Papers by Alejandro Fuentes
Biotechnology and applied biochemistry, 2004
A transient gene-expression system was developed and used to characterize promoter strength, to v... more A transient gene-expression system was developed and used to characterize promoter strength, to verify suitability of bacterial gene modifications for expression in plant cells, and to express active antibody molecules. The system is based on suspension tobacco cells transformed by Agrobacterium in a transient way. Conditions such as pre-culture of tobacco cells and the co-cultivation period were identified as determinants to achieve high expression levels. Under established conditions the activity strength of CaMV (cauliflower mosaic virus) 35 S and ToMoTV (tomato mottle taino virus) AL1 promoters were compared. A modified cry gene sequence from Bacillus thuringiensis was expressed and detected by Western-blot analysis. A monoclonal antibody against anti-(hepatitis B virus surface antigen) was produced in such quantities as to allow testing of biological activity and preliminary characterization.
Plant Disease, 2002
Lima beans are an important crop in Delaware and the Mid-Atlantic Region. In the summer of 2000, ... more Lima beans are an important crop in Delaware and the Mid-Atlantic Region. In the summer of 2000, five commercial cultivars (3-28, 184-85, C-elite Sel, Butter Bean, and Jackson Wonder) of lima bean in Delaware, Maryland, and New Jersey were observed with white, appressed mycelia on infected pods that appeared distinctly different from signs of downy mildew infection caused by Phytophthora phaseoli. Isolations were made by placing diseased pods between layers of rye media (1). A fungus that produced white mycelia with sporangia was consistently isolated. All Phytophthora isolates from the infected pods were heterothallic, grew at 35°C, had as much as 100 µm long pedicles on varying shapes of caducous sporangia with tapering base and >2 papillae, and were identified as P. capsici (2). Initially, three surface-disinfected pods from cv. Early Thorogreen plants grown in the greenhouse were floated on 20 ml of sterile water in a petri dish, and each was inoculated with a disk of P. capsici. This was repeated for nine isolates obtained from lima bean. After incubation for 7 days at room temperature, all 27 pods were infected, and P. capsici was reisolated from all the pods. A pathogenicity test was performed on the same cultivars from which the original field isolates were collected. Three seedlings and two plants with mature pods were inoculated with a sporangial suspension of each of the nine isolates and placed in a dew chamber for 5 days at 20 to 25°C and 100% relative humidity. White mycelial growth was observed on seedlings and mature pods. One inoculated plant developed brown-to-black stem lesions with white mycelia. All pods on the mature plants showed appressed, white mycelia identical to that observed in the commercial lima bean fields. P. capsici was consistently reisolated from all inoculated plants. In 2000, most infected pods in infested fields were observed low in the plant canopy or touching the soil. However, in 2001, infected pods were mostly in the lower and mid-portion of the plants observed in baby lima bean fields in Kent County, DE.
Methods in Molecular Biology, 2009
Biotechnology Letters, 2000
An anti-hepatitis B surface antigen (HBsAg), single-chain Fv antibody fragment (scFv) with a 6-hi... more An anti-hepatitis B surface antigen (HBsAg), single-chain Fv antibody fragment (scFv) with a 6-histidine N-terminal tag was produced in cultured transgenic tobacco cells. Western blot and antigen-specific chromatography showed high levels of biologically active scFv in the culture supernatant (1 mg l-1) and in cells (5 mg kg-1). A simple one-step scFv purification was developed using immobilized metal ion affinity
Virus Research, 2004
A 597 nt fragment from Tomato mottle Taino virus (ToMoTV) DNA-A, with 459 nt located upstream of ... more A 597 nt fragment from Tomato mottle Taino virus (ToMoTV) DNA-A, with 459 nt located upstream of the Replication-associated protein translation start codon, was tested for promoter activity in solanaceous plants. The promoter activity of this fragment (pRep 459::Rep ) was demonstrated when it was introduced upstream the uidA reporter gene into tobacco, potato and tomato plants by genetic transformation. It became active in 7-day-old transgenic tobacco seedlings as revealed by a vascular-specific pattern of gene expression which was maintained during the continued growth of the plant. Transformed potato and tomato plants also showed a vascular-specific pattern of expression. In comparative assays, pRep 459::Rep showed an expression activity 10-40-fold less than the 35S promoter from Cauliflower mosaic virus. To delimit the minimal cis-acting elements necessary for vascular specificity of this promoter, a set of PCR deletion mutants of pRep 459::Rep (pRep 459 , pRep 324 , pRep 203 , pRep 145 , pRep 132 and pRep 115 ), were generated and used to transform tobacco plants. Transgenic tobacco plants belonging to all the pRep versions were blue stained in the vascular system except those from the pRep 115 version. The results described in this report demonstrate that the minimal sequences necessary for the pRep promoter activity are confined in a segment of 132 nts (located between the nts 2454 and 2585 of the ToMoTV DNA A) and that this promoter harbors those elements sufficient for vascular-specific expression.
Transgenic Research, 2006
The whitefly-transmitted Tomato Yellow Leaf Curl Virus (TYLCV) is the major pathogen of tomato cr... more The whitefly-transmitted Tomato Yellow Leaf Curl Virus (TYLCV) is the major pathogen of tomato crop in Cuba and one of the most outstanding viral diseases of plants worldwide. In this work, we have developed transgenic tomato plants, transformed with an intron-hairpin genetic construction to induce post-transcriptional gene silencing against the early TYLCV replication associated protein gene (C1). The intron-hairpin RNA produced involves 726 nts of the 3¢ end of the TYLCV C1 gene as the arms of the hairpin, and the castor bean catalase intron. Transgenic tomato plants belonging to line 126, which harbor a single transgene copy, showed immunity to TYLCV, even in extreme conditions of infection (4-leaf-stage plants and 300 to many hundreds viruliferous whiteflies per plant during 60 days). Dot blot hybridization of these plants showed no TYLCV DNA presence 60 days after inoculation. Small interfering RNA molecules were detected in both inoculated and non-inoculated plants from line 126. These transgenic tomato plants of the otherwise very TYLCV-susceptible Campbell-28 tomato cultivar, are the first report of resistance to a plant DNA virus obtained by the use of the intron-hairpin RNA approach.
Biotecnol. Apl, 2007
This work describes the development of a fast and efficient methodology for Agrobacterium tumefac... more This work describes the development of a fast and efficient methodology for Agrobacterium tumefaciens-mediated genetic transformation of internodal stem segments from potato (Solanum tuberosum L.), cultivar Désirée, using phosphinothricin (PPT, ...
Biotecnología …, 2006
In this report we describe a methodology for rapid sugarcane shoot regeneration using meristemati... more In this report we describe a methodology for rapid sugarcane shoot regeneration using meristematic tissue from in vitro-grown plantlets. Shoot regeneration from previously induced meristematic tissue, in the presence of up to 22.62 µM of 2,4-D was evaluated. The explants were transferred, after a week induction, to a propagation medium supplemented with activated charcoal and then transferred to a regeneration medium for shoot development. The highest regeneration efficiency, averaging 9.34 shoots/explant, was obtained from cut meristematic tissue previously induced in the presence of 4.52 µM 2,4-D. We evaluated the first five stem segments from the basal stem to the leaves in two Cuban cultivars to determine their capacity for plant regeneration. The largest number of regenerated plantlets was obtained from the first two segments of the basal stem. For the first segment the regeneration frequency was 16.95 and 8.0 shoots/explant for C1051-73 and C86-12 varieties respectively. We concluded that this procedure is useful for the regeneration of large amounts of sugarcane plants thus minimizing production time.
Biotecnología …, 2003
When generating stably transformed transgenic plants, transient gene expression experiments are e... more When generating stably transformed transgenic plants, transient gene expression experiments are especially useful to confirm that the foreign molecule of interest is expressed with an adequate biological activity. In this paper we report the transient ...
Biotechnology …, 2000
An anti-hepatitis B surface antigen (HBsAg), single-chain Fv antibody fragment (scFv) with a 6-hi... more An anti-hepatitis B surface antigen (HBsAg), single-chain Fv antibody fragment (scFv) with a 6-histidine N-terminal tag was produced in cultured transgenic tobacco cells. Western blot and antigen-specific chromatography showed high levels of biologically active scFv in the ...
Biotechnology Journal, 2008
Agrobacterium tumefaciens technology is the battle horse for tomato genetic transformation. Howev... more Agrobacterium tumefaciens technology is the battle horse for tomato genetic transformation. However, tomato varieties with low regeneration capacity are very difficult to transform. In the past, tomato transformation through Agrobacterium infection was focused on varieties capable of high regeneration yield, while successful transformation of low regenerable cultivars has not been reported. The genotype response to tissue culture conditions is believed to drive the frequency of regeneration of transgenic plant, whereas the capacity for cell proliferation could determine the transformation efficiency through this technology. The Campbell-28 cultivar is an example of constraints arising from a high morphogenetic potential with low conversion compared to normal plants. In the present work the roles that contribute to improved transgenic plant recovery from this recalcitrant variety were explored for factors like Agrobacterium concentration and antibiotics for bacterial removal and transformant selection. Analysis of the efficiency from independent transformation experiments revealed a more than twofold increase of transformant regeneration after selection on ammonium glufosinate compared to kanamycin selection, showing a transformation efficiency of 21.5%.
Biotecnología …, 2003
In this work, we established a non-radioactive nucleic acid hybridization technique (Dot blot) fo... more In this work, we established a non-radioactive nucleic acid hybridization technique (Dot blot) for begomovirus detection. Two probes were constructed: a generic probe (TYLCV coat protein gene) and a specific probe (intergenic region and the 5' end of the TYLCV coat protein gene). The efficacy, analytical and diagnostic sensitivity, analytical and diagnostic specificity, positive and negative prediction values and repeatability were determined using the designed probes. Assays using the generic probe showed values of efficacy, sensitivity, specificity and repeatability between 85-100 percent, whereas the assays employing the TYLCV specific probe showed percentages higher than 98% for all parameters analyzed. The generic probe can be used to detect different begomoviruses, while the specific one can only be used to detect Tomato yellow leaf curl virus.
Biotecnología …, 2009
Tomato yellow leaf curl virus (TYLCV) is a major threat to tomato production in the tropics and s... more Tomato yellow leaf curl virus (TYLCV) is a major threat to tomato production in the tropics and sub-tropics around the world. The application of genetic engineering and pathogen derived resistance mechanisms to obtain tomatoes that are resistant to this pathogen is considered a promising alternative to the current protective practice against the virus. However, the development of transgenic tomato plants that are resistant to the virus is a resourceconsuming and time-consuming procedure, often with unpredictable efficiency, which hinders the evaluation of genetic designs. For this reason an assessment of the strategies against TYLCV replication preceding transgenic tomato development would ensure the protective potential of the candidate transgene. Attempting to circumvent this issue, the present study demonstrated the feasibility of using tobacco cell lines to study the consequences of c1 antisense expression on TYLCV replication. As a result, the transgenic tobacco cell lines were able to produce siRNA that is complementary to the c1 sequence and inhibited TYLCV multiplication, forecasting what would happen in transgenic plants harboring this antiviral strategy.
Annals of General Psychiatry, 2008
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Papers by Alejandro Fuentes