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GENETIC TRANSFORMATION OF PLANT MITOCHONDRIA : DEVELOPMENT OF A GENERAL STRATEGY

Objective

GIVEN THE PIVOTAL ROLE OF THE MITOCHONDRION IN THE ENERGY ECONOMY OF THE CELL, AND THE FACT THAT IT CONTAINS ITS OWN GENETIC SYSTEM, IT HAS BEEN NEGLECTED AS A SOURCE OF GENETIC DIVERSITY BY THE PLANT BREEDER.
THE ABILITY TO TRANSFORM PLANT MITOCHONDRIA WOULD ALLOW :
- THE DEVELOPMENT OF A BROADER BASE FOR THE BREEDING PROGRAMMES USED IN THE PRODUCTION OF HYBRID CROP PLANTS;
- AN EVALUATION TO BE MADE OF THE POTENTIAL FOR TRANSFERRING GENES WHICH MAY BE CAUSALLY RELATED TO THE CYTOPLASMIC MALE STERILE (CMS) PHENOTYPE; SUCCESS IN THIS AREA COULD SHORTEN THE LONG BREEDING PROGRAMME WITHIN SPECIES AND INTRODUCE THIS COMMERCIALLY USEFUL TRAIT (CMS) INTO CROPS NOT POSSESSING IT.
A genetic transformation system for plant mitochondria would allow functional analyses of the mitochondrial genome and its products, and would open the way for engineering of the genome to modify mitochondrial metabolism, or to introduce cytoplasmic male sterility (CMS) into new crops and varieties. Chimaeric chloramphenicol acetyltransferase (CAT) genes were constructed that would be expressed specifically in plant mitochondria and thereby confer resistance to an antibiotic (chloramphenicol), allowing a transformed cell to be selected. Established deoxyribonucleic acid (DNA) delivery systems (direct DNA transfer and Agrobacterium tumefaciens) were used in attempts to transform plant protoplasts, and potentially transformed, chloramphenicol (CAP) resistant calli were recovered.

CAP was chosen as the most suitable of the selective agents to recover transformed plants. However, CAP was shown to be a relatively inefficient agent for selection of transformed plant cells. Therefore, the conditions under which CAP can be used to recover large numbers of nuclear transformants following transformation of tobacco with chimaeric CAT genes were defined. Several chimaeric CAT genes were constructed containing the transcription and translation initiation signals from plant mitochondrial genes, together with sequences sharing sequence homology with the mitochondrial genome of tobacco to promote integration by homologous recombination. It was shown that these genes are not expressed by the nuclei of tobacco protoplasts and calli. More than 2E8 protoplasts were transformed by naked DNA and A tumefaciens mediated transformation, and potential transformants are being analysed for evidence of integrated foreign DNA.
EXPERIMENTAL EVIDENCE INDICATES THAT TRANSCRIPTION INITIATION SIGNALS INVOLVED IN GENE EXPRESSION IN MITOCHONDRIA ARE DIFFERENT FROM THOSE NEEDED FOR NUCLEAR GENE EXPRESSION. SELECTION FOR MITOCHONDRIAL TRANSFORMATION IN PLANT CELLS, USING AGROBACTERIUM TI-PLASMIDS-DERIVED VECTORS, SHOULD THEREFORE RELY ON GENES DESIGNED TO FUNCTION IN MITOCHONDRIA. A CHIMERIC GENE, ACTIVE IN MITOCHONDRIA ONLY, WILL BE CONSTRUCTED IN EDINBURGH BY THE COMBINATION OF A MITOCHONDRIAL PROMOTOR WITH DNA SEQUENCES CODING FOR AN ENZYME WHICH INACTIVATES A DRUG TOXIC TO MITOCHONDRIA (CHLORAMPHENICOL). THE LABORATORY OF KOLN WILL ADD HIS EXPERIENCE IN GENETIC TRANSFORMATION OF PLANTS WITH TI-PLASMIDS VECTORS.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

University of Edinburgh
Address
King's Buildings Mayfield Road
EH9 3JH Edinburgh
United Kingdom

Participants (1)

MAX-PLANCK-GESELLSCHAFT ZUR FOERDERUNG DER WISSENSCHAFTEN E.V.
Germany
Address
Carl-von-linné-weg 10
50829 Koeln