Georges Freyssinet

The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens.

Photorhabdus luminescens is a symbiont of nematodes and a broad-spectrum insect pathogen. The complete genome sequence of strain TT01 is 5,688,987 base pairs (bp) long and contains 4,839 predicted protein-coding genes. Strikingly, it encodes a large number of adhesins, toxins, hemolysins, proteases and lipases, and contains a wide array of antibiotic synthesizing genes. These proteins are likely to play a role in the elimination of competitors, host colonization, invasion and bioconversion of the insect cadaver, making P. luminescens a promising model for the study of symbiosis and host-pathogen interactions. Comparison with the genomes of related bacteria reveals the acquisition of virulence factors by extensive horizontal transfer and provides clues about the evolution of an insect pathogen. Moreover, newly identified insecticidal proteins may be effective alternatives for the control of insect pests.

The fate of recombinant plant DNA in soil

Abstract Genetically modified tobacco containing a bacterial gentamicin resistance gene ( aacC1 ) was developed and cultured in a field to monitor the fate of plant DNA in soil for up to 3 years. We determined whether DNA persisted as extracellular molecules or as genetic information in soil bacteria, by extracting total DNA after in situ lysis or bacterial DNA after physical extraction of bacteria from the soil. The recovered DNAs were used in PCR reactions with primers targeting the aacCl gene coding for gentamicin resistance, tobacco-specific sequences in the ribulose biphosphate carboxylase/oxygenase gene and sequences specific to genetically modified tobacco inside the T-DNA. Plant DNA was found to persist in the soil for several months, but no longer than 3 years. There was no evidence that the resistance gene from the plant had been transferred to soil bacteria, although primers targeting the aacC1 gene provided positive PCR signals in all soil samples.

Production of isolated somatic embryos from sunflower thin cell layers.

We describe here a two step procedure which allows the easy isolation of somatic embryos from Sunflower (Helianthus annuus L.) hypocotyl tissues. Thin cell layers composed of the epidermis plus 3 to 6 parenchyma cell layers were incubated for 5 days in a basal Murashige and Skoog medium using an auxin to cytokinin weight ratio of 1/1. The epidermis layers were then transferred to a Gamborg medium containing a high level of sucrose. After one week of incubation in this medium, many somatic embryos started to be released from the parental epidermal tissue. Even though the germination of these embryos is difficult, we have been able to induce secondary embryos and regenerate fertile plants.

Fertile plant regeneration from sunflower (Helianthus annuus L.) immature embryos

Abstract We describe here a simple technique which allows the production of fertile plants from the hypocotyls of immature embryos. Immature embryos of 0.1–5 mm are incubated on a medium which slows down the development of the zygotic embryo and allows the differentiation of somatic embryo-like structures. These structures are isolated and transferred to a medium allowing development of shoots and roots. Young plantlets, transferred to soil, develop to maturity and are then self-pollinated and seed-set. This technique has been tested for six different cultivars and might be general for immature sunflower embryos. Seeds have been tested in the field and no variants have been detected suggesting that with this technique extensive somaclonal variation does not occur.

Regulation of the maizerab17 gene promoter in transgenic heterologous systems

The maizerab17 gene is expressed in different plant parts in response to ABA and osmotic stress (J. Vilardellet al., Plant Mol Biol 14 (1990) 423–432). Here we demonstrate that 5′ upstream sequences of therab17 gene confer the appropriate patterns of expression on the chloramphenicol acetyl transferase (CAT) reporter gene in transgenic tobacco plants, as well as in protoplasts derived from cultured rice cells. Specifically, a CAT construct containing a large 5′ upstream fragment ofrab17 (−1330/+29) results in high levels of CAT activity in embryos, leaves and roots of transgenic plants subjected to water stress or ABA treatment. Transient expression assays in rice protoplasts transfected with CAT genes fused torab17 promoter deletions indicate that a 300 bp DNA fragment (−351/−102) is sufficient to confer ABA responsiveness upon the reporter gene. Furthermore, a 100 bp sequence (−219/−102) is capable of conferring ABA responsiveness upon a minimal promoter derived from the 35S CaMV promoter. Gel retardation experiments indicate that maize nuclear proteins bind to this fragment. This region of 100 bp contains a sequence (ACGTGGC) which has been identified as an abscisic acid response element in studies of other ABA-responsive plant genes.

Crops resistant to oxynils: from the laboratory to the market

Abstract Herbicide resistance was one of the first traits introduced to crops by transgenic methods. The main reason for this was the fact that it is generally controlled by a single gene and that a large body of biochemical information is available on the mode of action, mechanisms of resistance and metabolism of herbicide molecules. Oxynil herbicides, such as bromoxynil and ioxynil, destroy dicot plants. In order to extend the selectivity of these herbicides to some of the major dicot crops such as cotton and oilseed rape, a gene coding for a nitrilase which specifically detoxifies these herbicides was isolated in collaboration with Calgene (Davis, CA, USA). The coding region of the gene was linked to regulatory DNA sequences for expression in plants and transferred into various dicotyledonous species, such as tobacco, tomato, cotton, oilseed rape, carrot, potato and eggplant. All plants containing the bacterial nitrilase gained resistance to oxynil herbicides. Field experiments have already been conducted with cotton, oilseed rape, tobacco and potato. They show the transgenic cultivars behave identically as the non-transgenic ones and that herbicide resistance at the agronomic level has been achieved. Experiments are in progress for the commercial development of some of these lines.

High expression of transgene protein in Spirodela

The monocot family Lemnaceae (duckweed) is composed of small, edible, aquatic plants. Spirodela oligorrhiza SP is a duckweed with a biomass doubling time of about 2xa0days under controlled, axenic conditions. Stably transformed Spirodela plants were obtained following co-cultivation of regenerative calli with Agrobacterium tumefaciens. GFP activity was successfully monitored in different subcellular compartments of the plant and correlated with different targeting sequences. Transgenic lines were followed for a period of at least 18xa0months and more than 180 vegetative doublings (generations). The lines are stable in morphology, growth rate, transgene expression, and activity as measured by DNA–DNA and immunoblot hybridizations, fluorescence activity measurements, and antibiotic resistance. The level of transgene expression is a function of leader sequences rather than transgene copy number. A stable, transgenic, GFP expression level >25% of total soluble protein is demonstrated for the S. oligorrhiza system, making it among the higher expressing systems for nuclear transformation in a higher plant.

Nucleotide sequence and characterization of a maize cytoplasmic ribosomal protein S11 cDNA

We isolated a Zea mays cDNA encoding the 40S subunit cytoplasmic ribosomal protein S11. The nucleotide sequence was determined and the derived amino acid sequence compared to the corresponding Arabidopsis thaliana protein showing an homology of 90%. This ribosomal protein is encoded by a small multigene family of at least two members. The mRNA steady-state level is about one order of magnitude higher in rapidly growing parts of the plant such as the roots and shoots of seedlings compared to fully expanded leaf tissue.

Transformed calli obtained by direct gene transfer into sunflower protoplasts

Helianthus annuus protoplasts were transformed with the plasmid pCaMVNEO (Frommet al. 1986) conferring kanamycin resistance to plant. Transformed calli were selected with a frequency of 4 calli for 106 treated protoplasts. DNA was extracted from kanamycin resistant calli. Analysis of this DNA shows the presence of the NPTII gene.

Callus and embryoid formation from protoplasts of Helianthus annuus

Sunflower hypocotyl protoplasts have been isolated and cultured. Optimum plating density for cell division and colony formation was in the range of 5 to 7×104 cells/mi in an agarose medium supplemented with BAP (1 mg/l) and NAA (1 mg/l). Plating efficiency was 60% after 21 days of culture. In the resultant culture a mixed population of calli and embryoids was observed. Thirty seven percent of the cell clusters exhibited a developmental pattern similar to an embryoid. Many stages of embryogenesis were observed in the same cultures.