María Alejandra Alvarez

Effect of carbon and nitrogen sources on growth and solasodine production in batch suspension cultures of Solanum eleagnifolium Cav.

The effect of inoculum size, carbon sources (fructose, glucose, maltose, sucrose), nitrate and ammonia on solasodine production by Solanum eleagnifolium Cav. was studied. The specific growth rate was estimated to be 0.15–0.20 d-1 with all sugars tested at a concentration of 90 mM. Sucrose (180 mM) produced the highest biomass value (about 2.8 mg DW ml-1) while the lowest one was produced by maltose. Although solasodine productivity values after 11 days of culture were similar for all sugars tested, the maximum values of productivity (0.9 mg g-1 d-1) were achieved after 6 days of culture with sucrose (180 mM). Solasodine productivity of cultures conducted with a large inoculum (20% w/v fresh material) was double that with a small inoculum (10% w/v fresh material).

Peroxidase production in vitro by Armoracia lapathifolia (horseradish)-transformed root cultures: effect of elicitation on level and profile of isoenzymes.

Transformed roots of Armoracia lapathifolia (horseradish) were established by infection with Agrobacterium rhizogenes LBA 9402. They were used as a culture system in vitro for peroxidase production in vitro, to avoid many of the problems that affect the traditional production from field‐grown species of Armoracia sp. The time course of growth of these cultures showed that total peroxidase attained maximum levels at the end of the exponential growth phase. At this stage of culture, elicitation assays were performed with AgNO3 and CuSO4 as abiotic elicitors and with fungal extracts of Verticillum sp., Monodyctis cataneae and Aspergillus niger as biotic elicitors. The best results were obtained with Verticillum sp., 24 h after elicitation, with an increase of approx. 100% in peroxidase activity. The isoenzyme pattern analysed by isoelectric focusing revealed predominantly basic and acidic isoenzymes in both plant roots and transformed root cultures. Elicited samples showed a similar isoenzyme pattern with a slight increase in basic isoenzymes.

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.

Expression of the antibody 14D9 in Nicotiana tabacum hairy roots

Nicotiana tabacum hairy roots that express the antibody 14D9 were established. The 14D9 antibody yield obtained after 20 days of culture was 5.95 μg 14D9ml -1 . The addition of the reticulum endoplasmic retention sequence KDEL demonstrated a positive effect over the intracellular 14D9 amounts with a yield increase up to 20.82 µg ml -1 . DMSO increased the antibody amount in the biomass from 20.00 to 64.03 µg ml -1 while PVP (at 1.5 gl -1 ) and gelatine (at 5.0 gl -1 ) increased total 14D9 amounts in the culture medium to 25 µg and 14 µg respectively.

The influence of auxins, light and cell differentiation on solasodine production bySolanum eleagnifolium Cav. calli.

The effect of auxins, light and cellular production ofSolanum eleagnifolium Cav. calli were studied. 2,4-dichlorophenoxyacetic acid (4.5 μM) was the plant growth regulator used for calli initiation and this produced the highest solasodine concentration. The solasodine concentration in darkness was significantly lower than that achieved under a photoperiod of 16 h. Differentiated tissue obtained by adequate hormonal balance (several ratios of 3-indolebutyric acid to 6-benzylaminopurine) produced higher yields of solasodine than non-differentiated tissue. 3-indolebutyric acid (2.5 μM) and 6-benzylaminopurine (8.8 μM) increased the productivity of solasodine by 100%.

Solasodine Production by Solanum eleagnifolium Cav. in vitro Cultures: Influence of Plant Growth Regulators, Age and Inoculum Size. Large-Scale Production

Abstract The effect of different auxins (2,4-D, 2,4,5-T, NAA and IAA) on calli maintenance and plant cell suspension initiation as well as the influence of auxins in combination with citoquinines on growth and solasodine productivity by cell suspensions of Solanum eleagnifolium Cav. was studied. 2,4-D and 2,4,5-T produced a friable and productive calli line and a fine and homogeneous cell suspension cultures. 2,4-D (0.5 μM or 5 μM) or NAA (50 μM) produced the highest growth index and solasodine productivity with values of about 0.5 mg 1−1 day−1 to 0.8 mg 1−1 day−1. Simultaneous administration of auxin and cytokinin increased the solasodine productivity with a diminution of cell growth. The combination of NAA (50 μM) with kinetin (0.25 μM) produced an increase of solasodine productivity from 0.8 mg 1−1 day−1 to 2.54 mg 1−1 day−1. An inoculum size of 5% V/V and 19-day-old was selected to be used in fermentation processes in order to obtain solasodine production by S. eleagnifolium Cav. suspension cultures. ...

Genetic transformation of Calibrachoa excellens via Agrobacterium rhizogenes: changing morphological traits.

Summary Leaves and stem segments of in vitro-cultured Calibrachoa excellens were used as explants to obtain transgenic hairy roots after inoculation with the following strains of Agrobacterium rhizogenes: LBA 15834, LBA 8490, LMG 155, and A13. Only leaves showed sensitivity towards Agrobacterium infection. A. rhizogenes strain LBA 15834 was the most virulent among the four strains tested, with 33% of leaves showing root and callus proliferation, while only 10% of the explants showed root and/or callus development using strain A13, and no responses were observed in explants infected with the other two strains used. Two different transformation events were recovered as transgenic plants regenerated from hairy roots via spontaneous organogenesis. These plants showed, to different degrees, the classic symptoms of hairy root disease. PCR analysis of the recovered plants showed the presence of the rol A, rol B, rol C, and rol D genes, and the absence of the vir D1 gene, confirming the absence of Agrobacterium. Southern blotting indicated the presence of one inserted copy in one transformation event, and two copies in the other. Morphological and physiological differences were observed between the independent transformation events, as well as several differences such as flower colour and leaf shape, between plants of the same event. Pollen from one of the transformed plants was used to cross with commercial hybrids of Calibrachoa, and the presence of the rol A and rol B genes was confirmed in four out of 26 plants recovered that showed some hairy root characteristics. For the first time, transgenic hairy rootplants of the genus Calibrachoa have been regenerated, which would allow transgene incorporation into a breeding programme.

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.