Stable transformation of cultured cells of the liverwortMarchantia polymorpha by particle bombardment

Suspension-cultured cells (A-18 line) of the liverwortMarchanta polymorpha were bombarded by a pneumatic particle gun with plasmid pCH harbouring the hygromycin phosphotransferase (HPT) gene (hpt) under the control of the cauliflower mosaic virus (CaMV) 35 S promoter and the nopaline synthase polyadenylation region. Nine weeks after bombardments, 128 hygromycin-resistant calluses were obtained from an approximate total of 7×10⁶ cells. Ten cell lines chosen randomly were analysed further. Southern blot analysis showed that all of the ten lines contain thehpt gene in the genome, demonstrating that these lines are transformants. An HPT enzyme activity assay confirmed the expression of the gene in all of the transformant lines.     

Gene transfer to cereal cells mediated by protoplast transformation

Summary Direct gene transfer to cereal cells was achieved by transformation of protoplasts with naked DNA. Protoplasts isolated from cultured cells of Triticum monococcum were incubated in the presence of polyethylene glycol (PEG) with circular and linear plasmid DNA. The pBR322-derived plasmid, pBL1103-4, contained a selectable chimeric gene comprising the protein coding region of the Tn5 aminogly-coside phosphotransferase type II gene (NPT II), the nopaline synthase promoter (pNOS) and the polyadenylation signal of the octopine synthase gene. Transformed cells were selected in medium containing kanamycin and identified by detection of aminoglycoside phosphotransferase II activity.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Multiple resistance to sulfonylureas and imidazolinones conferred by an acetohydroxyacid synthase gene with separate mutations for selective resistance

Summary The acetohydroxyacid synthase (AHAS) gene from the Arabidopsis thaliana mutant line GH90 carrying the imidazolinone resistance allele imr1 was cloned. Expression of the AHAS gene under the control of the CaMV 35S promoter in transgenic tobacco resulted in selective imidazolinone resistance, confirming that the single base-pair change found near the 3 end of the coding region of this gene is responsible for imidazolinone resistance. A chimeric AHAS gene containing both the imr1 mutation and the csr1 mutation, responsible for selective resistance to sulfonylurea herbicides, was constructed. It conferred on transgenic tobacco plants resistance to both sulfonylurea and imidazolinone herbicides. The data illustrate that a multiple-resistance phenotype can be achieved in an AHAS gene through combinations of separate mutations, each of which individually confers resistance to only one class of herbicides.     

Expression of the Arabidopsis AtAux2-11 auxin-responsive gene in transgenic plants

Five constructions containing deletions of the promoter from an auxin-inducible gene of Arabidopsis thaliana, AtAux2-11, were fused to the coding region of the reporter gene LacZ, which encodes -galactosidase, and a polyadenylation 3-untranslated nopaline synthase sequence from Agrobacterium. These chimeric genes were introduced into Arabidopsis by Agrobacterium tumefaciens-mediated transformation, and expression of the gene was examined by spectrophotometric and histochemical analyses. A 600 bp fragment from the AtAux2-11 promoter conferred histochemical patterns of staining similar to the longest 5 promoter tested, a 3.0 kb fragment. Localization of AtAux2-11/LacZ activity in the transgenic plants revealed spatial and temporal expression patterns that correlated with tissues and cells undergoing physiological processes modulated by auxin. LacZ activity was expressed in the elongating region of roots, etiolated hypocotyls, and anther filaments. Expression was detected in the vascular cylinder of the root and the vascular tissue, epidermis, and cortex of the hypocotyl, and filament. The AtAux2-11/LacZ gene was preferentially expressed in cells on the elongating side of hypocotyls undergoing gravitropic curvature. Expression of the chimeric gene in the hypocotyls of light-grown seedlings was less than that in etiolated seedling hypcotyls. The AtAux2-11/LacZ gene was active in the root cap, and expression in the root stele increased at sites of lateral root initiation. Staining was evident in cell types that develop lignified cell walls, e.g. trichomes, anther endothecial cells, and especially developing xylem. The chimeric gene was not expressed in primary meristems. While the magnitude of expression increased after application of exogenous auxin (2,4-D), the histochemical localization of AtAux2-11/LacZ remained unchanged.Transgenic plants with a 600 bp promoter construct (–0.6 kb AtAux2-11/LacZ) had higher levels of basal and auxin-inducible expression than plants with a 3.0 kb promoter construct. Transgenic plants with a –500 bp promoter had levels of expression similar to the –3.0 kb construct. The –0.6 kb AtAux2-11/LacZ gene responded maximally to a concentration of 5 × 10^–6 to 5 × 10^–5 M 2,4-D and was responsive to as little as 5 × 10^–8 M. The evidence presented here suggests that this gene may play a role in several auxin-mediated developmental and physiological processes.co-first authors     

Heat-inducible hygromycin resistance in transgenic tobacco

We have constructed a chimaeric gene consisting of the promoter of the soybean heat shock (hs) gene Gmhsp17,6-L, the coding region of a hygromycin phosphotransferase (hpt) gene, and the termination sequence of the nopaline synthase (nos) gene. This gene fusion was introduced into tobacco by Agrobacterium-mediated gene transfer. Heat-inducible synthesis of mRNA was shown by northern hybridization, and translation of this RNA into a functional protein was indicated by plant growth on hygromycin-containing media in a temperature-dependent fashion. One hour incubation at 40 °C per day, applied for several weeks, was sufficient to express the resistant phenotype in transgenic plants containing the chimaeric hs-hpt gene. These data suggest that the hygromycin resistance gene is functional and faithfully controlled by the soybean hs promoter. The suitability of these transgenic plants for selection of mutations that alter the hs response is discussed.     

Gene inactivation in Arabidopsis thaliana is not accompanied by an accumulation of repeat-induced point mutations

Chromosomal integration of multicopy transgene inserts in higher plants is often followed by loss of expression. We have analysed whether this inactivation can trigger repeat-induced point mutations (RIP) as has been observed in Neurospora crassa. We have previously characterized transgenic lines of Arabidopsis thaliana containing the hygromycin phosphotransferase (hpt) gene either as a unique sequence in plants expressing the gene, or as multimeric, closely linked repeats in clones that were resistant to hygromycin directly after transformation but exhibited gene inactivation in the subsequent generation. At the sequence level, we have determined the mutation frequencies in the promoter and coding regions of active and inactive copies of transgene inserts after passage through three sexual generations. No RIP-like mutations were found in inactivated genes. Comparison of our data with those from Neurospora suggest that sequence divergence within plant repetitive DNA is either much slower than in Neurospora or is generated by a different mechanism.     

Complementation of anArabidopsis thaliana biotin auxotroph with anEscherichia coli biotin biosynthetic gene

TheArabidopsis thaliana biotin auxotrophbio1 was rendered prototrophic by transformation with a chimeric transgene containing theEscherichia coli bioA gene driven by a constitutive promoter. ThebioA gene encodes the biotin biosynthetic enzyme 7,8-diaminopelargonic acid aminotransferase. Unlike the untransformed control plants, transgenic plants expressing the bacterial transgene synthesized biotin and grew to maturity without biotin-deficiency symptoms. These findings demonstrate thatbio1/bio1 mutant plants are defective in the gene encoding 7,8-diaminopelargonic acid aminotransferase.     

Stable transformation and long-term expression of the gusA reporter gene in callus lines of perennial ryegrass (Lolium perenne L.)

Stable transformation of perennial ryegrass (Lolium perenne L.) was achieved by biolistic bombardment of a non embryogenic cell suspension culture, using the hpt and gusA gene. The transformation yielded on the average 5 callus lines per bombardment (1.4×10⁶ cells). Stable integration of the genes into the plant genome was demonstrated by Southern analysis of DNA, isolated from hygromycin-resistant callus lines. The gusA reporter gene, which was regulated by the constitutive promoter of the rice gene GOS2, was expressed in both transient and stable transformation assays, indicating that this promoter is suitable for expression of a transferred gene in perennial ryegrass. Long-term GUS expression was observed in ca. 40% of the callus lines, whereas the other callus lines showed instability after 6 months and 1 year of culture.     

High frequency stable transformation ofArabidopsis thaliana by particle bombardment

Root explants ofArabidopsis thaliana ecotype C24 were bombarded with the plasmid pCH harboring the hygromycin phosphotransferase gene (hpt). A selection condition with post-bombardment culture of 3 days followed by culture with 20 mgl⁻¹ hygromycin gave the highest yield of transformants. More than 44% of explant clumps formed transformant shoots.     

Transient gene expression in cassava using high-velocity microprojectiles

The bacterial gene encoding -glucuronidase (GUS) was transiently expressed in cassava leaves following the introduction of the gene by microparticle bombardment. The DNA expression vector used to introduce the reporter gene is a pUC 19 derivative and consisted of a CaMV 35S promoter (P35S), the GUS coding region and 7S polyadenylation region. Several other promoters and regulating sequences were tested for efficiency in cassava leaves. Two derivatives of the P35S, one including a partial duplication of the upstream region of the P35S and the other containing a tetramer of the octopine synthase enhancer, were found to be expressed at three times the level of the P35S in cassava leaves. The ubiquitin 1 promoter fromArabidopsis thaliana was expressed at the same level as the P35S. No influence on the level of expression was observed when different 3 ends were used. The biolistic transient gene expression system in cassava leaves allows rapid analysis of gene constructs and can serve as a preliminary screen for chimeric gene function in the construction of transgenic cassava plants.     

Development of flooding-tolerant Arabidopsis thaliana by autoregulated cytokinin production

Flooding is one of the most serious environmental stresses that affect plant growth and productivity. Flooding causes premature senescence which results in leaf chlorosis, necrosis, defoliation, cessation of growth and reduced yield. This study was conducted to determine the effects of autoregulated cytokinin production on the flooding tolerance of Arabidopsis thaliana plants. A chimeric gene containing the senescence-specific SAG12 promoter and the ipt gene coding for isopentenyl transferase, a rate-limiting enzyme in the cytokinin biosynthesis pathway, was constructed. The chimeric gene was introduced into Arabidopsis plants by Agrobacterium-mediated vacuum infiltration. Four transgenic lines were chosen for flooding tolerance determinations. DNA hybridization analysis and PCR confirmed that all four of the transgenic lines carried the ipt gene. The segregation of kanamycin resistance in the T₂ generation indicated 1 to 3 integration events. GUS expression and RT-PCR of the ipt gene confirmed the senescence-specificity of the SAG12 promoter. Morphologically, the transgenic lines appeared healthy and normal. Transgenic plants began to flower at the same time as wild-type plants, but the period from flowering to senescence was lengthened by 7 to 12 days. Tolerance of the transgenic plants to waterlogging and complete submergence was assayed in three independent experiments. All four transgenic lines were consistently more tolerant to flooding than wild-type plants. The results indicated that endogenously produced cytokinin can regulate senescence caused by flooding stress, thereby, increasing plant tolerance to flooding. This study provides a novel mechanism to improve flooding tolerance in plants.     

Chloroplast transformation by Agrobacterium tumefaciens

A chimeric gene consisting of the promoter region of the nopaline synthase gene (Pnos) fused to the coding sequence of the chloramphenicol acetyltransferase gene (cat gene) of Tn9 was introduced by co-cultivation in tobacco protoplasts followed by selection with 10 μg/ml chloramphenicol. The chloramphenicol-resistant plants derived from these selected calli were unable to transmit the Cm^R phenotype through pollen. A typically maternal inheritance pattern was observed. Southern blot analysis showed that the chimeric Pnos-cat gene was present in the chloroplasts of these resistant plants. Furthermore, the chloramphenicol acetyltransferase activity was shown to be associated with the chloroplast fraction. These observations are the first proof that the Agrobacterium Ti-plasmid vectors can be used to introduce genes in chloroplasts.     

Molecular analysis of the aspartate kinase-homoserine dehydrogenase gene from Arabidopsis thaliana

The gene encoding Arabidopsis thaliana aspartate kinase (ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) was isolated from genomic DNA libraries using the carrot ak-hsdh gene as the hybridizing probe. Two genomic libraries from different A. thaliana races were screened independently with the ak probe and the hsdh probe. Nucleotide sequences of the A. thaliana overlapping clones were determined and encompassed 2 kb upstream of the coding region and 300 bp downstream. The corresponding cDNA was isolated from a cDNA library made from poly(A)⁺-mRNA extracted from cell suspension cultures. Sequence comparison between the Arabidopsis gene product and an AK-HSDH bifunctional enzyme from carrot and from the Escherichia coli thrA and metL genes shows 80%, 37.5% and 31.4% amino acid sequence identity, respectively. The A. thaliana ak-hsdh gene is proposed to be the plant thrA homologue coding for the AK isozyme feedback inhibited by threonine. The gene is present in A. thaliana in single copy and functional as evidenced by hybridization analyses.The apoprotein-coding region is interrupted by 15 introns ranging from 78 to 134 bp. An upstream chloroplast-targeting sequence with low sequence similarity with the carrot transit peptide was identified. A signal sequence is proposed starting from a functional ATG initiation codon to the first exon of the apoprotein. Two additional introns were identified: one in the 5 non-coding leader sequence and the other in the putative chloroplast targeting sequence. 5 sequence analysis revealed the presence of several possible promoter elements as well as conserved regulatory motifs. Among these, an Opaque2 and a yeast GCN4-like recognition element might be relevant for such a gene coding for an enzyme limiting the carbon-flux entry to the biosynthesis of several essential amino acids. 3 sequence analysis showed the occurrence of two polyadenylation signals upstream of the polyadenylation site.This work is the first report of the molecular cloning of a plant ak-hsdh genomic sequence. It describes a promoter element that may bring new insights to the regulation of the biosynthesis of the aspartate family of amino acids.Abbreviations AK aspartate kinase - HSDH homoserine dehydrogenase - ID intermediate domain - Tp transit peptide     

High intensity and blue light regulated expression of chimeric chalcone synthase genes in transgenic Arabidopsis thaliana plants

Summary To establish a genetic system for dissection of light-mediated signal transduction in plants, we analyzed the light wavelengths and promoter sequences responsible for the light-induced expression of the Arabidopsis thaliana chalcone synthase (CHS) promoter fused to the -glucuronidase (GUS) marker gene. Transgenic A. thaliana lines carrying 1975, 523, 186, and 17 by of the CHS promoter fused to the GUS gene were generated, and the expression of these chimeric genes was monitored in response to high intensity light in mature plants and to different wavelengths of light in seedlings. Fusion constructs containing 1975 and 523 by of CHS promoter sequence behaved identically to the endogenous CHS gene under all conditions. Expression of these constructs was induced specifically in response to high intensity white light and blue light. The response to blue light was seen in the presence of the P_fr form of phytochrome. Fusion constructs containing 186 by of promoter sequence showed reduced basal levels of expression and only weak stimulation by blue light but were induced significantly by high intensity white light. These analyses showed that the expression of the A. thaliana CHS gene is responsive to a specific blue light receptor and that sequences between — 523 and — 186 by are required for optimal basal and blue light-induced expression of this gene. The experiments lay the foundation for a simple genetic screen for light response mutants.     

Molecular characterization and genetic origin of the Brassica napus acetohydroxyacid synthase multigene family

Summary The Brassica napus rapeseed cultivar Topas contains an acetohydroxyacid synthase (AHAS) multigene family consisting of five members (AHAS 1–5). DNA sequence analysis indicate that AHAS1 and AHAS3 share extensive homology. They probably encode the AHAS enzymes essential for plant growth and development. AHAS2 has diverged significantly from AHAS1 and AHAS3 and has unique features in the coding region of the mature polypeptide, transit peptide and upstream non-coding DNA, which raises the possibility that it has a distinct function. AHAS4 and AHAS5 have interrupted coding regions and may be defective. The complexity of the AHAS multigene family in the allotetraploid species B. napus is much greater than reported for Arabidopsis thaliana and Nicotiana tabacum. Analysis of the presumptive progenitor diploid species B. campestris and B. oleracea indicated that AHAS2, AHAS3 and AHAS4 originate from the A genome, whereas AHAS1 and AHAS5 originate from the C genome. Further variation within each of the AHAS genes in these species was found.     

The Transformation of the Unicellular Alga Chlamydomonas reinhardtii by Electroporation

The cell wall–lacking mutant CW-15 of the unicellular green alga Chlamydomonas reinhardtii was transformed by electroporation using plasmid pCTVHyg, which was constructed with the hygromycin phosphotransferase genehpt as the selective marker and the Tn₅ transposon of Escherichia coli under the control of the virus SV40 early gene promoter. Under optimal conditions (10⁶ mid-exponential cells/ml; electric field strength 1 kV/cm; and pulse length 2 ms), the transformation yielded 10³ Hyg^R transformants per 10⁶ recipient cells. The exogenous DNA integrated into the nuclear genome of Ch. reinhardtii was persistently inherited through more than 350 cell generations. The advantages of this system for the transformation ofCh. reinhardtii with heterologous genes are discussed.     

Non-recombinant background in gene targeting: illegitimate recombination between a hpt gene and a defective 5′ deleted nptII gene can restore a Kmr phenotype in tobacco

Previously we have demonstrated gene targeting in plants after Agrobacterium-mediated transformation. In these initial experiments a transgenic tobacco line 104 containing a T-DNA insertion with a defective neomycin phosphotransferase (nptII) gene was transformed with a repair construct containing an otherwise defective nptII gene. Homologous recombination between the chromosomally located target and the incoming complementary defective nptII construct generated an intact nptII gene and led to a kanamycin-resistant (Km^r) phenotype. The gene targeting frequency was 1×10^–5. In order to compare direct gene transfer and Agrobacterium-mediated transformation with respect to gene targeting we transformed the same transgenic tobacco line 104 via electroporation. A total of 1.35×10⁸ protoplasts were transformed with the repair construct. Out of nearly 221 000 transformed cells 477 Km^r calli were selected. Screening the Km^r calli via PCR for recombination events revealed that in none of these calli gene targeting had occurred. To establish the origin of the high number of Km^r calli in which gene targeting had not occurred we analysed plants regenerated from 24 Km^r calli via PCR and sequence analysis. This revealed that in 21 out of 24 plants analysed the 5-deleted nptII gene was fused to the hygromycin phosphotransferase (hpt) gene that was also present on the repair construct. Sequence analysis of 7 hpt/nptII gene fusions showed that they all contained a continuous open reading frame. The absence of significant homology at the fusion site indicated that fusion occurred via a process of illegitimate recombination. Therefore, illegitimate recombination between an introduced defective gene and another gene present on the repair construct or the chromosome has to be taken into account as a standard byproduct in gene targeting experiments.     

Exchange of gene activity in transgenic plants catalyzed by the Cre-lox site-specific recombination system

The Cre-lox site-specific recombination system of bacteriophage P1 was used to excise a firefly luciferase (luc) gene which had previously been incorporated into the tobacco genome. The excision event was due to site-specific DNA recombination between two lox sequences flanking the luc gene and was catalyzed by the Cre recombinase introduced by cross-fertilization. Recombination resulted in the fusion of a promoter with a distally located hygromycin phosphotransferase (hpt) coding sequence and the excision event was monitored as a phenotypic change from expression of luc to expression of hpt. The efficiency of recombination was estimated from the exchange of gene activity and confirmed by molecular analysis. The relevance to potential applications of site-specific deletion-fusion events for chromosome engineering are discussed.     

Chimeric RNA/DNA oligonucleotide-directed gene targeting in rice

Site-specific mutagenesis in a rice genome was obtained by introducing chimeric RNA/DNA oligonucleotides (COs) by means of particle bombardment. Three COs were designed to target the independent codons for Pro-171, Trp-548 and Ser-627 of the endogenous rice acetolactate synthase (ALS) gene so it would confer resistance to ALS-inhibiting herbicides. Sequencing of the ALS gene of herbicide-resistant plants demonstrated that the ALS sequence was modified in a site-specific fashion. The efficiency of gene conversion mediated by COs was estimated to be 1×10^-4. These results demonstrate that CO-directed gene targeting is feasible in rice.Abbreviations ALS Acetolactate synthase - BS Bispyribac-sodium - Cf Chlorsulfuron - CO Chimeric RNA/DNA oligonucleotide Communicated by H. Uchimiya