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Propagación in vitro de selecciones de guayabo (Psidium guajava L) y su respuesta a hormonas y periodos de subcultivo

LUIS ANTONIO DOMINGUEZ PERALES (2011)

Tesis (Maestría en Ciencias, especialista en Genética).- Colegio de Postgraduados, 2011.

La obtención de plantas de guayabo a partir de métodos de propagación como: hijuelos de raíz o por semilla, afecta sus rendimientos y su sanidad, considerando también que la propagación a partir de semillas genera una gran variación en la forma, tamaño y calidad de los frutos. La regeneración in vitro de plantas completas de guayabo puede ser la alternativa o complemento perfecto de los métodos convencionales de propagación, al obtener plantas a gran escala, uniformes y libres de microorganismos. En esta investigación se desarrollaron protocolos para la propagación in vitro de dos selecciones de Psidium guajava L. A partir de brotes obtenidos in vitro se consiguió establecer una metodología para su multiplicación en medio basal MS adicionado con sacarosa 30 g∙L-1, mio-inositol 100 mg∙L-1, tiamina HCl 1.0 mg∙L-1 y cuatro concentraciones distintas de 6-bencilaminopurina(BAP): 0.0, 2.2, 4.4 y 6.6 µmol∙L-1. Con la concentración de 4.4 µmol∙L-1 de BAP se obtuvieron los mejores resultados para la selección Roja Exterior Redonda, donde se tuvieron 1.69 brotes en promedio y una longitud de brotes promedio de 117 mm. Para la selección 17-06, los resultados más sobresalientes se obtuvieron con la concentración de 2.2 µmol∙L-1, ya que el número y la longitud de los brotes aumentaron en 46 y 15 %, respectivamente, comparados con el testigo (sin BAP). Para evaluar el efecto del tiempo de subcultivo en la selección Roja Exterior Redonda se utilizó medio basal MS adicionado con la mejor concentración de BAP (2.2 µmol∙L-1). Se observó que 45 días de tiempo de subcultivo favoreció el crecimiento y desarrollo de los explantes. De igual manera, se evaluó la respuesta del enraizamiento de explantes de guayabo expuestos al medio basal MS adicionado con dos reguladores de crecimiento de efecto auxínico: ácido indolacético (AIA) y ácido naftalenacético; el uso de AIA promovió numéricamente un incremento en la cantidad y la longitud de las raíces (1.69 y 116 mm, respectivamente) presentando 50 % de brotes con raíces. _______________ In vitro PROPAGATION OF SELECTIONS OF GUAVA (Pisidum guajava L.) AND ITS RESPONSE TO HORMONES AND SUBCULTURE PERIOD. ABSTRACT: Most of the guava plantations in Mexico have been established with conventional propagation methods, such as the production of plants from root tillers or by seed, which affects the performance and health. In addition , the propagation from seed generates great variation in shape, size and quality of fruit. That is why the in vitro regeneration of whole plants of guava may be the perfect alternative or complement to obtain uniform and free of microorganisms plants on a large-scale. In this research we developed protocols for in vitro culture of two selections of Psidium guajava L. By using shoots obtained in vitro we were able to establish a methodology for their in vitro multiplication in MS medium supplemented with 30 g∙liter-1 sucrose, 100 mg∙liter-1 myo-inositol, 1.0 mg∙liter-1 thiamine and four concentrations of 6-benzylaminopurine (0.0, 2.2, 4.4 and 6.6 µmol∙liter-1 ). The best results were achieved with a concentration of 4.4 µmol∙liter-1 BAP for the cultivar Roja Exterior Redonda, where the average number of outbreaks was 1.69 of three explants compared with control treatment (without BAP) with only 1.15 shoots with 117 mm of average length. In the case of the cultivar 17-06, the more outstanding results were obtained with the concentration of 2.2 µmol∙liter-1, as the number and length of shoots increased by 46 and 15% respectively, compared with the control treatment (without BAP). To evaluate the effect of time of subculture on the selection Roja Exterior Redonda, MS basal medium was used supplemented with the best concentration of BAP (2.2 µmol∙liter-1). It was noted that 45 days of subculture time favored the growth and development of the explants. Similarly, we assessed the response of the rooting of guava explants exposed to MS medium supplemented with two growth regulators with auxinic effect: indoleacetic acid (IAA) and naphthaleneacetic acid, IAA promoted an increase in the quantity and the length of roots (1.69 and 116 mm, respectively), showing 50% of shoots with roots.

Master thesis

Psidium guajava L. Micropropagación Organogénesis directa In vitro culture Shoot regeneration Genética Maestría CIENCIAS AGROPECUARIAS Y BIOTECNOLOGÍA

Un protocolo de embriogénesis somática para la regeneración y caracterización in vitro de Laelia anceps ssp. dawsonii

A protocol of somatic embryogenesis for the in vitroregeneration and characterization of Laelia anceps ssp. dawsonii

Hilda Eulalia Lee Espinosa ANTONIO LAGUNA CERDA Joaquín Murguía González Lourdes Georgina Iglesias Andreu BENJAMIN GARCIA ROSAS DIANA ESCOBEDO LOPEZ YOLANDA MARIA MARTINEZ OCAMPO FELIPE ALONSO BARREDO POOL NANCY SANTANA BUZZY (2010)

Laelia anceps ssp. dawsonii is one of the most appreciated Mexican wild orchids, with atractive characteristics for ornamental potential according to international standards. It has become an important economic species, subjected to excessive collection and risk of extinction. To ensure its conservation, it is necessary to develop efficient propagation protocols which allow its sustainable utilization and reduce collection. Somatic embryogenesis is an efficient technology for in vitro plant multiplication. In this study we evaluated in vitro conditions for developing a somatic embryogenesis protocol for Laelia anceps ssp. dawsonii. Embryogenic calli was induced from mature seeds, in a Murashige and Skoog medium supplemented with multiple combinations of naphthalene acetic acid (NAA), 6-benzylaminopurine (BAP), kinetin (Kin) and indole 3 acetic acid (IAA). The combination NAA+BAP+IAA (2.0 mg L-1 each) was optimal for callus induction, producing 611 embryoids in a 16 h photoperiod (33.8 µmol m-2 s-1) when subcultured every 45 d on the same medium. This conditions allowed for complete plantlet development. In approximately three months, plantlets achieved 4-5 cm in a V&W medium supplemented with BAP (2 mg L-1), AIA (1 mg L-1) and 0.2 % activated charcoal; after 30 d plantlets were acclimatized in a greenhouse with 95 % survival rate.

Article

LAELIA ANCEPS SSP. DAWSONII IN VITRO CULTURE SOMATIC EMBRYOGENESIS ORCHIDACEAE BIOLOGÍA Y QUÍMICA BIOLOGÍA Y QUÍMICA

Efficient plant regeneration from leaf explants of Solanum americanum

INGRID AILEEN O'CONNOR SANCHEZ ANGEL VIRGILIO DOMINGUEZ MAY MIGUEL ANGEL KEB LLANES TOMAS AUGUSTO GONZALEZ ESTRADA YURI JORGE JESUS PEÑA RAMIREZ (2010)

A very efficient system for direct plant regeneration from in vitro-derived leaf explants of Solanum americanum was developed. S. americanum is a tropical plant with important medical properties. The in vitro procedure that was established consists of (i) induction of shoots from leaf tissue, (ii) elongation of shoots, and (iii) rooting of plantlets. The induction of shoots was achieved on Murashige and Skoog solid medium supplemented with different combinations of zeatin riboside and 1-naphthalene acetic acid or 6-benzylaminopurine and 1-naphthalene acetic acid. The best combination for plant regeneration was MS with 5.7 μM zeatin riboside and 0.11 μM 1-naphthalene acetic acid. In the second step, the shoot clumps were transferred to MS basal medium without plant growth regulators, resulting in the differentiation of most of the shoot initials into well developed shoots. In the third stage, plantlets were efficiently rooted on half-strength MS basal medium supplemented with 58.5 mM sucrose. The rooted plants were established in soil with a 100% success rate. This system can be useful to perform further experiments to obtain transgenic plants of this species as well as for other biotechnological approaches. © 2010 Academic Journals.

Article

1-NAPHTHALENE ACETIC ACID AMERICAN NIGHTSHADE IN VITRO CULTURE MEDICINAL PLANT SPECIES ORGANOGENESIS SHOOTS REGENERATION ZEATIN RIBOSIDE BIOLOGÍA Y QUÍMICA

Efficient plant regeneration from leaf explants of Solanum americanum

INGRID AILEEN O'CONNOR SANCHEZ ANGEL VIRGILIO DOMINGUEZ MAY MIGUEL ANGEL KEB LLANES TOMAS AUGUSTO GONZALEZ ESTRADA YURI JORGE JESUS PEÑA RAMIREZ (2010)

A very efficient system for direct plant regeneration from in vitro-derived leaf explants of Solanum americanum was developed. S. americanum is a tropical plant with important medical properties. The in vitro procedure that was established consists of (i) induction of shoots from leaf tissue, (ii) elongation of shoots, and (iii) rooting of plantlets. The induction of shoots was achieved on Murashige and Skoog solid medium supplemented with different combinations of zeatin riboside and 1-naphthalene acetic acid or 6-benzylaminopurine and 1-naphthalene acetic acid. The best combination for plant regeneration was MS with 5.7 μM zeatin riboside and 0.11 μM 1-naphthalene acetic acid. In the second step, the shoot clumps were transferred to MS basal medium without plant growth regulators, resulting in the differentiation of most of the shoot initials into well developed shoots. In the third stage, plantlets were efficiently rooted on half-strength MS basal medium supplemented with 58.5 mM sucrose. The rooted plants were established in soil with a 100% success rate. This system can be useful to perform further experiments to obtain transgenic plants of this species as well as for other biotechnological approaches. © 2010 Academic Journals.

Article

1-NAPHTHALENE ACETIC ACID AMERICAN NIGHTSHADE IN VITRO CULTURE MEDICINAL PLANT SPECIES ORGANOGENESIS SHOOTS REGENERATION ZEATIN RIBOSIDE BIOLOGÍA Y QUÍMICA BIOLOGÍA Y QUÍMICA

In vitro LEAD AND NICKEL ACCUMULATION IN MESQUITE (Prosopis laevigata) SEEDLINGS

LETICIA BUENDIA GONZALEZ JUAN OROZCO VILLAFUERTE MARIA ELENA ESTRADA ZUÑIGA CARLOS EDUARDO BARRERA DIAZ EDUARDO JAIME VERNON CARTER FRANCISCO CRUZ SOSA (2010)

The growth, survival and Pb(II) and Ni(II) uptake of Prosopis laevigata seedlings were determined in order to evaluate their bioaccumulation capability. The seedlings were cultured during 50 days on modied Murashige and Skoog medium supplemented with 10 g.l-1 of sucrose, 0.0, 0.32, 0.63, 1.26, 2.10, 4.20 mM Ni(II), and 0.0, 0.23, 0.45, 0.90, 1.50, 3.0 mM Pb(II). None of the studied heavy metals avoided germination; however, both produced smaller plants with fewer leaves and secondary roots. Seedlings showed an accumulation of 2 582 and 3 895 mg Ni kg-1, and of 27 300 and 40 666 mg Pb kg-1, both in dry basis (d.b.), in shoot and root, when cultured with 1.26 mM Ni and 3.0 mM Pb, respectively. These results indicated that signicant translocation from the roots unto aerial parts took place. A bioaccumulation factor for Ni superior to 32 and for Pb over 21 was exhibited by the seedlings, so that P. laevigata can be considered as a viable accumulator species of Pb(II) and Ni(II) for phytoremediation purposes.

Article

Ingeniería Prosopis laevigata phytoremediation bioaccumulation heavy metals in vitro culture BIOLOGÍA Y QUÍMICA

Hongos micorrízicos ericoides y sus efectos en plantas de arándano azul (Vaccinium corymbosum L) cv BILOXI micropropagadas.

JORGE HUMBERTO LEÓN OSORIO (2017)

Tesis (Maestría en Ciencias, especialista en Fisiología Vegetal).- Colegio de Postgraduados, 2017.

Se realizó la búsqueda de hongos micorrízicos ericoides mediante aislamientos a partir de raíces de Vaccinium confertum HKB, de los aislamientos se identificó por métodos moleculares a Leptodontidium orchidicola, el cual se inoculó en el proceso de enraizamiento de microestacas de arándano azul Vaccinium corymbosum L. cv. Biloxi. Se desarrolló un protocolo para la obtención masiva de plantas de alta calidad fitosanitaria, por cultivo in vitro. La aplicación de 2iP ejerció un efecto favorable sobre la regeneración de brotes a partir de yemas axilares, así como el efecto de AIB en el proceso de enraizamiento de microestacas. A partir del aislamiento de hongos, se observó que L. orchidicola desarrolló un complejo de hifas dentro de las células de la epidermis de raíces de arándano azul, similar a los enrollamientos hifales típicos de la micorriza ericoide. La inoculación de este hongo también promovió el crecimiento radical y vegetativo; así como, la acumulación de mayor cantidad de biomasa en esta planta. Este es el primer reporte de L. corchidicola como endófito en raíces de V. confertum, y de su asociación con V. corymbosum. La inoculación de L. orchidicola podría utilizarse como herramienta biotecnológica importante para el enraizamiento plantas de arándano azul con alta calidad fitosanitaria, obtenidas por técnicas de cultivo in vitro utilizando el protocolo generado en esta investigación. _______________ ERICOID MYCORRHIZAL FUNGI AND EFFECTS ON MICROPROPAGATED BLUEBERRY (Vaccinium corymbosum L.) PLANTLETS, CV BILOXI. ABSTRACT: This research isolated potential ericoid mycorrhizal fungi from roots of Vaccinium confertum HKB, and Leptodontidium orchidicola was identified by means of molecular techniques and then, inoculated during the rooting of microcuttings of blueberry plants (Vaccinium corymbosum L. cv. Biloxi). First, an in vitro protocol was developed for the massive propagation of plantlets with high health quality. The application of 2iP had significant effects on shoot regeneration from axillary buds; the IBA also favored the rooting of microcuttings. Regarding fungi isolation, L. orchidicola developed hyphal complexes in epidermal cells resembling the typical hyphal coils of the ericoid mycorrhiza. The inoculation of this fungus resulted in improved vegetative and root growth, and plant biomass. Results provide the first report of L. corchidicola as an endophyte in roots of V. confertum, and as potential symbiont for V. corymbosum. The inoculation of L. orchidicola may be utilized as a biotecnhological tool for improving the rooting of blueberry plants obtained from the in vitro culture protocol developed in this research.

Master thesis

Cultivo in vitro Vaccinium Enraizamiento Hongos Leptodontidium Cenoccocum Micorriza ericoide Enrollamientos hifales In vitro culture Vaccinium Seed germination Rooting Fungi Ericoid mycorrhiza Hyphal coils Fisiología Vegetal Maestría CIENCIAS AGROPECUARIAS Y BIOTECNOLOGÍA CIENCIAS AGRARIAS AGRONOMÍA BOTÁNICA GENERAL

Especificidad potencial de hongos micorrícicos en el proceso de germinación y supervivencia in vitro de orquídeas terrestres

Nubya Edith Montes Villa (2016)

Instituto de Investigaciones Agropecuarias y Forestales. Facultad de Biología. Facultad de Medicina Veterinaria y Zootecnia. Facultad de Agrobiología. Facultad de Químico Farmacobiología. Programa Institucional de Maestría en Ciencias Biológicas

In Mexico the Orchidaceous family is taxonomically well cataloged. However, like it happens in others parts of world, in our country the natural populations of orchids have been decreased drastically, and knowledge about their current situation and the interaction they share with other organisms, like their mycorrhiza fungi, is scarce. These fungi are very important for the germination process of the seed and during different stages of the plant development. Therefore, studies about these topics are important because they will allow to determine ecological specificity situation (plant-fungus association in natural conditions), as well as potential specificity (plant-fungus association that produced in laboratory conditions), that would allow to determine the specificity that terrestrial orchids might have towards their fungal partner and efficiency of the symbiosis in the development processes and survival in in vitro cultures, as an alternative in conservation programs of this plants family in their natural habitats. The aim of this study was the morphological characterization and identification of mycorrhiza fungi of terrestrial orchids include in genus Bletia, Habenaria y Govenia, and assess their potential specificity, in vitro germination and seedling development. To achieve this, plants roots were collected and their mycorrhiza fungi were isolate and identified from the macroscopic and microscopic characteristics. In addition, capsules of the same genera were collected to perform viability and germination seeds´ tests.

En México la familia Orchidaceae se encuentra taxonómicamente bien catalogada. Sin embargo, como sucede en otras partes del mundo, en nuestro país las poblaciones naturales de orquídeas han disminuido drásticamente sin que se cuente con datos precisos de su situación actual y de la interacción que guardan con otros organismos, como son sus hongos micorrízicos, los cuales son muy importantes para los procesos de germinación de la semilla, así como en distintas etapas de desarrollo de la planta. Por lo que, estudios como el presente son importantes ya que permiten determinar la especificidad ecológica (asociación planta-hongo en condiciones naturales), como potencial (asociación planta-hongo que se producen en condiciones de laboratorio), que pudieran tener las orquídeas terrestres hacía su socio fúngico y la eficiencia de la simbiosis en los procesos de desarrollo y supervivencia en cultivos in vitro, como propuesta de conservación eficiente de esta familia de plantas en sus hábitats naturales. El objetivo de este estudio fue caracterizar e identificar morfológicamente los hongos micorrízicos de orquídeas terrestres de los géneros Govenia, Habenaria y Bletia, y evaluar su especificidad potencial en la germinación y desarrollo de plántulas in vitro. Para abordar esto, se colectaron raíces de las plantas y se identificaron sus hongos micorrízicos a partir de las características macroscópicas y microscópicas que presentaron, las cuales se obtuvieron de cultivos puros. Además, se colectaron cápsulas de los mismos géneros para llevar a cabo las pruebas de viabilidad de semillas y la germinación de estás en diferentes tratamientos.

Master thesis

CIENCIAS AGROPECUARIAS Y BIOTECNOLOGÍA FB-M-2016-1210 Opción en interacción planta-microorganismo-insecto Simbiosis Sebacina In vitro

Influence of dietary fiber upon in vitro microbial cecal fermentation in mexican hairless and mexican cuino pigs

IGNACIO ARTURO DOMINGUEZ VARA LUIS ANGEL LARA FUENTES CARLOS ALBERTO GARCIA MONTES DE OCA JOSE ROMERO BERNAL NAZARIO PESCADOR SALAS MANUEL GONZALEZ RONQUILLO (2010)

The objective of the present study was to evaluate and compare the in vitro cecal fermentation (by the gas production technique), in Mexican hairless pig (MHP) and Mexican cuino pig (MCP), adding cellulose or starch as substrates (0, 100, 200, 300 and 400 mg/g DM). 12 pigs were slaughtered (BW= 104±0.5 kg), six of each genotype were collected from the cecal contents and there was a pool for every two pigs in each genotype, and thereafter, for each substrate (cellulose or starch) in its different concentration, three flasks were incubated with inoculum for each pool and made three series of incubation. The experimental design use the effect of genotype, substrate and concentration of the substrate added on the variables of in vitro fermentation. The averages of the data were compared by Tukey's method. In vitro gas production was higher (P<0.05) (mL/g DM) for MHP (206.8) vs MCP (180.2). The degradation fractional rate rhythm (b, 0.094 and c, -0.0127) and lag time (1.79) of the MHP was higher than MCP (b, 0.074; c -0.102) and lag time (1.26); with the addition of carbohydrates as substrates, in vitro gas production of potato starch was higher (P<0.05) than cellulose (238.3 vs 148.7 mL/g DM); the fractions b, c and lag time, there were differences (P<0.05) between substrates. The increasing addition of cellulose or starch had a lineal effect (P<0.05) with the gas production, level cero mg (133.46) to 400 mg of substrate (263.16 mL/g DM). MHP had a higher cecal fermentation than MCP, being higher the gas production at 9 and 12 h, there was higher gas production (cecal fermentation) when added starch as substrate.

Article

Agrociencias Cellulose Digestibility Gas production In vitro Mexican cuino pig Mexican hairless pig Starch CIENCIAS AGROPECUARIAS Y BIOTECNOLOGÍA

Propagación in vitro de Dalbergia congestiflora (Campincerán) a partir de estacas cultivadas en invernadero

Alejandra Hernández García (2014)

In this research a method of clonal propagation in vitro of Dalbergia congestiflora (campincerán) was established, culturing shoots from cuttings grown in greenhouse culture. Shooting of 20 cm cuttings was evaluate with three diameters (0.5, 1 and 1.5 cm). These were obtain from an adult individual in field, applying three rooting: IBA (indole-3-butyric acid), Auxin-F (Raizone-Plus) and Auxin-NP (Pro-root) (1, 5 and 10 ppm). Cuttings were grown in peat moss-perlite (1:1 v/v) in greenhouse conditions and under continuous irrigation with running water (n=3), evaluating the percentage of sprouting, number and length of the shoots for 45 days. At this time it was observed a greater length of the shoots (10.5 cm per cutting) with 5 ppm of Auxin-F and a greater number of shoots per cutting (11.5 shoots) with a length of 8 cm, with Auxin-NP in 5 ppm. After 5 months of culture, 66% of the cuttings with 10 ppm of Auxin-F formed 4 roots and 40% of callus. The cuttings treated with Auxin-NP were used for the establishment of in vitro for producing the largest number of explants (29 buds/stake). The in vitro establishment of D. congestiflora was achieved with the bud explants in the Murashige and Skoog (MS) culture media without growth regulators. Four aseptic treatments were utilized and an 80% survival of the explants with a loss of 20% by necrosis was obtained with the selected treatment. This consisted in treat the explants with 20% of commercial sodium hypochlorite (6% chlorine active) for 5 min, detergent hyclin (15%) more Tecto 60 (5.0 g/L) for 5 min, and finally with 20% of sodium hypochlorite more Tecto 60 (5.0 g/L) for 20 min. At 45 days after MS cultivation with 0.05 mg/L benzyladenine (BA), 75% of the explants developed an initial shoot of 0.5 cm in length with two leaves. The largest number of shoots (4.6 shoots/explant) with a length of 1.63 cm was obtained in 100% of buds grown for 45 days in MS with 1.0 mg/L BA and 0.1 mg/L naphtalenacetic acid (NAA), in this medium was also observed a high percentage of callogenesis. Organogenesis in callus was obtained in MS with 1.0 mg/L BA and 0.1 mg/L of ANA, presenting 1.4 shoots/cm2 with a length of 1.6 cm.

En la presente investigación se estableció un método de propagación clonal in vitro de Dalbergia congestiflora (campincerán) por el cultivo de brotes a partir de estacas cultivadas en invernadero. Se evaluó la brotación en estacas de 20 cm de longitud con tres diámetros (0.5, 1 y 1.5 cm). Éstas fueron obtenidas de un individuo adulto en campo, aplicando tres enraizadores: AIB (ácido indol-3-butírico), Auxina-F (Raizone-Plus) y Auxina-NP (Pro-root) (1, 5 y 10 ppm). Las estacas fueron cultivadas en turba-agrolita (1:1 v/v) en condiciones de invernadero y bajo riego continuo con agua corriente (n=3), evaluando el porcentaje de brotación, número y longitud de los brotes durante 45 días. A este tiempo se observó una mayor longitud del brote (10.5 cm por estaca) con 5 ppm de Auxina-F y un mayor número de brotes por estaca (11.5 brotes) con una longitud de 8 cm, con Auxina-NP en 5 ppm. Después de 5 meses de cultivo, el 66% de las estacas con 10 ppm de Auxina-F formaron 4 raíces y 40% de callo. Las estacas tratadas con Auxina-NP fueron utilizadas para el establecimiento in vitro, por producir el mayor número de explantes (29 yemas/estaca). El establecimiento in vitro de D. congestiflora se logró con el cultivo de yemas en medio de cultivo de Murashige y Skoog (MS) sin reguladores de crecimiento. Se aplicaron cuatro tratamientos de asepsia, obteniendo un 80% de supervivencia de los explantes y una pérdida del 20% por necrosis con el tratamiento seleccionado, consistente en tratar los explantes con hipoclorito de sodio comercial (6% cloro activo) al 20% por 5 min, detergente hyclin (15%) más Tecto 60 (5.0 g/L) por 5 min y finalmente con hipoclorito de sodio másTecto 60 (5.0 g/L) al 20%por 20 min. A los 45 días del cultivo en MS con 0.05 mg/L de benciladenina (BA), el 75% de los explantes desarrolló un brote inicial de 0.5 cm de longitud con dos hojas.

Master thesis

INGENIERÍA Y TECNOLOGÍA FITECMA-M-2014-2029 Facultad de Ingeniería en Tecnología de la Madera Campincerán Callogénesis Micropropagación Yemas In Vitro Ciencias y Tecnología de la Madera