Comparison of somatic and sexual incompatibility between Datura innoxia and Atropa belladonna

After protoplast fusion somatic hybrid calli were obtained by complementation selection between an albino mutant of Datura innoxia and the wildtype of Atropa belladonna (Krumbiegel and Schieder, 1979. Planta 145, 371–375). In the present study experiments are described concerning leaf and shoot induction on several media supplemented with different combinations and concentrations of hormones. Except for fleshy leaves and embryos, no well-formed shoot could be obtained. However, under standard culture conditions after one and a half years, one line produced numerous green shoots, showing a reduced number of chromosomes from Atropa belladonna. The loss of some chromosomes decreased the degree of somatic incompatibility. The additional appearance of shoots with albino sectors, of total albino shoots, and of green shoots showing a different phenotype, demonstrated that the elimination of the chromosomes occurred not only once, but several times. At least one shoot nearly stable in chromosome content and green subline could be obtained possessing only 6 chromosomes of Atropa belladonna and the original chromosome number of Datura innoxia. Experiments were carried out to test the feasibility of producing sexual hybrids through in vivo and in vitro methods by cross pollination. However, no embryos, seeds, or plantlets were obtained, thus demonstrating that protoplast fusion is the only possibility for obtaining hybrids between these two species.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlor-phenoxyacetic acid - IAA indoleacetic acid - NAA -naphtaleneacetic acid - SDS sodiumdodecylsulfate     

Genetic evidence for the hybrid nature of somatic hybrids from Datura innoxia Mill.

The hybrid nature of tetraploid somatic hybrids of two genetically different chlorophyll-deficient mutants from Datura innoxia Mill. was demonstrated with the aid of anther culture. Green and chlorophyll-deficient androgenetic lines could be regenerated from the pollen grains.     

Spontaneous extensive chromosome elimination in somatic hybrids between somatically congruent species Nicotiana tabacum L. and Atropa belladonna L.

Summary Mesophyll protoplasts of the kanamycin-resistant nightshade, Atropa belladonna, were fused with mesophyll protoplasts of the phosphinothricin resistant-tobacco, Nicotiana tabacum. A total of 447 colonies resistant to both inhibitors was selected. Most of them regenerated shoots with morphology similar to one of the earlier obtained and described symmetric somatic hybrids Nicotiana + Atropa. However, three colonies (0.2%) regenerated vigorously growing tobacco-like shoots; they readily rooted, and after transfer to soil, developed into normal, fertile plants. Unlike their tobacco parental line, BarD, the obtained plants are resistant to kanamycin [they root normally in the presence of kanamycin (200 mg/1)] and possess activity of neomycin phosphotransferase (NPT II) with the same electrophoretic mobility as the one of the nightshade line. According to Southern blot hybridization analysis carried out with the use of radioactively labeled cloned fragments of the Citrus lemon ribosomal DNA repeat, as well as with Nicotiana plumbaginifolia genus-specific, interspersed repeat Inp, the kanamycin-resistant plants under investigation have only species-specific hybridizing bands from tobacco. Cytological analysis of the chromosome sets shows that plants of all three lines possess 48 large chromosomes similar to Nicotiana tabacum ones (2n = 48), and one small extra chromosome (chromosome fragment) similar to Atropa belladonna ones (2n = 72). Available data allow the conclusion that highly asymmetric, normal fertile somatic hybrids with a whole diploid Nicotiana tabacum genome and only part (not more than 2.8%) of an Atropa belladonna genome have been obtained without any pretreatment of a donor genome, although both these species are somatically congruent.     

Enzymes of N-methylputrescine biosynthesis in relation to hyoscyamine formation in transformed root cultures of Datura stramonium and Atropa belladonna

The activities of enzymes related to the biosynthesis of N-methylputrescine, a precursor of the alkaloid hyoscyamine, have been measured in root cultures of Datura stramonium L. and Atropa belladonna L. transformed with Agrobacterium rhizogenes. Ornithine -Nmethyltransferase and -N-methylornithine decafboxylase were undetectable, indicating that -N-methylornithine is an unlikely intermediate in the formation of N-methylputrescine. The activity of putrescine-N-methyltransferase (EC 2.1.1.53) was comparable to, or greater than, that of arginine decarboxylase (EC 4.1.1.19) or ornithine decarboxylase (EC 4.1.1.17). Radiolabel from dl-[5-¹⁴C]ornithine, l-[U-¹⁴C]arginine, [U-¹⁴C]agmaine and [1,4-¹⁴C]putrescine was incorporated into hyosyamine by Datura cultures. Hyoscyamine production by Datura cultures was substantially inhibited by the arginine-decarboxylase inhibitor, dl--difluoromethylarginine, but not by the corresponding ornithine-decarboxylase inhibitor, dl--difluoromethylornithine. Together with the demonstration that label was incorporated from [U-¹⁴C]agmatine, this indicates clearly that arginine is metabolised to hyoscyamine at least in part via decarboxylation to agmatine, even though a high activity of arginase (EC 3.5.3.1) was measurable under optimal conditions. The effect of unlabelled putrescine in diminishing the incorporation into hyoscyamine of label from dl-[ 5-¹⁴C] ornithine and l-[U-¹⁴C] arginine does not lend support to the theory that ornithine is metabolised via a bound, asymmetric putrescine intermediate.Abbreviations DFMA dl--difluoromethylarginine - DFMO dl--difluoromethylornithine We thank Miss E. Bent for valuable technical assistance and J. Eagles, K. Parsley and Dr. F. Mellon for mass-spectrometric analysis. We are grateful to Dr. A.J. Parr and Dr. M.J.C. Rhodes for helpful discussions. We are indebted to the Merrell Dow Research Institute, Cincinnati, Ohio, USA for supplying DFMA and DFMO.     

Novel class of rDNA repeat units in somatic hybrids between Nicotiana and Atropa

Summary Behavior of ribosomal RNA genes in the process of somatic hybridization was analyzed using hybrids Nicotiana tabacum + Atropa belladonna. Blothybridization of parental species DNAs to ³²P-rDNA specific probes revealed two classes of ribosomal repeats in both tobacco and nightshade; their length was 11.2 kb, 10.4 kb (tobacco) and 9.4 kb, 10.2 kb (night-shade). For analysis of hybrids, labelled ³²P rDNA specific probes were hybridized to DNA of parental species and somatic hybrids digested with restriction endonucleases EcoR1, EcoRV and BamH1. A new class of ribosomal DNA repeat, absent in parental species, was found in hybrid line NtAb-1. Possible mechanisms of appearence of a new rDNA class in the process of somatic cell fusion are discussed.     

Transformation of haploid Datura innoxia protoplasts and analysis of the plasmid integration pattern in regenerated transgenic plants

We developed a highly efficient transformation protocol for the PEG-mediated direct transfer of plasmid DNA into protoplasts of haploid Datura innoxia. Vectors harbouring a neomycin phosphotransferase II gene or a hygromycin B phosphotransferase gene under the control of different promoters were used in the transformation experiments. Various amounts of plasmid DNA were applied without any carrier DNA to show the direct influence of the plasmid DNA concentration on the transformation efficiency. Approximately 95% of the selected calli were regenerated to plants; 20% of them remained haploid. Total DNA of different transgenic plants was analysed with regard to the integration pattern of the plasmid DNA. Plants carrying only one or two copies of the vector DNA were observed as well as individuals with multi-copy integration (up to ten or more copies).Abbreviations ATF/RTF absolute/relative transformation frequency - BAP 6-benzylaminopurine - CaMV cauliflower mosaic virus - CTAB N-cetyl-N,N,N-trimethyl-ammonium bromide - HPT hygromycin B phosphotransferase gene - PEG polyethyleneglycol - MES 2-(N-morpholino) ethanesulfonic acid - NPT II neomycin phosphotransferase II gene     

Partial characterization of two stable auxotrophic cell strains of Datura innoxia Mill

A growth analysis of several presumptive leaky auxotrophs from Datura innoxia suspension cultures led to the discovery of an adenine-requiring cultures led to the discovery of an adenine-requiring cell strain (Ad1). Both Ad1 and Pn1, a pantothenate-requiring strain isolated earlier from these cultures, still require either adenine or pantothenate for growth after more than one year in culture. Attempts to select prototrophic revertants have failed. Ad 1 also grew well in a medium containing either 5-aminoimidazole-4-carboxamide ribotide or inosine instead of adenine; Pn 1 with pantoic acid alone but not at all in the presence of -alanine or -ketoisovalerate alone instead of pantothenate. Pn 1 cells starved of pantothenate for up to 4d and Ad1 of adenine for 10d or more resumed growth when transferred to appropriately supplemented media. Wild-type Datura cells grown on unsupplemented medium would not crossfeed the required nutrients to the auxotrophs. The starvation and cross-feeding experiment showed that both auxotrophs could be used in reconstruction experiments to develop enrichment-selection techniques for the isolation of more auxotrophs.     

Genetic processes in intergeneric cell hybrids Atropa + Nicotiana

Summary The genetic constitution of the cell hybrids Atropa belladonna + Nicotiana chinensis, obtained by cloning of individual heteroplasmic protoplast fusion products (Gleba et al. 1982) and cultured in vitro for 12 months, has been studied. The study comprised 11 hybrid cell clones of independent origin and included analysis of a) chromosome number, size, morphology, and relative position in metaphase plates, b) multiple molecular forms of the enzymes esterase and amylase, and c) relative nuclear DNA content. The data obtained permit us to conclude that, after one year of unorganized growth in vitro, the cells of most (8) clones had retained chromosomes of both parents, while species-specific elimination of nearly all Atropa chromosomes had occurred in three clones. About half of the non-segregating clones possess 120–150 chromosomes including 50–70 of Atropa and 50–90 of Nicotiana. Other clones are polyploid and possess 200–250 chromosomes with a predominance of either Atropa or Nicotiana chromosome types. Only a few chromosomal changes (reconstituted chromosomes, ring chromosomes) have been detected. In some metaphase plates, chromosomes of the two parents tend to group separately, indicating non-random arrangement of chromosomes of the two parents within the hybrid nucleus. Cytophotometric studies of the relative nuclear DNA content showed that distribution histograms for cell clones were similar to those of non-hybrid cultured cells. Cell populations were relatively homogenous and do not indicate any genetic instability as a result of hybridization between remote plant species. Biochemical analysis of isoenzyme patterns confirmed that in most cell clones, species-specific multiple molecular forms of esterase and amylase from both parents were present, i.e. genetic material of both parental species was expressed in the cell hybrids.Dedicated to Professor G. Melchers with gratitude     

The isolation of auxotrophs from Datura innoxia Mill. cell cultures following recovery of arsenate-treated cells on feeder plates

Wild-type (Ph1) and adenine-requiring (Ad1) cell lines of Datura innoxia Mill. were used in experiments to evaluate arsenate as a growth-lethal compound and its use as a counterselection agent. These experiments led to the devising of methods for the recovery of Ph1 and Ad1 cells after arsenate treatment when plated at low density on feeder plates. The modified counterselection technique was then used to isolate three new auxotrophs from mutagenized suspensions of Ph1 cells, two of which were partially characterized. One, C18, requires casein hydrolysate for growth and lacks an active nitrate reductase; the other, JM3, will grow only when the medium contains threonine.Abbreviation CFU colony-forming unit     

Functional cybrid plants possessing a Nicotiana genome and an Atropa plastome

Summary Mesophyll protoplasts of plastome chlorophyll-deficient, streptomycin-resistant Nicotiana tabacum were fused with those of wild type Atropa belladonna using the polyethylene-glycol/high pH/high Ca⁺⁺/dimethylsulfoxide method. Protoplasts were cultured in nutrient media suitable for regeneration of tobacco but not Atropa cells. In two experiments, a total of 41 cell lines have been selected as green colonies. Cytogenetic (chromosomal number and morphology) and biochemical (isozyme analyses of esterase, amylase and peroxidase) studies were used to evaluate the nuclear genetic constitution of regenerated plants. To study plastid genetic constitution, restriction endonuclease analysis of chloroplast DNA was performed. Three groups of regenerants have been identified: (a) nuclear hybrids (4 cell lines); (b) Atropa plants, most probably arising from rare surviving parental protoplasts (4 lines) and (c) Nicotiana/Atropa cybrids possessing a tobacco genome and an Atropa plastome (33 lines). Most of cybrids obtained were diploid, morphologically normal plants phenotypically similar to tobacco. Some plants flowered and yielded viable seeds. Part of cybrid regenerants were variegated, variegation being transmitted to sexual progeny. Electron microscopic analysis of the mesophyll cells of variegated leaves revealed the presence of heteroplastidic cells. Analysis of thylakoid membrane polypeptides shows that in the cybrids the content of at least one of the major polypeptides, presumably a chlorophyll a/b binding protein is drastically reduced.     

Nucleotide pools in suspension-cultured cells of Datura innoxia

A general picture of the metabolic events which govern to growth behaviour of a batch culture of suspended dedifferentiated cells of Datura innoxia is obtained by following both the uptake and accumulation of the medium phosphate and sucrose by the cells, and the synthesis of RNA, protein and starch. The results are compared with the changes in the nucleotide pools described in the preceding paper. The sequence of formation and the regulatory dependencies of cellular pools of phosphate, sucrose, nucleotides and RNA in the production of proteins, starch and cell mass, and in the control of proliferation and cell growth are discussed. Furthermore, the importance of the maintenance pools for metabolic survival during starvation is emphasized.     

Production and characterization of somatic hybrids between Solanum melongena L. and S. sisymbriifolium Lam.

Summary Protoplasts of 6-azauracil (AU) resistant cell lines of Solanum melongena L. were fused with protoplasts of S. sisymbriifolium Lam. to create somatic hybrids between these sexually-incompatible species. Following fusion, colonies were selected which were capable of growth in medium containing 1mM AU. These colonies were placed on medium containing zeatin which had been shown to stimulate anthocyanin production during shoot organogenesis in tissue explants of S. sisymbriifolium but not in S. melongena. A total of 37 anthocyanin-producing colonies were identified from which 26 hybrid plants were regenerated. The morphological traits intermediate to those of the parents included: flower colour, leaf shape, and trichome density. Cytogenetic analysis revealed that all hybrids were aneuploids but their chromosome numbers were close to the expected number of 48. Isozyme analysis revealed that nuclear genes of both parents were expressed in the hybrids. In addition, isoelectric focussing of the large subunit of ribulose 1,5-bisphosphate carboxylase (Rubisco) provided evidence that each hybrid expressed only the S. sisymbriifolium chloroplast genome. All hybrids regenerated thus far have been sterile.Contribution No. 787 Ottawa Research Station     

Complementation by intraspecific protoplast fusion of auxotrophic cell lines of Datura innoxia P. Mill.

It was determined using electrophoresis in polyacrylamide gels containing native DNA or RNA that sugar non-specific nuclease active at pH 5.2 was expressed in tobacco callus. The nuclease had a relative molecular mass of about 34.6 kDaltons and degraded substrates in the following order of decreasing rate: denaturated DNA>poly dA>UV-irradiated native DNA>native DNA>alkylated native DNA>apurinated native DNA>poly dGpoly dC. The nuclease activity changed during callus growth and plant regeneration, but no developmental changes in electrophoretic patterns were detected. The increase in specific DNAse activity of nuclease was maximal in the exponential phase of callus growth on both growth and regeneration media, except for activity in the cytokinin-independent cell strain grown on growth medium. The specific DNAse activity of nuclease decreased during the bud formation period, while total DNAse activity calculated per mg of dry weight was slightly higher in vegetative buds (9.1U) than in undifferentiated tissue of callus (8.5U). Specific DNAse activity was, on the average, several hundred-fold lower in the vegetative tissues of flowering tobacco plants than in calluses in the exponential phase of growth.     

Selection of a universal hybridizer in Sinapis turgida Del. and regeneration of plantlets from somatic hybrids with Brassica species

A double-mutant cell line, which was unable to grow in a medium with NO ₃ ^- as the sole nitrogen source and was resistant to 5-methyl-tryptophan (5MT), was selected from cell suspensions of Sinapis turgida Del. (Brassicaceae) by culturing the cells in AA medium (Toriyama and Hinata, 1985, Plant Sci. 41, 179–183) supplemented with 50 mM chlorate and 229 M 5MT. Protoplasts of this cell line were fused with mesophyll protoplasts of Brassica oleracea L. with dextran, and six somatic hybrids were selected initially by culture in the NO ₃ ^- medium and then by transfer to the NO ₃ ^- medium supplemented with 229 M 5MT. The somatic hybrids produced embryoids, leaves and plantlets on a regeneration medium. The hybrid characters were confirmed by investigations of acid phosphatase (EC 3.1.3.2) and peroxidase (EC 1.11.1.7) isoenzymes, chromosome number, growth on NO ₃ ^- medium, 5MT resistance, and capacity to regenerate plants. Somatic hybrids between S. turgida Del. and B. nigra (L.) Koch were also obtained using this method. These results indicate that the double-mutant cell line established here will be able to serve as a universal hybridizer to select somatic hybrids after protoplast fusion with any other wild-type partner.Abbreviations B5 medium of Gamborg et al. (1968) - MS medium of Murashage and Skoog (1962) - 5MT 5-thethyltryptophan     

Factors affecting the glucosylation capacity of cell cultures of Datura innoxia and Scopolia carniolica for monophenolic compounds

Suspension grown cells of Datura innoxia and Scopolia carniolica were tested for their glucosylation capacity and some factors affecting the efficiency of the reaction were studied.Cells at the end of the exponential growth phase showed a high glucosylation capacity. Light conditions had little effect on the bioconversion reaction. For the substrates hydroquinone and p-hydroxybenzoic acid the bioconversions were concentration-dependent. Permeabilization with propanol diminished the bioconversion capacity. Depending on the substrate used, relatively large amounts of substrate and product could not be recovered. Tannic acid could partly prevent decomposition of the compounds. The bioconversion capacity of cultures with a low glucosylation capacity could be enhanced by addition of uridine diphosphate-glucose, indicating that the sugar donor is a critical factor. From six substrates the natural compounds hydroquinone, p-hydroxybenzoic acid and vanillin were glucosylated. No glucosides were detected from tyramin and two synthetic aminotetralines.Abbreviations 5HAT 5-hydroxyaminotetralin - NO437 2-(N-propyl-N-2-thienylamino)-5-hydroxytetraline - pHBA p-hydroxybenzoic acid - UDP-glucose uridine diphosphate-glucose     

Regeneration of interspecific somatic hybrids between Helianthus annuus L. and Helianthus maximiliani (Schrader) via protoplast electrofusion

Helianthus maximiliani is one of the wild Helianthus species with the genes for resistance to many pathogens including Sclerotinia sclerotiorum. Unfortunately, a transfer of disease resistance genes from this species into the cultivated sunflower is limited by its poor crossability with the cultivated sunflower and sterility of interspecific hybrids. To overcome this problem, mesophyll protoplasts of Sclerotinia sclerotiorum-resistant clone of H. maximiliani were electrically fused with etiolated hypocotyl protoplasts of the cultivated sunflower inbred line PH-BC₁-91A. Fusion products were embedded in agarose droplets and subjected to different regeneration protocols. Developed microcalluses were released from the agarose and transferred into solid media. Shoot regeneration was achieved by culture of calluses on regeneration medium containing 2.2 mg l⁻¹ BAP and 0.01 mg l⁻¹ NAA after the treatment with a high concentration of 2,4 D for a limited period of time. A morphological and RAPD analysis confirmed a hybrid nature of the regenerated plants.     

Morphological and molecular characterization of fertile tetraploid somatic hybrids produced by protoplast electrofusion and PEG-induced fusion between Lycopersicon esculentum Mill. and Lycopersicon peruvianum Mill.

Summary Mesophyl protoplasts of two genotypes of cultivated tomato (Lycopersicon esculentum Mill.) and one of its wild relative species (Lycopersicon peruvianum Mill.) were fused by using electrofusion and polyethyleneglycol-induced fusion. Forty-three fertile tetraploid somatic hybrid plants, each deriving from separate calli, were recovered from both fusion procedures. Electrofusion appeared more efficient than chemical fusion for the production of somatic hybrids. These plants appeared morphologically similar, whatever the fusion procedure and tomato genotype. They had intermediate leaf, inflorescence, and flower morphology. After self-pollination, the hybrids set fruit of intermediate size and color. The hybrid nature of these plants was confirmed by isoelectric focusing of the Rubisco small subunits used as nuclear markers. L. esculentum and L. peruvianum were distinguished by means of two chloroplast markers: CF1-ATPase subunit as analyzed by isoelectro-focusing and ct DNA restriction patterns. All hybrids displayed both ct markers of only one parent with no biased transmission. Mitochondrial (mt) DNAs were prepared from flower buds by using miniaturized CsCl gradients. Preliminary analysis indicated that mt genomes from the hybrids all differed from those of both parents. mt DNA Sall restriction enzyme analysis revealed that all but two hybrids contained one novel fragment of 13.5 kb. Gene mapping experiments showed that the mt apocytochrome b and ATPase subunit 9 homologies in the somatic hybrid mt DNA resembled L. esculentum and L. peruvianum, respectively; the mt nad5 probe distinguished at least four distinct patterns in the hybrids. These results indicated that mt DNA rearrangements involving intergenomic recombinations occurred through protoplast fusion. A greater mt DNA polymorphism was induced with chemical fusion than with electrofusion.     

Production of somatic hybrids between Daucus carota L. and Nicotiana tabacum

Protoplasts of a kanamycin-resistant (KR, nuclear genome), streptomycin-resistant (SR, chloroplast genome) and chlorophyll-deficient (A1, nuclear genome) Nicotiana tabacum (KR-SA) cell suspension cultures or X-ray-irradiated mesophyll protoplasts of kanamycin- and streptomycin-resistant green plants (KR-SR) were fused with protoplasts of a cytoplasmic male-sterile (CMS) Daucus carota L. cell suspension cultures by electrofusion. Somatic hybrid plants were selected for kanamycin resistance and the ability to produce chlorophyll. Most of the regenerated plants had a normal D. carota morphology. Callus induced from these plants possessed 23–32 chromosomes, a number lower than the combined chromosome number (66) of the parents, and were resistant to kanamycin, but they segregated for streptomycin resistance, which indicated that N. tabacum chloroplasts had been eliminated. Genomic DNA from several regenerated plants was analyzed by Southern hybridization for the presence of the neomycin phosphotransferase gene (NPTII); all of the plants analyzed were found to contain this gene. Mitochondrial (mt) DNA was analyzed by Southern hybridization of restriction endonuclease digests of mtDNA with two DNA probes, PKT5 and coxII. The results showed that the two plants analyzed possessed the mitochondria of D. carota. These results demonstrate that the regenerated plants are interfamilial somatic hybrids.