Ricardo Ordas - Academia.edu (original) (raw)

Papers by Ricardo Ordas

Journal of virology, 1999

The major structural protein VP60 of rabbit hemorrhagic disease virus (RHDV) has been produced in... more The major structural protein VP60 of rabbit hemorrhagic disease virus (RHDV) has been produced in transgenic potato plants under the control of a cauliflower mosaic virus 35S promoter or a modified 35S promoter that included two copies of a strong transcriptional enhancer. Both types of promoters allowed the production of specific mRNAs and detectable levels of recombinant VP60, which were higher for the constructs carrying the modified 35S promoter. Rabbits immunized with leaf extracts from plants carrying this modified 35S promoter showed high anti-VP60 antibody titers and were fully protected against the hemorrhagic disease.

The effect of introns on gene expression was evaluated. Several intron-promoter combinations were... more The effect of introns on gene expression was evaluated. Several intron-promoter combinations were introduced by microparticle bombardment into two pine species, stone pine (Pinus pinea L.) and salgareño pine (Pinus nigra Arn. ssp. Salzmannii (Dunal) Franco). Gene expression was evaluated by measuring transient GUS expression. Two promoters (CaMV35S and double CaMV35S modified) and two introns (intron 1 from maize genes alcohol dehydrogenase-1 and Shrunken-1) were used in our study. In both pine species tested, the Sh1-int1 increased transient GUS expression from 2 to 6-fold compared to the intron-less construction. On the contrary, the inclusion of the Adh1-int1 associated with the double CaMV35S modified resulted in a dramatic decrease in the expression in both pine species analyzed. Our results suggest that Sh1-int1 may be useful for the acquisition of the required levels of genetic activity of new agronomic traits introduced into pines.

Transfer and expression of the β-glucuronidase gene (uidA) in cultured cotyledons of stone pine (... more Transfer and expression of the β-glucuronidase gene (uidA) in cultured cotyledons of stone pine (Pinus pinea L.) was obtained by microprojectile bombardment. Conditions for optimum transient expression were established by using plasmid pBI121 delivered by 1.0 µm-diameter gold particles, into 1-day-old cultured cotyledons. Helium pressure of 6.2 MPa, microcarrier travel distance of 6 cm, and 0.8 µg of plasmid DNA per bombardment, were the best parameters for high levels of transient uidA expression. By using these parameters, 98% of bombarded cotyledons showed β-glucuronidase activity, with a mean of 63 Gus foci per cotyledon. This system was used to study the expression of uidA gene driven by several heterologous promoters. The expression under the control of the sunflower polyubiquitin gene (UbB1) promoter ( 1 deletion) was higher (99% of GUS positive cotyledons) than under the control of the CaMV35S promoter, whereas the rice actin and the maize alcohol dehydrogenase gene promoters gave lower uidA expression, as determined histochemically. These results were confirmed by using the GUS fluorometric assay. Use of a deletion of the sunflower polyubiquitin promoter resulted in GUS activity detectable 35 days after bombardment, and significant levels of GUS activity were confirmed at the end of that period. The results will be useful to design protocols for stable transformation and high levels of transgene expression in P. pinea.

We have investigated the use of five different promoter-5%UTR leader combinations to develop a po... more We have investigated the use of five different promoter-5%UTR leader combinations to develop a potato plant-based expression system for the production of VP60 protein from rabbit haemorrhagic disease virus (RHDV). The relative efficiency of CaMV 35S, modified CaMV 35S, sunflower polyubiquitin and patatin promoters, as well as the addition of the 5% leader sequence from 10 gene of T7 phage, on VP60 gene expression in leaf and tuber tissues of plants, cultivated both in vitro and as potted plants, was investigated. Our results indicated that the B33 promoter with the 10 leader gave the highest level of expression in tubers. The rabbits immunised with tuber plant extracts containing VP60 elicited specific antibody responses and were protected against challenge with virulent RDHV.

Rabbit hemorrhagic disease, which is caused by a calicivirus, is a lethal infection of adult anim... more Rabbit hemorrhagic disease, which is caused by a calicivirus, is a lethal infection of adult animals that is characterized by acute liver damage and disseminated intravascular coagulation. In this study, we report the production of the major structural protein VP60 of rabbit hemorrhagic disease virus in transgenic tubers of potato plants and its use as an oral immunogen in rabbits.

Viral Immunology, 2001

We have recently communicated the oral and parental immunogenicity of the structural protein VP1 ... more We have recently communicated the oral and parental immunogenicity of the structural protein VP1 of foot and mouth disease virus (FMDV) expressed in different transgenic plants. Those results clearly indicated the necessity of increasing the expression of the foreign genes in the transgenic plant to avoid additional steps toward the purification and/or concentration of the antigen of interest. Here, we report the production of transgenic potatoes plants containing the VP1 gene cloned under the regulatory activity of either a single (pRok2) or a double (pRok3) copy of the S35 cauliflower mosaic virus (CaMV 35S) promoter, as a strategy for increasing the level of VP1 gene expression. The presence of the VP1 gene in the plants was confirmed by polymerase chain reaction (PCR) and its specific transcription activity was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that, although the immunized animals presented a FMDV VP1 specific antibody response and protection against the experimental challenge, no significant differences were demonstrated in the immunizing activity of plant extracts obtained from the pRok2 or pRok3 transformed plants. These results confirm those previously obtained using other plant species allowing the possibility of using plants as antigen expression vectors, and demonstrated that at least in the potato system, the use of double CaMV 35S promoter does not cause a significant increase in the level of the VP1 expressed.

Trees, 2009

This paper describes a micropropagation protocol for in vitro propagation of mature Stone Pine tr... more This paper describes a micropropagation protocol for in vitro propagation of mature Stone Pine trees. Axillary bud development was achieved by culturing bud explants in media containing various cytokinins. Experiments were conducted to test the effect of asepsis conditions, type and concentration of cytokinin and rooting protocol. Four cytokinins were tested, namely, benzyladenine, meta-topolin, N-benzyl-9-(2-tetrahydropyranyl)-adenine and thidiazuron (TDZ) of which TDZ gave the best results, as 59% shoot development was obtained following the application of 1 μM TDZ to the culture medium. The shoot development was significantly influenced by the genotype of the tree, but was effective in explants from all 20 genotypes used in the trial. In vitro rooting was, however, difficult to achieve and could only be induced at low rates. This protocol represents the first successful biotechnological approach to the micropropagation of adult Pinus pinea trees.

Plant Science, 1991

ABSTRACT Viable protoplasts were isolated from suspension culture cells of globe artichoke (Cynar... more ABSTRACT Viable protoplasts were isolated from suspension culture cells of globe artichoke (Cynara scolymus L.). Protoplast yield, cell wall regeneration and cell division were influenced by several factors, e.g. age of the cell culture, enzyme composition, culture density. First cell divisions were observed after 4–6 days of culture. Upon transfer to solid medium, the cell colonies gave rise to proliferating green calli. Globular structures formed on these calli but failed to develop further.

Plant Physiology and Biochemistry, 2001

Isolated cotyledons from Pinus pinea L. germinated embryos cultured in vitro showed shoot organog... more Isolated cotyledons from Pinus pinea L. germinated embryos cultured in vitro showed shoot organogenesis in response to treatment with 4.4 µM N 6 -benzyladenine (BA). In late germination phases, the extent of the response onset decreased and adventitious buds were redistributed to the basal zone of the cotyledons. To understand these two events, analyses of indole-3-acetic acid (IAA) and several cytokinins have been carried out in apical and basal portions of cotyledons taken at four different culture periods of the embryos on germination medium (0, 2, 4 or 6 d). The highest endogenous content of all hormones, except N 6 -isopentenyladenosine, was found in the most responsive explants, those that were taken from 0-d-old cultured embryos. Apical portions of cotyledons taken from 0 to 4-d-old cultured embryos showed a reduction in the content of IAA and several cytokinins, but basal portions did not show the same pattern. BA dose-response curves were made to explain the loss of caulogenic potential with the age of the explants. The results suggested the combination of two factors, changes in the endogenous hormonal content in original explants and loss of their sensitivity to exogenously applied BA, as the cause for the reduction of the ability of Pinus pinea cotyledons to form adventitious buds. © 2001 Éditions scientifiques et médicales Elsevier SAS adventitious bud / cytokinin / embryo culture / indole-3-acetic acid / Pinus pinea BA, N 6 -benzyladenine / BFC, bud forming capacity / DHZ, dihydrozeatin / DHZR, dihydrozeatin riboside / IAA, indole-3-acetic acid / iP, N 6 -isopentenyladenine / iPR, N 6 -isopentenyladenosine / ½LP, modified Le Proive medium / ½LPB, modified Le Proive medium + BA / ½LPC, modified Le Proive medium + activated charcoal / SEC, shoot elongation capacity / Z, zeatin / ZR, zeatin riboside *Correspondence and reprints: fax +34 985104867.

Plant Molecular Biology, 1991

Sequences encoding the immunoglobulin heavy-chain variable (VH) domains were engineered in a new ... more Sequences encoding the immunoglobulin heavy-chain variable (VH) domains were engineered in a new general purpose vector to transform plants via Agrobacterium. The expression of an isolated VH domain (IVD) after introduction into the plant genome has been monitored by northern, western and immunohistochemical analysis. Immunoblotting showed that the polypeptide was stably expressed and accounted for up to 1 ~o of the soluble protein fraction. It is therefore proposed that single immunoglobulin domains of suitable specificity expressed in plants may constitute an effective system to inhibit the activity of molecules involved in plant pathology or plant development.

Plant Cell, Tissue and Organ Culture, 2008

Organogenesis response of six selected half-sibling families of stone pine (Pinus pinea L.) has b... more Organogenesis response of six selected half-sibling families of stone pine (Pinus pinea L.) has been evaluated, showing genotype-dependent behaviour. The caulogenic phase was characterized by high values of Survival and Organogenesis, while the rooting phase (the bottleneck of many coniferous species) showed great variability among families. Provenance influence was also studied, and the rhizogenesis protocol was optimized for the selected families. The highest values were obtained with family 36, with 100% of Organogenesis, a Bud Formation Capacity (BFC) Index of 6.54 and 38.44% of Rooted Shoots; on the other hand, family 61 presented the worst results, with 83.64% Organogenesis, a BFC Index of 3.01 and a 29.69% Rooting Rate. According to these results, both families will be used in further experiments looking for the underlying bases of the different organogenic behaviour between both families under the same culture conditions. In addition to this, and for the first time in this species, random amplified polymorphism DNA (RAPD) analysis has been carried out to determine whether somaclonal variation had occurred. The results suggested an absence of variation during the whole in vitro process, although more thorough studies would be required for a conclusive answer.

Plant Cell, Tissue and Organ Culture, 2007

An optimized protocol for Agrobacterium tumefaciens-mediated transformation of mature Quercus sub... more An optimized protocol for Agrobacterium tumefaciens-mediated transformation of mature Quercus suber L. embryogenic masses is reported. In this work several variables were tested. Plant genotype, explant type and time elapsed between the last subculture and inoculation, i.e. the explant preculture period, were found to be very important. Interaction between inoculum density and cocultivation period influenced the transformation efficiency as well. A transformation efficiency (i.e. percentage of the inoculated explants that yielded independent transgenic embryogenic lines) of up to 43% was obtained, greatly improving the previously described method for plant transformation of adult-selected cork oak. It was also shown that this protocol could be applied to various genotypes.

Physiologia Plantarum, 2001

expressed in the cotyledons of germinating pine seeds trans-We have isolated and characterized a ... more expressed in the cotyledons of germinating pine seeds trans-We have isolated and characterized a genomic clone encoding formed by microprojectile bombardment. Stable transforma-Scots pine (Pinus syl7estris) cytosolic glutamine synthetase (GS). The clone contains the 5% end half of the gene including tion of Arabidopsis thaliana revealed the shoot apical meristem as the major region of heterologous permanent part of the coding region and 980 bp upstream of the translation initiation codon. The major transcription start site (+ 1) expression in Arabidopsis, in agreement with the expression of the GS gene in Pinus. Moreover, quantitative data derived was mapped around 180 nucleotides upstream of the translation initiation codon. Sequence analysis of the 5%-upstream from fluorometric -glucuronidase assays in control and conregion of the gene reveals the presence of putative regulatory tinuous light-grown transgenic Arabidopsis plants indicate that the isolated upstream region of the gene contains regula-elements including a poly-CT consensus sequence, a purinetory sequences involved in the response to light. rich tandem repeat and two AT-rich regions.

Physiologia Plantarum, 1996

... Factors affecting transient gene expression in cultured radiata piee cotyledons following par... more ... Factors affecting transient gene expression in cultured radiata piee cotyledons following particle bombardment Manuel Rey, Maria Victoria Gonzalez, Rjcardo J. Ordas, Raffaela Tavazza and Giorgio Ancora Rey, M., Gonzalez, MV, Ordas, RJ, Tavazza, R. and Ancora, G. 1996. ...

Journal of Plant Physiology, 2011

The responses of juvenile plants of forest crops to drought stress are a key stage in the surviva... more The responses of juvenile plants of forest crops to drought stress are a key stage in the survival of forest populations. In this work, a suitable experimental system to study the early drought resistance mechanisms and signaling in a drought-tolerant clone (C14) of Eucalyptus globulus Labill is proposed. This system, using hydroponic culture and an osmotic agent, polyethylene glycol 8000, was demonstrated to induce severe stress in the root area, affecting the responses of the plantlets at the aerial level. These responses were very fast, beginning only 3 h after the induction of stress, and the results highlight the roles of xylematic abscisic acid (ABA) and pH changes over other signals, such as cytokinins, as early chemical signals in rapid water stress. The relationship between these chemical factors, ABA and pH, and the physiological and water parameters observed were significant, supporting their proposed principal role. This work aids our understanding of underlying responses to hydrological limitations of forest crops, and provides valuable information for further physiological and molecular studies of water stress in this and other tree species.

Journal of Plant Physiology, 2010

Type-A response regulators play an important role in cytokinin-induced adventitious shoot formati... more Type-A response regulators play an important role in cytokinin-induced adventitious shoot formation, acting as negative regulators of cytokinin signal transduction. In this work, we obtained the full-length cDNA clone of a type-A response regulator from the conifer Pinus pinea, designated PipiRR1. The derived peptide sequence showed all the characteristic motifs found in angiosperms. Gene expression analysis showed that the gene was differentially expressed during adventitious shoot formation in P. pinea cotyledons, suggesting that PipiRR1 may play a role in caulogenesis in conifers. This is the first type-A response regulator identified in gymnosperms.

Journal of Plant Physiology, 2009

Adventitious bud formation in stone pine cotyledons cultured in the presence of benzyladenine (BA... more Adventitious bud formation in stone pine cotyledons cultured in the presence of benzyladenine (BA) has been proposed as a model for the study of in vitro shoot organogenesis in conifers. This is because of its advantageous characteristics including the requirement of only one plant growth regulator (BA), the synchronous fashion of its induction, and the homogeneity and low degree of differentiation of cotyledons. Although optimal culture conditions have been developed and are currently in use, we still lack data for BA dynamics in cotyledons cultured under these conditions, and the morphological description of the early induction stages has not, until now, been approached from a histological perspective. Consequently, this is the focus of the present report. Additionally, we examined uptake and metabolism of BA in cotyledons from two selected families, previously characterized by, and selected for, the difference in the magnitude of their organogenic response. Media transfer experiments established that cotyledons should be in contact with 44.4 microM BA for at least 6h to obtain any caulogenic response (minimum shoot-induction period). Histological observations, carried out here for the first time in this species, determined that meristemoid structures had already begun to appear in explants within 12 h of culture. Moreover, results from the BA uptake and metabolism experiments indicated that the point at which explants reached the maximum concentration of active forms of BA (276.60 microM at 6 h) and the onset of the determination phase of shoot organogenesis were directly related. A direct relationship was also observed between the intensity of the caulogenic response in cotyledons from families 36 and 61 and the endogenous concentration of BA and its riboside at the start of the induction phase. Hence, family 36, characterized by its higher bud production, reached concentrations of 251.56 microM, while family 61, selected for its low bud-producing trait, only attained 175.80 microM. Finally, a correlation was observed between 6-benzylamino-9-[O-glucopyranosyl-(1-->3)ribofuranosyl]-purine values and the magnitude of the shoot organogenesis response.

Journal of Plant Physiology, 2009

Journal of virology, 1999

The major structural protein VP60 of rabbit hemorrhagic disease virus (RHDV) has been produced in... more The major structural protein VP60 of rabbit hemorrhagic disease virus (RHDV) has been produced in transgenic potato plants under the control of a cauliflower mosaic virus 35S promoter or a modified 35S promoter that included two copies of a strong transcriptional enhancer. Both types of promoters allowed the production of specific mRNAs and detectable levels of recombinant VP60, which were higher for the constructs carrying the modified 35S promoter. Rabbits immunized with leaf extracts from plants carrying this modified 35S promoter showed high anti-VP60 antibody titers and were fully protected against the hemorrhagic disease.

The effect of introns on gene expression was evaluated. Several intron-promoter combinations were... more The effect of introns on gene expression was evaluated. Several intron-promoter combinations were introduced by microparticle bombardment into two pine species, stone pine (Pinus pinea L.) and salgareño pine (Pinus nigra Arn. ssp. Salzmannii (Dunal) Franco). Gene expression was evaluated by measuring transient GUS expression. Two promoters (CaMV35S and double CaMV35S modified) and two introns (intron 1 from maize genes alcohol dehydrogenase-1 and Shrunken-1) were used in our study. In both pine species tested, the Sh1-int1 increased transient GUS expression from 2 to 6-fold compared to the intron-less construction. On the contrary, the inclusion of the Adh1-int1 associated with the double CaMV35S modified resulted in a dramatic decrease in the expression in both pine species analyzed. Our results suggest that Sh1-int1 may be useful for the acquisition of the required levels of genetic activity of new agronomic traits introduced into pines.

Transfer and expression of the β-glucuronidase gene (uidA) in cultured cotyledons of stone pine (... more Transfer and expression of the β-glucuronidase gene (uidA) in cultured cotyledons of stone pine (Pinus pinea L.) was obtained by microprojectile bombardment. Conditions for optimum transient expression were established by using plasmid pBI121 delivered by 1.0 µm-diameter gold particles, into 1-day-old cultured cotyledons. Helium pressure of 6.2 MPa, microcarrier travel distance of 6 cm, and 0.8 µg of plasmid DNA per bombardment, were the best parameters for high levels of transient uidA expression. By using these parameters, 98% of bombarded cotyledons showed β-glucuronidase activity, with a mean of 63 Gus foci per cotyledon. This system was used to study the expression of uidA gene driven by several heterologous promoters. The expression under the control of the sunflower polyubiquitin gene (UbB1) promoter ( 1 deletion) was higher (99% of GUS positive cotyledons) than under the control of the CaMV35S promoter, whereas the rice actin and the maize alcohol dehydrogenase gene promoters gave lower uidA expression, as determined histochemically. These results were confirmed by using the GUS fluorometric assay. Use of a deletion of the sunflower polyubiquitin promoter resulted in GUS activity detectable 35 days after bombardment, and significant levels of GUS activity were confirmed at the end of that period. The results will be useful to design protocols for stable transformation and high levels of transgene expression in P. pinea.

We have investigated the use of five different promoter-5%UTR leader combinations to develop a po... more We have investigated the use of five different promoter-5%UTR leader combinations to develop a potato plant-based expression system for the production of VP60 protein from rabbit haemorrhagic disease virus (RHDV). The relative efficiency of CaMV 35S, modified CaMV 35S, sunflower polyubiquitin and patatin promoters, as well as the addition of the 5% leader sequence from 10 gene of T7 phage, on VP60 gene expression in leaf and tuber tissues of plants, cultivated both in vitro and as potted plants, was investigated. Our results indicated that the B33 promoter with the 10 leader gave the highest level of expression in tubers. The rabbits immunised with tuber plant extracts containing VP60 elicited specific antibody responses and were protected against challenge with virulent RDHV.

Rabbit hemorrhagic disease, which is caused by a calicivirus, is a lethal infection of adult anim... more Rabbit hemorrhagic disease, which is caused by a calicivirus, is a lethal infection of adult animals that is characterized by acute liver damage and disseminated intravascular coagulation. In this study, we report the production of the major structural protein VP60 of rabbit hemorrhagic disease virus in transgenic tubers of potato plants and its use as an oral immunogen in rabbits.

Viral Immunology, 2001

We have recently communicated the oral and parental immunogenicity of the structural protein VP1 ... more We have recently communicated the oral and parental immunogenicity of the structural protein VP1 of foot and mouth disease virus (FMDV) expressed in different transgenic plants. Those results clearly indicated the necessity of increasing the expression of the foreign genes in the transgenic plant to avoid additional steps toward the purification and/or concentration of the antigen of interest. Here, we report the production of transgenic potatoes plants containing the VP1 gene cloned under the regulatory activity of either a single (pRok2) or a double (pRok3) copy of the S35 cauliflower mosaic virus (CaMV 35S) promoter, as a strategy for increasing the level of VP1 gene expression. The presence of the VP1 gene in the plants was confirmed by polymerase chain reaction (PCR) and its specific transcription activity was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that, although the immunized animals presented a FMDV VP1 specific antibody response and protection against the experimental challenge, no significant differences were demonstrated in the immunizing activity of plant extracts obtained from the pRok2 or pRok3 transformed plants. These results confirm those previously obtained using other plant species allowing the possibility of using plants as antigen expression vectors, and demonstrated that at least in the potato system, the use of double CaMV 35S promoter does not cause a significant increase in the level of the VP1 expressed.

Trees, 2009

This paper describes a micropropagation protocol for in vitro propagation of mature Stone Pine tr... more This paper describes a micropropagation protocol for in vitro propagation of mature Stone Pine trees. Axillary bud development was achieved by culturing bud explants in media containing various cytokinins. Experiments were conducted to test the effect of asepsis conditions, type and concentration of cytokinin and rooting protocol. Four cytokinins were tested, namely, benzyladenine, meta-topolin, N-benzyl-9-(2-tetrahydropyranyl)-adenine and thidiazuron (TDZ) of which TDZ gave the best results, as 59% shoot development was obtained following the application of 1 μM TDZ to the culture medium. The shoot development was significantly influenced by the genotype of the tree, but was effective in explants from all 20 genotypes used in the trial. In vitro rooting was, however, difficult to achieve and could only be induced at low rates. This protocol represents the first successful biotechnological approach to the micropropagation of adult Pinus pinea trees.

Plant Science, 1991

ABSTRACT Viable protoplasts were isolated from suspension culture cells of globe artichoke (Cynar... more ABSTRACT Viable protoplasts were isolated from suspension culture cells of globe artichoke (Cynara scolymus L.). Protoplast yield, cell wall regeneration and cell division were influenced by several factors, e.g. age of the cell culture, enzyme composition, culture density. First cell divisions were observed after 4–6 days of culture. Upon transfer to solid medium, the cell colonies gave rise to proliferating green calli. Globular structures formed on these calli but failed to develop further.

Plant Physiology and Biochemistry, 2001

Isolated cotyledons from Pinus pinea L. germinated embryos cultured in vitro showed shoot organog... more Isolated cotyledons from Pinus pinea L. germinated embryos cultured in vitro showed shoot organogenesis in response to treatment with 4.4 µM N 6 -benzyladenine (BA). In late germination phases, the extent of the response onset decreased and adventitious buds were redistributed to the basal zone of the cotyledons. To understand these two events, analyses of indole-3-acetic acid (IAA) and several cytokinins have been carried out in apical and basal portions of cotyledons taken at four different culture periods of the embryos on germination medium (0, 2, 4 or 6 d). The highest endogenous content of all hormones, except N 6 -isopentenyladenosine, was found in the most responsive explants, those that were taken from 0-d-old cultured embryos. Apical portions of cotyledons taken from 0 to 4-d-old cultured embryos showed a reduction in the content of IAA and several cytokinins, but basal portions did not show the same pattern. BA dose-response curves were made to explain the loss of caulogenic potential with the age of the explants. The results suggested the combination of two factors, changes in the endogenous hormonal content in original explants and loss of their sensitivity to exogenously applied BA, as the cause for the reduction of the ability of Pinus pinea cotyledons to form adventitious buds. © 2001 Éditions scientifiques et médicales Elsevier SAS adventitious bud / cytokinin / embryo culture / indole-3-acetic acid / Pinus pinea BA, N 6 -benzyladenine / BFC, bud forming capacity / DHZ, dihydrozeatin / DHZR, dihydrozeatin riboside / IAA, indole-3-acetic acid / iP, N 6 -isopentenyladenine / iPR, N 6 -isopentenyladenosine / ½LP, modified Le Proive medium / ½LPB, modified Le Proive medium + BA / ½LPC, modified Le Proive medium + activated charcoal / SEC, shoot elongation capacity / Z, zeatin / ZR, zeatin riboside *Correspondence and reprints: fax +34 985104867.

Plant Molecular Biology, 1991

Sequences encoding the immunoglobulin heavy-chain variable (VH) domains were engineered in a new ... more Sequences encoding the immunoglobulin heavy-chain variable (VH) domains were engineered in a new general purpose vector to transform plants via Agrobacterium. The expression of an isolated VH domain (IVD) after introduction into the plant genome has been monitored by northern, western and immunohistochemical analysis. Immunoblotting showed that the polypeptide was stably expressed and accounted for up to 1 ~o of the soluble protein fraction. It is therefore proposed that single immunoglobulin domains of suitable specificity expressed in plants may constitute an effective system to inhibit the activity of molecules involved in plant pathology or plant development.

Plant Cell, Tissue and Organ Culture, 2008

Organogenesis response of six selected half-sibling families of stone pine (Pinus pinea L.) has b... more Organogenesis response of six selected half-sibling families of stone pine (Pinus pinea L.) has been evaluated, showing genotype-dependent behaviour. The caulogenic phase was characterized by high values of Survival and Organogenesis, while the rooting phase (the bottleneck of many coniferous species) showed great variability among families. Provenance influence was also studied, and the rhizogenesis protocol was optimized for the selected families. The highest values were obtained with family 36, with 100% of Organogenesis, a Bud Formation Capacity (BFC) Index of 6.54 and 38.44% of Rooted Shoots; on the other hand, family 61 presented the worst results, with 83.64% Organogenesis, a BFC Index of 3.01 and a 29.69% Rooting Rate. According to these results, both families will be used in further experiments looking for the underlying bases of the different organogenic behaviour between both families under the same culture conditions. In addition to this, and for the first time in this species, random amplified polymorphism DNA (RAPD) analysis has been carried out to determine whether somaclonal variation had occurred. The results suggested an absence of variation during the whole in vitro process, although more thorough studies would be required for a conclusive answer.

Plant Cell, Tissue and Organ Culture, 2007

An optimized protocol for Agrobacterium tumefaciens-mediated transformation of mature Quercus sub... more An optimized protocol for Agrobacterium tumefaciens-mediated transformation of mature Quercus suber L. embryogenic masses is reported. In this work several variables were tested. Plant genotype, explant type and time elapsed between the last subculture and inoculation, i.e. the explant preculture period, were found to be very important. Interaction between inoculum density and cocultivation period influenced the transformation efficiency as well. A transformation efficiency (i.e. percentage of the inoculated explants that yielded independent transgenic embryogenic lines) of up to 43% was obtained, greatly improving the previously described method for plant transformation of adult-selected cork oak. It was also shown that this protocol could be applied to various genotypes.

Physiologia Plantarum, 2001

expressed in the cotyledons of germinating pine seeds trans-We have isolated and characterized a ... more expressed in the cotyledons of germinating pine seeds trans-We have isolated and characterized a genomic clone encoding formed by microprojectile bombardment. Stable transforma-Scots pine (Pinus syl7estris) cytosolic glutamine synthetase (GS). The clone contains the 5% end half of the gene including tion of Arabidopsis thaliana revealed the shoot apical meristem as the major region of heterologous permanent part of the coding region and 980 bp upstream of the translation initiation codon. The major transcription start site (+ 1) expression in Arabidopsis, in agreement with the expression of the GS gene in Pinus. Moreover, quantitative data derived was mapped around 180 nucleotides upstream of the translation initiation codon. Sequence analysis of the 5%-upstream from fluorometric -glucuronidase assays in control and conregion of the gene reveals the presence of putative regulatory tinuous light-grown transgenic Arabidopsis plants indicate that the isolated upstream region of the gene contains regula-elements including a poly-CT consensus sequence, a purinetory sequences involved in the response to light. rich tandem repeat and two AT-rich regions.

Physiologia Plantarum, 1996

... Factors affecting transient gene expression in cultured radiata piee cotyledons following par... more ... Factors affecting transient gene expression in cultured radiata piee cotyledons following particle bombardment Manuel Rey, Maria Victoria Gonzalez, Rjcardo J. Ordas, Raffaela Tavazza and Giorgio Ancora Rey, M., Gonzalez, MV, Ordas, RJ, Tavazza, R. and Ancora, G. 1996. ...

Journal of Plant Physiology, 2011

The responses of juvenile plants of forest crops to drought stress are a key stage in the surviva... more The responses of juvenile plants of forest crops to drought stress are a key stage in the survival of forest populations. In this work, a suitable experimental system to study the early drought resistance mechanisms and signaling in a drought-tolerant clone (C14) of Eucalyptus globulus Labill is proposed. This system, using hydroponic culture and an osmotic agent, polyethylene glycol 8000, was demonstrated to induce severe stress in the root area, affecting the responses of the plantlets at the aerial level. These responses were very fast, beginning only 3 h after the induction of stress, and the results highlight the roles of xylematic abscisic acid (ABA) and pH changes over other signals, such as cytokinins, as early chemical signals in rapid water stress. The relationship between these chemical factors, ABA and pH, and the physiological and water parameters observed were significant, supporting their proposed principal role. This work aids our understanding of underlying responses to hydrological limitations of forest crops, and provides valuable information for further physiological and molecular studies of water stress in this and other tree species.

Journal of Plant Physiology, 2010

Type-A response regulators play an important role in cytokinin-induced adventitious shoot formati... more Type-A response regulators play an important role in cytokinin-induced adventitious shoot formation, acting as negative regulators of cytokinin signal transduction. In this work, we obtained the full-length cDNA clone of a type-A response regulator from the conifer Pinus pinea, designated PipiRR1. The derived peptide sequence showed all the characteristic motifs found in angiosperms. Gene expression analysis showed that the gene was differentially expressed during adventitious shoot formation in P. pinea cotyledons, suggesting that PipiRR1 may play a role in caulogenesis in conifers. This is the first type-A response regulator identified in gymnosperms.

Journal of Plant Physiology, 2009

Adventitious bud formation in stone pine cotyledons cultured in the presence of benzyladenine (BA... more Adventitious bud formation in stone pine cotyledons cultured in the presence of benzyladenine (BA) has been proposed as a model for the study of in vitro shoot organogenesis in conifers. This is because of its advantageous characteristics including the requirement of only one plant growth regulator (BA), the synchronous fashion of its induction, and the homogeneity and low degree of differentiation of cotyledons. Although optimal culture conditions have been developed and are currently in use, we still lack data for BA dynamics in cotyledons cultured under these conditions, and the morphological description of the early induction stages has not, until now, been approached from a histological perspective. Consequently, this is the focus of the present report. Additionally, we examined uptake and metabolism of BA in cotyledons from two selected families, previously characterized by, and selected for, the difference in the magnitude of their organogenic response. Media transfer experiments established that cotyledons should be in contact with 44.4 microM BA for at least 6h to obtain any caulogenic response (minimum shoot-induction period). Histological observations, carried out here for the first time in this species, determined that meristemoid structures had already begun to appear in explants within 12 h of culture. Moreover, results from the BA uptake and metabolism experiments indicated that the point at which explants reached the maximum concentration of active forms of BA (276.60 microM at 6 h) and the onset of the determination phase of shoot organogenesis were directly related. A direct relationship was also observed between the intensity of the caulogenic response in cotyledons from families 36 and 61 and the endogenous concentration of BA and its riboside at the start of the induction phase. Hence, family 36, characterized by its higher bud production, reached concentrations of 251.56 microM, while family 61, selected for its low bud-producing trait, only attained 175.80 microM. Finally, a correlation was observed between 6-benzylamino-9-[O-glucopyranosyl-(1-->3)ribofuranosyl]-purine values and the magnitude of the shoot organogenesis response.

Journal of Plant Physiology, 2009