Scielo RSS <![CDATA[Electronic Journal of Biotechnology]]> vol. 9 num. 3 lang. es <![CDATA[SciELO Logo]]> <![CDATA[<b>EDITORIAL</b> <b>ELECTRONIC JOURNAL OF BIOTECHNOLOGY</b> <b><i>....MOVING FROM SCIENCE TO DEVELOPMENT...</i></b>]]> <![CDATA[<b>A new variety of <i>Bacopa monnieri</i> obtained by <i>in vitro</i> polyploidization</b>]]> The commercial value of Bacopa monnieri, a widespread herbaceous plant in Argentina, can be substantially improved increasing its flower size by chromosome doubling with colchicine. MS supplemented with 0.25 mg/L of 6-benzylaminopurine proved to be an appropriate medium for the in vitro multiplication of nodal segments of B. monnieri. Polyploidization was achieved submerging nodal segments during 24 or 48 hrs in colchicine solution 0.001 and 0.01% P/V, in 1% DMSO. Segments submerged in water and in DMSO 1% aqueous solution were used as controls. DNA contents from recovered plants was measured and characterized and their phenotype described. Two tetraploid plants originated from independent events were detected. These plants showed significant differences in size and colour both in leaves and flowers compared to untreated controls. <![CDATA[<b>An alternative pathway for plant <i>in vitro</i> regeneration of chinaberry -tree <i>Melia azedarach</i> L. derived from the induction of somatic embryogenesis</b>]]> A highly efficient somatic embryogenesis system and subsequent plant regeneration of chinaberry (Melia azedarach L.) was developed. Plants were regenerated from indirect somatic embryogenesis induction. Novel features of this improved protocol, include: a) Embryogenic callus induction with no addition of 2, 4-D in the culture media; b) Somatic embryos differentiation was achieved by using high concentration of cytokinins (BAP 10 mg/L) and adenine; c) 100% conversion of somatic embryos to plants was practically obtained and 100% of plants survived under greenhouse conditions; d) Addition of putrescine improved somatic embryos germination. The amount of somatic embryos produced by the pathway of indirect somatic embryogenesis was 447 per gram of fresh weight callus. Regenerated plants were phenotypically normal. The developed protocol established the potential to produce plantlets from cotyledon explants through somatic embryogenesis. It also presents itself as a highly efficient method for mass clonal propagation and conservation of Melia azedarach. <![CDATA[<b>Analysis and sequencing of h6hmRNA, last enzyme in the tropane alkaloids pathway from anthers and hairy root cultures of<i> Brugmansia candida (Solanaceae)</i></b>]]> Brugmansia candida (Solanaceae) is a native tree distributed across South-American and produces the pharmacologically- important group of tropane alkaloids including scopolamine. This biocompound is synthesised from hyoscyamine by action of Hyoscyamine 6-&beta; hydroxylase (H6H, EC at the end of the tropane alkaloid pathway. Here are reported the tissue and organ-specific expression of h6hmRNA by RT-PCR analyses and the isolation, cloning and sequencing of the cDNA obtained from B. candida anthers and hairy root transformed cultures. Bioinformatic analysis of the nucleotide sequence revealed an uninterrupted ORF of 1038 bp and the predicted aminoacid sequence could be 344 aminoacid long. A database search showed that this sequence has high homology (97% identity) to Hyoscyamus niger H6H protein (Genbank accession number AAA33387.1). <![CDATA[<b>Analysis of the molecular basis of <i>Xanthomonas axonopodis</i> pv. <em>citri</em> pathogenesis in <i>Citrus</i> <i>limon</i></b>]]> Xanthomonas axonopodis pathovar citri (Xac) causes bacterial citrus canker, a serious disease of most citrus species. Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot disease in cruciferous plants. In Xcc, cell-cell signaling is mediated by diffusible signal factor (DSF). Synthesis of DSF depends on RpfB and RpfF. DSF perception and signal transduction have been suggested to involve a two-component system comprising RpfC and RpfG. It has been proposed that these proteins participate in a signal transduction system linking changes in the environment to the synthesis of DSF and the expression of virulence genes. Although the cluster of the rpf genes in Xac has synteny with the corresponding cluster in Xcc, two genes (rpfH and rpfI) are absent in Xac. To investigate DSF-mediated regulation during Xac-Citrus limon interaction, we constructed two strains of Xac, one with a mutation in the rpfF gene, leading to an inability to produce DSF, and one with a mutation in the rpfC gene leading to an overproduction of DSF. These mutants also show decreased levels of extracellular cyclic β-(1,2)-glucans and decreased production of endoglucanase and protease extracellular enzymes. The Xac DSF-deficient rpfF and the DSF-hyper producing rpfC mutants are both severely compromised in their ability to cause canker symptoms in lemon leaves compared to the wild-type. Here we provide evidence that rpf genes in Xac are involved in controlling virulence factors mediated by DSF. <![CDATA[<b>Antimicrobial effect of polyphenols from apple skins on human bacterial pathogens</b>]]> Apples possess many beneficial properties for the human health related with their high content in phenolic compounds. The antimicrobial effect of these compounds from the skin of two apple varieties, Royal Gala and Granny Smith, against human pathogens was examined. The phenolic compounds were extracted with the following solvents: A, acetone: water: acetic acid; B, ethyl acetate: methanol: water and C, ethanol: water. Total phenolic, flavonoid and non-flavonoid contents were analyzed in the extracts. The antimicrobial effect was determined using the agar diffusion method. The highest inhibitory effect of both apple varieties corresponded to extract A, which contained a high phenolic content. The Granny Smith extracts with higher phenolic content presented a superior antimicrobial effect against the selected microorganisms: Escherichia coli, Escherichia coli ATCC 25922, Escherichia coli ATCC 35218, Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212 and Listeria monocytogenes. The most sensitive microorganisms were Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853and Staphylococcus aureus ATCC 29213, whereas the most resistant strains were Escherichia coli ATCC 35218 and Escherichia coli. The results obtained demonstrate a direct relationship between the phenolic content of the extracts and the antimicrobial effect. <![CDATA[<b>Bioactivity of <i>Scytonema hofmanni </i>(Cyanobacteria) in <i>Lilium alexandrae in vitro</i> propagation</b>]]> Cyanobacteria produces bioactive compounds including plant growth regulators. Naphthalene acetic acid (NAA), a toxic substance, is a synthetic plant regulator used in micropropagation. The aim of this work was to evaluate morphogenetic and antioxidant effects produced by intra and extracellular substances from Scytonema hofmanni (Cyanobacteria) during the multiplication in vitro of Lilium alexandrae and to compare them to those produced by NAA. Intra and extracellular cyanobacterial products increased a) bulblets production reaching 83% and 78% of NAA effect, respectively; b) the bulblet diameter compared to NAA; and c) the bulblet survival due to the promotion of antioxidant activity measured as catalase, ascorbate peroxidase, and glutathione reductase activity. The cyanobacterial substances stimulated regeneration and delayed bulblet senescence. They could replace NAA, dangerous for the operator, not only during the regeneration phase but also during the storage of the viable bulblets cultivated in vitro. <![CDATA[<b>Biodegradation of agroindustrial wastes by<i> Pleurotus </i>spp for its use as ruminant feed</b>]]> The increasing expansion of agro-industrial activity has led to the accumulation of a large quantity of lignocellulosic residues all over the world. In particular, large quantities of rice straw (300.000 t) and citric bagasse (50.000 t) are annually produced in Uruguay. In this work we present the study of the bioconversion of these substrates with the edible mushroom Pleurotus spp so as to increase nutritional values and digestibility for its use as animal feed. The SSF process was optimized and the products after different periods of mushroom growth were evaluated. The microbial counts (cfu/g) for the inoculated substrates 44 days after incubation were 15 x 10(4), < 10 and < 10 for aerobic microorganisms, coliforms and E. coli, respectively. After 14 days of SSF the percentage of dry matter, ADF and NDF decreased, and the content of protein increased. These results show that vegetal cell-wall components were degraded during the period of mushroom incubation. PCR - RFLP analysis of the ITS region was used to characterize the Pleurotus species produced in Uruguay and discriminate between DNAs of Pleurotus ostreatus 814 and other fungi from the different substrates. <![CDATA[<b>Biosorption of heavy metals by <i>Talaromyces helicus</i></b>: <b>a trained fungus for copper and biphenyl detoxification</b>]]> At present, it is common to observe environments with organic and inorganic pollution, defined as co-contamination. Most industrial and urban effluents releases both pollutant types, leading to a complex environmental problem, as the biota must be tolerant to both xenobiotics. T. helicus, an efficient strain to degrade biphenyl, was trained with high copper levels, and became co tolerant to cobalt, lead and cadmium when was cultured in their presence. The copper adaptation was the result of physiological mechanisms, and the activated biochemical processes conferred resistance to Cu2+ as well as to other heavy metals. Furthermore, the Cu2+ adaptation of the mycelium was also transferred to the spores, that removed twice as much copper from solution than those of the no trained parentals. Interestingly, metals combinations were less toxic than single ones, and co tolerance development indicated that the cellular mechanisms that conferred resistance were non-specific, so the micobiota isolated from co contaminated environments often exhibited resistance to more than one ions. These results emphasized the detoxification abilities of T. helicus and the adaptation to heavy metals and biarylic compounds. This data is significant for the environmental biotechnology, suggesting that such tolerance and co tolerance could be acquired in natural environments. So a simple bioremediation strategy could enhance the detoxification of these polluted areas, as the degrader organisms could be present. <![CDATA[<b>Biotechnology in Argentine agriculture faces world-wide concentration</b>]]> In the 1980s, the technical pattern of production in agriculture changed due to the increasing design of genetically modified plants. Modern biotechnology thrived on events requiring certain thresholds of scientific and technological skills as well as scale economies usually seen in developed countries. The mergers and acquisitions during the mid-1990s led to a world-wide oligopoly composed of very few agri-biotechnological mega-corporations and the literature discusses the impact of the mergers and acquisitions on the agriculture of developing countries with comparative advantages in agriculture. This paper analyzes the world-wide process of agri-biotechnological mega-corporation mergers and acquisitions as well as its impact and interrelationships with Argentine agriculture using information from primary and secondary sources. Conclusions refer to the set-backs of endogenous agri-biotechnological development due to world-wide concentration in developing countries with comparative advantage in agriculture. <![CDATA[<strong>Biotransformation of 1,8-cineole, the main product of <i>Eucalyptus</i> oils</strong><strong> </strong>]]> The forest industry in Uruguay has grown considerably during the last decade. Eucalyptus plantations account for 74% of the forested land, with Eucalyptus globulus being the most widely distributed species. This industry is dedicated exclusively to the production of wood without exploiting the by-products (leaves and small branches). Eucalyptus leaves are known to contain important amounts of essential oils composed primarily of 1,8-cineole (1,3,3-trymethyl-2-oxabicyclo[2.2.2]octane). In this work, the biotransformation of 1,8-cineole, is achieved using a native bacterium (Rhodococcus sp.) which was isolated from the soil of Eucalyptus forest. A 98% of bioconversion was achieved. Three different optically pure compounds were obtained, and they were identified as 2-endo-hydroxy-1,8-cineole, 2-exo-hydroxy-1,8-cineole and 2-oxo-1,8-cineole. <![CDATA[<B>Callus culture for biomass production of milk thistle as a potential source of milk clotting peptidases</B>]]> The objective of this work was the optimization of the conditions of in vitro culture for callus production of Silybum marianum (L.) Gaertn. (Asteraceae). Sections of cotyledons, previously disinfected by washing successively with ethanol 70&ordm;, NaClO (10% w/v) and Tween 20 (0.05% v/v) and rinsing with sterile distilled water, were used as explants. For its initial culture, B5 medium supplemented with BA and 2,4-D solidified with phytagel was used, and a 63% survival was achieved. To obtain callus, two solid media were assayed (S1 and S2) using B5 medium supplemented with growth regulators (BA and 2,4-D or NAA and BA, respectively). The calli were grown at 25&ordm;C during 45 days in darkness. Growth kinetics was studied using S1 medium obtaining a typical growth curve with an exponential phase after 14 days of incubation (rate of growth 0.005 g dry weight/ day) and stationary phase after 35 days. The rate of growth in S2 medium was slower, and rhizogenesis was observed starting on the fifth week of incubation. From these results, the best culture medium for callus production of Silybum marianum was S1 medium. <![CDATA[Chlorinated biphenyl degradation by wild yeasts pre-cultured in biphasic systems]]> Environmental biotechnology has developed as an offshoot from sanitary engineering, and only recently the biological component of the ecosystems had been recognized as relevant when bioremediation strategies must be chosen to solve environmental problems. Yeasts were isolated on 2,4-dichlorobiphenyl, 2,3',4- and 2,4',5-trichlorobiphenyl, poorly soluble compounds in water, as carbon sources. Debaryomyces castelli, Debaryomyces maramus and Dipodascus aggregatus composed the mixed culture and represented 72% of the isolates; their degradation potential were studied in biphasic and monophasic systems. The biphasic cultures were obtained with phenol as the organic phase and MSM as the aqueous ones, the monophasic medium only with MSM. Both cultures were supplied with 50, 100, 150 and 200 ppm DCB, TCB-3' and TCB-4' as substrate. The growth rates varied with the dispersion degree, agitation rates and cell adhesion to the organic phase. The water-phenolic system improved yeasts selection in pollutant presence with low water solubilities, indeed, the adaptation and degradation were more slowly in the monophasic aqueous medium. Bioremediation is based on the presence of efficient microbial populations and pollutant availability; the tested yeasts and the organic-water system assayed put forward the possibility that hydrophobic substrates could be mineralized in natural habitats by wild yeast consortium. <![CDATA[<B>Construction of a molecular identification profile of new varieties of <I>Nierembergia linariaefolia</I> by anchored microsatellites</B>]]> The objective of the present work was to establish the molecular identification profile for six new varieties of Nierembergia linariaefolia to incorporate the fingerprint, as complementary information to the standard registration data. Total DNA was extracted from young leaves following the protocol of the cetyltrimethylammonium bromide. Anchored microsatellites were used as molecular markers. The amplification reactions were carried out with seven primers. A total of 251 loci were detected, 98% of them were polymorphic. The average of polymorphic loci was 35 loci per primer and, 41 loci per genotype. Six out of the seven primers used discriminated all the individuals involved in the present study; consequently, it was possible to generate the molecular identification profile for the six new varieties. This result, supported together with our previous reports, indicates that the anchored microsatellites are a very useful technique for the fingerprints generation in N. linariaefolia. <![CDATA[<b>Effect of different metals on protease activity in sunflower cotyledons </b>]]> The objective of the present work was to establish the molecular identification profile for six new varieties of Nierembergia linariaefolia to incorporate the fingerprint, as complementary information to the standard registration data. Total DNA was extracted from young leaves following the protocol of the cetyltrimethylammonium bromide. Anchored microsatellites were used as molecular markers. The amplification reactions were carried out with seven primers. A total of 251 loci were detected, 98% of them were polymorphic. The average of polymorphic loci was 35 loci per primer and, 41 loci per genotype. Six out of the seven primers used discriminated all the individuals involved in the present study; consequently, it was possible to generate the molecular identification profile for the six new varieties. This result, supported together with our previous reports, indicates that the anchored microsatellites are a very useful technique for the fingerprints generation in N. linariaefolia. <![CDATA[<b>Establishment of an<i> in vitro</i> micropropagation protocol for <i>Mecardonia tenella</i></b>]]> Mecardonia tenella is an attractive herbaceous native plant, characterized by their intense green foliage and their abundant yellow flowers. It is a very interesting plant for pot and/or garden. A trait to improve in this species is the size of the flowers. The goal of the present paper is to study the in vitro behaviour of M. tenella and to establish a routine protocol for its micropropagation. For the in vitro establishment of M. tenella, nodal segments were disinfected by standard methods using ethanol/sodium hypochloride and cultured on hormone-free MS medium, supplemented with a mixture of antibiotics and an antifungal. In order to study the hormonal requirements of the species, nodal segments were cultured on basal MS supplemented with antibiotic/antifungal mixture and the following concentrations of BAP and NAA (mg/L): 0.0; 0.25; 0.5 and 1.0. These plant regulators were tested in all possible combinations. In vitro plants growing in hormone-free medium were used as explant source. The best results were obtained in the treatments containing 0.25 and 0.5 mg/l BAP with a multiplication rate of 32 shoots per explant. The regenerated shoots rooted spontaneously. When transferred to the greenhouse, the ex vitro plants grew and flowered normally. <![CDATA[<b>Identification of microsatellites linked to <i>Lr47</i></b>]]> Leaf rust resistance gene Lr47 is located within a interstitial segment of Triticum speltoides Taush. 7S chromosome translocated to the short arm of chromosome 7A of bread wheat. This gene is resistant against currently predominant races of leaf rust from Argentina. The objectives of this study were to identify microsatellites linked to this source of resistance as a potential tool to introgress this source of resistance. Isogenic lines with and without Lr47 developed from 10 cultivars/breeding lines as well as 10 microsatellites previously mapped in 7AS chromosome were used in this study. Microsatellite gwm 60 was the only marker that co-segregated completely linked to Lr47. These data indicate that gwm 60 could be a valuable marker to introgress Lr47 in wheat germplasm. <![CDATA[<B><I>In vitro </I>culture response of barley (<I>Hordeum vulgare</I>) ethylene synthesis mutant MC 169</B>]]> Although it is generally accepted that plant in vitro culture response is influenced by the donor genotype, the genetic and molecular bases of this phenomenon are barely known. As a consequence, the optimization of tissue culture protocols is mainly empirically done. Researchers of the IGEAF studied the genetic basis of the in vitro regeneration of various plant species, including the tissue culture response of artificially induced barley mutants. One barley mutant, MC 169, carries a nuclear gene, recently described controlling the root growth in hydroponic cultivation. Under this condition, the roots of MC 169 mutant plants were longer than those of the original wild type line MC 182, a fact that was associated with a reduced ethylene biosynthesis. On the other hand, it is known that ethylene accumulation is inhibitory for in vitro regeneration of several plant species. In this study, we compared the in vitro culture response of mutant MC 169 with that of its mother line MC 182. The data about induction and regeneration of calli as well as those of habituated calli formation demonstrated that mutant MC 169 and its mother line MC 182 show a similar in vitro behaviour. <![CDATA[<b>Meiotic study of <i>Zea mays</i> ssp. <i>mays </i>(2n = 40) x <i>Tripsacum dactyloides </i>(2 n = 72) hybrid and its progeny</b>]]> Maize (2n = 40) x Tripsacum dactyloides (2n = 72) F1 hybrid plants (2n = 56) were obtained by embryo rescue and induction of somatic embryogenesis/organogenesis. Hybrid plants showed Tripsacum-like phenotypes, tolerance to stresses such as NaCl salinity and low temperatures. The more frequent meiotic configurations were 28II (24%), 24II + 2IV (19%) and 26II + 1IV (12%), with an average per cell of 0.55I + 25.18II + 1.19IV. Significant differences between plants were not observed. Pollen fertility ranged from 0% to 50%. After pollination with maize or Tripsacum, 20% of F1 plants have developed viable seeds, which originated the progeny. Thirty five percent of the progeny showed 2n = 56 chromosomes and F1 like-phenotypes, which suggests they have apomictic origin. The remaining plants were fertile and they showed maize-like phenotypes and different chromosome numbers (2n = 22, 24, 26, 28 and 30), because they kept the complete maize chromosome complement and some of the Tripsacum chromosomes. Meiotic cells showed pairing between chromosomes from both parental species, which suggests the possibility of genetic recombination between them. <![CDATA[<b>Morphological and chemical diversity in the Type IV glandular trichomes of Solananeae (<i>S. sisymbrifolium</i> and <i>N. glauca</i>) as germplasm resources for agricultural and food uses</b>]]> Morphological variation in type IV trichomes in Ss and Ng was studied through SEM. The differences can be related to chemical differences in the excreted sugar esters. Ng trichomes exude two fractions, one of glucose tri-esters and the other one of sucrose tetra-esters, in a 3:7 ratio. The main acid found forming these esthers, is 3-methylvalerianic acid, in consonance to those secreted by other Solanaceae. Esters from Ss are novel structures, which can also be separated into three fractions, two of arabinoxylans, and the other one of arabinose, all glycosilated with &beta;-hydroxipalmitic acid and sterified with the C12-C16 acids. All five fractions have antifungic activity at μg/cm² concentrations, both against common and mycotoxigenic fungi, such as A. niger, A. flavus, P. chrysogenum y P. expansum. A. flavus does not grow in the presence of the SE of Ss and is not insensible to those from Ng, but these last are more effective in the inhibition of P. expansum, the other mycotoxigenic fungi studied. The differential antifungal activity observed gives the plant protection against a wide spectrum of fungi, resulting in a better adaptation to the environment. Both plants are common weeds, with the potential of contributing to germplams lines in the improvement programmes of crops such as L. esculentum, and their extracts can be used as natural fungicides to protect crops and plantations. <![CDATA[<b>NaCl effects in <i>Zea mays</i> L. x <i>Tripsacum dactyloides</i> (L.) L. hybrid calli and plants</b>]]> High salt concentrations in soils negatively affect maize growth. Techniques such as remote hybridization and in vitro selection have been extensively used to accelerate breeding processes. In order to determine the usefulness of Tripsacum to improve salt tolerance in maize, the effects of NaCl, in vitro and in vivo, were evaluated in an intergeneric hybrid (MT) obtained from crossing Zea mays with Tripsacum dactyloides. Organogenic calli, induced from immature MT hybrid embryos, were exposed to different NaCl concentrations and the survival and regeneration percentages were calculated. Plants of the MT hybrid, obtained from the organogenic calli, were exposed to NaCl concentrations considered harmful for maize. The shoot dry weights of plants exposed to 250 mM NaCl did not show significant differences respect to the control ones. Although sodium content in shoots was incremented 2,5 fold, it was not toxic for this material. The MT hybrid showed better behavior, in vitro and in vivo, that maize genotypes exposed to similar conditions. <![CDATA[<b>Production of recombinant enzymes of wide use for research</b>]]> For biotechnological purposes, protein expression refers to the directed synthesis of large amounts of desired proteins. The aim of the present work was to produce reverse transcriptase Moloney murine Leukaemia Virus retro-transcriptase and Taq DNA polymerase, as bioactive products. In the present paper, we report the preparation of recombinant enzymes, expressed in E. coli strains. The enzymes produced exhibited quite good activity, compared with commercial enzymes, allowing us to replace the last ones for several lab applications. We are reporting changes and modifications to standard protocols described. The standard protocols were modified, i.e. for the purification step of Taq, a temperature dependent procedure was designed. The enzymes produced were used in different applications, such as PCR, RT-PCR, PCR Multiplex and RAPDs molecular markers. <![CDATA[<b>Radioactive trace in semi natural grassland</b>: <b>Effect of <sup>40</sup>K in soil and potential remediation</b>]]> The uptake of radionuclides by plant roots constitutes the main pathway for the migration of radiocaesium from soil to humans, via food chain. In this study we assessed radiocaesium uptake by plant in order to piece together information on factors affecting uptake processes, particularly K supply and differential uptake among different plant species. Soil contaminated by the Chernobyl accident and forage from a semi-natural alpine grassland, situated in Tarvisio, Italy, were sampled during 1999. Under field conditions, 137Cs uptake for Graminaceae and Taraxacum officinale seem to behave in a comparable way. Higher 40K soil activity concentration leads to a lower 137Cs plant uptake, suggesting an inhibitory pattern of potassium on radiocaesium plants uptake. For forage samples, a similar tendency was observed. We analyzed the influence of the ratio of 137Cs/ 40K in soil on 137Cs plant uptake. Under field conditions, the ratio observed varied in a range of 0.5 to 1.3. For most of the species, at higher 40K soil concentration a lower 137Cs uptake was observed, a fact that reflects the resulting effect of the complexity of factors controlling ion absorption from soil. <![CDATA[<B>RAPD and freezing resistance in <I>Eucalyptus globulus</B></I>]]> Eucalyptus globulus is the second most important forest species in Chile, after Pinus radiata. The main advantages of E. globulus are its fast growth (25 m³/ha/year) and its excellent wood quality for kraft pulp production. On the negative side, its low freezing tolerance has been an obstacle for the expansion of plantations, specifically on the foothills of the Andes. The difference in the freezing resistance between clones of E. globulus has a genetic base and, therefore, it could be detected through DNA molecular markers. Fifteen clones of E. globulus, eight classified as freezing resistant and seven as freezing sensitive were analyzed using the technique of RAPD, in order to obtain molecular markers that could differentiate between freezing sensitive and resistant clones. Eighteen primers amplified reproducible bands. Three bands were only present in freezing resistant clones, two bands of 768 bp, 602 bp obtained with UBC 218 primer and one band of 248 bp obtained with UBC 237 primer. The preliminary results indicate that polymorphism can be observed with the primers employed, but it is not possible to associate the bands with the cold resistance or susceptibility in E. globulus. <![CDATA[<B>Resistance to freezing in three <I>Eucalyptus globulus </I>Labill subspecies</B>]]> The resistance to low temperatures was assessed in seedlings of three subspecies of E. globulus (ssp globulus, ssp bicostata and ssp maidenii) of two different provenances. Lethal temperature 50 (LT50) was obtained by measuring the electrolytic conductivity, nucleation and freezing temperatures were obtained by thermal analysis and the total soluble carbohydrates concentration was determined through the phenol-sulphuric method. Results showed that the most resistant provenance corresponded to Bolaro Mountain of ssp maidenii with a LT50 lower than -9&ordm;C. The provenance Moogora of ssp globulus, had a LT50 of -8.47&ordm;C, which situates it, as the second most resistant to low temperatures. According to the nucleation and freezing temperatures, the results indicate that all the provenances analyzed evaded the formation of ice, except for Bolaro Mountain of ssp maidenii which was tolerant to freezing. Finally, an inverse correlation (r = -0.89) between the content of total soluble carbohydrates and the LT50 was found, indicating the cryoprotection effect of these in the resistance to low temperatures. <![CDATA[<B>Rooting in Km selective media as efficient <I>in vitro</I> selection method for sunflower genetic transformation</B>]]> Despite of numerous publications in sunflower genetic transformation, there is no efficient or reproducible protocol with low number of escapes. The latter would indicate that the selection method is not effective. In this work we used Km as selective agent, Agrobacterium tumefaciens EHA105 strain and a vector with the nptII gene under the nos promoter and uidA gene under 35S promoter. The response of agroinfected (A) and control (C) explants during the in vitro culture was studied and in both cases in presence or absence of Km in order to assign a differential morphologic response between transformed and non-transformed plants. The characteristics analyzed were: height, colour/aspect of the plantlets, in vitro rooting and in vitro bud-flower development. Selection was applied from the third regeneration media. Among the A plantlets two were capable of rooting, being positive by PCR, whereas the C were unable to root in presence of Km. One of them gave 6 seeds and in these plants, it was determined the presence of the transgene by PCR and GUS staining. This work shows that in Km selection, colour/aspect of shoots is not useful as selection criteria whereas rooting is an effective selection method in which no escapes were obtained. <![CDATA[<B>Semiquantitative analysis of genetically modified maize and soybean in food</B>]]> The aim of this study was to analyze quantitatively the presence of genetically modified organism in food with different composition and degree of processing. Total DNA was extracted by Dellaporta's method and GMO analysis was performed using two consecutives PCR reactions with specie specific primers (IVR and LE), screening primers (35S) and transgen specific primers (CRY and EPSPS). The quantification within the sensitivity establish by the EU was possible only in some foods (ice-cream, flours, soybean isolates and concentrates, starch). Samples with high lipid content or subjected to intense thermal treatments (such as some snacks, mayonnaise, creamy soup) could not be amplified mainly due to the presence of PCR inhibitors. Therefore the method was adequate for identification of food as GM, within the limits establish by EU, only for some Argentinean commercial food products. These findings showed that the develop method was satisfactory only for simple food that were not subject to intense thermal treatments and that do not have high lipid content and that the main limitation of the method is DNA purity. <![CDATA[<B>Sunflower storage proteins are transported in dense vesicles that contain proteins homologous to the pumpkin vacuolar sorting receptor PV 72</B>]]> Storage proteins are transported to a special storage compartments in seeds by Golgi dependent or independent pathways depending on the plant species. The aim of this work was to study the sunflower storage protein transport pathway and identified component of the sorting machinery. Immature sunflower seeds were analyzed by subcellular fractionation (using percoll and sucrose gradients) and electron microscopy. The vesicles isolated with percoll, have precursors of 11S globulins, α-TIP, δ-TIP, BiP, and two proteins that have homology to the pumpkin vacuolar sorting receptor PV72. Sucrose isolated vesicles have the same composition than percoll ones, except for the lack of BiP and the presence of only one protein that has reactivity with pea VSR BP80. Electronic micrographies of developing seeds show that the formation of electron dense aggregates starts in the endoplasmic reticulum, and that these aggregates are very abundant in the trans-Golgi apparatus, where release of dense vesicles happens. These vesicles contain a homolog of PV72 in their membranes. Storage proteins are also detected in multivesicular bodies whose membranes have reactivity with PV72 serum. All these results indicated that sunflower storage proteins are transported to protein storage vacuoles by a Golgi dependent pathway in a process in which homologous of PV72 are involved. <![CDATA[<B>Towards genetic transformation of local onion varieties</B>]]> The aim of this work was to explore the possibility of obtaining transgenic plants of onion varieties cultivated in Argentina, starting from calli induced from mature zygotic embryos, using two strains of Agrobacterium tumefaciens as transfection vectors. Mature embryos from three varieties of 'Valenciana' onion, Torrentina, Cobriza INTA and Grano de oro were in vitro cultivated for callus induction. After three to four months an average of 57.4% success for the three varieties was reached. Transformation was carried out with Agl1 or LBA 4404 A. tumefaciens strains, both carrying a binary vector containing the marker gene gus a and the selection gene nptII. Selection was done in callus induction medium containing 10 mgL-1 geneticin during three subcultures. At the end of the selection period, 342 portions of calli were recovered and transferred to regeneration medium. Of the selected calli evaluated by the expresión of the β-glucuronidase enzyme, 42% presented extensive blue areas or were completely blue. At the end of the first subculture in the regeneration medium, 54 calli were considered potentially organogenic because of the green areas observed. At the end of the wole regeneration period, just one normal plant was obtained, that was negative to PCR analysis using specific primers for gus a and nptII. All selected calli came from the Torrentina variety and the highest quantity of them were transformed with the strain LBA 4404. <![CDATA[<B>Transgenic trees and forestry biosafety</B>]]> The benefits from the development of transgenic trees are expected from the improvement of traits as growth and form, wood quality, industrial processes, disease and insect resistance, herbicide tolerance, ecological restoration, rooting ability, etc. One of the first reported field trials with genetically modified forest trees was established in Belgium in 1988 and the characteristic evaluated was herbicide tolerance in poplars. Since then, there have been more than 200 reported trials, involving at least 15 forest species. The majority of the field trials have been carried out in the USA (64%). More than 50% of the field trials are done with Populus species and the main target traits are herbicide tolerance (31%), followed by marker genes (23%) and insect resistance (14%). Until today, there is only one report on commercial-scale production of transgenic forest trees which is Populus nigra with the Bt gene release in China in 2002 and established on commercial plantations in 2003. Operational application of GMO's in forestry depends on technical, economical, political and public aspects, but the development of adequate regulatory frameworks and public acceptance of transgenic trees will define the future of this technology in forestry.