Peter Medgyesy

Stimulation of shoot regeneration in Triticum aestivum and Nicotiana plumbaginifolia Viv. tissue cultures using the ethylene inhibitor AgNO3

Silver nitrate effectively promoted shoot regeneration in wheat (Triticum aestivum L.) callus cultures derived from immature embryos. This effect could be observed in both weakly and strongly regenerating cultivars, and in using material from both field and greenhouse grown plants. The role of silver ions as an inhibitor of ethylene action was supported by a reversal of the inhibitory effects of 2,4-D and ethylene on morphogenesis in wheat callus cultures.Enhancement of shoot regeneration by silver nitrate was also observed in callus cultures of non-regenerating or weakly regenerating mutants of Nicotiana plumbaginifolia Viv. derived from cell cultures.

The use of cytoplasmic streptomycin resistance: Chloroplast transfer from Nicotiana tabacum into Nicotiana sylvestris, and Isolation of their somatic hybrids

SummaryLeaf protoplasts of Nicotiana tabacum SR1 (2n=4x=48) treated with iodoacetate (10 mM; 25 C; 30 min) and consequently unable to divide, and untreated leaf protoplasts of Nicotiana sylvestris (2n=2x=24) were fused using polyethylene glycol (PEG). The SR1 line is resistant to streptomycin because of a maternally inherited mutation, and has streptomycin-insensitive chloroplast ribosomes.After 1 month of growth in the absence of streptomycin protoplast-derived calli were plated into selective medium (1,000 μg ml-1 streptomycin) and the resistant clones were isolated. Out of 106 PEG-treated protoplasts (1:1 mixture of parental types) 137 resistant (green) clones were obtained, whereas in the same number of parental cells, not subjected to fusion induction, no resistant callus was found.At least four plants were regenerated from each of the clones. The regenerates were identified as somatic hybrids (H), N. sylvestris (Ns) or N. tabacum (Nt) by looking at esterase and peroxidase isoenzymes and morphology. The three types of regenerates were distributed amongst the clones as follows: H only (105 clones); Ns (16 clones); Ns+H (6 clones); Nt only (3 clones); Nt+H (6 clones); Nt+Ns (1 clone). The high proportion of hybrid regenerates indicates that nuclear fusion has occured in the overwhelming majority of the heterokaryocytes. Cytoplasmic mutations in combination with inactivation by iodoacetate, therefore, are suitable markers to produce somatic hybrids. Segregation of nuclei after fusion resulted in new combinations of organelles and nuclei, the final outcome being the transfer of resistant chloroplasts into N. sylvestris, some of which have the original diploid (2n=24) chromosome number. Data suggest that segregants were in most cases obtained from multiple fusions. Streptomycin resistance was inherited maternally in the N. sylvestris (six clones) tested and the hybrid (three clones) regenerates.

Transmission of paternal chloroplasts in Nicotiana

SummaryTransmission of paternal chloroplasts was observed in Nicotiana, considered to inherit organelles in a strictly maternal way. Plants carrying streptomycin resistant plastids were used as pollen donors. Cell lines with paternal plastids in the offspring were selected as green (resistant) sectors on calli induced from the seedlings on streptomycin-containing media. The presence of paternal plastids in the regenerated plants was confirmed by restriction analysis. In the Nicotiana plumbaginifolia ♀xN. plumbaginifolia Np(SR1)3 ♂ and the N. plumbaginifolia Np(gos)29 ♀xN. tabacum SR1 ♂ crosses 2.5% and 0.07% of the offspring were found to contain paternal (tabacum) plastids, respectively. These plants, however, carried maternal mitochondria exclusively. This sexual cybridization method offers a simple way to transfer chloroplasts solely, a goal not accessible by protoplast fusion.

Limited chloroplast gene transfer via recombination overcomes plastomegenome incompatibility between Nicotiana tabacum and Solanum tuberosum.

Green cybrids with a new nucleus-chloroplast combination cannot be selected after protoplast fusion in the intersubfamilial Nicotiana-Solanum combination. As an approach to overcome the supposed plastomegenome incompatibility, a partial plastome transfer by genetic recombination has been considered. After fusions of protoplasts of a light-sensitive Nicotiana tabacum (tobacco) plastome mutant and lethally irradiated protoplasts of wild-type Solanum tuberosum (potato), a single green colony was recovered among 2.5×104 colonies. The regenerated plants had tobacco-like (although abnormal) morphology, but were normally green, and sensitive to tentoxin, demonstrating chloroplast markers of the potato parent. Restriction enzyme analysis of the chloroplast DNA (cpDNA) revealed recombinant, nonparental patterns. A comparison with physical maps of the parental cpDNA demonstrated the presence of a considerable part of the potato plastome flanked by tobacco-specific regions. This “potacco” plastome proved to be stable in backcross and backfusion experiments, and normally functional in the presence solely of N. tabacum nucleus.

T7 RNA polymerase-directed expression of an antibody fragment transgene in plastids causes a semi-lethal pale-green seedling phenotype

A T7 promoter-controlled transgene, AbL, encoding a camel single-domain antibody fragment that binds to the model antigen chicken egg-white lysozyme was introduced into the plastid genome of tobacco. AbL expression was activated in the transplastomic line by introducing a nuclear transgene, ST7, encoding a light-regulated plastid-targeted T7RNAP by cross-pollination. The resulting AbL × ST7 progeny seedlings developed a pale-green phenotype and ceased growth soon after germination. High levels of AbL transcripts accumulated in AbL × ST7 seedlings and expression of functional AbL antibody was detected by ELISA. Transplastomic AbL plants were also crossed with nuclear-transformed tobacco plants containing a salicylic acid-inducible transgene encoding a plastid-targeted T7RNAP (PR-T7 transgene). The resulting AbL × PR-T7 progeny were wild-type in appearance but were slow growing and prone to wilting even when provided with adequate water. Although AbL transcription was inducible by treating AbL × PR-T7 leaves with salicylic acid, high levels of T7RNAP-dependent AbL transcripts also accumulated in the absence of induction. However, AbL antibody did not accumulate at levels detectable by immunoblotting or ELISA in AbL × PR-T7 plants despite the fact that total leaf RNA containing AbL transcripts was capable of directing AbL antibody synthesis in an E. coli-derived in vitro translation system.

Transplastomic tobacco plants expressing a fatty acid desaturase gene exhibit altered fatty acid profiles and improved cold tolerance

The possibility of altering the unsaturation level of fatty acids in plant lipids by genetic transformation has implications for the stress tolerance of higher plants as well as for their nutritional value and industrial utilisation. While the integration and expression of transgenes in the plastome has several potential advantages over nuclear transformation, very few attempts have been made to manipulate fatty acid biosynthesis using plastid transformation. We produced transplastomic tobacco plants that express a Δ9 desaturase gene from either the wild potato species Solanum commersonii or the cyanobacterium Anacystis nidulans, using PEG-mediated DNA uptake by protoplasts. Incorporation of chloroplast antibiotic-insensitive point mutations in the transforming DNA was used to select transformants. The presence of the transcript and the Δ9 desaturase protein in transplastomic plants was confirmed by northern and western blot analyses. In comparison with control plants, transplastomic plants showed altered fatty acid profiles and an increase in their unsaturation level both in leaves and seeds. The two transgenes produced comparable results. The results obtained demonstrate the feasibility of using plastid transformation to engineer lipid metabolic pathways in both vegetative and reproductive tissues and suggest an increase of cold tolerance in transplastomic plants showing altered leaf fatty acid profiles. This is the first example of transplastomic plants expressing an agronomically relevant gene produced with the “binding-type” vectors, which do not contain a heterologous marker gene. In fact, the transplastomic plants expressing the S. commersonii gene contain only plant-derived sequences, a clear attraction from a public acceptability perspective.

Intertrubal chloroplast transfer by protoplast fusion between Nicotiana tabacum and Salpiglossis sinuata

SummaryChloroplast tranfer was achieved by protoplast fusion between Nicotiana tobacum (Cestreae, Cestroideae) and Salpiglossis sinuata (Salpiglossideae, Cestroideae) in the family Solanaceae. Isolation of cybrid clones was facilitated by irradiation of the cytoplasm donor protoplasts, and the use of appropriate plastid mutants, streptomycin-resistant as donor, or light-sensitive as recipient. Cybrid colonies were selected by their green colour against the background of bleached (light-sensitive or streptomycin-sensitive) colonies. In the Nicotiana (Salpiglossis) cybrid plants possessing normal tobacco morphology and chromsome number, the presence of Salpiglossis, plastids was verified by restriction analysis of the chloroplast DNA. A similar analysis of the mitochondrial DNA of these lines revealed unique, recombinant patterns in the case of both fertile and sterile plants. Progeny showed no appearance of chlorophyll-deficiency in F1 and an additional back-cross generation. Attempts at transfer of entire chloroplasts between Nicotiana tabacum and Solanum nigrum (Solaneae, Solanoideae) did not result in any cybrid cell lines in a medium suitable for green colony formation of both species. These results suggest that fusion-mediated chloroplast transfer can surmount a considerable taxonomical distance, but might be hampered by a plastome-genome incompatibility in more remote combinations.

Triazine-resistant Nicotiana mutants from photomixotrophic cell cultures

SummaryTriazine-resistant mutants have been isolated in photomixotrophic cell cultures of Nicotiana plumbaginifolia. Triazine herbicides inhibit photosynthesis and cause extensive photodestruction of chloroplasts (bleaching) in sensitive plants. Selection was based on the greening ability of the resistant cells in the presence of 10-4 M terbutryn, under normal culture conditions, but in a medium containing a low sugar concentration. In the mutant plants, as compared to wild type, two to three orders of magnitude higher concentrations of triazines resulted in inhibition of photosynthetic electron transport and greening. The resistance was inherited maternally.

A light sensitive recipient for the effective transfer of chloroplast and mitochondrial traits by protoplast fusion in Nicotiana

SummaryA light sensitive mutant was used as a recipient in the transfer of chloroplasts from a wildtype donor. Gamma irradiated (lethal dose) mesophyll protoplasts of Nicotiana gossei were fused with mesophyll protoplasts of a N. plumbaginifolia line carrying light sensitive plastids from a N. tabacum mutant. After fusion, colonies containing wild-type plastids from the cytoplasm donor were selected by their green colour. Most of the regenerated plants had N. plumbaginifolia morphology, but were a normal green in colour. The presence of donor-type plastids was confirmed by the restriction pattern of chloroplast DNA in each plant analysed. These cybrids were fully male sterile with an altered flower morphology typical of certain types of alloplasmic male sterility in Nicotiana. The use of the cytoplasmic light sensitive recipient proved to be suitable for effective interspecific transfer of wild-type chloroplasts. The recombinant-type mitochondrial DNA restriction patterns and the male sterility of the cybrids indicated the co-transfer of chloroplast and mitochondrial traits.

Non-Photochemical Reduction of Intersystem Electron Carriers in Chloroplasts of Higher Plants and Algae

Besides reactions involved in the « Z » scheme of photosynthesis, additional electron transport pathways, such as cyclic electron flow around PSI or chlororespiration, have been reported in algal and in higher plant chloroplasts (1-4). These pathways generally involve a reduction of the intersystem electron transport chain (plastoquinones and/or the cyt b 6 f complex) by stromal components such as NAD(P)H or reduced ferredoxin. Different electron carriers have been proposed to mediate the non-photochemical reduction of inter-system electron transport chain. The discovery of chloroplast genes showing homologies with genes encoding subunits of the mitochondrial complex I (ndh genes), was taken as an evidence for the existence of a NADH dehydrogenase complex I in chloroplasts. Since, the existence of such a complex has been reported from non-denaturing gel electrophoresis (5) and recently, the NDH complex has been purified and partially characterized from pea chloroplasts (6). Inactivation of chloroplast ndh genes by plastid transformation has been undertaken in order to assess the role of the NDH complex during photosynthesis. However, if the disruption of ndh genes allowed to show that the NDH complex is functional in vivo and is involved in the dark re-reduction of the PQ pool after a period of illumination (7) a participation of the NDH complex during photosynthesis was not shown. Surprisingly, no clear phenotype was observed in inactivated transformants (7), suggesting that the NDH complex has a marginal role during photosynthesis. Other mediators have been proposed to be involved in the non-photochemical reduction of inter-system electron carriers: these include the putative ferredoxinquinone-oxidoreductase activity (FOR), FNR (4) and more recently a NAD(P)H-PQ oxidoreductase activity characterized in potato chloroplasts that would be different from the NDH complex, from FNR, and from FQR (8).