Hybrids between tobacco crown gall cells and normal somatic cells of Atropa belladonna

Protoplast fusion of Nicotiana tabacum (B6S3) crown gall cells and Atropa belladonna leaf mesophyll cells was carried out. Hybrids were selected for their capacity to grow on hormone-free media and to green in light. Shoots incapable of rhizogenesis were regenerated on the same media and grafted onto normal plants of different species. 57 hybrid cell lines differing in their genetic constitution were produced. Analysis of hybrid lines involved the determination of the lysopine dehydrogenase (LpDH) activity and the molecular forms of esterase and amylase, a restriction analysis of chloroplast DNA and a cytogenetic study.Abbreviations LS-H Linsmaier and Skoog (1965) hormone-free medium - LpDH lysopine dehydrogenase     

Activity of the corn Spm transposon system in transgenic plants Orychophragmus violaceus (L.) O.E. Schulz obtained by both direct transfer of DNA to protoplasts and agrobacterial transformation of root explants

Transposon mediated insertional mutagenesis is one of the approaches for the unique gene cloning. A wild species of Cruciferae family Orychophragmus violaceus (L.) O.E. Schulz, which is of interest for practical breeding as a donor of improved plant oil, was an object of the investigation. Plasmid construction used in the experiments included selective NPT II gene, reported GUS gene serving as an excision marker, structural BAR gene located within the dSpm element and Spm transposase. The GUS gene of this plasmid had not his own promoter and became functional only after Spm-transposition. Transformed Orychophragmus violaceus (L.) O.E. Schulz. plants were obtained by direct mesophyll protoplast transformation as well as Agrobacterium tumefaciens-mediated root explant transformation. Gene transfer and the transposition event were confirmed by the GUS activity and the PCR analysis. Relative transformation efficiency using protoplasts was 5.8%.     

Construction of the cybrid transplastomic Brassica napus plants containing Lesquerella fendleri chloroplasts

Transferring of Lesquerella fendleri genetically transformed plastids to Brassica napus plants has been performed with the somatic hybridization method. The plastome of the previously engineered transplastomic L. fendleri plants contained the aadA16gfp selective marker gene conferring spectinomycin/streptomycin resistance and green fluorescence under UV light. The protoplasts of B. napus chlorophyll-deficient plants were fused with gamma-irradiated protoplasts of L. fendleri transplastomic plants. A total of 59 green hybrid colonies have been isolated followed by spectinomycin/streptomycin selection. Shoot regeneration has been observed for two cell lines. Morphologically normal plants have been regenerated for one of them. PCR and isozyme analyses showed that the plants were transplastomic cybrids containing B. napus nuclei and L. fendleri transformed chloroplasts.     

The antidepressant-like effect of Ocimum basilicum in an animal model of depression

We investigated the efficacy of Ocimum basilicum (OB) essential oils for treating depression related behavioral, biochemical and histopathological changes caused by exposure to chronic unpredictable mild stress (CUMS) in mice and to explore the mechanism underlying the pathology. Male albino mice were divided into four groups: controls; CUMS; CUMS plus fluoxetine, the antidepressant administered for pharmacological validation of OB; and CUMS plus OB. Behavioral tests included the forced swim test (FST), elevated plus-maze (EPM) and the open ?eld test (OFT); these tests were performed at the end of the experiment. We assessed serum corticosterone level, protein, gene and immunoexpression of brain-derived neurotropic factor (BDNF) and glucocorticoid receptors (GRs) as well as immunoexpression of glial fibrillary acidic protein (GFAP), Ki67, caspase-3 in the hippocampus. CUMS caused depression in the mice as evidenced by prolonged immobility in the FST, prolonged time spent in the open arms during the EPM test and reduction of open field activity in the OFT. OB ameliorated the CUMS induced depressive status. OB significantly reduced the corticosterone level and up-regulated protein and gene expressions of BDNF and GR. OB reduced CUMS induced hippocampal neuron atrophy and apoptosis, and increased the number of the astrocytes and new nerve cells. OB significantly increased GFAP-positive cells as well as BDNF and GR immunoexpression in the hippocampus.     

Fertile asymmetric somatic hybrids between Lycopersicon esculentum Mill. and Lycopersicon peruvianum var. dentatum Dun.

Summary Thirteen nuclear asymmetric hybrids were regenerated under selective conditions following fusion of chlorophyll-deficient protoplasts from cultivated tomato (Lycopersicon esculentum Mill.) and -(-irradiated protoplasts from the wild species Lycopersicon peruvianum var. dentatum Dun. All hybrid plants were classified as being asymmetric based on morphological traits, chromosome numbers and isozyme patterns. The majority of the hybrids inherited Lycopersicon peruvianum var. dentatum chloroplasts. Mitochondrial DNA analysis revealed mixed mitochondria populations deriving from both parents in some of the hybrids and rearranged mitochondrial DNA in others. The asymmetric hybrids express some morphological traits that are not found in either of the parental species. Fertile F₁ plants were obtained after self-pollination of the asymmetric hybrids in four cases. The results obtained confirm the potential of asymmetric hybridization as a new source of genetic variation, and as a method for transferring of a part of genetic material from donor to recipient, and demonstrate that it is possible to produce fertile somatic hybrids by this technique.     

Intertribal hybrid cell lines of Atropa belladonna (X) Nicotiana chinensis obtained by cloning individual protoplast fusion products

Summary After fusion of isolated mesophyll protoplasts of belladonna (Atropa belladonna) with callus protoplasts of Chinese tobacco (Nicotiana chinensis) followed by mechanical isolation and cloning of individual heteroplasmic fusion products, 13 cell clones were obtained. The hybrid nature of most of the clones has been confirmed by biochemical (studies of amylase isozymes), cytogenetic (size and morphology of chromosomes) and physiological (peculiarities of cell-growth in vitro) analyses. Study of chromosomes and isozyme patterns in the hybrid cell lines revealed the presence of both parental genomes, without an indication of chromosome elimination, six months after hybridization. In 4 cell lines shootlike structures and plantlets have been produced by means of transfer to organogenesis-inducing media. The data obtained are interpreted as new evidence for the possibility of using non-sexual hybridization for the production of intergeneric, intertribal plant hybrids which cannot be obtained by sexual crossing. From these results the potential of Atropa (X) Nicotiana hybrids as a model system for genetic studies of distantly related plant species is discussed.This work is part of a joint project between Institute of Botany of the Ukrainian Academy of Sciences, Kiev, USSR, and Institute of Pharmaceutical Biology, University of Munich, FRG     

Transfer of transformed <Emphasis Type="Italic">Lesquerella fendleri</Emphasis> (Gray) Wats. chloroplasts into <Emphasis Type="Italic">Orychophragmus violaceus</Emphasis> (L.) O.E. Schulz by protoplast fusion

Asymmetric intergeneric hybrid plants were obtained through protoplast fusion between Orychophragmus violaceus (L.) O.E. Schulz and Lesquerella fendleri (Gray) Wats. The latter carried chloroplasts transformed with the fused aadA16gfp gene construct, conferring streptomycin–spectinomycin resistance and UV-induced green fluorescence. The somatic hybrids were selected using the properties of spectinomycin-induced plastid defects in “albino” O. violaceus plants (chloroplast recipient) combined with the γ-irradiation-induced inactivation of nuclei in plastid donor L. fendleri. The morphology and esterase isozyme pattern of the hybrid plant as well as the results of the PCR analysis of internal transcribed spacer of nuclear ribosomal DNA proved that the regenerated hybrids carried O. violaceus nuclei, while PCR amplification of the atpB– rbcL spacer and aadA16gfp gene fragments confirmed the presence of the transformed L. fendleri chloroplasts in these plants. Expression of the fused aadA16gfp gene construct was confirmed by sodium dodecylsulfate–polyacrylamide gel electrophoresis analysis and the resistance of the obtained plants to both streptomycin and spectinomycin.     

Alterations in chlorophyll a/b binding proteins in Solanaceae cybrids

In this study we have constructed a number of plants (cybrids), in which the nuclear genome of Nicotiana plumbaginifolia is combined with the plastome of Atropa belladonna, or the nuclear genome of N. tabacum with plastomes of Lycium barbarum, Scopolia carniolica, Physochlaine officinalis or Nolana paradoxa. Our biochemical and immunological analyses prove that in these cybrids the biogenesis of the chlorophyll a/b binding proteins (CAB) of the light harvesting complex II (LHCII) is altered. Besides normal sized CAB polypeptides of 27, 25.5 and 25 kDa, which become less abundant, the cybrids analyzed have additional polypeptides of 26, 24.5 and 24 kDa. Direct protein micro-sequencing showed that at least two truncated 26 kDa CAB polypeptides in plant cells containing a nucleus of N. plumbaginifolia and plastids of A. belladonna are encoded by the type 1 Lhcb genes. These polypeptides are 11–12 amino acids shorter at the N-terminus than the expected size. Based on the available data we conclude that the biogenesis of the LHCII in vivo may depend on plastome-encoded factor(s). These results suggest that plastome-encoded factors that cause specific protein degradation and/or abnormal processing might determine compartmental genetic incompatibility in plants.