Patricia L. Marconi

In vitro recurrent selection of potato: production and characterization of salt tolerant cell lines and plants

A stable salt-tolerant potato cell line, able to grow on media containing 60–450 mM NaCl (i.e. low to high salinity) was selected. Callus grown on 120 or 150 mM NaCl showed higher fresh weights than the rest of the treatments. Replacing NaCl by KCl or Na2SO4 showed that reductions in fresh weight were mainly due to the presence of Na+ ions. When PEG 6000 was added to the medium instead of salt, the salt tolerant cell lines were unable to overcome the PEG-induced water stress. Whole plants, regenerated from salt tolerant callus, exhibited salt stress tolerance as evidenced by their higher fresh and dry weights when watered with 90 mM NaCl, and they also produced more tubers per plant under salt stress. Salt-tolerant plants differed phenotypically from control plants both in terms of leaf shape, tuber flesh and skin colour, which was reddish. In addition, DNA fingerprinting by RAPDs, with 70 different primers, confirmed that the salt tolerant regenerants also differed genotypically from the control, salt sensitive Kennebec potato plants from which they had been selected.

Immunocompetent truncated E2 glycoprotein of bovine viral diarrhea virus (BVDV) expressed in Nicotiana tabacum plants: a candidate antigen for new generation of veterinary vaccines.

The bovine viral diarrhea virus (BVDV) is the etiological agent responsible for a wide spectrum of clinical diseases in cattle. The glycoprotein E2 is the major envelope protein of this virus and the strongest inductor of the immune response. There are several available commercial vaccines against bovine viral diarrhea (BVD), which show irregular performances. Here, we report the use of tobacco plants as an alternative productive platform for the expression of the truncated version of E2 glycoprotein (tE2) from the BVDV. The tE2 sequence, lacking the transmembrane domain, was cloned into the pK7WG2 Agrobacterium binary vector. The construct also carried the 2S2 Arabidopsis thaliana signal for directing the protein into the plant secretory pathway, the Kozak sequence, an hexa-histidine tag to facilitate protein purification and the KDEL endoplasmic reticulum retention signal. The resulting plasmid (pK-2S2-tE2-His-KDEL) was introduced into Agrobacterium tumefaciens strain EHA101 by electroporation. The transformed A. tumefaciens was then used to express tE2 in leaves of Nicotiana tabacum plants. Western blot and ELISA using specific monoclonal antibodies confirmed the presence of the recombinant tE2 protein in plant extracts. An estimated amount of 20 μg of tE2 per gram of fresh leaves was regularly obtained with this plant system. Injection of guinea pigs with plant extracts containing 20 μg of rtE2 induced the production of BVDV specific antibodies at equal or higher levels than those induced by whole virus vaccines. This is the first report of the production of an immunocompetent tE2 in N. tabacum plants, having the advantage to be free of any eventual animal contaminant.

In vitro shoot culture and antimicrobial activity of Berberis buxifolia Lam

Berberis buxifolia Lam., known as “Calafate”, is a plant native to Argentina that exhibits antimicrobial activity. This biological activity is attributed to the isoquinoline alkaloid berberine. The aim of this research was to test the antimicrobial properties of different extracts of this species, taking berberine as the reference molecule, and to examine if the expression of bacterial multidrug resistance (MDR) efflux pumps could be responsible for possible resistance mechanisms. To this end, a wild-type and a mutant strain of Staphylococcus aureus with a defective MDR efflux pump were used and the minimum inhibitory concentrations of the extracts were determined. The studies were carried out with infusions of in vivo shoots and “Calafate” commercial tea, as well as with the media derived from shoot cultures incubated with different plant growth regulators (thidiazuron, picloram, and jasmonic acid). As far as antimicrobial activity is concerned, all the extracts tested were significantly more effective than berberine standard. “Calafate” commercial tea and shoot tea had inhibitory concentrations similar to the one observed for ampicillin standard. The media from the shoot cultures, however, were significantly more effective than all the others, particularly the one derived from jasmonic acid, suggesting the presence of compounds that could be acting synergistically with berberine. There were no differences in antimicrobial activity against the wild-type and the mutant S. aureus; no definite conclusions could be drawn concerning the relationship between MDR pumps and possible pathogen resistance to extracts of B. buxifolia.

How polyamine synthesis inhibitors and cinnamic acid affect tropane alkaloid production.

Hairy roots of Brugmansia candida produce the tropane alkaloids scopolamine and hyoscyamine. In an attempt to divert the carbon flux from competing pathways and thus enhance productivity, the polyamine biosynthesis inhibitors cyclohexylamine (CHA) and methylglyoxal-bis-guanylhy, drazone (MGBG) and the phenylalamine-ammonia-lyase inhibitor cinnamic acid were used. CHA decreased the specific productivity of both alkaloids but increased significantly the release of scopolamine (approx 500%) when it was added in the mid-exponential phase. However, when CHA was added for only 48 h during the exponential phase, the specific productivity of both alkaloids increased (approx 200%), favoring scopolamine. Treatment with MGBG was detrimental to growth but promoted release into the medium of both alkaloids. However, when it was added for 48 h during the exponential phase, MGBG increased the specific productivity (approx 200%) and release (250–1800%) of both alkaloids. Cinnamic acid alone also favored release but not specific productivity. When a combination of CHA or MGBG with cinnamic acid was used, the results obtained were approximately the same as with each polyamine biosynthesis inhibitor alone, although to a lesser extent. Regarding root morphology, CHA inhibited growth of primary roots and ramification. However, it had a positive effect on elongation of lateral roots.

State of the Art on Plant-Made Single-Domain Antibodies

State of the Art on Plant-Made Single-Domain Antibodies In addition to conventional antibodies (with heavy and light chains), camelids also produce functional antibodies devoid of light chains (HCAbs) without the first constant domain (CH1). Their variable domains (VHH) have binding properties, high stability and solubility, and are considered the smallest available intact antigen–binding fragment derived from a functional immunoglobulin. For their practical utilities VHHs have been expressed in different platforms. This review aims to provide an update in the field of plant-made VHHs, their applications and limitations, and a discussion about the challenges for the near future in this field.

The expression of the 14D9 catalytic antibody in suspended cells of Nicotiana tabacum cultures increased by the addition of protein stabilizers and by transference from erlenmeyer flasks to a 2‐L bioreactor

The effect of two protein stabilizers (polyvinylpyrrolidone [PVP] and gelatine) on growth and 14D9 yield of Nicotiana tabacum cell suspension cultures (Ab‐KDEL and sec‐Ab) was analyzed. The addition of PVP at a concentration of 1.0 g L−1 produced the highest total 14D9 yield (biomass + culture medium) in the Ab‐KDEL line (4.82% total soluble protein [TSP]). With the addition of gelatine, the highest total 14D9 yield (2.48% TSP) was attained in the Ab‐KDEL line at 5.0 g L−1 gelatine. When the Ab‐KDEL suspended cells were cultured in a 2‐L bioreactor, the highest 14D9 yield was 8.1% TSP at a 5% w/v inoculum size, which was the best 14D9 yield so far obtained in the platforms tested (E. coli, N. tabacum leaves and seeds, N. tabacum hairy roots, and cell suspension cultures).

Genetic Transformation for Metabolic Engineering of Tropane Alkaloids

1.1 Tropane alkaloids: history and relevance Historically, plants have been the major source of active compounds for the pharmaceutical industry. Today, plant extracts and its active principles represent 25% of the annual commercialized drugs in the United States (Kinghorn and Seo, 1996, Buttler 2004, Prakash Rout et al., 2009, Qurishi et al., 2010). Moreover, the World Health Organization has estimated that more than the 80% of the population in developing countries relies for their health care on traditional medicines mostly from plant origin (Canter et al., 2005). The plant active principles are, in general, a product of the plant secondary metabolism; they are lowmolecular-weight compounds that in general participate in defence mechanisms against diseases (phytoalexins) or as attractants for pollinator insects (pigments and fragrances) (Canter et al., 2005). The production of each group of secondary metabolites, as counterpart of primary metabolites, is in general restricted to a limited number of species, and they usually are organ-specific and furthermore, tissue-specific. According to their chemical properties, secondary metabolites could be classified in three main groups: a) terpenes, b) phenols and c) alkaloids. Terpenes and terpenoids are the primary constituents of the essential oils, widely used as natural flavour additives for food and fragrances. Phenols are a class of chemical compounds consisting of a hydroxyl group directly bonded to an aromatic hydrocarbon group that seem to be universally distributed in plants. They are essential for the growth and reproduction of plants, and are produced as a response for defending injured plants against pathogens. Finally, alkaloids are characterized for being alkaline nitrogenated organic compounds, derived from aminoacids, generally insoluble in water and soluble in alcohol, ether, chloroform, etc. Among the alkaloids, those derived from tropane have received particular attention for their properties.