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THE TRANSFORMATION OF CHLOROPLASTS WITH AGROBACTERIUM TUMEFACIENS AND NAKED DNA

Objective

THE TRANSFORMATION OF CHLOROPLASTS WILL CREATE NEW AND INDISPENSABLE MEANS FOR THE FUNDAMENTAL RESEARCH OF CHLOROPLAST PHYSIOLOGY AND DEVELOPMENT. BECAUSE THE CHLOROPLAST GENOME IS RATHER SMALL AND WELL CHARACTERIZED, THE GENETIC ENGINEERING OF CHLOROPLASTS WILL HAVE DIRECT ECONOMIC APPLICATIONS (HERBICIDE RESISTANCES, AMINO ACIDS METABOLISM, IMPACT ON PHOTOSYNTHESIS,...)
Manipulation of chloroplasts by genetic engineering would enable modification of the photosynthesis system. There are 2 ways to do so: transport proteins from the cytoplasm into the chloroplast; change the chloroplast genetic material by transformation.

Various genes and vector constructions that could be used to select for rare chloroplast genome transformants were tested. This was tried by addition of a selectable marker gene to the chloroplast genome or by the exchange of a resident gene for its mutant allele conferring elevated tolerance to antibiotics. For the transformation experiments, direct deoxyribonucleic acid (DNA) transfer by conventional techniquesas well as by the biolistic approach and Agrobacterium mediated transformation were used.

With the different methods and gene constructs, resistant candidates were recovered. The recovery frequency for proposed gene insertions was very high; that of proposed gene conversions very low and close to that expected for spontaneous mutations. DNA analysis demonstrated that all candidates with proposed insertions were nuclear transformants, whereas the second class of candidates was spontaneous mutants tolerant of the selective agent. It can be concluded that chloroplast transformation must be a very rare event occurring at an even lower frequency than spontaneous mutation if Agrobacteria or conventional direct DNA delivery experiments are used. However, initial results with the shotgun approach suggest that here the frequency of tolerant candidates is significantly higher than that for spontaneous mutants.
THERE ARE NOW EVIDENCES THAT AGROBACTERIUM CAN TRANSFER DNA INTO CHLOROPLASTS. THIS PROJECT IS CONCERNED BY THE STANDARDIZATION OF A SYSTEM TO ENGINEER CHLOROPLAST DNA.

THE LABORATORY IN GENT WILL CONSTRUCT SEVERAL CHIMERIC GENES COMBINING CHLOROPLAST-SPECIFIC PROMOTORS AND ANTIBIOTIC RESISTANCE GENES. THE LABORATORY IN KOLN WILL CLONE THE CHLOROPLAST MUTANT GENES THAT CONFER RESISTANCE TO LINCOMYCIN AND STREPTOMYCIN. AGROBACTERIUM STRAINS HARBOURING TI-PLASMIDS ENGINEERED WITH THESE SEQUENCES WILL BE USED IN GENT TO INFECT PLANT PROTOPLASTS AND LEAF DISKS. THE SAME CONSTRUCTS WILL BE USED IN KOLN TO DIRECTLY TRANSFORM PLANT PROTOPLASTS USING NAKED PLASMIDS. BOTH LABORATORIES WILL ANALYZE TRANSFORMED PRODUCTS FOR THE REGULATION OF EXPRESSION AND STABILITY OF GENES INSERTED INTO THE CHLOROPLAST GENOME.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

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

Participants (1)

PLANT GENETIC SYSTEMS N V
Belgium
Address

Gent