THE CELL CULTURE AND GENETIC MANIPULATION OF 4 ECONOMICALLY IMPORTANT CROP PLANTS WILL BE EXAMINED, AND ALL DEVELOPMENTS ACHIEVED WITH A MODEL SPECIES (NICOTIANA PLUMBAGINIFOLIA) WILL BE APPLIED TO THE CROP SPECIES BY THE FOLLOWING LABORATORIES :
- POTATO AND RELATED SPECIES : HARPENDEN, GRONINGEN, WAGENINGEN
- TOMATO : PAVIA.
- SUGAR BEET : BRUSSELS.
- RAPE AND RELATED SPECIES :VERSAILLES.
THE ULTIMATE OBJECTIVE BEING THE INTRODUCTION INTO CLASSICAL PLANT BREEDING PROGRAMMES OF UNCONVENTIONAL CELLULAR AND MOLECULAR APPROACHES OF GENETIC TRANSFORMATION.
A methodology for the induction of micronuclei, containing one of a few metaphase chromosomes, has been developed. Micronucleated protoplasts can be fractionated using gradient centrifugation, yielding microprotoplasts with only a fraction of the total genome. Microprotoplasts can be fused with recipient protoplasts in order to achieve partial genome transfer.
New techniques of manipulation of the genome of higher plants are being developed and adapted to plants of agronomic importance. In an integrated approach, the following cell biotechnology methods were developed:
regeneration systems from single cells to plants;
introgression of foreign genetic material via protoplast fusion;
isolation of single chromosomes;
selection of mutant cell lines and plants;
elaboration of direct gene transfer techniques to introduce deoxyribonucleic acid (DNA) into plant following these manipulations;
application of methods developed with a model system (essentially Nicotiana plumbaginifolia) to the crop species Solanum sp, tomato and cereals.
Using these techniques the following results were obtained: regeneration of plants from explants of various crops; production of useful somaclonal variants of tomato and potato; isolation, culture and regeneration from protoplasts (single, wall free cells), especially in the case of barley for which green plantlets have been obtained; production of both symmetric and asymmetric (partial) somatic hybrids, using both chemical fusion and electrofusion.
Such methods were used to introduce useful agronomic characters (virus resistance genes in potato) and to develop asymmetric hybrids where only a limited amount of the donor genome is present. Further research involved the direct transfer of genes into protoplasts (DGT) by electroporation and chemical treatment, associated with transient expression study and analysis of the stable integration of the introduced genes. Techniques for the isolation of micronuclei as well as methods for transfer of organelles by microinjection were also perfected.
GENETIC MANIPULATION OF PLANT CELLS EMPLOYS AN ARRAY OF METHODS AND TECHNIQUES FOR THE TRANSFER OF GENETIC ELEMENTS AND COMPLEXES VARYING FROM WELL DEFINED DNA FRAGMENTS INCORPORATED INTO PLANT DNA VECTORS TO COMPLETE NUCLEI BY MEANS OF CELL FUSION. IN BETWEEN THE TWO EXTREMES, OPTIMAL TRANSFER OF E.G. NAKED DNA AND CELL ORGANELLES REQUIRES THE FURTHER DEVELOPMENT OF CELLULAR ENGINEERGING FOLLOWED BY INVESTIGATIONS INTO INTEGRATION AND EXPRESSION OF THE TRANSFERRED MATERIAL IN THE RECIPIENT CELL. THE DEVELOPMENT OF THIS APPROACH IS THE CENTRAL PART OF THIS PROPOSAL.
IN AN INTEGRATED APPROACH, THE FOLLOWING METHODS FOR GENETIC MANIPULATION OF CROP PLANTS WILL BE EXAMINED :
- ISOLATION AND CHARACTERIZATION OF SELECTABLE MUTANTS (BRUSSELS, PAVIA, VERSAILLES),
- ANALYSIS OF THE CELL CYCLE AND SYNCHRONISATION (WAGENINGEN, PAVIA, VERSAILLES),
- CELL-MEDIATED TRANSFER (HARPENDEN, GRONINGEN, BRUSSELS, PAVIA),
- CHROMOSOME-MEDIATED TRANSFER (WAGENINGEN),
- DNA-MEDIATED TRANSFER (HARPENDEN, GRONINGEN, BRUSSELS, PAVIA),
- LIPOSOME-MEDIATED TRANSFER (WAGENINGEN, VERSAILLES),
- REGENERATION TECHNIQUES (HARPENDEN, PAVIA),
- CYTOLOGICAL AND MOLECULAR ANALYSIS OF PUTATIVE TRANSFORMANTS (ALL LABORATORIES).
Funding SchemeCSC - Cost-sharing contracts
AL5 2JQ Harpenden, Herts