A regeneração de plantas, por organogênese ou embriogênese somática, a partir do cultivo de células e tecidos vegetais in vitro é a base para a utilização da biotecnologia no melhoramento. Realizaram-se estudos com cinco cultivares de laranja doce (Citrus sinensis L. Osbeck), Pêra, Natal, Lima Verde, Hamlin e Westin. Este trabalho objetivou a avaliação da eficiência de isolamento de protoplastos das cultivares de laranja doce; o estudo da eficiência de plaqueamento em função de cinco densidades de protoplastos e diferentes meios de cultura e avaliação da embriogênese somática em função da composição dos meios de cultura e concentração da fonte de carboidrato. As soluções enzimáticas testadas para o isolamento de protoplastos foram: 1. Grosser e Chandler (1987), composta de 1% de celulase Onozuka RS (Yakult), 1% macerase R- 10 (Yakult Honsha) e 0,2% de pectoliase Y-23 (Seishin); 2. Grosser e Chandler (1987) modificado, composta de 1% de celulase Onozuka RS (Yakult), 1% macerase R-10 (Yakult Honsha); 3. Solução enzimática composta de 4% de celulase Onozuka R-10 (Yakult), 1% macerase R-10. O plaqueamento dos protoplastos foi realizado em cinco densidades, 2 x 104, 5 x 104; 105; 2x 105 e 3 x 105 protoplastos . mL-1,nos meios de cultura EME 0,7M, BH3 0,7M e BH3 + EME 0,7M em ausência de luz, a 25 ± 1 ºC. A solução enzimática 2 proporcionou um maior rendimento no isolamento de protoplastos das cultivares Hamlin, Natal e Pera e solução enzimática 1 foi melhor para a cultivar Westin. A eficiência final de plaqueamento avaliada aos 90 dias foi superior nas densidades de de 3 x 105 e 2 x 105 protoplastos. mL-1 para as cultivares Hamlin, Natal e Lima Verde, e na densidade de 2 x 105 e 105 protoplastos. mL-1 para a cultivar Westin. A indução da embriogênese somática ocorreu em meio de cultura MT modificado com 500 mg.L-1 de extrato de malte, acrescido de sacarose, galactose, glicose, sorbitol, lactose e maltose, nas concentrações de 18, 37, 75, 110 e 150 mM à temperatura de 27 °C. A formação de embriões somáticos variou com o genótipo, sendo a cultivar Lima Verde e Westin apresentaram menor número de embriões somáticos. As melhores fontes de carboidratos foram a maltose, seguida pela lactose nas concentrações de 37 e 75 mM para a cultivar Pêra, 37 mM para a cultivar Natal e 37, 75 e 110 mM para a cultivar Hamlin. / Plant regeneration, by organogenesis or somatic embryogenesis from cell cultures and in vitro plant tissue culture is the basis for the use of biotechnology in plant breeding. Studies were conducted with five cultivars of sweet orange (Citrus sinensis L. Osbeck), Pêra, Natal, Lima Verde, Hamlin and Westin. This work aimed to evaluate the isolation efficiency of protoplasts, to evaluate platting efficiency of protoplasts based on five densities of cells and different culture media and to evaluate somatic embryogenesis based on culture medium composition and concentration. The enzymatic solutions tested were: 1. Grosser and Chandler (1987): 1% de cellulase Onozuka RS (Yakult), 1% macerase R-10 (Yakult Honsha) and 0,2% de pectoliase Y-23 (Seishin); 2. Grosser and Chandler (1987) modified: 1% de cellulase Onozuka RS (Yakult) and 1% macerase R-10 (Yakult Honsha); 3. Enzimatic solution containing 4% cellulase Onozuka R-10 (Yakult) and 1% macerase R-10. Protoplasts were cultured at densities of 2 x 104; 5 x 104; 105; 2 x 105 e 3 x 105 protoplasts.mL-1 in EME 0,7M, BH3 0,7M and BH3 + EME 0,7M, in the dark, at 25 ± 1 ºC. The enzymatic solution 2 provided higher yield for the cultivars Hamlin, Natal and Pêra, and enzymatic solution 1 resulted in better protoplast isolation for cultivar Westin. Final platting efficiency, evaluated 90 days after culture, was higher at the densities of 3 x 105 e 2 x 105 protoplasts.mL-1 for Hamlin, Natal and Lima Verde, and at the density of 2 x 105 e 105 protoplasts.mL-1 for Westin. Somatic embryogenesis stimulation occurred in cultured medium MT (MURASHIGE AND TUCKER, 1969) modified with 500 mg. L-1 of malt extract, supplemented with sucrose, galactose, glucose, maltose, lactose and sorbitol at concentrations of 18, 37, 75, 110 and 150 mM, at 27 ± 1 ºC. Somatic embryos produced varied with the genotype, the smaller number of somatic embryos was observed in cultivars Lima Verde and Westin. The best source of carbohydrate were maltose, followed by lactose at concentrations of 37 and 75 mM for cultivar Pêra, 37 mM for cultivar Natal, and 37, 75 and 110 mM for cultivar Hamlin.

A regeneração de plantas, por organogênese ou embriogênese somática, a partir do cultivo de células e tecidos vegetais in vitro é a base para a utilização da biotecnologia no melhoramento. Realizaram-se estudos com cinco cultivares de laranja doce (Citrus sinensis L. Osbeck), Pêra, Natal, Lima Verde, Hamlin e Westin. Este trabalho objetivou a avaliação da eficiência de isolamento de protoplastos das cultivares de laranja doce; o estudo da eficiência de plaqueamento em função de cinco densidades de protoplastos e diferentes meios de cultura e avaliação da embriogênese somática em função da composição dos meios de cultura e concentração da fonte de carboidrato. As soluções enzimáticas testadas para o isolamento de protoplastos foram: 1. Grosser e Chandler (1987), composta de 1% de celulase Onozuka RS (Yakult), 1% macerase R- 10 (Yakult Honsha) e 0,2% de pectoliase Y-23 (Seishin); 2. Grosser e Chandler (1987) modificado, composta de 1% de celulase Onozuka RS (Yakult), 1% macerase R-10 (Yakult Honsha); 3. Solução enzimática composta de 4% de celulase Onozuka R-10 (Yakult), 1% macerase R-10. O plaqueamento dos protoplastos foi realizado em cinco densidades, 2 x 104, 5 x 104; 105; 2x 105 e 3 x 105 protoplastos . mL-1,nos meios de cultura EME 0,7M, BH3 0,7M e BH3 + EME 0,7M em ausência de luz, a 25 ± 1 ºC. A solução enzimática 2 proporcionou um maior rendimento no isolamento de protoplastos das cultivares Hamlin, Natal e Pera e solução enzimática 1 foi melhor para a cultivar Westin. A eficiência final de plaqueamento avaliada aos 90 dias foi superior nas densidades de de 3 x 105 e 2 x 105 protoplastos. mL-1 para as cultivares Hamlin, Natal e Lima Verde, e na densidade de 2 x 105 e 105 protoplastos. mL-1 para a cultivar Westin. A indução da embriogênese somática ocorreu em meio de cultura MT modificado com 500 mg.L-1 de extrato de malte, acrescido de sacarose, galactose, glicose, sorbitol, lactose e maltose, nas concentrações de 18, 37, 75, 110 e 150 mM à temperatura de 27 °C. A formação de embriões somáticos variou com o genótipo, sendo a cultivar Lima Verde e Westin apresentaram menor número de embriões somáticos. As melhores fontes de carboidratos foram a maltose, seguida pela lactose nas concentrações de 37 e 75 mM para a cultivar Pêra, 37 mM para a cultivar Natal e 37, 75 e 110 mM para a cultivar Hamlin. / Plant regeneration, by organogenesis or somatic embryogenesis from cell cultures and in vitro plant tissue culture is the basis for the use of biotechnology in plant breeding. Studies were conducted with five cultivars of sweet orange (Citrus sinensis L. Osbeck), Pêra, Natal, Lima Verde, Hamlin and Westin. This work aimed to evaluate the isolation efficiency of protoplasts, to evaluate platting efficiency of protoplasts based on five densities of cells and different culture media and to evaluate somatic embryogenesis based on culture medium composition and concentration. The enzymatic solutions tested were: 1. Grosser and Chandler (1987): 1% de cellulase Onozuka RS (Yakult), 1% macerase R-10 (Yakult Honsha) and 0,2% de pectoliase Y-23 (Seishin); 2. Grosser and Chandler (1987) modified: 1% de cellulase Onozuka RS (Yakult) and 1% macerase R-10 (Yakult Honsha); 3. Enzimatic solution containing 4% cellulase Onozuka R-10 (Yakult) and 1% macerase R-10. Protoplasts were cultured at densities of 2 x 104; 5 x 104; 105; 2 x 105 e 3 x 105 protoplasts.mL-1 in EME 0,7M, BH3 0,7M and BH3 + EME 0,7M, in the dark, at 25 ± 1 ºC. The enzymatic solution 2 provided higher yield for the cultivars Hamlin, Natal and Pêra, and enzymatic solution 1 resulted in better protoplast isolation for cultivar Westin. Final platting efficiency, evaluated 90 days after culture, was higher at the densities of 3 x 105 e 2 x 105 protoplasts.mL-1 for Hamlin, Natal and Lima Verde, and at the density of 2 x 105 e 105 protoplasts.mL-1 for Westin. Somatic embryogenesis stimulation occurred in cultured medium MT (MURASHIGE AND TUCKER, 1969) modified with 500 mg. L-1 of malt extract, supplemented with sucrose, galactose, glucose, maltose, lactose and sorbitol at concentrations of 18, 37, 75, 110 and 150 mM, at 27 ± 1 ºC. Somatic embryos produced varied with the genotype, the smaller number of somatic embryos was observed in cultivars Lima Verde and Westin. The best source of carbohydrate were maltose, followed by lactose at concentrations of 37 and 75 mM for cultivar Pêra, 37 mM for cultivar Natal, and 37, 75 and 110 mM for cultivar Hamlin.

Thesis (M. Sc. (Agricultural Science)) -- University of Limpopo, 2001 / Refer to document

This work was aimed to investigate the basic viral infection protocols mainly focusing on Nicotiana benthamiana hairy root cultures and wild-type tobacco mosaic virus (TMV). The application of transgenic virus containing the gene for green fluorescent protein (GFP) for foreign protein production in plant tissue cultures and whole plants was also studied. The effect on viral accumulation of the form of plant tissue culture used, such as hairy roots, shooty teratomas and suspended cells, was investigated. Viral infection was shown to have no effect on culture growth and morphology. Hairy root cultures are a superior host for viral propagation and production in vitro. The maximum specific rate of viral accumulation occurred mainly during the root growth phase. The average maximum virus concentration in the hairy roots was 0.82 ?? 0.14 mg g-1 dry weight and virus protein represented a maximum of approximately 6% of total soluble protein in the root biomass. Proportional scale-up of TMVinfected hairy roots in shake flasks and bioreactors can be achieved without changing the average virus concentration accumulated in the hairy roots. The level of viral accumulation was much lower in N. benthamiana hairy roots infected with transgenic virus containing GFP (TMVGFPC3) compared with TMV and low levels or no GFP was detected. Viral accumulation and GFP production in whole plants was studied using different generations of transgenic TMV-GFPC3 virus. Hybrid viruses with the foreign gene GFPC3 deleted may have been formed in successive TMV-GFPC3 generations, resulting in the loss of GFP production and enhanced viral infectivity. In vitro generated RNA transcript and first generation TMV-GFPC3 were found to be more suitable for infection than the second generation TMV-GFPC3. However, the accumulation of GFP and virus concentration did not occur at the same ratio. Provided a more genetically stable transgenic viral vector is used for infection, transient viral infection of hairy roots can be a potential alternative system for foreign protein production than plants grown in the field as the containment or safety issues can be addressed.

published_or_final_version / Ecology and Biodiversity / Master / Master of Philosophy

Shoot cultures of Aloe polyphylla were initiated from young shoot explants of in vitro grown plants. The basal medium was MS medium (MURASHIGE and SKOOG, 1962), supplemented with 100 mgl ¯¹ myo-inositol, and 30 gl ¯¹ sucrose. Agar (0.8 %) was used as the gelling agent. Different cytokinins, singly or in combination with auxins (IBA and NAA), were tested for shoot proliferation activity. All the cytokinins tested (kinetin, zeatin, iP, and BA) gave a good shoot proliferation response. The optimal concentrations for shoot proliferation of each of the cytokinins tested were: zeatin (0.5 mgl ¯¹), kinetin (1.5 mgl ¯¹), iP (1.0 mg ¯¹) and BA (1.5 mgl ¯¹). In combination with auxins, the optimal combinations were kinetin/NAA (2.0/0.1 mgl ¯¹), kinetin/lBA (1.5/1.0 mgl ¯¹), zeatin/lBA (1.0/0.5 mgl ¯¹), zeatin/NAA (1.0/1.0 mgl ¯¹), BA/IBA (1.0/1.0 mgl ¯¹), BA/NAA (1.5/0.1 mgl ¯¹). Although it gave the highest number of shoots per explant, BA was responsible for hyperhydricity. Temperature and sucrose also influenced shoot proliferation. The optimal temperature was 25°C, while 30 gl ¯¹ was the optimal concentration of sucrose for shoot proliferation. Plants rooted well in plant growth regulator-free MS medium. Amongst the potting mixtures tested, soil: sand: vermiculite (1:1:1 v/v) was the best with 98 % plantlet survival. In the second part of this project, Platycerium bifurcatum cultures were established using leaf explants. The basal medium was MS medium (MURASHIGE and SKOOG, 1962), supplemented with 100 mgl ¯¹ myo-inositol and 30 g l ¯¹ sucrose. For bud initiation, 1.0 mgl ¯¹ BA was used, while 0.8 % agar was used as the gelling agent. Three different strengths of MS medium (full, half, and one-quarter strength) without plant growth regulators were tested for further bud growth and development. Half-strength MS proved to be the best for further bud growth and development. Rooting was best achieved in one-quarter strength MS medium without plant growth regulators. In vitro grown plantlets were successfully acclimatized using peat as the potting medium. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2001.

Hypoxoside, a phenolic diglucoside, with a diarylpentane-type structure, is thought to be the medicinally active constituent of corm extracts of Hypoxis hemerocallidea Fisch. & Mey. which are reputed to alleviate the symptoms of prostate hypertrophy and urinary infections. The biosynthes is and production of this unique phytochemical were investigated in H. hemerocallidea using both in vivo and in vitro systems. It was found, in root-producing callus, that [l4]C-phenylalanine and [14]C-t-cinnamic acid were efficient precursors for hypoxoside in comparison to [14]C-sodium acetate and [14]C-acetyl coenzyme-A, which were not incorporated into the phenolic compound. Thus, at least one aryl moiety of hypoxoside was derived, via phenylalanine and t-cinnamic acid, from the shikimate pathway. The acetate pathway did not appear to be involved in the biosynthetic process. The data supports the hypothesis that the molecule is formed from two cinnamate units with the loss of a carbon atom, in opposition to the proposal that the molecule is derived from head-to-tail condensation of acetate units onto a propenylic moiety. Despite the structural similarities between hypoxoside and caffeic and p-coumaric acids, these two hydroxycinnamic acids were not efficient precursors for hypoxoside in vivo or in vitro. A number of reasons are put forward to explain this finding. It was found that the greatest concentration of hypoxoside was located in the corms of intact plants. The major biosynthetic site of the molecule was also found to be located in this organ. Since the roots did accumulate the phytochemical to a small extent, the biosynthetic potential of these organs has not been disregarded. That of the leaves has been, however. The report by PAGE (1984) that the upper region of the corm contained a greater con cent ration of hypoxoside than the lower portion, is substantiated in this study, where this region was found to be more biosynthetically active than the lower half. Light microscopic and electron microscopic studies revealed that starch storing cells, which accumulated phenolics in their vacuoles, contained seemingly synthetically active tubular endoplasmic reticulum in their cytoplasm. A greater number of these cells were concentrated in the upper region as opposed to the lower half of the corm. It is postulated that these cells are the site for biosynthesis and accumulation of hypoxoside. The shikimate pathway, from which the precursors for hypoxoside are derived, was found, through the exposure of intact plants to [14]C-carbon dioxide, to be located mainly in the leaves. It is postulated from the above study and one in which [14]C-phenylalanine, [14]C-t-cinnamic acid, [14]C-p-coumaric acid and [14]C-caffeic acid were applied to intact plants, that phenylalanine and/or cinnamic acid are the transported form of the shi kimate derivatives. p-Coumaric and caffeic acids, which are metabolically more stable, are envisaged to be the sequestering forms. The investigation of the seasonal production of hypoxoside revealed that most of the synthesis and accumulation occurred after the corms had broken winter dormancy and after the flush of leaf growth had slowed down. During dormancy the production of hypoxoside appeared to cease. The in vjtro studies, where the effects of light, temperature, nutrients, plant growth regulators and supply of potential precursors, on hypoxoside production by root-producing callus were investigated, indicate that this metabolite is not simply a "shunt" metabolite. A number of factors other than precursor availability enhanced, or reduced the jn vjtro production of this phytochemical. Furthermore, production of the phytochemical and growth were not always antagonistic. Hypoxoside, the biosynthesis of which requires a more thorough investigation, is, however, according to this investigation, a typical secondary metabolite in many respects. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1989.

Multiple shoots were produced from nodal explants of thirty-day-old in vitro grown seedlings and from pretreated three, five- and nine-month-old greenhouse grown Acacia mearnsii plants, respectively. Explants were sterilized for 15 minutes using 0.1 % HgCl₂ for the three-month-old explants and 0.2 % for the five and nine-month-old explants. Nodal explants were induced to form multiple shoots when placed on Murashige and Skoog (MS) medium supplemented with 2.0 mg l ¯¹ benzyladenine (BA). Rooting of these shoots was achieved on MS medium supplemented with 1.0 mg l ¯¹ indole-3-butyric acid (IBA). Plantlets were acclimatized in transparent plastic containers under greenhouse conditions with a 90 % success rate. These plantlets were successfully acclimatised under greenhouse conditions and planted in the field together with plants regenerated by cuttings. In an attempt to overcome maturation effects and loss of juvenile characteristics, when using adult plant material in vitro, investigations were undertaken into the use of coppice material, as an alternative explant source. A. mearnsii trees from five ages (two, four, six, eight and ten-years old, respectively) were decapitated to a height of 1.5 m. After three weeks, coppice was noted on the stumps of trees from all ages. A linear response to coppice production was noted, with the greatest coppice production being on the two-year-old tree stumps and the least on the ten-year old tree stumps. Decontamination of the coppice was successful and multiple shoot production was obtained from coppice taken from all age groups on MS medium supplemented with 2.0 mg l ¯¹ BA. The effect of various sucrose concentrations were investigated. Greater shoot production occurred with increased sucrose concentrations (20 and 30 g l ¯¹). It was evident that rejuvenation of mature tissue could be achieved through the use of coppice material. A second approach to rejuvenate adult material and to overcome the deleterious effects of maturation, was in the use of apical meristems. Meristems were taken from 30-day-old in vitro grown plants, from coppice (rejuvenated tissue) and adult material of five various tree ages (two, four, six, eight and ten-years-old, respectively). Plant material were taken over two seasons (1997 to 1999) and the use of agar and liquid support media were tested under both light and dark conditions. The coppice and adult material was successfully decontaminated in both seasons. In the first season (1997/1998), shoot production was obtained from meristems of in vitro grown plants, coppice and adult material from all trees on MS medium alone or MS medium supplemented with 2.0 mg l ¯¹ BA. In the following season (1998/1999), the use of a solidified agar medium was superior to the use of a liquid culture. There appeared to be no significant difference (p<0.05) between the use of light or dark culture conditions. Various media were tested and maximum shooting occurred on half-strength MS medium and Woody Plant Medium (WPM). However, once multiple shoot primordia were initiated, shoot elongation posed a problem. It was for this reason that the size of the meristems excised from the coppice material was increased from 0.5 mm to 1.0 mm in the 1997/1998 season, to 1.0 to 2.0 mm in the 1998/1999 season. The use of gibberellic acid and 100 ml jars were also investigated to see if this might enhance shoot elongation. Sufficient plant material was not available for a thorough investigation. Environmental conditions under which the plant material (adult or coppice) was harvested was similar in both seasons, with respect to temperature, but differed in rainfall. Rainfall was high (105.1 mm) in 1997/1998 season and low (ranging from 59.8 to 71.45 mm) in the 1998/1999 season. Shoot production from meristems taken from coppice material in the 1998/1999 season was significantly greater (p>0.05) than that in the 1997/1998 season, whereas shooting from the adult plant material remained unchanged. The disadvantage with using coppice material is that its production on decapitated tree stumps is dependant on rainfall, which is unpredictable. The differences in results from coppice material could be attributed to the fact that the trees felled in the two seasons were not related to each other in any way. In both seasons meristems, tree age was not a limiting factor, for meristems from adult and from coppice material. Meristems from the ten-year-old trees were as productive as those taken from the two-year-old trees. In the 1997/1998 season the results from the meristems from the adult material was equal if not greater than those obtained from the coppice material. In the 1998/1999 season, there was no significant difference (p<0.05) in percentage shoot production between the meristems from the adult and coppice material throughout the age groups. This suggests that the use of rejuvenated tissue in the form of coppice is not essential. This re-emphasized the advantage of using meristems taken from adult plant material. This study provided suitable protocols for the micropropagation of both in vitro and ex vitro grown nodal explants of A. mearnsii. However, as the plant material obtained from the field matures so the ease of obtaining sterile material decreased, thus reducing the chances of in vitro micropropagation. For this reason suitable pretreatments and rejuvenation methods are necessary if explants from mature field tissue are to be introduced into culture and successfully micropropagated. This study has shown that through the use of nodal material (taken from coppice produced on adult tree stumps) and apical meristems taken from both coppice and mature plant material, adult material can be successfully decontaminated, introduced into culture and stimulated to produce shoots. Analysis of tannin production was conducted to see if there was any indication that the presence of tannins in the plant material effected in vitro culture of nodal explants. However, no trends were obtained suggesting any influence of tannins on in vitro performance. In future years after further optimisation, these techniques could be incorporated in an A. mearnsii clonal programme, with the advantage of possibly eliminating maturation effects, commonly noted in vegetative practices. This will allow for easy manipulation and amplification of superior quality adult material. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1999.

This work was aimed to investigate the basic viral infection protocols mainly focusing on Nicotiana benthamiana hairy root cultures and wild-type tobacco mosaic virus (TMV). The application of transgenic virus containing the gene for green fluorescent protein (GFP) for foreign protein production in plant tissue cultures and whole plants was also studied. The effect on viral accumulation of the form of plant tissue culture used, such as hairy roots, shooty teratomas and suspended cells, was investigated. Viral infection was shown to have no effect on culture growth and morphology. Hairy root cultures are a superior host for viral propagation and production in vitro. The maximum specific rate of viral accumulation occurred mainly during the root growth phase. The average maximum virus concentration in the hairy roots was 0.82 ?? 0.14 mg g-1 dry weight and virus protein represented a maximum of approximately 6% of total soluble protein in the root biomass. Proportional scale-up of TMVinfected hairy roots in shake flasks and bioreactors can be achieved without changing the average virus concentration accumulated in the hairy roots. The level of viral accumulation was much lower in N. benthamiana hairy roots infected with transgenic virus containing GFP (TMVGFPC3) compared with TMV and low levels or no GFP was detected. Viral accumulation and GFP production in whole plants was studied using different generations of transgenic TMV-GFPC3 virus. Hybrid viruses with the foreign gene GFPC3 deleted may have been formed in successive TMV-GFPC3 generations, resulting in the loss of GFP production and enhanced viral infectivity. In vitro generated RNA transcript and first generation TMV-GFPC3 were found to be more suitable for infection than the second generation TMV-GFPC3. However, the accumulation of GFP and virus concentration did not occur at the same ratio. Provided a more genetically stable transgenic viral vector is used for infection, transient viral infection of hairy roots can be a potential alternative system for foreign protein production than plants grown in the field as the containment or safety issues can be addressed.

Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2016-12-19T12:44:53Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) rafaela_naiara_oliveira.pdf: 828917 bytes, checksum: 4fca08e2402e176f89301c9fc6e5d35b (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2016-12-19T16:54:47Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) rafaela_naiara_oliveira.pdf: 828917 bytes, checksum: 4fca08e2402e176f89301c9fc6e5d35b (MD5) / Made available in DSpace on 2016-12-19T16:54:47Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) rafaela_naiara_oliveira.pdf: 828917 bytes, checksum: 4fca08e2402e176f89301c9fc6e5d35b (MD5) Previous issue date: 2016 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Este trabalho teve como objetivo desenvolver procedimentos de propaga??o in vitro de Eremanthus incanus e controlar a hiperidricidade em explantes durante o cultivo. Foram realizados cinco experimentos, que envolveram as etapas de germina??o, multiplica??o e alongamento. No experimento um, avaliou-se a influ?ncia dos meios de cultura MS e WPM (25, 50, 75 e 100% dos sais e vitaminas) no percentual de germina??o e na altura, n?mero de folhas e peso de mat?ria seca das pl?ntulas produzidas. Nos experimentos dois, tr?s e quatro os tratamentos consistiram de dois tipos de recipientes (tubos de ensaio e frascos de cultura) e quatro formas de veda??o (pel?cula de PVC, papel celofane, fita microporosa e tampas espec?ficas). No experimento dois, avaliou-se a influ?ncia dos recipientes e das formas de veda??es sobre o percentual de germina??o e de contamina??o, altura, n?mero de folhas e peso de mat?ria seca de pl?ntulas. No experimento tr?s, foram avaliados o n?mero de brota??es e a hiperidricidade em explantes na fase de multiplica??o, em tr?s subcultivos. J? no experimento quatro, a fase de alongamento foi avaliada em fun??o dos recipientes e formas de veda??o, em rela??o ?s vari?veis altura, hiperidricidade e peso de mat?ria seca. No experimento cinco, foram testadas quatro concentra??es de BAP e de TDZ e avaliados o n?mero de brota??es, hiperidricidade e calosidade, em dois subcultivos. No experimento um, o meio WPM75 apresentou o maior percentual de germina??o, enquanto o meio MS75 apresentou maior altura e n?mero de folhas, e o WPM100 maior peso de mat?ria seca. No experimento quatro, as combina??es tubo+fita, tubo+PVC e frasco+PVC proporcionaram os maiores percentuais de germina??o, enquanto os menores percentuais de contamina??o foram observados nos tratamentos tubo+fita e tubo+tampa. A combina??o tubo+celofane apresentou maior valor de altura e peso de mat?ria seca, e frasco+PVC maior n?mero de folhas. Observou-se no experimento tr?s, com rela??o ao n?mero de brota??es, que no subcultivo um a combina??o frasco+celofane foi superior, enquanto nos subcultivos dois e tr?s o tratamento tubo+celofane se destacou. Para a hiperidricidade, no subcultivo um, a combina??o tubo+tampa foi a que apresentou menor hiperidricidade, no subcultivo dois os tratamentos tubo+PVC e tubo+tampa se destacaram, e no subcultivo tr?s o melhor tratamento foi tubo+celofane. Na fase de alongamento (Experimento quatro), a combina??o tubo+celofane foi a que apresentou maior altura m?dia de explantes. Nos tr?s subcultivos, n?o ocorreu hiperidricidade na combina??o frasco+celofane, sendo tamb?m nessa combina??o observado o maior peso de mat?ria seca. No experimento cinco, o tratamento 0,75 mg L-1 BAP apresentou o maior n?mero de brota??es, nos dois subcultivos. Para a hiperidricidade, em ambos subcultivos, o BAP apresentou plantas com menor n?vel de hiperidricidade. Conclui-se que, na germina??o de sementes de Eremanthus incanus, o meio WPM com 75% de sais e vitaminas ? o mais indicado, enquanto para o estabelecimento da cultura o melhor ? o MS 75%. O tipo de recipiente e veda??o influenciam na multiplica??o, no alongamento e na hiperidricidade dos explantes, sendo que a combina??o do recipiente tubo de ensaio com a veda??o papel celofane transparente proporcionou, em geral, os melhores resultados. Quando comparadas as citocininas BAP e TDZ na multiplica??o, indica-se 0,75 mg L-1 de BAP. Mais estudos envolvendo a propaga??o in vitro devem ser realizados, principalmente relacionados ?s etapas de enraizamento e aclimata??o, de forma a consolidar uma metodologia para E. incanus. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Ci?ncia Florestal, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016. / This study aimed to develop procedures for in vitro propagation of Eremanthus incanus and control the vitrification in explants during cultivation. Five experiments were carried out, involving the stages of germination, multiplication and stretching. In experiment 1, we evaluated the influence of culture medium MS and WPM (25, 50, 75 and 100% of salts and vitamins) in the percentage of germination and height, number of leaves and dry weight of the produced seedlings. The experiments 2, 3 and 4 treatments consisted of two types of containers (test tubes and culture vials) and four types of sealing (PVC film, cellophane, micropore tape and specific covers). In experiment 2, we evaluated the influence of the containers and sealing forms on the percentage of germination and contamination, height, number of leaves and dry weight of seedlings. In experiment 3, we evaluated the number of shoots and vitrification in explants in multiplication phase in three subcultures. In the experiment 4, the elongation phase was assessed according to the containers and sealing forms in relation to height variables, vitrification and dry matter weight. In experiment 5, four concentrations of BAP and TDZ were tested and the number of shoots, vitrification and callus were evaluated in two subcultures. In experiment 1, the culture medium WPM75 had the highest percentage of germination, while the MS75 medium showed higher height and number of leaves, and the WPM100 presented the greater weight of dry matter. In experiment 2, the combinations tube + tape, PVC pipe + tube and PVC + vial provided the highest percentage of germination, while the lowest percentage of contamination were observed in the treatments tube + tape and tube + cover. The tube combination tube + cellophane showed higher height and dry matter weight, and vial + PVC larger number of leaves. It was observed in the experiment 3, in relation with the number of sprouts, that in the subculture 1 the combination vial + cellophane was superior, while in subcultures 2 and 3 the treatment tube + cellophane stood out. For vitrification, in subculture 1 the combination tube + cover showed the lowest vitrification, in subculture 2 the treatments tube + PVC tube and cover + tube stood out, and in subculture 3 the best treatment was tube + cellophane. In the elongation phase (Experiment 4), the combination tube + cellophane showed the highest average height of explants. In the three subcultures, there was no vitrification in combination vial + cellophane, also being observed in this combination the greater weight of dry matter. In experiment 5, treatment 0.75 mg L-1 BAP had the highest number of shoots in the two subcultures. For vitrification, in both subcultures, BAP presented plants with lower vitrification. In conclusion, for the germination of Eremanthus incanus seeds, WPM medium with 75% of salts and vitamins is the most suitable, while for crop establishment the best medium is MS 75%. The type of container and sealing influence the multiplication, on elongation and vitrification of explants, being that the combination of the test tube container with transparent cellophane sealing provided, in general, the best results. When comparing the cytokinins BAP and TDZ in proliferation it is indicated 0.75 mg L-1 BAP. Further studies involving the in vitro propagation must be carried out, mainly related to steps of rooting and acclimatization to consolidate a methodology for E. incanus.