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

Summary 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.Abbreviations BAP 6-benzylaminopurine - NAA -napthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Kin kinetin - Zea zeatin - Adn S adenine sulphate - GA ₃ gibberellic acid     

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     

Analysis of characters coded for by T-DNA using hybridization between tumorous and normal plant cells

In somatic hybrids between tumourous Nicotiana tabacum (B6S3) and normal mesophyll Atropa belladonna cells, the following traits, directly or indirectly connected with T-DNA gene expression and tumourous growth, were analysed: lysopine dehydrogenase activity (LpDH), shoot suppression, root suppression, ability to grow on media with D-lactose as a sole carbon source and resistance to 2-aminoethylcysteine, 5-bromodeoxyuridine and 5-methyltryptophan. Dominant (semidominant) expression was observed for all but one trait studied, e.g. shoot suppression which behaved as a recessive character.Abbreviations LS-H Linsmaier and Skoog (1965) hormone-free medium - NAA Naphtaleneacetic acid - BAP 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - 5-BUdR 5-Bromodeoxyuridine - 2-AEC 2-Aminoethylcysteine - 5-MT 5-Methyltryptophan     

Increase of callus and embryoid production from hypocotyl protoplasts of sunflower (Helianthus annuus L.) by culture in microdrops

Hypocotyls of seedling plants of the sunflower inbred line HNK-81 were used as a source of protoplasts. Optimal conditions of isolation and culture of protoplasts were established. Using the method of individual culture in microdrops, a 77% final plating efficiency was achieved. Cytokinins, especially zeatin, showed a significant influence on the formation of globular embryo-like structures, but these failed to develop into mature embryos. Calli obtained in liquid media containing auxins and cytokinins grew on agar solidified media without growth regulators. Communicated by M. ONDŘEJ     

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.     

New CMS-associated phenotypes in cybrids Nicotiana tabacum L. (+Hyoscyamus niger L.)

Morphological characteristics were studied in cytoplasmic male sterile (CMS) cybrids possessing the tobacco nuclear genome, Hyoscyamus niger plastome and recombinant mitochondria. After backcrosses with tobacco, new flower modifications were found, including: conversions of stamens into branched filamentous structures; alterations in the shape of petals and the corolla limb; and high degrees of reduction in most flower organs. Vegetative alterations (leaf elongation and stem branching) occurred in some cybrids. Results confirmed that a protoplast fusion-based alloplasmic cytoplasm transfer, followed by conventional backcrosses, is a useful tool for generating alternative CMS sources with novel nucleo-cytoplasmic compositions. These alterations in the genetic status were accompanied by modified floral and vegetative phenotypes.     

Bioengineering of a phytoremediation plant by means of somatic hybridization

Phytoremediation is a technology that exploits a plant's ability to remove contaminants from the environment or render toxic compounds harmless. An efficient metal phytoremediating plant must combine high biomass production and established cultivation methods with high tolerance to a specific contaminant and ability for root uptake, translocation, and compartmentalization of contaminants in the above-ground biomass. Symmetric and asymmetric somatic hybridizations were used to introduce toxic metal-resistant traits from Thlaspi caerulescens into Brassica juncea. B. juncea hypocotyl protoplasts were fused with T. caerulescens mesophyll protoplasts. The hypocotyl protoplasts of B. juncea were stained with CFDA before fusion and thus fluoresced green under UV, whereas the mesophyll protoplasts of T. caerulescens had red autofluorescense. Heteroplasmic fusion products were identified and selected by flow cytometry and cell sorting. All putative hybrids grown in the greenhouse had morphological characteristics of B. juncea. A Thlaspi-specific repetitive sequence was hybridized to total DNA of plants, including the parental species. All plants from both symmetric and asymmetric fusions showed Thlaspi-specific hybridization patterns while B. juncea did not exhibit any hybridization signal. Hybrid plants, produced by asymmetric somatic hybridization between the two species, demonstrated high metal accumulation potential, tolerance to toxic metals, and good biomass production.     

Obtaining and analysis of intergeneric somatic hybrids between Brassica napus and "albino" line of Orychophragmus violaceus

The Orychophragmus violaceus chlorophylldefective line of "albino" type has been obtained by spectinomycin treatment. Somatic hybridization between Orychophragmus violaceus and Brassica napus was performed by fusion of green mesophyll protoplasts of rape and callus protoplasts of the O. violaceus "albino" line. Near two hundred of regenerant plants were selected according to the regeneration type and ability to become green, and were determined as hybrids. Chloroplast DNA in selected hybrids was identical to rape chlDNA, which was confirmed by the PCR-RFLP analysis of plastid DNA fragments. Fragments of hybrid mitochondrial DNA analyzed by the PCR-RFLP analysis were identical to fragments of O. violaceus. The nuclear genome of the majority of hybrids was represented by the O. violaceus genome, which was demonstrated by analyses of isoenzymes, DNA telomeric sequences, ribosomal and satellite DNAs, and the RAPD analysis. The cytogenetic analysis of a number of lines has shown variability in the number of chromosomes in the obtained lines.     

Analysis of nuclear and mitochondrial genomes of transplastomic Salpiglossis sinuata plants obtained by transfer of transformed plastids from N. tabacum (+S. sinuata) cybrid

New model system of plastid transformation has been proposed using a wild representative of Solanaceae family--S. sinuata. Earlier obtained cybrid plants N. tabacum (+ S. sinuata) were used for transformation experiments by PEG treatment of protoplasts with aadA gene that confers resistance to spectinomycin. Transformed S. sinuata plastome was transferred from N. tabacum (+ S. sinuata) cybrid to S. sinuata wild type plants by somatic hybridization. Molecular analysis of nuclear and mitochondrial DNA has been performed.     

Plant-made trastuzumab (herceptin) inhibits HER2/Neu+ cell proliferation and retards tumor growth

Background

Plant biotechnology provides a valuable contribution to global health, in part because it can decrease the cost of pharmaceutical products. Breast cancer can now be successfully treated by a humanized monoclonal antibody (mAb), trastuzumab (Herceptin). A course of treatment, however, is expensive and requires repeated administrations of the mAb. Here we used an Agrobacterium-mediated transient expression system to produce trastuzumab in plant cells.

Methodology/Principal Findings

We describe the cloning and expression of gene constructs in Nicotiana benthamiana plants using intron-optimized Tobacco mosaic virus- and Potato virus X-based vectors encoding, respectively, the heavy and light chains of trastuzumab. Full-size antibodies extracted and purified from plant tissues were tested for functionality and specificity by (i) binding to HER2/neu on the surface of a human mammary gland adenocarcinoma cell line, SK-BR-3, in fluorescence-activated cell sorting assay and (ii) testing the in vitro and in vivo inhibition of HER-2-expressing cancer cell proliferation. We show that plant-made trastuzumab (PMT) bound to the Her2/neu oncoprotein of SK-BR-3 cells and efficiently inhibited SK-BR-3 cell proliferation. Furthermore, mouse intraperitoneal PMT administration retarded the growth of xenografted tumors derived from human ovarian cancer SKOV3 Her2+ cells.

Conclusions/Significance

We conclude that PMT is active in suppression of cell proliferation and tumor growth.