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Abrahamsson M.,Swedish University of Agricultural Sciences | Valladares S.,Swedish University of Agricultural Sciences | Valladares S.,Plant Biotechnology Laboratory | Merino I.,Swedish University of Agricultural Sciences | And 2 more authors.
In Vitro Cellular and Developmental Biology - Plant | Year: 2017

Somatic embryos can be used for propagating forest trees vegetatively, which is of great importance for capturing the genetic gain in breeding programs. However, many economically important Pinus species are difficult or impossible to propagate via somatic embryogenesis. In order to get a better understanding of the difficulties to propagate Pinus species via somatic embryogenesis, we are studying the developmental pathway of somatic embryos in different cell lines. In a previous study, we showed that the morphology of early somatic embryos in Scots pine (Pinus sylvestris) differs between cell lines giving rise to normal or abnormal cotyledonary embryos. In this study, we have compared the proliferation and degeneration pattern of early and late embryos in a normal and abnormal cell line. In both cell lines, a high frequency of the embryos degenerated. Among the degenerating embryos, two main degeneration patterns could be distinguished. In the normal cell line, the embryos degenerated similar to how the subordinate embryos are degraded in the seed. In the abnormal cell line, the degeneration of the embryos resulted in a continuous loop of embryo degeneration and differentiation of new embryos. We observed a similar degeneration pattern when embryogenic tissue was initiated from megagametophytes containing zygotic embryos at the stage of cleavage polyembryony. Based on our results, we suggest that the degeneration pattern in abnormal cell lines starts during initiation of embryogenic cultures. © 2017 The Author(s)


Nunes C.F.,Plant Biotechnology Laboratory | Ferreira J.L.,Plant Biotechnology Laboratory | Fernandes M.C.N.,Plant Biotechnology Laboratory | Breves S.S.,Plant Biotechnology Laboratory | And 6 more authors.
Ciencia Rural | Year: 2011

Several extraction methods of genomic DNA for identification and characterization of genetic diversity in different plant species are routinely applied during molecular analysis. However, the presence of undesirable compounds such as polyphenols and polysaccharides is one of the biggest problems faced during the isolation and purification of high quality DNA in plants. Therefore, achievement of fast and accurate methods for DNA extraction is crucial in order to produce pure samples. Leaves of strawberry genotypes (Fragaria ananassa) have high contents of polysaccharides and polyphenols which increase the sample viscosity and decrease the DNA quality, interfering with the PCR performance. Thereby, in this study we evaluated the quality and amount of genomic DNA extracted from young leaves of strawberry after tissue lyophilization and maceration in presence of polivinilpirrolidone (PVP). The CTAB method was used as reference procedure and it was modified to improve the DNA extraction. The modifications consisted of tissue lyophilization overnight until it was completely freeze-dried and addition of PVP during the tissue maceration in liquid nitrogen. The results showed the efficiency and reliability of the modified method compared to the unmodified method, indicating that combination of lyophilization and PVP improve the quality and amount of the DNA extracted from strawberry leaves.


Martins P.K.,Genetics and Biotechnology Laboratory | Nakayama T.J.,Federal University of Viçosa | Nakayama T.J.,Plant Biotechnology Laboratory | Ribeiro A.P.,Genetics and Biotechnology Laboratory | And 6 more authors.
Biotechnology Reports | Year: 2015

Abstract Setaria viridis was recently described as a new monocotyledonous model species for C4 photosynthesis research and genetic transformation. It has biological attributes (rapid life cycle, small genome, diploid, short stature and simple growth requirements) that make it suitable for use as a model plant. We report an alternative method of S. viridis transformation using floral dip to circumvent the necessity of tissue culture phase for transgenic plant regeneration. S. viridis spikes at boot stage were selected to be immersed in Agrobacterium suspension. T1 seeds could be identified in 1.5-2 months after floral dipping. We demonstrated through molecular analysis and RFP expression that seeds and resulting plants from dipped inflorescences were transformed. Our results suggest the feasibility of S. viridis floral dip transformation as a time-saving and cost-effective compared with traditional methods. To our knowledge, this is the first report using floral dip in S. viridis as an Agrobacterium-mediated transformation method. © 2015 The Author.


Alemu Setotaw T.,Federal University of Viçosa | Alemu Setotaw T.,Plant Biotechnology Laboratory | Teixeira Caixeta E.,Embrapa Cafe UFV | Alves Pereira A.,Empresa de Pesquisa Agropecuaria de Minas Gerais EPAMIG | And 5 more authors.
Crop Science | Year: 2013

The coefficient of parentage among 121 cultivars of Coffea arabica L. in Brazil released from 1939 to 2009 was estimated and used to study the genetic diversity and the breeding pattern of the breeding programs. A low genetic diversity was observed within the C. arabica cultivars of Brazil. The genetic base of 121 cultivars released in Brazil between 1939 and 2009 was defined by 13 ancestors. Seven ancestors contribute with 97.55% of the genetic base of C. arabica cultivars. Bourbon Vermelho contributed with 52.76% for the genetic pool of the C. arabica cultivars of Brazil followed by Sumatra (19.05%) and Híbrido de Timor (11.59%). Mundo Novo and Icatu Vermelho contributed with 87.65% for the genetic base of the C. arabica cultivars. It was calculated that 97.55% of the genetic base of the Brazilian C. arabica cultivars is derived from seven ancestors, indicating a narrow genetic base. Among the first progenies, Mundo Novo contributed with 69.39% of the genetic base of C. arabica cultivars in Brazil. The increase in the genetic diversity among C. arabica cultivars observed in recent decades is due to the introduction of parental lines with diverse genetic base. High genetic diversity was observed among cultivars released by Empresa de Pesquisa Agropecuária de Minas Gerais/Universidade Federal de Viçosa, Fundação Procafé, and Instituto Agronômico do Paraná. The 121 Brazilian cultivars were clustered into four groups based on coefficient of parentage. The distributions of genotypes over the cluster groups showed the effect of parental line contribution. © Crop Science Society of America.


Ferreira A.M.,University of Évora | Ferreira A.M.,New University of Lisbon | Marques A.T.,Institute Investigacao Cientifica Tropical | Marques A.T.,University of Milan | And 16 more authors.
PLoS ONE | Year: 2015

Bitter taste has been extensively studied in mammalian species and is associated with sensitivity to toxins and with food choices that avoid dangerous substances in the diet. At the molecular level, bitter compounds are sensed by bitter taste receptor proteins (T2R) present at the surface of taste receptor cells in the gustatory papillae. Our work aims at exploring the phylogenetic relationships of T2R gene sequences within different ruminant species. To accomplish this goal, we gathered a collection of ruminant species with different feeding behaviors and for which no genome data is available: American bison, chamois, elk, European bison, fallow deer, goat, moose, mouflon, muskox, red deer, reindeer and white tailed deer. The herbivores chosen for this study belong to different taxonomic families and habitats, and hence, exhibit distinct foraging behaviors and diet preferences. We describe the first partial repertoires of T2R gene sequences for these species obtained by direct sequencing. We then consider the homology and evolutionary history of these receptors within this ruminant group, and whether it relates to feeding type classification, using MEGA software. Our results suggest that phylogenetic proximity of T2R genes corresponds more to the traditional taxonomic groups of the species rather than reflecting a categorization by feeding strategy. © 2015, Public Library of Science. All rights reserved. This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.


Reis R.R.,Genetics and Biotechnology Laboratory | Reis R.R.,Federal University of Lavras | Andrade Dias Brito da Cunha B.,Genetics and Biotechnology Laboratory | Martins P.K.,Genetics and Biotechnology Laboratory | And 16 more authors.
Plant Science | Year: 2014

Drought is one of the most challenging agricultural issues limiting sustainable sugarcane production and, in some cases, yield losses caused by drought are nearly 50%. DREB proteins play vital regulatory roles in abiotic stress responses in plants. The transcription factor DREB2A interacts with a cis-acting DRE sequence to activate the expression of downstream genes that are involved in drought-, salt- and heat-stress response in Arabidopsis thaliana. In the present study, we evaluated the effects of stress-inducible over-expression of AtDREB2A CA on gene expression, leaf water potential (ΨL), relative water content (RWC), sucrose content and gas exchanges of sugarcane plants submitted to a four-days water deficit treatment in a rhizotron-grown root system. The plants were also phenotyped by scanning the roots and measuring morphological parameters of the shoot. The stress-inducible expression of AtDREB2A CA in transgenic sugarcane led to the up-regulation of genes involved in plant response to drought stress. The transgenic plants maintained higher RWC and ΨL over 4 days after withholding water and had higher photosynthetic rates until the 3rd day of water-deficit. Induced expression of AtDREB2A CA in sugarcane increased sucrose levels and improved bud sprouting of the transgenic plants. Our results indicate that induced expression of AtDREB2A CA in sugarcane enhanced its drought tolerance without biomass penalty. © 2014 Elsevier Ireland Ltd.


Belintani N.G.,Plant Biotechnology Laboratory | Guerzoni J.T.S.,Plant Biotechnology Laboratory | Moreira R.M.P.,State University Londrina | Vieira L.G.E.,Plant Biotechnology Laboratory
Biologia Plantarum | Year: 2012

Sugarcane is cultivated in tropical and subtropical regions where cold stress is not very common, but lower yields and reduced industrial quality of the plants are observed when it occurs. In our efforts to enhance cold tolerance in sugarcane, the gene encoding the enzyme isopentenyltransferase (ipt) under control of the cold inducible gene promoter AtCOR15a was transferred via biolistic transformation into sugarcane (Saccharum spp.) cv. RB855536. Semiquantitative RT-PCR using GAPDH encoding glyceraldehyde-3-phosphate dehydrogenase as the normalizer gene showed the increased expression of the ipt gene under cold stress. The detached leaves of genetically modified plants subjected to low temperatures showed visible reduction of leaf senescence in comparison to non-transgenic control plants. Induced overexpression of ipt gene also enhanced cold tolerance of non-acclimated whole plants. After being subjected to freezing temperature, leaf total chlorophyll contents of transgenic plants were up to 31 % higher than in wild type plants. Also, lower malondialdehyde content and electrolyte leakage indicated less damage induced by cold in transgenic plants. Thus, the expression of ipt driven by the stress inducible COR15a promoter did not affect plant growth while providing a greater tolerance to cold stress. © 2012 Springer Science+Business Media B.V.


Dibax R.,Federal University of Paraná | Deschamps C.,Federal University of Paraná | Filho J.C.B.,Federal University of Paraná | Vieira L.G.E.,Plant Biotechnology Laboratory | And 3 more authors.
Biologia Plantarum | Year: 2010

The purpose of this research was Eucalyptus saligna in vitro regeneration and transformation with P5CSF129A gene, which encodes Δ1-pyrroline-5-carboxylate synthetase (P5CS), the key enzyme in proline biosynthesis. After selection of the most responsive genotype, shoot organogenesis was induced on leaf explants cultured on a callus induction medium (CI) followed by subculture on a shoot induction medium (SI). Shoots were subsequently cultured on an elongation medium (BE), then transferred to a rooting medium and finally transplanted to pots and acclimatized in a greenhouse. For genetic transformation, a binary vector carrying P5CSF129A and uidA genes, both under control of the 35SCaMV promoter, was used. Leaves were co-cultured with Agrobacterium tumefaciens in the dark on CI medium for 5 d. The explants were transferred to the selective callogenesis inducing medium (SCI) containing kanamycin and cefotaxime. Calli developed shoots that were cultured on an elongation medium for 14 d and finally multiplied. The presence of the transgene in the plant genome was demonstrated by PCR and confirmed by Southern blot analysis. Proline content in the leaves was four times higher in transformed than in untransformed plants while the proline content in the roots was similar in both types of plants. © Springer Science+Business Media B.V 2010.

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