Coupled transcript and metabolite identification: insights on induction and synthesis of resveratrol in peanut, wild relatives and synthetic allotetraploid.

Resveratrol is an antioxidant that is a promising antitumoral, cardioprotective and neuroprotective agent. It has been found in a restricted number of plants including peanut (Arachis hypogaea L.) and its wild relatives. The objective of this study was to understand the relationship between resveratrol content and the expression of putative resveratrol synthase genes in four Arachis genotypes. Two diploids and two tetraploid were analyzed. Contents of resveratrol on non- and UV-treated leaves were estimated using HPLC. Resveratrol synthase (RS) was analyzed using RT-qPCR with primers developed in this study. Sequences of six Arachis species were amplified using two degenerated primer pairs that were designed based on Arachis and general RS available at GenBank. Those sequences were used to qPCR primers design. Test and control leaves were collected from plants cultivated in greenhouse and three biological replicates were evaluated for each genotype. The synthesis of resveratrol in leaves was induced by treatment with UV for 2.5 h. All genotypes studied synthesized resveratrol. Concentrations ranged from 193.66 µg/g in synthetic allotetraploid to 371.97 µg/g in A. duranensis. Natural and induced allotetraoploids showed lower levels of resveratrol than their diploid parents. Untreated samples did not produce significant amounts of resveratrol. The analysis of resveratrol content and levels of RS mRNA allowed the identification of one gene induced by the UV treatment. The data showed different amounts of RS in the different genotypes suggesting early and late response to the UV induction in the different species. The understanding of the variation found among species will help to identify species that have high resveratrol content and their ideal pos-induction times. This also will allow analysis of other tissues where high levels resveratrol would be very important, such as in seeds.

Inheritance of foreign genes in transgenic bean (Phaseolus vulgaris L.) co-transformed via particle bombardment.

Exploiting the biolistic process we have generated stable transgenic bean (Phaseolus vulgaris L.) plants with unlinked and linked foreign genes. Co-transformation was conducted using plasmid constructions containing a fusion of the gus and neo genes, which were co-introduced with the methionine-rich 2S albumin gene isolated from the Brazil nut and the antisense sequence of AC1, AC2, AC3 and BC1 genes from the bean golden mosaic geminivirus. The results revealed a co-transformation frequency ranging from 40% to 50% when using unlinked genes and 100% for linked genes. The introduced foreign genes were inherited in a Mendelian fashion in most of the transgenic bean lines. PCR and Southern blot hybridization confirmed the integration of the foreign genes in the plant genome.

Oncogene arrangement in a shooty strain of Agrobacterium tumefaciens.

The Agrobacterium tumefaciens nopaline strain 82.139 induces non-teratogenic shooty tumours on several plant species. We have determined the position of the T-region oncogenes in a 11.4 kb Xba I fragment which shows a general organization similar to its pTiC58 counterpart. Sequence analysis of the 4.7 kb right part of this fragment allowed us to identify the pTi82.139 ipt, 6b and nos coding sequences. pTi82.139 lacks the 6a gene, which lies between the ipt and 6b genes in pTiC58. The intervening region between the 6b and the nos genes contains an additional ORF with homology to ORF 21 (transcript 3') from the TR-DNA of octopine strain pTi15955.

Factors influencing transient gene expression in bean (Phaseolus vulgaris L.) using an electrical particle acceleration device.

The parameters influencing transient expression of the betaglucuronidase gene in bean embryonic axes, cotyledons, apical meristems and leaves were evaluated after gene delivery with an electrical particle acceleration device. A calciumspermidine procedure for coating gold particles with DNA resulted in higher levels of GUS expression with lower concentrations of gold particles compared with a calcium phosphate procedure. The DNA concentration, distance between the discharge chamber and the retaining screen and the vacuum in the apparatus also influenced gene delivery. Sections prepared for light and electron microscopy showed the localisation, within target cells, of gold particles used to deliver the DNA. Immunolocalization of foreign gene expression within cells confirmed an even distribution of gene product throughout the cell cytoplasm.

Transgenic poplars: expression of chimeric genes using four different constructs.

Leaf or stem explants of a hybrid poplar clone (Populus tremula X Populus alba), sensitive to Agrobacterium tumefaciens, were co-cultivated either by an octopine or a nopaline disarmed A. tumefaciens modified strain. Transformed poplar shoots were readily regenerated from explants. The protocol was improved using the nopaline disarmed strain C58/pMP90 with the binary vector pBI121. This protocol was then used to test three other vectors. The first one, possessing a nptII gene fused to the CaMV 19S promoter, permitted regeneration of transformed shoots in presence of 50 to 100 mg/l kanamycin. The two other vectors carried an additional nptII gene under the control of the CaMV 35S or CaMV 35S promoter with a double enhancer sequence (CaMV 70). CaMV 70 promoter provided consistently higher level of gene expression than the other promoters in both callus and leaf tissues.

An alternative approach for gene transfer in trees using wild-type Agrobacterium strains.

Micropropagated shoots of three forest tree species, poplar (Populus tremula x P. alba), wild cherry (Prunus avium L.) and walnut (Juglans nigra x J. regia), were inoculated each with six different wild-type Agrobacterium strains. Poplar and wild cherry developed tumors that grew hormone-independently, whereas on walnut, gall formation was weak. On poplar and wild cherry, tumors induced by nopaline strains developed spontaneously shoots that had a normal phenotype and did not carry oncogenic T-DNA. From these observations, we have established a co-inoculation method to transform plants, using poplar as an experimental model. The method is based on inoculation of stem internodes with an Agrobacterium suspension containing both an oncogenic strain that induces shoot differentiation and a disarmed strain that provides the suitable genes in a binary vector. We used the vector pBI121 carrying neo (kanamycin resistance) and uidA (beta-glucuronidase) genes to facilitate early selection and screening. Poplar plants derived from kanamycin-resistant shoots that did not carry oncogenic T-DNA, were shown to contain and to express neo and uidA genes. These results suggest that wild-type Agrobacterium strains that induce shoot formation directly from tumors can be used as a general tool for gene transfer, avoiding difficult regeneration procedures.

Identification of different agrobacterium strains isolated from the same forest nursery.

Several Agrobacterium strains isolated from the same forest nursery from 1982 to 1988 were compared by serological, biochemical, and DNA-DNA hybridization methods. Similarities among strains belonging to biovar 2 were observed by indirect immunofluorescence, whereas biovar 1 strains showed serological heterogeneity. Electrophoretic analysis of bacterial envelope-associated proteins showed that few bands appeared in the strains belonging to biovar 1, whereas many proteins appeared in the case of biovar 2 strains. Chromosomal DNA was analyzed with six random C58 chromosomal fragments. None of the six probes hybridized to the DNA of the two biovar 2 strains. One of the probes gave the same hybridization pattern with all biovar 1 strains, whereas the other probes yielded different patterns. The vir regions were closely related in the different pathogenic strains. The T-DNA and replication regions were less conserved and showed some variations among the strains.