Isolation of rapeseed genes expressed early and specifically during development of the male gametophyte.

A cDNA subtraction and differential hybridization strategy was used to isolate cDNAs expressed early during male gametophyte development in the important crop species Brassica napus. Three cDNAs, corresponding to genes highly and specifically expressed at the tetrad and microspore stages, are presented here. The analysis of one of them, named BnM3.4, by in situ hybridization, showed that it is expressed specifically and at a high level in the rapeseed microspore. The specificity in its profile of expression is most likely transcriptionally controlled as a similar pattern of expression was also observed in Arabidopsis thaliana plants transformed by the BnM3.4 promoter fused to the reporter GUS-coding sequence. The putative BnM3.4 promoter contains three dispersed copies of a motif described previously in the promoters of several genes expressed in the male gametophyte. The BnM3.4 gene encodes a predicted novel proline-rich protein of 23.4 kDa which may interact with cytoskeletal components or have a structural role in the cell wall.

Biosynthesis of gamma-linolenic acid in developing seeds of borage (Borago officinalis L.).

delta 6-desaturation of [14C]linoleoyl-CoA or [14C]oleoyl-CoA leading to the synthesis of gamma-linolenic acid was studied in vitro with microsomal fractions from developing seeds of Borago officinalis. Time course of the reaction, effects of protein and precursor concentrations and nucleotide requirements were examined. These parameters allowed us to improve the in vitro delta 6-desaturation assay. We observed that the precursors were acylated mainly in phosphatidylcholine, diacylglycerol and triacylglycerol, and then desaturated. NADH was absolutely required when [14C]oleoyl-CoA was the precursor, but not when [14C]linoleoyl-CoA was the precursor although it stimulated the reaction. The in vitro delta 6-desaturase activity was found mainly in phosphatidylcholine, associated with enriched endoplasmic reticulum membranes (ER) from embryos. No activity was observed in ER from seed coat or seedling. During maturation of the seeds, delta 6-desaturase reached its highest activity 14 to 16 days after pollination.

Agrobacterium-mediated transformation of Asparagus officinalis L. long-term embryogenic callus and regeneration of transgenic plants.

Twenty-three independent kanamycin resistant lines were obtained after cocultivation of longterm embryogenic cultures of three Asparagus officinalis L. genotypes with an Agrobacterium tumefaciens strain harboring ß-glucuronidase and neomycin phosphotransferase II genes. All the lines showed ß-glucuronidase activity by histological staining. DNA analysis by Southern blots of the kanamycin resistant embryogenic lines and of a plant regenerated from one of them confirmed the integration of the T-DNA.

Stable Accumulation of Modified 2S Albumin Seed Storage Proteins with Higher Methionine Contents in Transgenic Plants.

We present the results of two sets of experiments designed to express high methionine proteins in transgenic seeds in three different plant species. In the first approach, two chimeric genes were constructed in which parts of the Arabidopsis 2S albumin gene 1 (AT2S1) were fused at different positions to a Brazil nut 2S albumin cDNA clone. Brazil nut 2S albumin was found to accumulate stably in transgenic Arabidopsis, Brassica napus, and tobacco seeds. In the second approach, methionine-enriched AT2S1 genes were constructed by deleting sequences encoding a region of the protein which is not highly conserved among 2S albumins of different species and replacing them with methioninerich sequences. Introduction of the modified AT2S1 genes into three different plant species resulted in the accumulation of the methionine-enriched 2S albumins in all three species at levels reaching 1 to 2% of the total high salt-extractable seed protein.

Expression of the 2S albumin from Bertholletia excelsa in Brassica napus.

The methionine rich 2S albumin seed storage protein of Bertholletia excelsa has been expressed in seeds of Brassica napus (rapeseed). A chimeric gene driven by the soybean lectin 5' flanking regions was used to produce a fusion protein consisting of the soybean lectin signal peptide and the propeptide of the Brazil nut 2S albumin. Several transgenic plants were studied at the RNA and protein levels; in each case the chimeric gene was expressed and the protein detected at levels ranging from 0.02% to 0.06% of total protein. Transcriptional studies in a particular transgenic plant show that expression of the gene is tissue specific and developmentally regulated during seed maturation. The endogenous napin genes and the introduced gene are regulated differently, with expression of the chimeric gene paralleling that seen when the soybean lectin gene is expressed in other plant species. Western analysis using antibodies to Brazil nut 2S albumins resulted in the detection of a protein whose size is consistent with correct processing of the precursor.

Differential Expression of the Arabidopsis 2S Albumin Genes and the Effect of Increasing Gene Family Size.

We studied the expression of the four genes encoding 2S albumin seed storage proteins (at2S1 to at2S4) in Arabidopsis thaliana. All four genes followed similar temporal profiles throughout development, but at2S2 and at2S3 were expressed at significantly higher levels than at2S1 or at2S4. In situ hybridization showed that at2S2 to at2S4 mRNAs were present throughout the embryo, whereas at2S1 was expressed at levels similar to at2S2 and at2S3 in the embryo axis but at only insignificant levels in the cotyledons. The different members of the gene family are, thus, likely to be regulated by different combinations of cis-acting elements, but it cannot be ruled out that post-transcriptional factors play a role. We studied the effect of enlarging the gene family by introducing an extra, nearly identical gene driven by the promoter of at2S1. The data were consistent with a model in which the expression of at2S2 to at2S4 is not affected by that of at2S1, and in which, at least at low copy numbers of the introduced gene, there is no limit on the overall amount of RNA that the at2S gene family can produce.

Selection of somatic hybrids between diploid clones of potato (Solanum tuberosum L.) transformed by direct gene transfer.

Five diploid potato clones have been transformed by electroporation of protoplasts with different selectable markers. The resulting diploid regenerated plants have been used in somatic hybridization. It has been shown that hybrid cell selection on the basis of antibiotic or herbicide resistances brought by the two parents of fusion is an efficient method for the recovery of tetraploid somatic hybrids.

Use of a transient expression assay for the optimization of direct gene transfer into tobacco mesophyll protoplasts by electroporation.

Plasmid DNA was transfected into tobacco mesophyll protoplasts by electroporation. Transfection efficiency was estimated, using a transient expression assay based on the measurement of chloramphenicol transacetylase activity or by scoring colonies expressing resistance to paromomycin, an aminoglycoside related to kanamycin. Under conditions of cell survival superior to 50% after electroporation, transient expression signals and transformation efficiencies were found to be proportional. Factors affecting the efficiency of transformation were studied. A clear-cut optimum voltage (250-300 V/cm) was detected. Among various salts tested, potassium chloride was the best electrolyte. No improvement of electroporation efficiency was obtained by a heat-shock (45 degrees C/5 min) treatment prior to electroporation or by the presence of polyethylene glycol in the electroporation medium. The physiological state of plants used as the protoplast source significantly affected the transfection ability of the resulting protoplasts. These results are discussed and compared to previously published procedures.