Ballistic transformation of Caenorhabditis elegans.

A novel method to transform the nematode Caenorhabditis elegans is described. DNA coprecipitated with gold particles is shot at worms by means of a helium beam. Transformed worms are either identified by a dominant visible marker or selected by a conditional lethal system.

Plant regeneration from leaf protoplasts of evening primrose (Oenothera hookeri).

A protocol is presented for regenerating plants from leaf protoplasts of Oenothera. The method uses (1) embedding of isolated protoplasts at high cell densities in thin alginate layers, (2) initial culture in B5 medium containing 3 mg l-1 α-naphthaleneacetic acid (NAA) and 1 mg l-1 6-benzylaminopurine (BAP), (3) reduction of the osmotic pressure of the culture medium at early stages of culture and (4) plating of microcolonies recovered from the alginate onto solid B5 medium with 3 mg l-1 NAA and 1 mg l-1 BAP. The shortest time required from protoplast isolation to the appearance of shoot initials was 7 weeks. The efficiency of the procedure for protoplast to cell line formation is high (about 80%).

Tissue culture of wild-type, interspecific genome/plastome hybrids and plastome mutants of evening primrose (Oenothera): controlled morphogenesis and transformation.

A favourable combination of genetic features in the genus Oenothera offers access to fundamental biological aspects that are not readily approached with other materials. We have developed protocols for cell and tissue culture as well as for transformation, in order to establish the basis for a comprehensive cell and molecular biology of Euoenothera species, their genome/plastome hybrids and plastome mutants. Regeneration of plants from excised seedling parts (roots, hypocotyl, cotyledons, shoot tips) and leaf explants was optimal on NT medium containing 1 mg ⋅ l-1 6-benzylaminopurine and 3 mg ⋅ l-1 α-naphtalene acetic acid. This medium also proved to be efficient in the propagation of various wild-type genotypes, interspecific hybrids and plastome mutants. Using Ti-based approaches we also succeeded in generating transgenic Oenothera plants with relatively high efficiency.

Germination of cysts in acetabularia mediterranea.

Techniques for the growth of uniformly reacting populations of cysts of Acetabularia mediterranea and for quantitative measurement of cyst germination have been developed. Cysts of A. mediterranea can be induced to germinated by exposure to the atmosphere. Germination rates are very low in young cysts. They increased during exposure to total darkness. This "maturation of cysts" is found to be completed after a period of 12-15 weeks. Germination rates of cysts that have passed the maturation period exceed 90 percent in continuous white light and 80 percent in darkness. Cysts germinate in less than two days in darkness and less than four days in light. The influence of temperature at a range of 15 degrees C to 25 degrees C on germination kinetics is studied in light and darkness. Germination is accelerated with increasing temperature up to 21 degrees C. At higher temperature germination is delayed in light but the time of germination remains constant in darknesss. Rates of germination are not altered by the influence of temperature in light while in darkness there is a dramatic decrease at temperatures higher than 21 degrees C. From these findings it is concluded that cyst germinationA. mediteranea does not need any light but is influenced by light dependent systems. The influence of light is strongest at elevated temperatures.

[The influence of actinomycin D, puromycin, cycloheximide, chloramphenicol, and rifampicin on gamete formation in Acetabularia mediterranea]. / Der Einfluß von Actinomycin D, Puromycin, Cycloheximid, Chloramphenicol und Rifampicin auf die Gametenbildung von Acetabularia mediterranea.

Cysts of Acetabularia mediterranea were induced to germinate in the presence of various inhibitors of protein synthesis. Actinomycin D (50 µg/ml), puromycin (10 µg/ml) and cycloheximide (0.1 µg/ml) were found to be strong inhibitors of gamete formation, while chloramphenicol (up to 100 µg/ml) and rifampicin (10 µg/ml) did not show any effect. Malformations were found after treatment with puromycin and very high (200 µg/ml) concentrations of chloramphenicol.

Transfer of defined numbers of chloroplasts into albino protoplasts by subprotoplast/protoplast microfusion: chloroplasts can be "cloned", by using suitable plastome combinations or selective pressure.

Defined numbers (1-5) of (donor) chloroplasts were transferred into (acceptor) protoplasts of plastid albino mutants by subprotoplasts/protoplast microfusion. Single transferred plastids gave rise to new organelle populations in the progeny of the fusion products when suitable combinations of plastomes were used or when selective pressure for the plastome transferred was applied. This process is termed "chloroplast cloning" and is the first reported case of "cloning" a cell organelle. The plastome combination and the presence or absence of selective pressure were found to influence the frequencies with which cell lines, containing both plastomes or acceptor or donor only, were obtained, and the number of cell generations needed for complete segregation - as measured by the duration of culture before the green donor plastome could be detected. The high frequency of cell lines and regenerated shoots recovered with donor plastome only, even when only a single chloroplast was transferred, leads to the conclusion that all organelles present in the fusion product contribute to the organelle population of the progeny, i.e. organelle death or loss are not regularly occurring events during plant regeneration from protoplasts in Nicotiana tabacum.

Extraplastidic site-specific factors mediate RNA editing in chloroplasts.

Single nucleotides in higher plant organellar mRNAs are subject to post-transcriptional alterations by RNA editing, typically resulting in changes of the encoded protein sequence. Although some information has been acquired on the general features of the editing processes in both plastids and plant mitochondria, the mechanisms and factors involved in the selective recognition of the nucleotide to be edited are still unknown. To gain a better understanding of how an editing site is specifically selected by the organellar RNA editing machinery, we have attempted to rescue a previously generated tobacco plastid editing mutant. Using an interspecific protoplast fusion approach, we were able to restore RNA editing activity for a specific site in the psbF transcript that otherwise remained unedited. Our results suggest (i) that site-specific trans-acting factors mediate chloroplast editing site recognition and (ii) that these factors are of extraplastidic origin.

Somatic embryogenesis in cultured immature zygotic embryos and leaf protoplasts of Arabidopsis thaliana ecotypes.

Immature zygotic embryos of six ecotypes (Nd-0, Ler, C24, Col-0, Nossen, Ws-2) of Arabidopsis thaliana (L.) Heynh. were cultured in vitro. The same ecotypes, except Nossen, were used for studies on leaf protoplast culture. Experimental conditions for the induction of somatic embryos were established in both culture systems. In the case of immature zygotic embryos, the parameters investigated were the influence of developmental stage of the explant, the ecotypes used, and various concentrations and combinations of growth regulatory substances (phytohormones). In the ecotype Ler, structures were discovered which were very similar to those found in the early stages of zygotic embryo-genesis: globular structures at the end of a suspensor-like single file of cells were frequently observed. In the case of leaf protoplasts, high efficiencies of colony formation and plant regeneration occurred in Ws-2 and C24. A novel type of cell division pattern was found in Col-0 and C24, again highly reminiscent of the early division patterns in zygotic embryos. Similarities and differences between zygotic and somatic embryogenesis are discussed.

Lipofectin: direct gene transfer to higher plants using cationic liposomes.

It has recently been shown that lipofectin, a commercially available preparation of cationic liposomes is capable of animal and plant cell line transfection. Here, it is analyzed with respect to its toxicity for higher plant protoplasts and used for transient expression and stable transformation experiments with mesophyll protoplasts of Nicotiana tabacum and Nicotiana plumbaginifolia. Transient expression of the ß-glucuronidase gene (GUS) under control of the CaMV-35S-promoter was lower than after introduction of the same gene by polyethylene glycol. By transferring the neomycin phosphotransferase gene (NPTII) and subsequent culture and regeneration under selection with kanamycin, stably transformed plants were recovered after using Lipofectin in various protocols with or without additional application of electroporation. Efficiencies of stable transformation were comparable to those achieved with PEG and/or electroporation. Confirmation of transformants included assaying the enzyme activity of the gene product, genomic blotting, and transfer of the resistant phenotype to the progeny produced from selfed primary transformants.

Callus formation and plant regeneration in standard and microexplants from seedlings of barley (Hordeum vulgare L.).

Callus was induced in standard (1 mm) leaf base explants and in cross sections (microexplants) through the seedling axis from seedlings of Hordeum vulgare L.. Reduced callus formation was observed with increasing distance from the leaf base, and explants from the first and second leaves gave the best response. In serial hand sections of the seedling axis frequency of callus formation decreased from 100% in the apical region to 5% in the basal region. Callus formed from all tissues outside the central vascular elements, except for the coleoptile and the scutellum. Plants were regenerated from callus induced from both types of explants.

Transfer of defined numbers of chloroplasts into albino protoplasts using an improved subprotoplast/protoplast microfusion procedure: transfer of only two chloroplasts leads to variegated progeny.

A procedure is described by which it is possible to perform controlled microfusion of microscopically selected protoplast fusion partners with high efficiencies. The procedure is applied to fusion of Nicotiana tabacum (line 92V37. N. undulata cytoplasm) plastid albino protoplasts as a recipient and spontaneously formed subprotoplasts of green N. tabacum (line SR1) as donor. Products of individual electrofusion events are cloned via single cell nurse culture and the derived cell lines are analysed for the occurrence of variegated or green regenerating shoots, which are indicative of the establishment of the transferred organelles in the cell progeny. The plastid population in green regenerants recovered after the transfer of only two chloroplasts was demonstrated to have originated from the donor subprotoplast organelles by restriction analysis of total DNA using a plastome-specific probe.

Mutagenesis of the genes encoding subunits A, C, H, I, J and K of the plastid NAD(P)H-plastoquinone-oxidoreductase in tobacco by polyethylene glycol-mediated plastome transformation.

Plastids contain a NAD(P)H-plastoquinone-oxidoreductase (NDH complex) which is homologous to the eubacterial and mitochondrial NADH-ubiquinone-oxidoreductase (complex I), but the metabolic function of the enzyme is unknown. The enzyme consists of at least eleven subunits (A-K), which are all encoded on the plastid chromosome. We have mutagenized ndhC and ndhJ by insertion, and ndhK and ndhA-I by deletion and insertion, of a cassette which carried a spectinomycin resistance gene as a marker. The transformation was carried out by the polyethylene glycol-mediated plastid transformation method. Southern analysis revealed that even after repeated regeneration cycles each of the four different types of transformants had retained 1-5% of wild-type gene copies. This suggests that complete deletion of ndh genes is not compatible with viability. The transformants displayed two characteristic phenotypes: (i) they lack the rapid rise in chlorophyll fluorescence in the dark after illumination with actinic light for 5 min; in the wild-type this dark-rise reflects a transient reduction of the plastoquinone pool by reduction equivalents generated in the stroma; and (ii) transformants with defects in the ndhC-K-J operon accumulate starch, indicating inefficient oxidation of glucose via glycolysis and the oxidative pentose phosphate pathway. Both observations support the theory of chlororespiration, which postulates that the NDH complex acts as a valve to remove excess reduction equivalents in the chloroplast.

The stem-loop region of the tobacco psbA 5'UTR is an important determinant of mRNA stability and translation efficiency.

Regulation of chloroplast gene expression involves networked and concerted interactions of nucleus-encoded factors with their target sites on untranslated regions (UTRs) of chloroplast transcripts. So far, only a few cis-acting elements within such 5'UTR sequences have been identified as functional determinants of mRNA stability and efficient translation in Chlamydomonas in vivo. In this study, we have used chloroplast transformation and site-directed mutagenesis to analyse the functions of the 5'UTRs of tobacco psbA and rbcL fused to the coding region of the reporter gene uidA. Various mutant versions of the psbA leader, as well as rbcL/psbA hybrid leader elements, were investigated. Our results showed a 1.5- to 3-fold decrease in uidA mRNA levels and a 1.5- to 6-fold reduction in uidA translation efficiency in all psbA 5'UTR stem-loop mutants generated by sequence deletions and base alterations. This indicates that the correct primary sequence and secondary structure of the psbA 5'UTR stem-loop are required for mRNA stabilisation and translation. The 5'-terminal segment of the rbcL 5'UTR did not enhance the stability or translational activity of chimeric uidA mRNA under the standard light-dark regime of 16 h light and 8 h dark. Stabilising effects were, however, observed when the cells were kept continuously in the dark. Possible reasons for the influence of the 5'UTR of the tobacco psbA on mRNA stability and translation efficiency are discussed.

PEG- and electroporation-induced transformation in Nicotiana tabacum: influence of genotype on transformation frequencies.

Experimental parameters for direct gene transfer with recombinant DNA encoding neomycin phosphotransferase II (NPTII) under control of eukaryotic expression signals were established. The introduced gene was shown by the growth of transformants on media containing kanamycin, by genomic blotting and by assaying NPTII activity. Leaf protoplasts from three green genotypes of varieties xanthii and petit havanna, and from four plastome-encoded albino genotypes of Nicotiana tabacum were analyzed with respect to cell division kinetics and yield of kanamycin-tolerant colonies after direct gene transfer. No clear correlation was found between the time of onset of cell division and transformation frequency.

Efficient regeneration from cotyledon protoplasts in Arabidopsis thaliana.

An efficient and fast regeneration system from cotyledon protoplasts was established for Arabidopsis thaliana accessions C24, Columbia, and Wassilewskija. Culture conditions and media compositions were optimised for the development of protoplasts embedded in thin alginate layers. Unexpectedly, the absence of cytokinins had a positive effect on cell development. Moreover, combined adjustment of alpha-naphthylacetic acid and dicamba concentrations resulted in high plating efficiencies of up to 30%, followed by shoot regeneration within only 19 days after protoplast isolation. The protocol is reproducible, efficient, extremely fast, and regenerated plants are fertile. Thus, this cotyledon-based system could prove useful for studying plant cell and molecular biology in A. thaliana.

PEG-mediated plastid transformation: a new system for transient gene expression assays in chloroplasts.

Evidence is presented for the introduction of functional copies of the GUS-reporter gene with plastid regulatory signals into chloroplasts after treatment of Nicotiana plumbaginifolia leaf protoplasts with PEG. GUS-activity is found in cells derived from protoplasts treated with PEG in the presence of plasmids harbouring the GUS-gene under the control of plastid promoter and terminator signals (plastid-specific reporter gene constructions). The activity is maintained after chloroplast isolation and incubation with the protease thermolysin under conditions sufficient to completely remove the much higher transient nuclear/cytoplasmic expression of a GUS-gene carrying the CaMV 35S-promoter. Likewise, GUS-activity derived from a plasmid coding for the nuclear/cytoplasmic expression of the reporter gene with a plastid transit presequence is also maintained after these procedures. These results indicate that PEG-treatment is a suitable protocol by which to introduce DNA into chloroplasts for the study of transient gene expression.

Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation.

A new vector, pFaadAII, for transformation of plastids of Nicotiana tabacum L. has been developed. It harbours a chimeric gene consisting of the aadA coding region from Escherichia coli, the 16S rDNA promoter from tobacco combined with a synthetic ribosome-binding site, a 500-bp fragment containing the 3' untranslated transcript region (UTR) of the Chlamydomonas rbcL gene and 3.75-kb (5') and 0.95-kb (3') tobacco plastome sequences allowing for targeting the foreign sequences to the intergenic region between the rpl32 and trnL genes of the tobacco plastome. The vector thus targets foreign sequences to the small single-copy region of the plastome, which has so far not been modified by transformation. Leaf protoplasts of Nicotiana tabacum L. were treated with polyethylene glycol (PEG) in the presence of the vector. The protocol for PEG treatment aiming at plastome transformation was optimized. Cell lines were cultured in the presence of spectinomycin and streptomycin using a novel and efficient protoplast culture and selection system. Regenerants were characterized by polymerase chain reaction (PCR) analysis, Southern hybridization and reciprocal crossing. The transformation procedure is described in detail and parameters influencing its efficiency are presented. Special effort is placed on analyzing suitable selection conditions. Only a proportion of the cell lines with a resistant phenotype could be confirmed by molecular analysis and/or reciprocal crossings to represent plastome transformants. Integration of the plastome specific aadA cassette into the nuclear genome accounted for a fraction of the resistant cell lines. Still, as many as 20-40 plastome transformants can be expected from the treatment of 10(6) protoplasts. Therefore, the improved protocol for PEG-mediated plastome transformation in combination with the new aadA-vector supplies a simple, reproducible and cost-efficient alternative to the biolistic procedure.

Somatic hybridization of two selected single cells.

Two single mesophyll-protoplasts of Nicotiana tabacum cv. xanthi were selected into a 100 nl microdroplet of 0.4 M mannitol. Two cylindrical platinum electrodes were inserted into the microdroplet to align the two single cells via dielectrophoresis in an AC-field (1 MHz, about 120 V X cm-1). A single square DC-pulse of about 1.5 KV X cm-1 was applied to induce protoplast fusion.

Polyester synthesis in transplastomic tobacco (Nicotiana tabacum L.): significant contents of polyhydroxybutyrate are associated with growth reduction.

The pathway for synthesis of polyhydroxybutyrate (PHB), a polyester produced by three bacterial enzymes, was transferred to the tobacco plastid genome by the biolistic transformation method. The polycistronic phb operon encoding this biosynthetic pathway was cloned into plastome transformation vectors. Following selection and regeneration, the content and structure of plant-produced hydroxybutyrate was analysed by gas chromatography. Significant PHB synthesis was limited to the early stages of in vitro culture. Within the transformants, PHB synthesis levels were highly variable. In the early regeneration stage, single regenerates reached up to 1.7% PHB in dry weight. At least 70% of plant-produced hydroxybutyric acid was proven to be polymer with a molecular mass of up to 2,500 kDa. PHB synthesis levels of the transplastomic lines were decreasing when grown autotrophically but their phb transcription levels remained stable. Transcription of the three genes is divided into two transcripts with phbB being transcribed separately from phbC and phbA. In mature plants even low amounts of PHB were associated with male sterility. Fertility was only observed in a mutant carrying a defective phb operon. These results prove successful expression of the entire PHB pathway in plastids, concomitant, however, with growth deficiency and male sterility.