Colette Tourneur

Transformation of Pakchoi (Brassica rapa L. ssp. chinensis) by Agrobacterium infiltration

Transgenic pakchoi (Brassica rapa L. ssp. chinensis) plants were obtained in the progeny of plants infiltrated by an Agrobacterium tumefaciens strain carrying a gene for resistance to the herbicide phosphinotricin (Basta). Genetic analysis demonstrates the transmission of the herbicide resistant trait to the progeny. Molecular analyses show that the transgene was inserted in the plant genome and expressed. This work demonstrates that the infiltration transformation method originally devised for Arabidopsis thaliana can be adapted for other crucifer species and opens up the possibility of genetic engineering of pakchoi, an important vegetable plant.

Expression of the mutantArabidopsis thaliana acetolactate synthase gene confers chlorsulfuron resistance to transgenic poplar plants

The mutant acetolactate synthase (crs1-1) gene fromArabidopsis thaliana, which confers resistance to the herbicide chlorsulfuron, was transferred to a hybrid poplar (Populus tremula×P. alba) using twoAgrobacterium-mediated transformation methods (co-inoculation and co-cultivation). Two different constructs were used. In one, the mutantcrs1-1 gene was placed under the control of its own promoter, and, in the other, this gene was under the control of the duplicated cauliflower mosaic virus 35S promoter (70 promoter). The transformation efficiency ranged from 22 to 32% of the tumours in co-inoculation and from 67 to 77% of the stem explants in co-cultivation experiments. The usefulness of the herbicide chlorsulfuron as a selectable marker gene was also demonstrated. Successful genetic transformation was verified by Southern and northern analyses and enzyme activity. Plants carrying thecrs1-1 mutant gene under the control of the 70 promoter showed high levels of transcription and activity whereas plants carrying the nativecrs1-1 gene showed low levels of expression. However, transgenic plants expressing each of the chimaericcrs1-1 genes are completely resistant to high doses of chlorsulfuron in greenhouse tests.

Restriction maps and homologies of the three plasmids of Agrobacterium rhizogenes strain A4

Agrobacterium rhizogenes strain A4 is a virulent agropine-type strain possessing three plasmids: plasmid a (pArA4a, 180 kb) is not necessary for plant transformation, plasmid b (250 kb) is the root-inducing plasmid (pRiA4), and plasmid c (pArA4c) is a cointegrate of pArA4a and pRiA4. The total plasmid DNA (pArA4) of strain A4 was cloned in the cosmid pHSG262 and the library obtained was used to establish BamHI maps of the three plasmids. The plasmids a and Ri have an apparently identical region and a partly homologous region, and are different in the remaining regions including their origins of replication. Another agropine-type A. rhizogenes strain, HRI, bears only one plasmid, which is the Ri plasmid (pRiHRI). pRiHRI and pRiA4 present the same restriction maps for a great part, but are different in a region of 48 kb; however, this region of pRiHRI is found unmodified in pArA4a and may have a role in the virulence of the bacteria. The comparison between the restriction maps of the plasmids of strain A4 leads us to propose that the recombination event leading to pArA4c formation occurs within the identical regions of pArA4a and pRiA4. In addition, the comparison with the already established map of pRiHRI suggests that strain HRI could have been derived from a recombination event between the two homologous regions of pArA4c with subsequent loss of the smaller plasmid.

Transfer of a 4.3-kb fragment of the TL-DNA of Agrobacterium rhizogenes strain A4 confers the pRi transformed phenotype to regenerated tobacco plants

Abstract Two strategies were used to transfer into tobacco a 4.3-kb fragment of the TL-DNA of the Ri plasmid of Agrobacterium rhizogenes strain A4. In the liposome-mediated procedure a plasmid containing a neomycin phosphotransferase II (NPT II) gene conferring kanamycin resistance and another plasmid containing the 4.3-kb Eco RI fragment (pRiA4 Eco RI-15) were co-transferred into the tobacco genome. In the Agrobacterium transformation procedure, a micro-Ri vector containing a kanamycin resistance gene and the same pRiA4 fragment was used to transform tobacco leaf fragments. Kanamycin resistant plants were regenerated in both cases. They present a phenotype similar to that of plants regenerated from hairy roots induced by A. rhizogenes , that is wrinkled leaves, reduced apical dominance and ability to form hairy root on leaf fragments. In one plant (Ka158), the organization, expression and transmission to the progency of the inserted foreign DNA were analyzed more precisely.

Over-expression of acetolactate synthase confers resistance to valine in transgenic tobacco

Abstract Previous reports described a high level of resistance to chlorsulfuron resulting from the transfer into tobacco of the mutated acetolactate synthase (ALS) gene from the csr-l mutant of Arabidopsis. Here we report a comparison of metabolic changes resulting from the expression of the csr-l gene in transgenic tobacco, either under the control of its own promoter or under the control of the 35S promoter with a duplicated enhancer (p70). The Arabidopsis csr-l gene with the native promoter confers an average 300-fold increase in resistance to chlorsulfuron in tobacco without significant modification of the amount of ALS activity in the absence of the herbicide and without cross-resistance of valine, a feed-back inhibitor of the enzyme. The expression in tobacco of the csr-l coding sequence under the control of the p70 promoter confers an average 1500-fold increase in resistance to chlorsulfuron, with an increase by up to 12-fold in the amount of ALS activity in the absence of the herbicide and resistance to exogenous supply of valine, with an increase by up to 10-fold in the amount of valine required to inhibit bud regeneration from leaf discs. However, no correlated increase in the content of free branched chain amino acids in leaves was observed, suggesting that subsequent steps of the metabolic pathway might limit the synthesis of valine, leucine and isoleucine.

Some Biological and Molecular Properties of Pepper Veinal Mottle Virus Isolates Occurring in Tunisia

The potyviruses, PVY (potato virus Y) and PVMV (pepper veinal mottle virus) were identified by serological and symptom diagnosis in pepper (Capsicum annuum L.) in Tunisia during 1995–1997. Both were known to be major constraints to pepper production in North Africa. Pepper veinal mottle virus (PVMV) has not been reported previously in North Africa, but PVMV was found to be widely spread in peppers throughout Tunisia. By using reverse transcription followed by polymerase chain reaction (RT-PCR), we have now amplified the PVMV coat protein gene of two Tunisian isolates. DNA polymorphism, determined by restriction fragment length polymorphism (RFLP) of the coat protein gene, revealed two patterns distinguishing PVMV from PVY. Within PVMV isolates, molecular and biological properties showed no polymorphism suggesting that all isolates are representative of one PVMV strain occurring in Tunisia.

Expression of genes in transgenic plants from bicistronic transcriptional units

Abstract Bicistronic mRNAs encoding either the potato virus Y (PVY) coat protein or the Bacillus thuringiensis CryIC protein followed by the selectable marker gene neomycin phosphotransferase II ( npt II) were introduced by electroporation into tobacco protoplasts. The initiation codon for the npt II gene was located 44 or 39 nucleotides downstream of the termination codon of the PVY coat protein or B. thuringiensis cryIC gene, respectively. Plants selected using paromomycin (50 μ g/ml) were shown to express the PVY coat protein by Western blot analysis or the B. thuringiensis CryIC protein by an insect feeding bioassay. Transgenic plants were shown, by Northern analysis, to produce transcripts of either construct without rearrangement. A significantly reduced level of npt II activity was demonstrated in transgenic seedlings of the T1 and T2 generation either by germination on agar containing 75 μ g/ml kanamycin or by npt II activity assays. These results demonstrate that transgenes of interest can be expressed from a bicistronic transcriptional unit and that plants expressing the transgene can be selected by monitoring for activity of a distal marker gene.

Nucleotide sequence comparison of the 3′ terminal region of the genome of pepper vein mottle virusisolates from Tunisia and Ivory Coast

Summary. Three Tunisian PVMV isolates identified in pepper and tomato fields and one isolate from Ivory Coast were submitted to biological and molecular analysis. Phenotypically, Tunisian isolates induced mild symptoms while the Ivory Coast one is more aggressive on tobacco. As no PVMV sequence data are available, detailed sequence comparisons of coat protein gene (CP) were made. No nucleotide or amino acid changes in this region could be related to the pathogenicity of the isolates analysed. With the aim to increase our molecular understanding of the biological properties, we have sequenced the 3′-non translated region (3′NTR). Results suggest that this region of the RNA genome may be involved in the modulation of disease symptoms.