Objective
THE STRUCTURE AND FUNCTION OF THE GENETIC SYSTEMS OF PLANT MITOCHONDRIA (MT) IS ONLY BEGINNING TO BE ELUCIDATED, BUT THE TECHNOLOGIES OF MOLECULAR BIOLOGY AND OF SOMATIC CELL GENETICS ARE NOW ALLOWING RAPID ADVANCES TO BE MADE. FROM THIS WILL COME THE ABILITY TO MANIPULATE THESE GENETIC SYSTEMS FOR CROP PLANT IMPROVEMENT. THE FIRST BENEFITS WILL BE SEEN IN THE ABILITY TO INDUCE AND MANIPULATE CYTOPLASMIC MALE STERILITY (CMS) WHICH IS OF CRITICAL IMPORTANCE IN THE PRODUCTION OF HYBRID FORMS OF MANY IMPORTANT AGRICULTURAL AND HORTICULTURAL CROPS WITHIN THE EEC. IN THE LONGER TERM WILL COME A MORE DETAILED UNDERSTANDING AND ULTIMATELY IMPROVEMENT OF THE METABOLIC PROCESSES WHICH UNDERLIE CELL ENERGETICS AND IN WHICH THE MITOCHONDRIA PLAY A CRITICAL ROLE.
The mitochondrial genome of higher plants cooperates with the nuclear information to drive the mitochondrial functions, which are determinant for the plant energy and metabolism. Cytoplasmic male sterility (CMS) is a mitochondrially encoded dysfunction, which is routinely used by plant breeders to produce economically important F1 seeds (cereals, vegetables).
For a better understanding of the real information encoded by the mitochondrial genome research focussed on:
basic knowledge, such as molecular organisation, expression, recombination and replication mechanisms in fertile plants;
the comparison of fertile and cms lines of economically important plants;
the development of cytoplasmic selectable markers;
the design of new strategies to modify the mitochondrial content of a higher plant;
combined classical molecular biology techniques supplemented with somatic cell fusion, somaclonal variations and mechanical microinjection.
The research gave the following results:
identification of plasmid and native deoxyribonucleic acid (DNA) molecules;
characterization of sequences involved in replication (ORI, D) and recombination;
molecular characterization of CMS plants at the DNA level and protein level;
development of cytoplasmic selectable markers.
Mechanical microinjection of total organelles and mitochondria into protoplasts was used to construct new plants. A nuclear gene alters the maternal inheritance of organelles. In vitro culture of immature embryos induced reorganizations of the mitochondrial genome through nuclear genes and led to regenerated plants with permanently altered genome. This reorganization is related to regeneration capacity. The recently demonstrated ribonucleic acid (RNA) editing mechanism was confirmed at the protein level.
THE RESEARCH WILL BE CARRIED OUT AS PART OF AN INTEGRATED EFFORT BETWEEN 5 LABORATORIES (UCL, LOUVAIN; INRA; DIJON, FRANCE; JOHN INNES INSTITUTE, NORWICH, UNITED KINGDOM; FREE, UNIVERSITY, AMSTERDAM, NEDERLAND; UNIV. PARIS, ORSAY, FRANCE)
THIS JOINT A EFFORT INVOLVES THE FOUR ESSENTIAL STEPS :
A) STUDY OF THE STRUCTURE AND EXPRESSION OF THE MITOCHONDRIAL GENOME IN DIFFERENT PLANT SPECIES.
B) TRANSFER OF MITOCHONDRIA.
FOR PET UN IA BOR.ETUNIA WITH A LOW FREQUENCE CHLOROPLASTARE TRANSFERED THROUGH THE FERTILIZATION PROCESS. THIS HAS BEEN DEMONSTRATED USING EITHER THE PLASTID INHERITED YELLOW VIRESCENT MUTANT OR A CT DNA BAMH| FRAGMENT FROM P. PARODII. THREE ASPECTS ARE UNDER INVESTIGATION :
1- IS THE GENETICCONTROL OF THE ORGANELLE WETHER OF THE SPOROPHYTIC OR OF THE GAMETIC ONE. THE SEGREGATIONS ARE NOW IN AGREEMENT WITH A GAMETOPHYTIC CONTROL. MOREOVER IT SEEMS THE CONTROL IS UNDER ONE SINGLE GENE.
2- THE MAPPING OF THE TRANSMITTOR-GENES LOCATED ON THE SEVEN CHROMOSOMES OF PETUNIA.
3- IN ORDER TO CHECK TRANSMISSION OF MITOCHONDRIA WITH CHLOROPLAST A RECEPTOR GENOTYPE WAS CONSTRUCTED HAVING YV AND A CMS CYTOPLASM-SELECTION FOR STABILIZATION OF REVERTED FERTILITY IS STILL UNDER WORK.
C) TRANSFORMATION.
D) CHARACTERIZATION OF PLASMIDS AND RNA CONTAININGPARTICLES.
1- CHARACTERIZATION OF PLASMIDS FROM BEET SPECIES
ALL THE BEET SPECIES SCHECKED DISPLAYED A PLASMID SET ALLOWING US TO RECOGNIZE THE PATELLARES WITH ABOUT 1-1.2KB LONG CIRCULAR PLASMID FROM THE VULGARES WITH ABOUT 1.5KB LONG CIRCULAR PLASMID.
AMONG THE VULGARES SECTION THE GARDEN BEETSDISPLAYED A PLASMID OF THE SAME SIZE AS PO BUT STAINING NO HOMOLOGY WITH IT. THAT PLASMID AS A PROBE.
THE TEST IS RAPID, CHEAPER THAN FIELD CONTROL AND CAN BE USED BY BEET BREEDERS TO CONTROL THE COMMERICAL SEED STOCK PURITY.
2- RNA CONTAINING PARTICLES IN VICIA FABA
AN ELISA TEST WAS DEVELOPPED TO DETECT SPECIFICALLY THE PRESENCE OF VIRUS LIKE PARTICLE (VLP) ANTIGENS. THE TEST IS NOW USED TO CONTROL CMS DURING THE BACK CROSS PROCESS TO CONVERT MALE LINES INTO CMS ONES.
INSITU IMMUNOLABELLING WITH THE PROTEIN A GOLD LABELLING TECHNIQUE WAS SET UP. BECAUSE OF NON REACTING PARTICLES A STATISTICAL MODEL WAS DEVELOPPED TO POINT OUT THE HIGHER AFFINITY OF ANTIGENS (X30) TO VLPS THAN TO UNSPECIFIC PARTICLES.
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Data not availableCall for proposal
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CSC - Cost-sharing contractsCoordinator
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France