDs, AFLPs, PCR-RFLP, RFLP, PCR-SEQ, SSRs, in some cases more than one technique is being compared in the same lab. All techniques have been successfully applied and have revealed high levels of polymorphism. The techniques, however, differ in their sensitivity, accuracy and informativeness at the different taxonomic levels. By the end of the programme some 50 genotypes of Rhododendron will have been studied and data will be contrasted and compared (a) between labs for the same techniques, (b) between techniques in the same lab and (c) betwen different techniqes in different labs.
In the final coordination programme it was felt that some efforts in the programme should be directed towards screening for "useful" variation. "Useful" here is simply meant to include variation of agronomic significance. Ahmed Jahoor and co-workers at Munich University, Germany are screening accessions with RFLP markers linked to traits of agronomic importance such as powdery mildew resistance, whilst Robbie Waugh and colleagues at SCRI, Scotland are similarly using RAPD markers. In a completely contrasting approach plant tissue cultures and their regenerants are being screened using RAPDS, any variant bands cloned and their polymorphisms confirmed (or otherwise) by southern hybridisations. Any hypervariable probes thus identified are then being tested against cultivars and species. Using this approach, Ana Vazques and co-workers at the University Computense de Madrid have identified hypervariable sequences in rye which are polymorphic among cultivars and species. Francesco Sala and colleagues at the University of Pavia in Italy have similarly identified a hypervariable region in the cp genome of rice and Marcello Buiatti and co-workers at Florence University have identified variable fragments in kiwi fruit and tomato, which are currently being cloned and investigated. All these tissue-culture derived sequences are being tested out by others in the project to assess their broad-range usefulness. In addition, Nelson Marmiroli and colleagues at the University of Parma, Italy are using primers for stress responsive genes, such as heat shock proteins and dehydrins, to screen material in CPs E, F and G.
For further information please contact the coordinator: dr. A. Karp, IACR-Long Ashton, Department of Agricultural Sciences, University of Bristol, Bristol, BS18 9AF UK, phone: (+44)-1275-392-181, fax: (+44)-1275-394-281.
How and Why Do Companies Link with Public Sector Research
Dr. J. Senker
Countries around the world, recognising the importance of academic expertise in the commercialisation of biotechnology, have promoted links between public sector research (PSR) and industry. Before discussing how and why small and large companies link with public sector biotechnology research, it is important to see this phenomenon in its broader context.
Since the early 1980s, the amount of money spent by industry on research in university and government laboratories has been increasing world-wide and in 1992/3 industry provided 11 per cent of British universities' income. Three factors account for this increase:
1. "Supply push": the inability of governments in industrialised countries to sustain previous growth levels for research expenditure. Universities or laboratories which want to maintain or expand their research activities have to look for new non-government sources of funding.
2. "Demand pull": industry in research-intensive sectors is getting involved in university collaborations because the underlying science is extremely dynamic with new knowledge emerging all the time; development sometimes happens at the interface between different disciplines and fields and companies need to cover more fields than can be covered by company R&D alone. Their 'search' activities' require that they are plugged in to PSR to be aware of the new knowledge and opportunities arising.
3. Government, both at European and national level, has played the role of match-maker between academic science and companies through schemes such as the EU's Framework Programmes and the UK's LINK and SMART programmes. Some programmes aim to help small firms and others have a technology focus, but none appear to have been designed specifically to promote innovation by biotechnology SMEs.
Two recent studies have shown that large and small firms involved in biotechnology, though having similar motivations for linking with university science, have not been similarly well served by these schemes. I will first report on the problems which small biotechnology firms have in making use of university science, and then discuss the nature and purpose of large pharmaceutical companies' PSR links.
A study was undertaken to identify small biotechnology firms awareness of schemes for technology transfer, and the relevance of these schemes to their needs because a British scheme for technology transfer was working well for large firms, but few small firms were taking advantage of it. 39 firms were interviewed in a broad range of sectors: pharmaceuticals, plant agriculture, food and drink, chemicals, environment and process equipment. The majority had under 100 employees. The firms were very research intensive. 97% conducted in-house development or applied research and 15% were involved in state-of the-art basic research. 64% had R&D employment of 5 or more staff and in seven firms more than 50% of the staff were in R&D. More than one-fifth of the companies had a Director who was a senior member of university faculty. Moreover, 74% of these firms had links with university research. Links ranged from direct, fully-funded research contracts and consultancy arrangements to licensing agreements, academic members on firms' Scientific Advisory Boards and informal links, including academics acting as evaluators of a company's products.
SMEs would have liked even more links with PSR but were not helped by government-subsidised schemes. They did not have either the financial or manpower resources required to enter these schemes, and the time frame was unrealistic to their circumstances. Their investors usually demand a return on investments within three years. Companies can fund external research if they have a steady flow of income. Unlike large firms, which can invest in long-term, strategic research out of profits on current products, many SMEs are still developing some or all of their product range and have no flow of income to support linkage.
Lack of knowledge of people with the specific skills and knowledge needed was a second barrier to links, and even where SMEs had links they did not know if they were working with the best academic experts. Sometimes, where appropriate academics existed, their expertise was totally committed to large firms. Thirdly, in some areas there was no relevant expertise. For instance SMEs involved in micropropagation said that there is a lack of generic scientific knowledge on how to grow a whole plant through tissue culture and woody plants have been neglected. For hardwood species there is a lack of basic information or ground rules. For instance, why do oak trees grow in flushes? Very little is understood about the way in which plant pathogens infect plants and how to control them. At the institutional level, PSR research is fragmented between various departments, with no sharing of knowledge or equipment.
SMEs prefer to do research in-house, in order to control its rate of progress and direction, but if they wish to use programmes for technology transfer, they face many problems. For any scheme two questions are central: How much will it cost? How long will it take? This is difficult to discover from the mass of information they receive about a vast plethora of schemes; nor do they know which schemes are relevant. If they decide to apply, the long, bureaucratic application forms are a nightmare! Unlike large firms, they lack staff or time to understand schemes and how to apply. The length and high cost of schemes are other barriers, especially EU schemes which seem to be geared towards the needs of large firms.
A second study looked at the PSR links in biotechnology of four large pharmaceutical companies; it followed an earlier project which had investigated these companies' links in the early 1980s. Interviews were conducted with 12 R&D staff, both Research Directors and bench scientists, and investigated linkage and knowledge flows to R&D from all sources: from in-house research, other companies and PSR. We investigated the extent of companies' interaction with PSR, its nature and the reasons for such linkage.
Companies' early links with PSR had been to understand more about the potential of biotechnology, and to find out who the experts were. By the late 1980s all had recruited large teams of biotechnologists to apply their techniques to drug discovery, and biotechnology is now regarded as central to future innovation and growth. British PSR was considered to have good scientists, novel ideas, research ability and to be accessible to industry, but to lack funding and equipment.
These companies were spending 1-3% of their research budgets on links with PSR, which amounted to several million pounds. Internal sources contributed most knowledge towards innovation, but PSR was the most important source of external knowledge. PSR contributes to innovative R&D in two distinct ways. Firstly, it is a source of new knowledge in specialist fields of science. Secondly, it provides practical help and assistance in response to specific problems, often in the area of experimental methodologies. The majority of such knowledge is gained through the dual use of personal contacts and the published literature. Reading the literature helps researchers to keep up with the latest developments, but they need to supplement their reading with discussions of issues arising from the literature, often about methods and applications. Alternatively, personal contacts may suggest papers or experts relevant to solving company problems. It is especially important for companies to be on the 'unpublished grapevine' and hear about research before it is published. They do this through their personal contacts and by acting as referees for papers submitted to scientific journals.
Formal links take the form of research contracts, campus laboratories, consultancies and participation in government schemes for linkage. High cost direct linkages are used to access new expertise, to acquire new techniques and skills or to develop gaps in underpinning knowledge. Government sponsored linkage tends to be exploratory, or are used to keep the company abreast of emerging knowledge and to generate new contacts. However, it is clear that the number of informal communication channels between industry and PSR far exceed the number of formal linkages. Moreover, strong informal linkage is essential to success in any formal linkage. Often, collaborations which fail to meet their objectives are those where relations between the bench workers in the collaborating organisations are not strong and friendly. Mutual respect and trust are vital ingredients and therefore must be built up carefully.
The findings of both studies indicate that the direct exploitation of EC research programme results by companies is rare. Companies tend to make a creative synthesis of in-house and external knowledge and techniques to produce innovations, and a major source of external knowledge comes from the recruitment of trained researchers and from reading the literature. However, the importance of informal links for the flow of knowledge from PSR to industry must not be under-estimated. It is therefore important to create many opportunities where researchers from both sides of the divide can meet informally and exchange ideas. This could also be encouraged by schemes which allow for the secondment of scientists between the public and private sectors. Publishing research results in scientific journals also helps the flow of knowledge. Finally, perhaps a more targeted effort needs to made to help SMEs enter technology transfer schemes. As suppliers to large firms, take-over targets or collaborators, they make a vital contribution to the competitiveness of European industry.
For further information, please contact Dr. J. Senker, Science Policy Research Unit, University of Sussex, Mantell Building, Falmer, Brighton, E. Sussex BN1 9RF, UK, phone: (+44)-1273-686-758, fax: (+44)-1273-685-865.
Plant Growth Factors and Development
Dr. S. de Vries
The research as carried out under theme A1 of the PTP project Network A concerns the following topics:
1. Embryo and seed development. In this part of the project, we are searching for the natural substrates of two plant secreted enzymes, an endochitinase and a peroxidase, both of which were shown to positively influence the information of embryogenic cells and somatic embryos in carrot tissue culture. The chitinase gene is expressed in the endosperm of carrot and Arabidopsis (Terzi, Fry and de Vries). The knolle gene, which identifies epidermis formation in Arabidopsis zygotic embryos was cloned, and the epidermis-specific marker gene AtEP2 was used to help characterize mutant zygotic embryos (Jrgens, de Vries). Genes that are involved with seed formation and embryo maturation such as transparent testa and abi3 are being cloned by transposon mutagenesis (Koornneef).
2. Light control and leaf development. The possibility to use transgenic plants overexpressing phytochrome genes as a means to eliminate the shade avoidance reaction and as a result of that to increase the amount of harvestable compounds is being investigated in both Solanum and Arabidopsis. Transgenic plants that have an over 10-fold reduction in the amount of phyA have been produced and these exhibited a partial shade-avoidance reaction (Smith, Thomas, Gatz). Several genes involved in leaf morphogenesis were cloned and are under analysis (Hernalsteens), while leaf morphology mutants in Arabidopsis have been identified in EMS screens. Cloning of genes involved in leaf venation and xylem formation is in progress. The pfl gene (pleiotropic effects on leaf morphology) is in progress (van Lijsebettens). 3. Flower development.Arabidopsis homologues of genes encoding proteins binding to the promoter of the Antirrhinum meristem identity gene squamosa have been isolated and are being analyzed (Huijser), while additional MADS-box containing transcription factors were isolated. Experiments to identify mutant phenotypes of these genes by a reverse genetics approach are in progress (Schwartz-Sommer, Coen). The barley Hooded mutation was shown to result from inactivation of a homeobox containing knotted homologue, the BKn3 gene. Heterologous expression in tobacco results in homeotic phenotypes (Salamini). To identify genes that interact with the Antirrhinum gene floricaula, a transposon strategy is initiated to identify modifiers of the flo pnenotype in a line that has a weak flo allele (Coen). In Arabidopsis, two VRN loci have been identified that reduce the vernalization response of the late flowering mutant fca. The VRN1 locus is being cloned by transposon tagging from a close Ds donor element (Dean).
A clear advantage of a diverse and large project as the A1 theme is that contacts with scientists in other areas is easier and there is access to strategies and materials not normally available.
An introduction was also given into research aimed to identify molecular markers for cells that are undergoing the transition between somatic and embryogenic cell in carrot suspension cultures. This is of interest due to often-observed failure in the initiation of embryogenic cultures in crop species. In order to identify embryogenic cells, semi-automatic video cell tracking was used to catalogue single suspension cells. Results obtained for over 30,000 individual cells obtained from and established suspension culture showed that somatic embryos develop from all morphologically distinguishable cells including small cytoplasmic cells and elongated vacuolated cells (Toonen et al., Planta 194, 565-572, 1994) It appears therefore, that the morphology of cells is a poor criterion to determine whether an individual cell is capable of embryo formation or not. This necessitates the development of reliable single cell markers that can be used to pinpoint individual embryogenic cells. Several genes have now been cloned that are expressed only in single cells present in cultures that are in the process of acquiring embryogenic potential (Schmidt, Guzzo, Toonen and the Vries, in preparation).
For further information, please contact the coordinator Dr. S.C. de Vries (and J. Schell), Agricultural University Wageningen, Department of Molecular Biology, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands, phone: (+31)-8370-83584, fax: (+31)-8370-83584.
Molecular basis of Pathogenicity, Avirulence and Resistance in the interaction between the fungal pathogen Cladosporium fulvum and Tomato
Dr. P. de Wit
The interaction between the pathogenic fungus Cladosporium fulvum and its only host tomato is a well established model system to study plant-fungus interaction that comply with a gene-for-gene relationship (De Wit, 1992). The fungus C. fulvum only invades the intercellular space of tomato leaves without forming specialized feeding structures. Apoplastic fluid (AF) isolated from infected leaves contains proteins that play a role in the communication between the two interacting organisms. We assume that the fungus requires pathogenicity factors to grow in planta and to prevent, or to inactivate, host defence responses. On the other hand, host resistance is based on recognition of the pathogen, probably resulting from a direct interaction between avirulence gene (Avr) products and resistance gene (Cf) products. How the fungus accomplishes and maintains compatibility during a successful infection, and which molecular processes determine host resistance are the main questions our laboratory focuses on.
Pathogenicity: Two putative C. fulvum pathogenicity genes are currently under investigation in our research group. These genes: ecp1 and ecp2 (Van Den Ackerveken et al., 1993) have been obtained following determination and purification of fungal proteins which are specifically found in apoplastic fluids of compatible interactions (Joosten and De Wit, 1988; Wubben et al., 1994). The expression of both ecp1 and ecp2 is strongly induced in C. fulvum while growing in planta (Wubben et al., 1994). The experimental procedures to obtain gene disruption via a double homologous recombination transformation system were first set up in C. fulvum to disrupt the Avr9 gene (Marmeisse et al., 1993). This allowed us to perform disruption on both ecp genes (Marmeisse et al., 1994; Van Den Ackerveken et al., unpubl. results). The resulting disruptants were first tested on tomato plantlets where no clear effects on pathogenicity were detected. We are now studying the disruptants on adult plants which represent a more natural situation for the fungus. In these studies, both disruptants, although still pathogenic, showed clearly altered phenotypes leading to a reduction in their pathogenic abilities. Thus the ecp genes seem to play a role in pathogenicity C. fulvum on adult tomato plants.
The results obtained with ecp1 and ecp2 disruptants support a "multigenic pathogenicity" theory where the fungus will be drastically affected in pathogenicity only with a cumulative knock-out of several pathogenicity genes. To test this hypothesis, we are currently constructing a double ecp1/ecp2 disruptant. For that purpose we used the parasexual cycle with both single disruptants as parental strains. Stable diploid colonies have been obtained following protoplast fusion and putative rehaploidized colonies are under investigation.
We are also in the process of isolating a third ecp gene: ecp3 whose product is also specifically found in apoplastic fluids of compatible interactions. In parallel, we have developed experimental procedures to separate chromosomes of C. fulvum by Pulse Field Gel Electrophoresis. First, this has allowed us to assign the chromosome on which the genes, we have isolated, is located. Secondly, this expertise can provide us with information about possible chromosomal rearrangements in transformants and disrupted strains.
Host Resistance: Two avirulence genes, Avr4 and Avr9, have been cloned from C. fulvum (Joosten et al. 1994a, Van Kan et al. 1991). The Avr4 and Avr9 genes are only expressed in planta and encode pre-pro-proteins of 135 and 63 amino acid (aa) residues, respectively, each containing a signal sequence for extracellular targeting. Following secretion, the proteins are proteolytically converted into cysteine-rich mature of AVR4 and AVR9 elicitor peptides of 105 and 28 amino acid residues, respectively. Analysis of C. fulvum strains virulent on Cf4 tomato genotypes showed the presence avr4 alleles carrying a point mutation which in most races, results in a replacement of one of the eight cysteine residues by a tyrosine residue. In only a few cases another aa residue, located between the fourth and fifth cysteine residue of the AVR4 peptide, was replaced, while in one case a nucleotide deletion was found giving rise to a frame shift (Joosten et al., unpubl. results). Although during infection all avr4 alleles of races virulent on Cf4 genotype are transcribed, none of the proteins encoded by these avr4 alleles have been detected in AF by western analysis (Joosten et al., unpubl. results). C. fulvum races virulent on Cf9 tomato genotypes lack the Avr9 gene completely (Van Kan et al., 1991).
The AVR9 peptide, isolated in large quantities from culture filtrate of an Avr9+ gene transgenic race of C. fulvum which constitutively produces AVR9 (Van den Ackerveken, 1993), is used for structure analysis and binding studies. Preliminary data on the tertiary structure of the AVR9 peptide reveal a compact barrel-like structure comprising 3 antiparallel b-strands connected with 2 loops and three disulfide bridges connecting all six cysteine residues (J. Vervoort, Dept of Biochemistry, WAU, unpubl. results). Using Potato Virus X (PVX)-derived constructs to express modified Avr9 genes, the relative importance of a specific aa residue is determined. Some modifications do not influence the necrosis-inducing activity of the modified AVR9 peptide, whereas others result in a decreased or increased elicitor activity (Vogelsang et al., unpubl. results). In order to study specific binding of the AVR9 elicitor to plasma membranes from leaves of Cf0 and Cf9 tomato genotypes, the peptide has been iodinated and plasma membranes have been isolated according to a two-phase-partitioning method. Initial results showed that high affinity binding sites for AVR9 are present on plasma membranes of both Cf9 and Cf0 tomato genotypes (Kooman-Gersmann et al., unpubl. results). Although Cf0 and Cf9 genotypes respond very differentially after AVR9 challenge, this differential response is not reflected in qualitative and quantitative differences in binding, but results from differential post-binding reactions. Currently, AVR9 binding to tomato membranes is investigated in more detail and a search has been started for high affinity binding sites for AVR9 in other plant species.
Injection of the purified AVR9 elicitor in Cf9 leaves induces acidic and basic PR protein accumulation, but basic PR proteins accumulated non-specifically probably in response to the injection event (Wubben et al., 1994). Immunolocalization studies showed that in incompatible interactions chitinases and 1,3-b-glucanases accumulate faster to higher levels than in compatible interactions (Wubben et al., 1992). However, in in vitro assay, germinated conidia of C. fulvum were not affected by chitinases and 1,3-b-glucanases (Joosten et al., 1994b). Thus the role of these PR proteins in the defence response of tomato against of C. fulvum, remains unclear. Cell suspension cultures produced from tomato lines, near-isogenic for a Cf resistance gene have been raised. Together with purified AVR4 and AVR9 elicitors, resistance gene-mediated, early defence responses will be studied, and components of the signal transduction pathway conducting defence responses will be identified.
The Avr9 gene is exploited to develop pathogen resistant plants according to a HR-based strategy (De Wit, 1992). Introduction of the Avr9 gene into a Cf9 tomato plant, under the transcriptional control of a pathogen inducible promoter might result in local HR and necrosis eventually leading to a pathogen resistant plant. Transgenic tobacco and tomato plants without the Cf-9 gene produce active AVR9 elicitor peptide (Hone et al., unpubl. results). Progeny plants derived from crosses between wild-type Cf9 tomato plants and transgenic Cf0 tomato plants expressing Avr9 show reduced plant growth, necrosis or complete plant death. Currently, transgenic tomato plants containing the Avr9 gene under the transcriptional control of a pathogen-inducible promoter are developed.
References:
De Wit PJGM. Molecular characterization of gene-for-gene systems in plant-fungus interactions and the application of avirulence genes in control of plant pathogens. Ann Rev Phytopathol 1992;30:391-418.
Joosten MHAJ, Cozijnsen TJ, De Wit PJGM. Host resistance to a fungal tomato pathogen lost by a single base-pair change in an avirulence gene. Nature 1994a;367: 384-386.
Joosten MHAJ and De Wit PJGM. Isolation, purification and preliminary characterization of a protein specific for compatible Cladosporium fulvum (syn. Fulvia fulva)-tomato interactions. Physiol Mol Plant Pathol 1988;33:241-253.
Joosten MHAJ, Verbakel HM, Nettekoven ME, Van Leeuwen J, Van Der Vossen RTM, De Wit PJGM. The fungal pathogen Cladosporium fulvum is not sensitive to chitinase and 1,3-b-glucunase defence proteins of its host, tomato. 1994b, submitted.
Marmeisse R, Van den Ackerveken GFJM, Goosen T, De Wit PJGM, Van Den Broek HWJ. Disruption of the avirulence gene avr9 in Cladosporium fulvum causes virulence on tomato genotypes with the complementary resistance gene Cf9. Molec Plant-Microbe Interac 1993;6:412-417.
Marmeisse R, Van den Ackerveken GFJM, Goosen T, De Wit PJGM, Van Den Broek HWJ. The in planta induced ecp2 gene of the tomato pathogen Cladosporium fulvum is not essential for pathogenicity. Curr Genet 1994;26:245-250.
Van den Ackerveken GFJM, Van Kan JAL, Joosten MHAJ, Muisers JM, Verbakel HM, De Wit PJGM. Isolation and characterization of two putative pathogenicity genes of Cladosporium fulvum. Molec Plant-Microbe Interact 1993;6:210-215.
Van Kan JAL, Van den Ackerveken GFJM, De Wit PJGM. Cloning and characterization of cDNA of avirulence gene avr9 of the fungal tomato pathogen Cladosporium fulvum, causal agent of tomato leaf mold. Molec Plant-Microbe Interact 1991;4:52-59.
Wubben JP, Joosten MHAJ, De Wit PJGM. Expression and localization of two in planta induced extracellular proteins of the fungal pathogen Cladosporium fulvum Molec Plant-Microbe Interact 1994;7:516-524.
Wubben JP, Joosten MHAJ, Van Kan JAL, De Wit PJGM. Subcellular localization of plant chitinases and 1,3-b-glucanases in Cladosporium fulvum (syn Fulvia fulva)-infected tomato leaves. Physiol Molec Plant Pathol 1992;41:23-32.
Wubben JP, Lawrence CB, MHAJ, De Wit PJGM. Differential induction of PR protein gene expression in tomato by Cladosporium fulvum and its race-specific elicitors. 1995, submitted.
For further information, please contact:
Prof.dr. P.J.G.M. de Wit, (or Richard Laug, Guy Hone, Matthieu H.A.J. Joosten, Paul Vossen, Miriam Kooman-Gersmann, Ralph Vogelsang, Ton J. Cozijnsen, Jacques Vervoort and Jos P. Wubben), Department of Phytopathology, Wageningen Agricultural University, Binnenhaven 9, 6709 PD Wageningen, The Netherlands, phone: (+31)-8370-83130, fax: (+31)-8370-83412. Biological Seed Coatings: the ECLAIR project and an industrial perspective
Mr. R.J. Scheffer
Four industries and four government-funded groups combined forces in an ECLAIR project to address the main factors relevant for practical application of rhizobacteria as inoculants for seed/plant establishment.
The project 'Biological inoculants for seed/plant establishment' had a total budget of 3.7 million ecu, with an EC contribution of 1.9 million ecu. Bacterial strains have been developed with strong biocontrol or growth improving qualities. Extensive greenhouse and field tests were carried out, and main factors relevant for production and formulation studied. It was shown that opportunities exist for practical biological control of Fusarium wilt diseases and of damping off caused by Pythium ultimum. The complexity of biological control became especially clear for the studies on the use of Burkholderia
(Pseudomonas) cepacia, which for corn have an extreme root colonizing potential. Seed treatments can result in an enhancement of emergence and growth of corn in field soils, which coincides with a decreased infection of roots by Pythium and Fusarium species.
Major steps have been taken into understanding synthesis and uptake of siderophores, molecules vital for competition for iron (Fe3+), a rare ion in the soil and on the root surface. Analysis of one specific strain of Pseudomonas putida revealed an extreme specialization in competition for iron, through a multitude of receptor genes and a complex network of signal perception and regulation of gene expression. The strain clearly gets a competitive advantage from this specialization. The complex nature of siderophore biosynthesis makes it difficult to increase production by a strain or to transfer the biosynthesis genes to other strains. However, the outer membrane receptors which mediate uptake of siderophore-bound iron can well be transferred and stably maintained in many pseudomonads. This can enhance their ability to utilize alternative siderophores and therefore increase their competitive ability under low iron conditions.
Also the genes controlling synthesis of a potent antifungal agent (2,4-diacetylphloroglucinol have been characterized. This metabolite exerts significant control of the damping off pathogen Pythium ultimum. At least one biosynthetic locus and two regulatory loci are relevant; their transfer into another strain converted this strain from an ineffective biocontrol strain into one that did control Pythium damping off in sugarbeet.
ECLAIR projects were pre-competitive in nature, therefore products and processes developed during the course of the program will typically require a further two to three years development before commercialization. Products based on the project will aim at two of the main scopes of the ECLAIR program: increasing the use and value of agricultural produce and/or improvement of the cost-effectiveness and quality of agricultural production. However, development of commercially successful 'biologicals' (being any biological product aiming at growth/yield improvement or biological control) is far from trivial. A few of the hurdles to be tackled during development of biological seed treatments are outlined below.
One key issue is the efficacy of biologicals in comparison with alternatives such as resistance in the host plant or chemical control. Resistance in the plant is attractive because of its often absolute character, but sources of resistance have to be available, the time frame for a breeding success is long and of course only the newly bred varieties express the desired resistance.
Novel agrochemicals combine a very good efficacy with a relatively limited environmental impact. However, replacement of older chemicals, with stronger negative environmental side effects, is difficult as long as they are available, especially because they tend to be off-patent and (therefore) cheap.
Biologicals in general have some very desirable properties of which two main ones of relevance here are their limited environmental impact and the ability of especially bacterial and fungal strains studied for seed and soil treatments to colonize the roots of their target crop. Colonization of roots brings the biological control agent at the place where it has to do its work: the interface between plant and environment, which is the main pocket of nutrient availability in the soil ecosystem and of course the place where any soil-borne plant pathogen will enter the plant root.
The fact that in natural ecosystems only a very limited amount of the plant biomass is being destroyed by pests and pathogens shows the potential for biological control; disease-suppressive soils are the well-studied examples where the soil ecosystem develops an effective control of plant pathogens even in agricultural fields. Pseudomonas
bacteria as studied in the ECLAIR project discussed above are thought to be of major importance in these disease-suppressive soils. Applications of such isolated strains have convincingly shown the possibilities for biological control by one or very few strains.
Problems encountered seem to be centred around two related demands for commercial biological control: it has to be reliable and it has to be robust. Two terms I use deliberately to emphasize that biological control should give not only a comparable degree of control in repeated experiments, but also under a variety of environmental conditions.
Clearly, biological seed treatments should, and will, find their place where alternatives are either not cost effective or not available. Combinations of efficacy, cost price and last but not least legislative restrictions do currently determine the products to be developed.
For more information, please contact: Mr. R.J. Scheffer, S&G Seeds B.V., P.O.Box 26, 1600 AA Enkhuizen, the Netherlands, phone: (+31)-2280-66178, fax: (+31)-2280-17144.
The Agro-Industrial Research Programmes
Dr. J. Azcon-Bieto
The application of modern biotechnologies to agriculture and agro-industry is a key area for research needs in Europe. Recent advances in plant physiology, biochemistry, genetics and molecular biology, particularly recombinant DNA technology, provide new opportunities for breeders, farmers and industrialists. It is expected that these technologies can have in the near future an important impact on the improvement of food and non-food crops, also by complementing traditional plant breeding activities.
The commercial possibilities of plant biotechnology appear to be rather high in the medium or long-term, but European companies are still hesitant to fully exploit these market possibilities due to uncertainties about political and regulatory issues, and also due to problems raised by the poor acceptability of biotechnology. Other problems can also be related to the efficiency of technology transfer from research projects to the industry, which is the main issue of this meeting.
Many international research projects related to plant biotechnology are funded by the EU, being included in programmes such as ECLAIR, FLAIR and BRIDGE (Second Framework Programme - FP2), BIOTECH and AIR (FP3), and important funding for this area will continue under the Fourth Framework Programme (1994-1998), particularly in the FAIR and BIOTECH II programmes. Specific research about "applications" of biotechnology to agriculture and agro-industry (e.g. food and non-food sectors) has been mainly catered for by the ECLAIR and AIR programmes, and currently by the AIR programme, complementing more fundamentally oriented research funded in Biotech programmes.
Efficient knowledge and technology transfer within EU research projects is normally increased by having an international partnership which includes on the one hand, research groups from universities and public research institutions, and on the other hand, groups from the private sector (e.g. large companies, SMEs, cooperatives). The direct involvement of one or more industrial partners in the projects can greatly facilitate the rapid communication of the results generated by the academic partners to the industrial partners and vice-versa, and can also significantly improve the strategic position of European companies towards future market possibilities of plant biotechnology. Exchanges of personnel between partners are also a very effective mechanism for knowledge transfer and for establishing good personal relationships.
A higher communication level in EU projects can take place when several individual projects can be linked through a formally established network around a common working theme. This "thematic network" could organize large coordination meetings with participants of several projects, workshops, training courses on technical and regulatory matters (e.g. patents), links with industrial platforms, etc. Dissemination activities can also be organized very effectively within thematic networks, overcoming difficulties which often occur in individual research projects, associated to the relatively small size of the projects. The FAIR programme offers possibilities to organize these thematic networks, and an example of theme could be the applications of plant biotechnology to improve food raw materials.
For more information, please contact: Dr. J. Azcon-Bieto, Commission of European Communities, DG XII-E2, SDME 2/30, AGRO-Ind.Res.Div., Rue de la Loi 200, B-1049 Brussels, Belgium, phone: (+32)-2296-6213, fax: (+32)-2296-4322.
In the discussion yesterday evening, we became aware that the information flow between industry and academia was not yet as frequent as we wished to, and we hope to find ways to stimulate this exchange. Therefore, in order to ameliorate the information flow, I would like to take the chance to present you three measures which are established in Biotechnology under the 4th Framework programme, however, we have the impression that they are not yet very well diffused in the scientific community of plant biotechnology.
These, as I call them "untapped possibilities" are the following:
1. Special measure for SMEs (Small and Medium sized Enterprises). The Commission has implemented preparatory awards (also called exploratory awards) in order to encourage and facilitate participation of small and medium sized enterprises in research activities covered by the Biotechnology programme. These preparatory awards help SMEs to set up future research projects in looking for partners, in designing a RTD project, in verifying novelty and in studying technical feasibility. A maximum amount of 45.000 ECUS can be granted by this award given that 2 independent SMEs from different EU member States are submitting the proposal. It seems very likely that in future a continuous review and granting mechanism will be installed by the Commission services for this kind of award. (Please contact Mr. R. Van Vliet: tel: (+32)-2295-3574, for detailed information).
2. Demonstration projects. For the first time in the 4th Framework programme, demonstration activities are implemented. The formal definition of a demonstration project is: "proving the technical viability of a new technology, together with, as appropriate, its possible economic advantage. The actions will be pre-competitive, and should as such focus on the application of new technologies and involve participation by both producers and users". The main purpose of demonstration projects is to help to exploit and disseminate scientific results in the European Community. All 8 areas of the Biotechnology programme are open for demonstration projects for the four calls under Framework-4. A total budget of 30 million ECUs is foreseen. Demonstration projects differ from research projects in their objectives:
to prove superiorness/pre-established level of performance of the new technology.
to demonstrate economical advantages.
These advantages can be the profit in industry, the efficiency of public services, the real benefits for consumers and even the improvement of public perception. (Please contact Mr. A. Herrero phone: (+32)-2295-4683 for further information).
3. Training grants. Continuous exchange of know-how and expertise is very important for researchers, especially in the rapidly changing field like Biotechnology. Therefore, for the period 1995-1998, a total budget of around 30 MECUs is available for training activities in Biotechnology. It is expected that roughly 350 grants can be awarded during this period. Grants include fellowships for pre-doctorates and post-doctorates as well as grants to establish researchers who come from a less-favoured region and who wish to return there after their post-doctorate abroad. Moreover, specially only in plant biotechnology the AMICA-project has a special budget for short term training fellowships allocated between one and six months duration.
All these grants are open for industry and offer the unique possibility that highly skilled personal is financed for his/her work in industry over a maximum period of 2 years. (Please contact Mr. A. Vassarotti, phone: (+32)-2295-8309, for further information).
We hope that these specific measures will increase the mobility of plant biotechnology industry across national boundaries and will promote full participation of companies in research and/or demonstration projects in EU funded contracts in the field of plant science.
For more information, please contact: Prof.dr. A. Hoeveler, Commission of European Communities, DG XII, Division Biotechnology, Rue de la Loi 200, B-1049 Brussels, Belgium, phone: (+32)-2295-6801, fax: (+32)-2295-5365.
Initiatives suggested by the Plant Industrial Platform.
1. A proposal (to be submitted to the EC) for a structured dissemination of scientific results will be prepared. A PIP task force will secure proper industrial input in this initiative. Goal is also to develop dissemination scenario's which avoid an overload of information, which are profitable for all participants and which do not obstruct research developments. Thematic networks could be the instrument of choice.
2. A PIP task force will be assigned to feed the discussion on industrial involvement in the definition of future Framework programmes and individual EU research programmes or projects. This activity will be the active follow-up of the PIP document: 'FW-4, Comments from the Plant Industrial Platform' (July 1994) and the discussion started during the May 1995 PIP workshop. This is considered a high priority in the light of the definition of Framework V.
3. PIP will continue to:
stimulate the formation of scientific/industrial consortia, and
to include industrial representatives in the board of such consortia.
At present PIP is represented in the PTP-AMICA board and, when funded during Framework-4 in FAIR, in the board of the NEO-DIET thematic network. An important responsibility of a board in these consortia is the proper dissemination of results, also to SMEs.
Commission contact person: Mr. Ciaran Mangan, Life Sciences and Technologies, Agro-Industrial Research, DGXII E-2, SDME 2/27, rue de la Loi, B-1049 Brussels.
In the PIP Newsletter #6 an overview was listed of the relevant titles of research projects from 20 programme clusters. Below, further details of projects of one of these clusters, Agriculture, is given. Information on other clusters, such as Biomass production, Crop inputs and Food safety, will be included in future PIP Newsletters.
Title: Lettuce for the next century: improved culture and product through genetic engineering. Coordinators: Dr. Ir.A.M.M. de Laat, D.J. van der Have B.V., Research Department Rilland, P.O. Box 1, NL-4410 AA RILLAND Tel.: (+31).1135.2151, Fax.: (+31).1135.2237 Objectives: Lettuce is an important vegetable crop throughout the E.C. As the crop is sensitive to attack by viruses, fungi and insects, current practice for lettuce culture needs massive chemical inputs. On 100,000 ha about 4,000 tons of agrochemicals are used with an estimated costs of 180 M ECU per annum.
As a matter of fact such measures are undesirable because of the negative environmental impact and risk residues in the final product. In addition, it is well documented that winter-grown greenhouse lettuce causes health constraints, due to high nitrate contents. To solve this problem imports over long distance has poor prospects due to the limited storability and high consumer standards for "quality". Over the past decades conventional plant breeding has made a contribution to reduce the problems mentioned above. Cultivars resisting various strains of Bremia (downy mildew) and viruses were widely introduced. Due to:
the fast-growing public concern about environmental pollution and food quality,
limited possibilities of conventional breeding to solve some of these problems and
the slow progress made by this approach, alternative methods should be investigated.
The proposed research envisaged to:
Reduce the need for insecticides by genetic protection against aphids, miners and viruses.
Reduce the need for fungicides (used mainly to control Bremia) by broad and durable resistance against fungi.
Improved quality of lettuce by reduced nitrate content.
Reduce losses/improve quality by increased storability.
Investigate the safety aspects of the resulting "novel food".
To exploit this research successfully, a multidisciplinary approach is essential. The different partners are highly complementary in their expertise and contribution to this project.
Title: ENITA -European network for Information technology in agriculture. Coordinators: Mr. Iver Thysen, Research Centre for Agriculture, DK -Tjele. Abstract: It is proposed to organize an open network for individuals and organisations working with research on and applications of information technology in agriculture. The purpose of the network is to facilitate communication and cooperation among researchers software house workers advisers and end-user representatives.
A suitable acronym for the network is ENITA: European Network for Information Technology in Agriculture.
Communication within ENITA will primarily be by the existing international electronic networks. The ENITA network will contain a bulletin board for communication between users information databases concerning ENITA members (individuals and organisations) and inventories concerning research and applications in information technology simulation models farm management programs etc. within agriculture. The information will be entered interactively and unsupervised by the users.
The information on each item in the database will be kept to a minimum in order to secure an easy access and communication from different computer types and communication links. The network will be implemented during the first year of the project and tested on a full operational scale during the following two years. A sub-contractor may be chosen for this purpose. At the end of the project the benefits in terms attracting potential users of the network will be evaluated.
Title: Development and testing of quantitative methods for research on agricultural systems and the environment. Coordinators: Mr. F.W.T. Penning de Vries, Dienst Landbouwkundig Onderzoek Centrum voor Agrobiologisch Onderzoek, NL - Wageningen. Abstract: To advance possibilities for quantitative research on agricultural systems and their environment, by improving systems research in participating institutes through exchange and standardization of concepts, approaches, knowledge, computer programs and data.
To reach the objectives, three actions are proposed:
creation of a small network of research groups already actively involved in quantitative research on agricultural systems; network members exchange information, set guidelines for standardization of modelling, participate in workshops, give seminars at laboratories of other members, and stimulate short visits of junior staff and students to other participants.
informing all European agricultural scientists actively and passively interested in systems research by a Newsletter presenting relevant progress, announcements of published methods, models, data sets and meetings.
The core of the Newsletter will be produced by the research groups participating in the network. It will be investigated whether the Newsletter, once well established, can also cover relevant news from other continents.
setting up and publishing a comprehensive inventory of relevant simulation models and simulation techniques on crops (without constraints, with water and/or nutrient shortage, with biological constraints), animal production, cropping systems, and farming systems. The inventory will include inventories of crop models already made in some countries, and will be initiated in the countries of the network participants and later expanded to other European countries.
Inclusion of models developed elsewhere in the world will be considered.
Title: Development of a strategy for cooperation and optimal documentation and supply of literature. Coordinators: Mr. M.B. Duisendstraal, Library Wageningen Agricultural University Dep of Ecological Agriculture, NL-Wageningen. Abstract: The overall aim is to improve possibilities for the development of ecological agriculture inside and outside the EU countries, by optimizing working conditions for researchers in the field, in terms of improving utilization of research results produced on ecological agriculture.
Furthermore, the following aims are stated:
To inventorize and to evaluate existing documentation practices for ecological agriculture.
To improve literature documentation services and document supply in the field of ecological agriculture.
To discuss and to clarify needs and demands concerning documentation services on ecological agriculture, and to include both producers (=documentation services) and consumers (=user groups in the field of research, education, extension service) in this discussion.
To develop future strategies for literature documentation on ecological agriculture.
To ensure that initiatives (databases, networks a.o.), which are and which will be established, are linked, in order to improve utilization and efficiency of offered efforts.
Furthermore, the aim is to emphasize the possibilities for continuation of initiatives by means of cooperation with CABI, Agris a.o..
Title: Research for the adaptation of oilseed crops management to them new requirements of the common agricultural policy: seed quality, environment. Cooperators: Mr. Andre Pouzet, Centre Technique interprofessionnel des oleagineux metropolitains, Paris, France. Objectives: The objectives of the concerted action are:
to improve the efficiency of national research on oilseed crops by scientific exchanges about methods and results,
to make easier the transfer of existing knowledge and know-how from one country to the others.
to help farmers in their adaptation to the new Common Agricultural Policy objectives and
to identify national and European Union oilseeds R&TD priorities and requirements.
Title: Control and assessment of agricultural data with a GPS- supported data collecting system (=CADCOS) Cooperators: Steffen Kuntz, Erwin Kayser-Threde GMBH, Munich, Germany. Abstract: One of the most cost intensive and time consuming tasks in environmental monitoring and control and/or the update of landuse databases in general is the field survey. A problem of similar importance is the quality control and the reliability of the data collected.
In this context the project is aimed to develop CADCOS to minimize such problems. The system will combine D-Gps with a PENTOP including GIS-facilities.
Research
Contributions
Nutrient Enhancement of Dietary Ingredients in European Trade (NEO-DIET).
A EU-FAIR Thematic Network Proposal:
Objectives. As the relationship between individual diet and health becomes clearer, the potential is growing for reducing morbidity and mortality, and consequently for increasing the quality of life, through appropriate and judicious food choices. There is clear evidence of strong links between diet and health in humans and of the complex manner in which the quantity, physical structure and chemical nature of food can all affect and determine health. In particular, an increasing number of minor dietary components [plant secondary metabolites] are known to be involved in the control of disease and the modulation of healthy physiological performance.
Much of this evidence is epidemiological, rather than clinical, and as such offers little, if any, explanation of the relationships between diet and health at the cellular, genetic and molecular level. Until these relationships are better elucidated, reliable and specific advice cannot be provided to those consumers for whom it would be most appropriate; the absence of such mechanistic knowledge also inhibits the ability of the food industry in developing and exploiting products with enhanced nutritional benefit. There is, however, sufficient evidence to suggest that if such dietary advice is provided for consumers, and followed, then there could be a major impact on the social and economic costs of degenerative disease.
Specific objectives of this Thematic Network, within a pan-European context, are to:
1. increase understanding of the mechanisms determining the physiological and metabolic effects of individual plant-derived components of the diet, and to better understand the effects of structure and composition of such individual component on human health and well-being,
2. use such information to enable:
plant breeders, biotechnologists, agronomists and the primary production sector to provide material with enhanced nutritional benefit.
the food manufacturing/retail industry to more effectively exploit healthy product opportunities.
consumers to receive appropriate information so as to make informed decisions on their dietary habits.
3. to create an effective and responsive Network structure which will assist the Commission in its future development of diet and health RTD components at a European level.
Understanding these aims, which taken together would provide a basis for increasing the competitiveness of the European agri-food industry and reducing the costs of health care within the Community, will be a further integration of European activities across the spectrum of plant, food and nutrition research. This will encourage the participation of industry and, in particular SMEs, at an early stage and provide an effective platform for exploiting opportunities which the Commission provides for trans-national training and collaboration.
For further information, please contact: dr. Roger Fenwick, Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, UK, phone: (+44)-1603-507-723, fax: (+44)-1603-255-000.
The role of cyclic nucleotide systems in higher plant signal transduction mechanisms.
Request for industrial partners.
It is intended to submit a proposal under the Plant Cell and Molecular Biology section of the Biotechnology Programme of Framework IV to examine the role of cyclic nucleotide systems in higher plant signal transduction mechanisms. The existing consortium of partners, comprising four university research groups led by Drs. Chiatante (Campobasso, Italy), Newton (Swansea, U.K.), Palme (Koln, Germany) and van Onckelen (Antwerpen, Belgium), possesses extensive experience of cyclic nucleotide research and the requisite facilities for research in this area, ranging from the physiological and molecular biological aspects to the biochemical and mass spectrometric levels.
While cyclic nucleotide systems are well established as key metabolic regulators in mammals and are the targets of a large number of highly profitable pharmaceutical products, there is no clear understanding of cyclic nucleotide function in plants. Indeed many of the early publications were somewhat ambiguous and as a result for many years the mere existence of cyclic nucleotides in plants was a controversial topic. However recent unequivocal evidence that cyclic nucleotides and the related synthetic and degradative enzymes are endogenous to plants has been reported, and in the last 18 months evidence of plant cyclic nucleotide functions, for example in mediating phytochrome action in chloroplast development, in stimulating phytoalexin production in cellular defence against fungal pathogens, and in regulation of ion-channels, has been published.
The aim of our programme is to elucidate the functions of cyclic nucleotides in Arabidopsis by monitoring changes in cyclic nucleotide concentrations under manipulated conditions, and by purifying and characterising the cyclase, phosphodiesterase, kinase and binding proteins. Knowledge of the regulatory roles and of the sensitivity of the enzymes to effectors will provide a basis for manipulation of the cyclic nucleotide systems, of potential value in applications relating for example to growth optimization and resistance to environmental stress and pathogens.
For further information please contact the group coordinator: Dr. R.P. Newton, Biochemistry Group, School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, United Kingdom. Fax: (+44)-1792-295-447, Email: r.p.newton@swansea.ac.uk
A consortium of European laboratories was formed with a common interest in signal transduction in plants. The aim of this consortium is to concentrate on a limited number of signal transduction pathways controlling specific developmental processes. The different members of the consortium possess a wide range of expertise and the sharing of these expertise between laboratories is expected to greatly accelerate the elucidation of each of the chosen pathways. By concentrating on signal transduction from plant growth regulators and on the well characterised self-incompatibility system, it is hoped to identify key control points for a wide range of developmental processes in plants. Once identified, these control points will be of great interest from an applied point of view as targets for modulating plant growth and development.
Examples of some of the processes influenced by the pathways under investigation and of potential applications are: plant shape, flowering time, plant breeding (F1 hybrids), plant reproduction, plant-pathogen interactions, hybrid seeds, senescence, somatic embryogenesis, juvinility, micropropagation, rooting of cuttings, artificial seeds (dormancy), seed germination, brewing.
The consortium is very interested in interacting with industrial companies on this project. Such interactions could potentially be funded via the EU, indeed an application for funding through the Framework-4 programme is being prepared. A second meeting of consortium members will take place in Orsay, Paris on the 7th of July. For further information, contact dr. Mark Cock, UMR 9938 INRA-CNRS-ENS-Lyon, Reconnaissance Cellulaire et Amelioration des Plantes, Ecole Normale Superieure de Lyon, 46 Allee d'Italie, F-69364 Lyon Cedex 07, France, phone: (+33)-7272-8611, fax: (+33)-7272-8600, Email: mark.cock@ens-lyon.fr
Molecular exploitation of a plant pararetrovirus: cauliflower mosaic virus
Our research objectives are to exploit cauliflower mosaic virus (CaMV) for basic research in plant molecular biology and pathology and its applications in biotechnology. We have considerable experience and a long background in this area and view fundamental research as an essential foundation for industrial application.
CaMV offers much scope for exploitation as a research and biotechnological tool. It has already provided one of the most widely used genetic elements in transgene technology (the 35S promoter) which our group played a major role in identifying (Covey et al., 1981. Nucl. Acids Res. 9, 6735-6747). Other useful viral genetic elements await further development and exploitation. CaMV also infects the Cruciferae which includes major crop species (eg oilseed rape) and Arabidopsis. CaMV has potential for delivering foreign genes to plants for short term expression utilising new strategies. CaMV offers many possibilities to study the molecular pathology of host/virus interaction and resistance phenomena. It is also a unique molecular tool for understanding fundamental molecular process of gene expression and replication. Since CaMV replicates by reverse transcription, it can provide a new angle to investigate replication of important human and animal viruses such as HIV and hepatitis B virus. Specific research areas are:
Plant resistance. Identification of crucifer genes involved in viral pathogenesis and characterisation of host factors involved in terminating CaMV replication (see Covey et al. 1990. Proc. Natl. Acad. Sci. 87, 1633-1637).
Exploitation of CaMV genetic elements in biotechnology including CaMV replicon-based plant gene vectors as molecular tools and for manufacture of novel products in plants.
Development of methods and processes to suppress multiplication of retroviruses and pararetroviruses.
For more information, contact: Dr. S.N. Covey, Department of Virus Research, John Innes Centre, Norwich NR4 7UH, UK, phone: (+44)-1603-452-571, fax: (+44)-1603-456-844, E-mail: covey@bbsrc.ac.uk.
Development of probes to monitor the process of somatic embryogenesis in crop species
The Embryogenesis group in the Department of Molecular Biology started in 1984. At present it consists of 2 undergraduate students, 1 visiting scientist, 3 PhD students, 3 post-docs and 1 technician, all with temporary contracts. Permanent positions are 1 technician and 1 group leader. Funding is primarily through the Dutch Organisation of Fundamental Research and the EC. Primary research interests and projects currently in progress are:
The cloning and characterization of genes expressed in single embryogenic cells in carrot. In this project we have generated a series of marker genes for which we are currently developing vital detection systems.
Mode of action of secreted proteins that influence somatic embryogenesis. In this project we concentrate on the plant substrates for endochitinases that have been shown to rescue embryogenesis in the carrot cell line ts11.
Development of reliable tissue culture and somatic embryogenesis systems in Arabidopsis with the ultimate goal of a genetic dissection of the formation of somatic embryos.
Employing enhancer trap/gene trap lines to obtain genes expressed in and effecting the formation of the protoderm in Arabidopsis zygotic embryos.
An area of research that we want to develop is to study particular zygotic embryo mutants of Arabidopsis under in vitro conditions. Development of accurate and reliable probes to monitor the process of somatic embryogenesis in crop species. Additional contacts are Dr. A.J. Schram, S&G Seeds, Enkhuizen, the Netherlands and prof.dr. G. Jrgens, Univ. Tbingen, Germany.
For further enquiries, please contact dr. Sacco C. de Vries, Agricultural University Wageningen, Department of Molecular Biology, Dreijenlaan 3, 6703 HA Wageningen, the Netherlands,
phone: (+31)-8370-84325 / 82036, fax: (+31)-8370-83584, E-mail: sacco.de.vries@mac.mb.wau.nl
Transgenic approaches to reduce nitrate accumulation in the vegetative tissue of legumes and other crops.
Nitrate can be accumulated in the vegetative tissues of plants used for human consumption. It would be desirable to reduce this nitrate accumulation, in order to increase the nutritional quality and to fulfil the EU regulations on maximal nitrate contents of foods. A concerted action program on biotechnology approaches to control nitrate content is already developed on target species such as lettuce, or under preparation for other crops such as endive, spinach, or carrot. The goals of the research project will use two different approaches: reduction of nitrate storage by competition with the nitrate assimilation process (plant overexpressing the metabolic pathway) or reduction of nitrate uptake (antisense inhibition of one of the molecular events involved in nitrate uptake or translocation in vegetative tissue). In the species under study the identification of appropriate promoters conferring the stable and specific expression of transgenes in the tissue of interest will be screened and tested by reporter gene techniques. Selected promoters will then be used to drive the expression of nitrate reductases or antisense constructs against transporters in the different crops. Proper evaluation by field trial experiments will be required to assess the efficiencies of the two technologies.
For more information, contact dr. Michel Caboche, Laboratoire de Biology Cellulaire, INRA Versailles, Rue de Saint-Cyr, F-78026 Versailles Cedex, phone: (+33)-13083-3060, fax: (+33)-13083-3111, Email: caboche@versailles.inra.fr
Identification and Characterization of Plant Defense Genes.
The aim of our project is the identification and characterization of plant defense genes. In particular, we are interested in genes whose expression is induced in response to infection by fungal pathogens, and/or by wounding. In this respect, we have already identified several defense genes of maize, genes whose expression is induced either during seed germination or in leaves of adult plants.
Currently we are involved in the following studies:
cloning of new genes induced by fungal infection and by wounding. Analysis of the coordinated expression among them.
characterization of DNA regulatory sequences (promoters) by transient expression after transformation of plant protoplasts, and bombardement of plant tissues.
function of defense proteins by expression in E.coli and "in vitro" assays for determination of antifungal activities.
Results obtained, besides the basic interest of knowing the genes involved in the plant defense response, have potential biotechnological applications for the improvement through genetic engineering of the properties of resistance to pathogen infection (fungi) or insect attack (wound-inducible genes) in plants.
A second subject of research interest is the development of methods for detection of DNA of plant pathogenic fungi by Polymerase Chain Reactions. At the present moment we have designed PCR primers that specifically detect the DNA from the fungus Fusasium moniliforme, a natural pathogen of maize (unpublished results). This methodology can be successfully used be seed producers. Important crop losses are caused by fungal diseases, and many of them are seed transmitted. The extent to which seed contamination can be reduced is dependent upon the development of efficient screening systems.
For more information, contact: dr. Blanca San Segundo, Departamento Genetica Molecular, Centro de Investigacion y Desarrollo de Barcelona, Consejo Superior de Investigaciones Cientificas (CSIC), Jordi Girona 18, E-08034 Barcelona, Spain, phone: (+34)-3-400-6128, fax: (+34)-3-204-5904.
Our group is integrated in the Department of Plant Biology at the University of Salamanca, in the Plant Physiology Section, which has a long expertise in the physiology of germination. Recently, we have cloned several cDNAs to mRNAs induced and repressed during the germination of chickpea.
cDNAs encoding two cysteine proteinases are induced during germination (Plant Molec Biol 25, 207-205, 1994). The clone CAPR10a, coding for a member of the IPR (Intracellular Pathogenesis Related) proteins, is induced during seed development at the time of desiccation probably by the phytohormone ABA and repressed in the first four days of germination. ABA and ethylene are antagonists in its regulation during germination. Also induced during germination and before radicle emergence is a mRNA coding for the enzyme S-adenosyl methionine (SAM) synthetase. SAM synthetase is involved in the aminoacid metabolism but also in ethylene, polyamine and lignin biosynthesis. Induction of this mRNA before germination may be a common mechanism for increased lignin synthesis prior to radicle emergence, well conserved in seed plants. The cloning of the promoters of these genes may be of interest to direct the synthesis of desired proteins in different stages of seed development or germination.
For more information, contact Dr. Emilio Cervantes, Departemento de Biologia Vegetal (Fisiologia Vegetal), Facultad de Biologia Universidad de Salamanca, E-37007, Spain, phone: (+34)-23-294-400, ext. 1951, fax: (+34)-23-294-682, E-mail: ecervant@gugu.usal.es
Since 1988 TNO the Netherlands Organisation for Applied Scientific Research is active in the research area Plant Biotechnology. The research activities are concentrated in the Centre for Phytotechnology which covers the collaboration between TNO and Leiden University in this field. Both funded research projects e.g. by EU, and contract research for companies is performed. The projects vary from routine analysis to custom-made long term research programmes. The research activities are communicated to the market in three products:
Seed Physiology,
Plant Product Quality
Crop Improvement.
This contribution will deal with the product Crop Improvement. This research area includes the following subjects:
in vitro plant production form various tissues resulting in e.g. clonal propagation, somatic embryos and/or artificial seeds, (doubled) haploids (DH)
in vitro development of e.g. embryos (rescued), seeds, flowers
in vitro selection of traits
somatic hybridization
stable genetic modification using the particle bombardment approach or application of Agrobacterium spp., specialised on monocotyledonous and recalcitrant dicotyledonous species
molecular, biochemical, immunological and microscopical analysis of plants for quality determination
development of (molecular) markers for desired characteristics
isolation of genes coding for traits of interest
In this issue only one of the above-listed research topics will be highlighted and will deal with microspore-derived plant formation and its applications.
Anther and microspore culture can be of interest for plant breeding with one or more of the following purposes. First of all fixation of traits is possible in homozygous regenerants, and molecular analysis can be performed on segregating haploid populations. Further, microspore-derived plants can be used as homozygous parental lines of F1 hybrids. Moreover, genetic variability can be generated by sexual crossings at the diploid level of tetraploid crops. Besides, microspores or microspore-derived embryos can be used as target cells for genetic transformation or in vitro selection.
In general, there are two approaches for production of DH. The method of choice will depend in part on the purpose of application. A first approach is to culture as many anthers as possible, with the expectation that some will yield DH. This approach can be followed, if the aim is production of DH for e.g. increased genetic variability. However, the alternative approach should be seriously considered, especially when feasibility of DH application in breeding is concerned. The alternative approach is to define the material used and to characterise key factors involved in induction of plant formation. The ultimate aim is to search for that specific microspore, which is competent of plant formation under specific conditions. In TNO we perform research since 1988 with the model plant barley according to this alternative; since more recently a cell bio