125 years of virology and ascentof biotechnologies based on viral expression.

The study of viruses lasts for more than a century since their discovery in 1892. In recent decades, viruses are also being actively exploited as a biotechnological tool. Plant-virus-driven transient expression of heterologous proteins is an actively developing production platform; it is the basis of several industrial processes that are currently being used for the production of multiple recombinant proteins. Viral vectors have also become useful tools for research. Viral vectors delivered by Agrobacterium (magnifection) provide for high pro-tein yield, rapid scale up and fast manufacturing. In this review, we explore modern approaches for bio technological production of recombinant proteins in plants using viral vectors.

Rapid, high-yield production in plants of individualized idiotype vaccines for non-Hodgkin's lymphoma.

BACKGROUND: Animal and clinical studies with plant-produced single-chain variable fragment lymphoma vaccines have demonstrated specific immunogenicity and safety. However, the expression levels of such fragments were highly variable and required complex engineering of the linkers. Moreover, the downstream processing could not be built around standard methods like protein A affinity capture. DESIGN: We report a novel vaccine manufacturing process, magnifection, devoid of the above-mentioned shortcomings and allowing consistent and efficient expression in plants of whole immunoglobulins (Igs). RESULTS: Full idiotype (Id)-containing IgG molecules of 20 lymphoma patients and 2 mouse lymphoma models were expressed at levels between 0.5 and 4.8 g/kg of leaf biomass. Protein A affinity capture purification yielded antigens of pharmaceutical purity. Several patient Igs produced in plants showed specific cross-reactivity with sera derived from the same patients immunized with hybridoma-produced Id vaccine. Mice vaccinated with plant- or hybridoma-produced Igs showed comparable protection levels in tumor challenge studies. CONCLUSIONS: This manufacturing process is reliable and robust, the manufacturing time from biopsy to vaccine is <12 weeks and the expression and purification of antigens require only 2 weeks. The process is also broadly applicable for manufacturing monoclonal antibodies in plants, providing 50- to 1000-fold higher yields than alternative plant expression methods.

Magnifection--a new platform for expressing recombinant vaccines in plants.

Today, plant biotechnology relies on two processes for delivery and expression of heterologous genes in plants: stable genetic transformation and transient infection with viral vectors. Although much faster, the transient route until recently was limited because of virus' low infectivity and its inability to carry average-size or larger transgenes. A recently developed new generation transfection technology overcomes these limitations by relying on Agrobacterium as an infective systemic agent that delivers viral replicons. This improved process is being used to simultaneously start transient gene amplification and high-level expression in all mature leaves of a plant, and such a transfection can be done on an industrial scale. This eclectic technology, called 'magnifection', combines advantages of three biological systems: vector efficiency and efficient systemic DNA delivery of Agrobacterium, speed and expression level/yield of a plant RNA virus, as well as posttranslational capabilities and low production costs of a plant. The proposed process allows for industrial production that does not require genetic modification of plants, that is much faster than previous methods, and that is biologically safe. Numerous applications in the area of vaccine manufacturing are being discussed.

Extensive developmental and metabolic alterations in cybrids Nicotiana tabacum (+ Hyoscyamus niger) are caused by complex nucleo-cytoplasmic incompatibility.

The genetic basis of multiple phenotypic alterations was studied in cell-engineered cybrids Nicotiana tabacum (+ Hyoscyamus niger) combining the nuclear genome of N. tabacum, plastome of H. niger and recombinant mitochondria. The plants possess a complex, maternally inheritable syndrome of nucleo-cytoplasmic incompatibility, severely affecting growth, metabolism and development. In vivo, the syndrome was manifested as: late germination of seeds; dramatic decrease of chlorophyll and carotenoids in cotyledons and leaves; altered morphology of cotyledons, leaves and flowers; and dwarfism. The leaf phenotype depended on light intensity. In 'green flowers' (an extreme phenotype), homeotic function B was downregulated. In vitro, the incompatibility syndrome was restricted to the pigment deficiency of cotyledons. Electron microscopy revealed perturbations in the differentiation of chloroplasts and palisade parenchyma cells in bleached leaves. The pigment deficiency accompanied by retarded growth is discussed as a result of plastome-genome incompatibility, whereas other features are likely to be due to nucleo-mitochondrial incompatibilities.

Tobacco ribosomal DNA spacer element stimulates amplification and expression of heterologous genes.

Here we show that the cis-acting genetic element aps (amplification-promoting sequence), isolated from the nontranscribed spacer region of tobacco ribosomal DNA (rDNA), increases the level of expression of recombinant proteins. Transgenic tobacco plants, transformed with expression cassettes containing the herbicide-resistant acetolactate synthase (hr-ALS) gene or the green fluorescent protein (GFP) gene fused to the aps sequence, had greater levels of corresponding messenger RNAs (mRNAs) and proteins compared to transformants lacking aps. Analysis of transgenic plants showed that aps increased the copy number and transcription of the adjacent heterologous genes and, in the case of hr-ALS, enhanced the herbicide resistance phenotype. Both the increased transgene copy number and enhanced expression were stably inherited. These data provide the first evidence that the aps sequence can be used for gene amplification in transgenic plants and possibly other multicellular organisms.

Functional cybrid plants of Lycopersicon peruvianum var 'dentatum ' with chloroplasts of Lycopersicon esculentum.

Fertile cybrid plants of three subclones, B1A, B3A, B4A were regenerated from the single colony obtained after the fusion of mesophyll protoplasts of plastome chlorophyll-deficient mutant Lycopersicon peruvianum var 'dentatum' (line 3767) and γ-irradiated mesophyll protoplasts of L. esculentum (cv 'Quedlinburger Frühe Liebe'). Cytogenetic, isozyme, RAPD, morphological and restriction analyses all showed that the subclones had the nuclear genome of L. peruvianum var 'dentatum' and plastome genome of L. esculentum, while the mitochondrial genome was altered. No phenotypical traits that could be taken as evidence of plastome-genome incompatibility in the cybrid subclones were observed. Genetic functionality of all subclones was proven by the backcrossing analysis. To study the functionality of the cybrid plants we also carried out an analysis of their photosynthetic system. Data on chlorophyll-a and -b content, analyses of the fluorescence induction curves, intensity of CO2 assimilation, pigment-protein complexes and polypeptides of thylakoid membranes showed the absence of structural and functional abnormalities in the photosynthetic apparatus of the cybrid plants. We concluded that the plastome of L. esculentum is able to effectively interract with the nuclear genome of L. peruvianum var 'dentatum' and together with the recombined chondriome can support genetic functionality of cybrid plants of the peruvianum tomato.

Production of recombinant proteins in plant root exudates.

The large-scale production of recombinant proteins in plants is limited by relatively low yields and difficulties in extraction and purification. These problems were addressed by engineering tobacco plants to continuously secrete recombinant proteins from their roots into a simple hydroponic medium. Three heterologous proteins of diverse origins (green fluorescent protein of jellyfish, human placental alkaline phosphatase [SEAP], and bacterial xylanase) were produced using the root secretion method (rhizosecretion). Protein secretion was dependent on the presence of the endoplasmic reticulum signal peptide fused to the recombinant protein sequence. All three secreted proteins retained their biological activity and, as shown for SEAP, accumulated in much higher amounts in the medium than in the root tissue.

Use of plant roots for phytoremediation and molecular farming.

Alternative agriculture, which expands the uses of plants well beyond food and fiber, is beginning to change plant biology. Two plant-based biotechnologies were recently developed that take advantage of the ability of plant roots to absorb or secrete various substances. They are (i) phytoextraction, the use of plants to remove pollutants from the environment and (ii) rhizosecretion, a subset of molecular farming, designed to produce and secrete valuable natural products and recombinant proteins from roots. Here we discuss recent advances in these technologies and assess their potential in soil remediation, drug discovery, and molecular farming.

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.

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.

Cybrid production based on mutagenic innactivation of protoplasts and rescuing of mutant plastids in fusion products: Potato with a plastome fromS. bulbocastanum andS. pinnatisectum.

A procedure for cybrid production, based on double treatment of donor protoplasts by physical and afterwards chemical mutagens at superlethal doses (γ-irradiation at a dose of 1000 Gy was applied for the inactivation of nuclei; 3-5 mMN-nitroso-N-methylurea was used for the efficient induction of plastome mutation) and the rescuing of mutant plastids after fusion with untreated recipient protoplasts, was developed. For identification of mutant donor-type plastids in fusion products a selection for streptomycin was performed. In two sets of experiments, in whichS. tuberosum served as the recipient of foreign cytoplasm with the wild tuber-bearing speciesS. bulbocastanum andS. pinnatisectum as donors, a total of about 40 streptomycin-resistant colonies was isolated. Eight regenerants from theS. tuberosum+S. bulbacastanum fusion combination and four fromS. tuberosum+S. pinnatisectum were further investigated using chromosome counting, analysis of esterase isoenzymes, restriction analysis of organelle DNA, and blot hybridization. All but one plant from both combinations were characterised as potato cybrids possessing exclusively foreign plastids and retaining a morphology typical of the recipient. Only in one line was rearranged mtDNA detected. The availability of potato cybrids facilitates the analysis of plastome-encoded breeding traits and the identification of the most valuable source of cytoplasm among the wild potato species. The described system for producing cybrids without genetic selectable markers in the parental material offers the possibility for the rescue of cytoplasmic mutations which are impossible to isolate by conventional approaches.

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. sylvestris. 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.

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

Thirteen nuclear asymmetric hybrids were regenerated under selective conditions following fusion of chlorophyll-deficient protoplasts from cultivated tomato (Lycopersicon esculentum Mill.) and gamma-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 mitochondrial 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.

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

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.

Genome reorganization in Nicotiana asymmetric somatic hybrids analysed by in situ hybridization.

In situ hybridization was used to examine genome reorganization in asymmetric somatic hybrids between Nicotiana plumbaginifolia and Nicotiana sylvestris obtained by fusion of gamma-irradiated protoplasts from one of the parents (donor) with non-irradiated protoplasts from the other (recipient). Probing with biotinylated total genomic DNA from either the donor or the recipient species unequivocally identified genetic material from both parents in 31 regenerant plants, each originating from a different nuclear hybrid colony. This method, termed genomic in situ hybridization (GISH), allowed intergenomic translocations containing chromosome segments from both species to be recognized in four regenerants. A probe homologous to the consensus sequence of the Arabidopsis thaliana telomeric repeat (5'-TTTAGGG-3')n, identified telomeres on all chromosomes, including 'mini-chromosomes' originating from the irradiated donor genome. Genomic in situ hybridization to plant chromosomes provides a rapid and reliable means of screening for recombinant genotypes in asymmetric somatic hybrids. Used in combination with other DNA probes, it also contributes to a greater understanding of the events responsible for genomic recovery and restabilization following genetic manipulation in vitro.

Isolation and culture of protoplasts of pigeonpea (Cajanus cajan L.).

High yields of protoplasts were obtained from leaves of aseptically grown plants and calli originated from different explants, in several cultivars of Cajanus cajan L. The protoplasts divided to form cell clusters in modified KM 8p medium and developed to protocolonies after dilution with liquid Caboche's medium within three to four weeks of culture. The protocolonies proliferated to form green calli on solid Caboche's medium. No shoots or plants were obtained.

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

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.

Nucleo-cytoplasmic incompatibility in cybrid plants possessing an Atropa genome and a Nicotiana plastome.

Twenty-nine cybrids possessing an Atropa belladonna nuclear genome and a Nicotiana tabacum plastome were selected from two independent protoplast fusion experiments. In contrast to the previously described reciprocal, green and fertile cybrids with a Nicotiana nuclear genome and an Atropa plastome (Kushnir et al. 1987), the plants obtained were totally chlorophyll-deficient. An Atropa nuclear genome and a Nicotiana plastome from these chlorophyll-deficient cybrids were combined with an Atropa or a Scopolia plastome and a Nicotiana nuclear genome, respectively, in control fusion experiments. All of these nuclear genome/plastome combinations gave rise to normal, green plants. Therefore, we conclude that an N. tabacum plastome is incompatible with an A. belladonna nuclear genome.

Isolation and characterization of intergeneric somatic hybrids in the Apocynaceae family.

Protoplasts were isolated from callus cultures of Rauwolfia serpentina Benth., Rhazya stricta Decaisne, and Catharanthus roseus (L.) G. Don, or from leaves of Vinca minor L. Protoplast isolation, culture, and fusion techniques as well as hybrid screening systems were developed for these species, and hybrids were obtained. Hybrid combinations were Rauwolfia + Vinca, Rauwolfia + Catharanthus, Rauwolfia + Rhazya, and Catharanthus + Vinca. For hybrid isolation, the physiological complementation method was utilized. Analyses of the material obtained included a cytogenetic study of the chromosomes, a study of multiple molecular forms of transferases and esterases, and the blot hybridization of restricted nuclear DNA using ribosomal DNA as a probe. Hybrids were identified in all species' combinations tried. A ten-fold increase in the accumulation of raucaffricine (relative to the parental Rauwolfia strain) was observed in one cell line of the Rauwolfia + Vinca hybrid. Our studies indicated the genetic stability of the great majority of the hybrid cell lines over a period of more than 20 months of in vitro growth. No shoot morphogenesis has so far been observed in this material.