The fate of recombinant chromosomes and genome interaction in Nicotiana asymmetric somatic hybrids and their sexual progeny

Genomic in-situ hybridization (GISH) was used to monitor the behaviour of parental genomes, and the fate of intergenomic chromosome translocations, through meiosis of plants regenerated from asymmetric somatic hybrids between Nicotiana sylvestris and N. plumbaginifolia. Meiotic pairing in the regenerants was exclusively between chromosomes or chromosome segments derived from the same species. Translocation (recombinant) chromosomes contained chromosome segments from both parental species, and were detected at all stages of meiosis. They occasionally paired with respectively homologous segments of N. sylvestris or N. plumbaginifolia chromosomes. Within hybrid nuclei, the meiotic division of N. plumbaginifolia lagged behind that of N. sylvestris. However, normal and recombinant chromosomes were eventually incorporated into dyads and tetrads, and the regenerants were partially pollen fertile. Recombinant chromosomes were transmitted through either male or female gametes, and were detected by GISH in sexual progeny obtained on selfing or backcrossing the regenerants to N. sylvestris. A new recombinant chromosome in one plant of the first backcross generation provided evidence of further chromosome rearrangements occurring at, or following, meiosis in the original regenerants. This study demonstrates the stable incorporation of chromosome segments from one parental genome of an asymmetric somatic hybrid into another, via intergenomic translocation, and reveals their transmission to subsequent sexual progeny.     

Protoplast culture and somaclonal variability of species of series Juglandifolia

Mesophyll protoplasts of species of series Juglandifolia (Solanum rickii, S. lycopersicoides, S. ochranthum and S. juglandifolium) were isolated and cultured in liquid nutrient media TM-2 or KM8P. The cell colonies formed were transferred onto agar-solidified media TM-3 or GM, and 10 to 15 days later onto TM-4, PRM, MS3ZG, KK or C regeneration media. Formation of the shoots for S. rickii and S. lycopersicoides was observed after 30 to 35 days on regeneration medium. The regenerated shoots were rooted on hormone-free MS medium. Morphological and cytogenetic analyses have shown that somaclonal variants might arise in the course of plant regeneration from protoplasts of these species.Abbreviations BAP 6-benzylaminopurine - 2, 4-D 2, 4 dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige and Skoog medium - B5 Gamborg medium - TRIS tris(hydroxymethyl)aminomethane     

A novel and fully scalable Agrobacterium spray‐based process for manufacturing cellulases and other cost‐sensitive proteins in plants

Transient transfection of plants by vacuum infiltration of Agrobacterium vectors represents the state of the art in plant‐based protein manufacturing; however, the complexity and cost of this approach restrict it to pharmaceutical proteins. We demonstrated that simple spraying of Nicotiana plants with Agrobacterium vectors in the presence of a surfactant can substitute for vacuum inoculation. When the T‐DNA of Agrobacterium encodes viral replicons capable of cell‐to‐cell movement, up to 90% of the leaf cells can be transfected and express a recombinant protein at levels up to 50% of total soluble protein. This simple, fast and indefinitely scalable process was successfully applied to produce cellulases, one of the most volume‐ and cost‐sensitive biotechnology products. We demonstrate here for the first time that representatives of all hydrolase classes necessary for cellulosic biomass decomposition can be expressed at high levels, stored as silage without significant loss of activity and then used directly as enzyme additives. This process enables production of cellulases, and other potential high‐volume products such as noncaloric sweetener thaumatin and antiviral protein griffithsin, at commodity agricultural prices and could find broad applicability in the large‐scale production of many other cost‐sensitive proteins.     

The role of survivin in angiogenesis during zebrafish embryonic development

Background  

Survivin is the smallest member of the inhibitor of apoptosis (IAP) gene family. Recently, the zebrafish survivin-1 gene has been cloned, showing remarkable sequence identity and similarity over the BIR domain compared with human and mouse survivin gene. Here we investigated the role of survivin in angiogenesis during zebrafish development. Morpholinos (MOs) targeting the 5' untranslated region (UTR) (Sur_UTR) and sequences flanking the initiation codon (Sur_ATG) of zebrafish survivin-1 gene were injected into embryos at 1–4 cell stage. Vasculature was examined by microangiography and GFP expression in Tg(fli1:EGFP) ^y1 embryos. Results: In embryos co-injected with Sur_UTR and Sur_ATG-MOs, vasculogenesis was intact but angiogenesis was markedly perturbed, especially in the inter-segmental vessels (ISV) and dorsal longitudinal anastomotic vessels (DLAV) of the trunk, the inner optic circle and optic veins of developing eyes and the sub-intestinal vessels. Apoptosis was increased, as shown by TUNEL staining and increase in caspase-3 activity. Efficacy of Sur_UTR and Sur_ATG-MOs was demonstrated by translation inhibition of co-injected 5'UTR survivin:GFP plasmids. The phenotypes could be recapitulated by splice-site MO targeting the exon2-intron junction of survivin gene and rescued by survivin mRNA. Injection of human vascular endothelial growth factor (VEGF) protein induced ectopic angiogenesis and increased survivin expression, whereas treatment with a VEGF receptor inhibitor markedly reduced angiogenesis and suppressed survivin expression. Conclusion: Survivin is involved in angiogenesis during zebrafish development and may be under VEGF regulation.     

Transfer of amiprophosmethyl resistance from a Nicotiana plumbaginifolia mutant by somatic hybridisation

Transfer of resistance to the phosphorothioamidate herbicide, amiprophosmethyl (APM), from the β-tubulin mutant of Nicotiana plumbaginifolia to the interspecific N. plumbaginifolia (+) N. sylvestris and to the intertribal N. plumbaginifolia (+) Atropa belladonna somatic hybrids has been demonstrated. Transfer to the recipient species was accomplished by: (1) symmetric hybridisationand (2) asymmetric hybridisation using γ-irradiation of donor protoplasts. Cytogenetic analysis confirmed the hybrid origin of the hybrids obtained. It was established that most of them typically inherited no more than three donor chromosomes, although it was possible to obtain symmetric hybrids in the case of symmetric fusion. Immunofluorescent microscopy analysis has shown that protoplasts of the mutant, and of the N. plumbaginifolia (+) N. sylvestris and N. plumbaginifolia (+) A. belladonna hybrids, retained the normal structure of interphase microtubule (MT) arrays and mitotic figures after treatment with 5 μM APM, whereas MTs of protoplasts of the recipients were destroyed under these conditions. It was also shown that hybrid clones contained an altered β-tubulin isoform originating from the N. plumbaginifolia mutant. The selected hybrid clones were characterised by cross-resistance to trifluralin, a dinitroaniline herbicide with the same mode of anti-MT action. Some of the somatic hybrids which could flower were fertile. It was established that seeds of some fertile hybrids were able to germinate in the presence of 5 μM APM. The results obtained thus support the conclusion that the technique of somatic hybridisation, especially asymmetric fusion, can be used to transfer APM resistance from the N. plumbaginifolia mutant to different (related and remote) plant species of the Solanaceae, including important crops. Received: 22 December 1997 / Accepted: 27 July 1999     

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     

Amplification of repetitive DNA from Nicotiana plumbaginifolia in asymmetric somatic hybrids between Nicotiana sylvestris and Nicotiana plumbaginifolia

Summary Asymmetric somatic hybrids were obtained between a chlorophyll-deficient mutant of Nicotiana sylvestris (V42) and a nitrate-reductase (NR)-deficient line of N. plumbaginifolia (cnx20 or Nia26), using each of the parents alternately as the irradiated donor. Irradiation doses applied ranged from 10 to 1,000 Gy of gamma-rays. Hybrid selection was based on complementation of NR deficiency with wild-type NR genes. To aid in the analysis of somatic hybrids, species-specific repetitive DNA sequences from N. plumbaginifolia (NPR9 and NPR18) were cloned. NPR18 is a dispersed repetitive sequence occupying about 0.4% of the N. plumbaginifolia genome. In turn, NPR9, which is part of a highly repetitive DNA sequence, occupies approximately 3% of the genome. The species-specific plant DNA repeats, together with cytological analysis data, were used to assess the relative amount of the N. plumbaginifolia genome in the somatic hybrids. In fusion experiments using irradiated N. plumbaginifolia, an increase in irradiation dose prior to fusion led to a decrease in N. plumbaginifolia nuclear DNA content per hybrid genome. For some hybrid lines, an increase in the quantity of repetitive sequences was detected. Thus, hybrid lines 1NV/21, 100NV/7, 100NV/ 9, and 100NV/10 (where N. plumbaginifolia was the irradiated donor) were characterized by amplification of NPR9. In the reverse combination (where N. sylvestris was the irradiated donor), an increase in the copy number of NPR18 was determined for hybrid clones 1VC/2, 1VC/3, 100VC/2 and oct100/7. Possible reasons for the amplification of the repeated sequences are discussed.     

A new middle repetitive sequence of Nicotiana plumbaginifolia genome

A middle repetitive sequence NPR18 was isolated from Nicotiana plumbaginifolia nuclear genome [8]. Sequences homologous to the repeat are dispersed through genomes of several Nicotiana species. compute-assisted data analysis of NPR18 primary sequence reveals several features attributed to mobile genetic elements: an AT content higher than average for nuclear DNA of genus Nicotiana plants; a number of direct and inverted repeats. Some of the repeats displayed homology to the terminal and subterminal repeats of Ac/Ds-like plant elements.     

Characterization of the Nicotiana tabacum L. genome by molecular cytogenetics

Nicotiana tabacum (2n=48) is a natural amphidiploid with component genomes S and T. We used non-radioactive in situ hybridization to provide physical chromosome markers for N. tabacum, and to determine the extant species most similar to the S and T genomes. Chromosomes of the S genome hybridized strongly to biotinylated total DNA from N. sylvestris, and showed the same physical localization of a tandemly repeated DNA sequence, HRS 60.1, confirming the close relationship between the S genome and N. sylvesfris. Results of dot blot and in situ hybridizations of N. tabacum DNA to biotinylated total genomic DNA from N. tomentosiformis and N. otophora suggested that the T genome may derive from an introgressive hybrid between these two species. Moreover, a comparison of nucleolus-organizing chromosomes revealed that the nucleolus organizer region (NOR) most strongly expressed in N. tabacum had a very similar counterpart in N. otophora. Three different N. tabacum genotypes each had up to 9 homozygous translocations between chromosomes of the S and T genomes. Such translocations, which were either unilateral or reciprocal, demonstrate that intergenomic transfer of DNA has occurred in the amphidiploid, possibly accounting for some results of previous genetic and molecular analyses. Molecular cytogenetics of N. tabacum has identified new chromosome markers, providing a basis for physical gene mapping and showing that the amphidiploid genome has diverged structurally from its ancestral components.     

High-yield production of authentic human growth hormone using a plant virus-based expression system

We describe here a high-yield transient expression system for the production of human growth hormone (hGH, or somatotropin) in transfected Nicotiana benthamiana leaves. The system is based on a recently described plant virus-based modular expression vector [Gleba, Y., Marillonnet, S. and Klimyuk, V. (2004) Engineering viral expression vectors for plants: the 'full virus' and the 'deconstructed virus' strategies. Curr. Opin. Plant Biol. 7, 182-188; Marillonnet, S., Giritch, A., Gils, M., Kandzia, R., Klimyuk, V. and Gleba, Y. (2004) In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium. Proc. Natl. Acad. Sci. USA, 101, 6852-6857], and represents a simple and fast alternative to stable transformation. By using various combinations of provector modules, hGH was produced in three compartments of the cell: the apoplast, the chloroplast and the cytosol. We found that targeting to the apoplast provided the highest amount of correctly processed and biologically active hGH, with a yield of up to 10% of total soluble protein or 1 mg per gram of fresh weight leaf biomass. These results indicate that the use of viral vectors for high-yield production of human therapeutic proteins in plants by transient expression provides an attractive alternative to production protocols using standard expression vectors in transgenic or transplastomic plants.