Made available in DSpace on 2014-06-11T19:26:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-06-22Bitstream added on 2014-06-13T19:33:23Z : No. of bitstreams: 1 santos_rb_me_jabo.pdf: 1467164 bytes, checksum: 5cce9136001027d260fc80ff1c5229f4 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os objetivos do presente trabalho foram (i) identificar uma cultivar de soja (Glycine max) com melhor resposta in vitro para indução de calos e regeneração; (ii) otimizar o processo de transformação genética via biobalística em embriões oriundos de cotilédone maduros de soja. Os experimentos de indução de calos e transformação genética foram realizados com as cultivares IAS-5, BR16 e MSOY6101. Para indução de calos foram utilizadas altas doses de auxinas [5 mg/L 2,4D (2,4-diclorofenoxiacético- 2,4D), 5 mg/L 2ip (2-isopenteniladenina) e 10 mg/L ANA (ácido-naftalenacético)] e para regeneração de 0 a 2 mg/L de BAP (6-benzylaminopurine), adicionando-se 0,5 mg/L de ANA em todos os tratamentos. Vários parâmetros foram testados na transformação: concentração do tungstênio; BAP (0 a 5 mg/L) no co-cultivo e a concentração de canamicina para seleção de plantas. A construção utilizada foi constituída pelo vetor pCAMBIA 2301, contendo o gene repórter GUS, regulado pelo promotor constitutivo 35SCaMV e adição do gene de seleção NPTII que confere resistência a canamicina. Foram observadas diferenças para a indução e regeneração via embriogênese somática entre as cultivares de soja, resultando na seleção da cultivar 'IAS-5' para avaliação do potencial embriogênico quanto à diferenciação e regeneração de plantas. Essa cultivar apresentou alta taxa de indução, proliferação e regeneração estável. Os resultados mostraram que a adição de 0,5 mg/L de BAP ao meio de regeneração e 5 mg/L de 2,4D ao meio de indução aumentaram as taxas de diferenciação dos embriões de 0 para 27,77%. A partir dos resultados observados na transformação genética em N. benthamiana, adotou-se 24 mg/μL de tungstênio por membrana para as três cultivares de soja. Para regeneração em embrião maduro de soja o melhor resultado... / The present work aimed to (i) identify the best soybean (Glycine max) cultivar for in vitro response for callus induction and plant regeneration; (ii) optimize genetic transformation of soybean mature cotyledon embryo via biobalistic. The callus induction and genetic transformation experiments were performed using IAS- 5, BR16 and MSOY6101 soybean cultivars. High auxin levels (5 mg/L 2,4- diclorofenoxiacetic, 5 mg/L 2-isopenteniladenine and 10 mg/L acid-naftalenacetic) were used for callus induction and to regeneration, adding 0,5 mg/L of acid-naftalenacetic and 0 to 2 mg/L of 6-benzylaminopurine (BAP), in all the treatments of regeneration. The following parameters were tested in order to optimize the transformation: tungsten concentration; BAP concentration (0 to 5 mg/L) in the co-cultivation medium and Kanamycin concentration in the selection medium. The genetic construction used was comprised of pCAMBIA2301 vector, containing gusA gene reporter, under the regulation of 35SCaMV constitutive promoter and NPTII selection gene that provides resistance to Kanamycin. Some differences were observed at the induction and regeneration of somatic embryogenesis among the three cultivars and only IAS-5 cultivar was selected for the assessment of embryogenic potential regarding plant differentiation and regeneration. This cultivar presented high level of callus induction, stable proliferation and stable regeneration. The results showed that the addition of 5 mg/L of 2,4D to the induction medium and 0,5 mg/L of BAP to the regeneration medium increased the levels of differentiation of the IAS-5 soybean embryos from 0 to 27,77%. Based on the results observed in the genetic transformation in N. benthamiana, 24 mg/μL of tungsten per membrane is now being used in the bombardment for the three soybean cultivars... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:32:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2003-06-23Bitstream added on 2014-06-13T20:23:47Z : No. of bitstreams: 1 bordayepez_cc_me_botfca.pdf: 605083 bytes, checksum: 56a87160ec1d63940d534d9e6a51cff7 (MD5) / O presente trabalho teve como objetivo estabelecer o protocolo de desinfestação de estacas e sernentes e germinação de Pothomorphe umbeilata (L) e adaptar o protocolo de micropropagação de Pothomorphe umbeilata (L), incluindo a produção de calos e organogênese, além de comparar o teor de carotenóides entre calos e plântulas. O experimento foi conduzido no Laboratório de Biotecnologia Vegetal do Departamento de Química e Bioquímica, do Instituto de Biociências da UNESP - Botucatu, SP e o material vegetal (sementes e estacas) foi obtido no município de Adrianópolis-PR. Semente germinadas de P. umbeilata foram inoculadas em diferentes concentrações de BAP (0,5 mg.L ; 1,0 mgL ; 1,5 mg.L) e NAA (0,4 mg.L 0,6 mg.L ; 0,6 mg.L ) respectivamente, visando estimular a produção de calos e dosar o carotenóides. Após 60 dias do cultivo, os calos contendo algumas brotações, foram transferidos para meio de diferenciação das plântulas (GA3 0,1 mg.L , BAP 0,5 mgL ) por um período de 40 dias, para logo serem transferidos para meio de diferenciação de plântulas. Calos (coletados aos 60 dias) e plântulas (coletadas aos 140 dias) foram congelados em nitrogênio líquido e mantidos em freezer a 80°C para posteriores análises de carotenóides. O melhor tratamento para a produção de calos e formação de gemas foi NAA 0.6 mgL em combinação com BAP 10 mg.L . Nas plântulas sem adição de reguladores vegetais foi encontrada uma maior concentração de carotenõides, em comparação aos calos. / The present research aimed at establishing a protocol of stalks and seed desinfectation, and seed germination of Pothomorphe umbeilata (L.). A protocol of micropropagation including the induction of callus formation and organogenesis was adapted. Besides, it was compared the carotenoids quantity between Porhomorphe umbeliata calius and plantlets. This experiment was carried out iii Biotechnology Vegetal Laboratory of the Chernistr and Biochemistry Department at the Instituto de Biociências of Universidade Estadual Paulista, Botucatu campus. The vegetal matenal (seed and stalks) was obtained from Adranópolis - PR. Pothomorphe umbeilata germinated seeds were inoculated in different concentrations of BAP (0,5 mg.L-l; 1,0 mg.L.-l; 1,5 mg.L-l) and NAA (0,4 mg.L-l; 0,6 mg.L-1; 0,6 mg.L-l) respectively, in order to stimulate calius induction and increase quantity of earotenoids. Afler 60 days, calius which contained shoots were inoculated in plantlets diferenciation medium GA3 0,1 mg.L4, BAP 0,5 mg.U ) during 40 days and transferred to plantlets growth medium. Plantlets were acclimatized Calius collected after 60 days and plantlets were collected afier 140 days. There were frozen in liquid nitrogen and maintained in freezer 80°C to be used in further carotenoids test. The best treatment for callus production and shoots elongation was NAA 0.6 mg.L in association with BAP 1.0 mg.L . The higher carotenoids coneentration was in plantlets without growth regulators, compared with calius.

Orientador: Janete Aparecida Desidério / Coorientador: Sandra Helena Unêda Trevisoli / Banca: Newton Cavalcante de Noronha Junior / Banca: Sônia Marli Zingaretti / Resumo: Os objetivos do presente trabalho foram (i) identificar uma cultivar de soja (Glycine max) com melhor resposta in vitro para indução de calos e regeneração; (ii) otimizar o processo de transformação genética via biobalística em embriões oriundos de cotilédone maduros de soja. Os experimentos de indução de calos e transformação genética foram realizados com as cultivares IAS-5, BR16 e MSOY6101. Para indução de calos foram utilizadas altas doses de auxinas [5 mg/L 2,4D (2,4-diclorofenoxiacético- 2,4D), 5 mg/L 2ip (2-isopenteniladenina) e 10 mg/L ANA (ácido-naftalenacético)] e para regeneração de 0 a 2 mg/L de BAP (6-benzylaminopurine), adicionando-se 0,5 mg/L de ANA em todos os tratamentos. Vários parâmetros foram testados na transformação: concentração do tungstênio; BAP (0 a 5 mg/L) no co-cultivo e a concentração de canamicina para seleção de plantas. A construção utilizada foi constituída pelo vetor pCAMBIA 2301, contendo o gene repórter GUS, regulado pelo promotor constitutivo 35SCaMV e adição do gene de seleção NPTII que confere resistência a canamicina. Foram observadas diferenças para a indução e regeneração via embriogênese somática entre as cultivares de soja, resultando na seleção da cultivar 'IAS-5' para avaliação do potencial embriogênico quanto à diferenciação e regeneração de plantas. Essa cultivar apresentou alta taxa de indução, proliferação e regeneração estável. Os resultados mostraram que a adição de 0,5 mg/L de BAP ao meio de regeneração e 5 mg/L de 2,4D ao meio de indução aumentaram as taxas de diferenciação dos embriões de 0 para 27,77%. A partir dos resultados observados na transformação genética em N. benthamiana, adotou-se 24 mg/μL de tungstênio por membrana para as três cultivares de soja. Para regeneração em embrião maduro de soja o melhor resultado... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The present work aimed to (i) identify the best soybean (Glycine max) cultivar for in vitro response for callus induction and plant regeneration; (ii) optimize genetic transformation of soybean mature cotyledon embryo via biobalistic. The callus induction and genetic transformation experiments were performed using IAS- 5, BR16 and MSOY6101 soybean cultivars. High auxin levels (5 mg/L 2,4- diclorofenoxiacetic, 5 mg/L 2-isopenteniladenine and 10 mg/L acid-naftalenacetic) were used for callus induction and to regeneration, adding 0,5 mg/L of acid-naftalenacetic and 0 to 2 mg/L of 6-benzylaminopurine (BAP), in all the treatments of regeneration. The following parameters were tested in order to optimize the transformation: tungsten concentration; BAP concentration (0 to 5 mg/L) in the co-cultivation medium and Kanamycin concentration in the selection medium. The genetic construction used was comprised of pCAMBIA2301 vector, containing gusA gene reporter, under the regulation of 35SCaMV constitutive promoter and NPTII selection gene that provides resistance to Kanamycin. Some differences were observed at the induction and regeneration of somatic embryogenesis among the three cultivars and only IAS-5 cultivar was selected for the assessment of embryogenic potential regarding plant differentiation and regeneration. This cultivar presented high level of callus induction, stable proliferation and stable regeneration. The results showed that the addition of 5 mg/L of 2,4D to the induction medium and 0,5 mg/L of BAP to the regeneration medium increased the levels of differentiation of the IAS-5 soybean embryos from 0 to 27,77%. Based on the results observed in the genetic transformation in N. benthamiana, 24 mg/μL of tungsten per membrane is now being used in the bombardment for the three soybean cultivars... (Complete abstract click electronic access below) / Mestre

Please read the abstract in the 00front part of this document / Thesis (PhD (Botany))--University of Pretoria, 2006. / Plant Science / unrestricted

Monterey pine (Pinus radiata), a native tree to California and two Mexican islands, is important both ecologically and economically. Outside native stands, Monterey pines are grown for landscaping in California and on plantations around the world. Pitch canker, a disease caused by the fungus Gibberella circinata Nirenberg & O’Donnell (Fusarium circinatum Nirenberg and O'Donnell) is threatening the survival of Monterey pines. The disease currently affects Monterey pines in many parts of the world including the native stands. No effective chemical or biological control is available but some Monterey pines show resistance to the disease. The purpose of this project was to develop a working protocol for producing genetic clones of the resistant pines through micropropagation. These genetic clones will be used for outplanting in places outside the native stands for ornamental and plantation purposes. This project analyzes the results of ten trials with varied parameters and bases the final protocol on the parameters used in the trial that induces the growth of new shoots. The final protocol developed in this project describes, step-by-step, the media preparation for the initiation, plant material collection, surface sterilization of plant material, plating in media and initiation of shoots on explants. The protocol calls for collecting shoot tips with hardened buds that have not yet elongated, then washing the shoot tips in sterile water with Tween 20 for 15 minutes. The shoots tips are then surface sterilized in a 50% bleach solution for 20 minutes. The explants are broken into disks (to minimize damage to the cells) by inserting the tip of a scalpel and tilting it slightly. The initiation media shown to induce growth consists of ½ strength LePoivre basal salt mixture, 5mg/L benzylaminopurine, 3% sucrose and 0.8% agar and is adjusted to a pH of 5.7, then autoclaved for 20 minutes. The explants are inserted into solidified media and incubated in a growth chamber programmed for 16 hours of light and 8 hours of dark with temperatures of 27ºC and 22ºC and light irradiance of 80µEm-2s-1. After 1 month the protocol calls for transferring the growing shoots to elongation media with full LP basal salts and transferring every month. When the number of desired shoots has been reached the forthcoming protocol for rooting can be followed.

The variation for embryo production in anther culture of Solanum phureja was examined as a function of maximum greenhouse temperature prior to bud harvest and innate responsiveness among anthers within a bud. S. phureja clones PP5, AD2-4, A3P2-6 and AD3-4 were grown in a greenhouse under a 16 h photoperiod. The temperature was monitored continuously using a thermograph. Buds were collected from PPS and AD2-4 and the anthers were cultured in two groups of five flasks. In the first group, each flask contained the 30 anthers from 6 buds; the second group, each flask contained 1 anther from each of 30 buds -- a total of 30 anthers per flask. Significantly smaller coefficients of variation were observed for the second group, suggesting that the variation for embryogenic capacity among buds was greater than that among anthers within a bud. Variation in embryo yield as a function of greenhouse temperature for clones A3P2-6 and AD3-4 was examined by stepwise regression analysis. Embryogenic capacity of clone A3P2-6 was adversely affected by high temperatures (31-37°C) that occurred 2 and 7 days before bud harvest. However similarly high temperatures appeared to enhance the androgenic response of clone AD3-4. Regeneration rate of anther-derived embryos over three subcultures to fresh regeneration medium was examined as a function of anther donor or clone, cold pretreatment of embryos, and morphological classification of embryos. Only clonal origin significantly affected regeneration. Regeneration rate declined on each serial subculture. The frequency of regenerable embryos varied from 12.5% for clone BARD 1-3 to 46.0% for clone A3P2-6. Flow cytometric analyses were performed on several anther-derived monoploids of S. phureja to examine the frequency of nuclei at the 1x, 2x, and 4x levels within and among clones. Significant variation was found among duplicate cultures of the individual clones, but this variation was small enough to allow the detection of significant differences among the clones. Monoploid cell frequency ranged from 22.3% to 35.7%. Diploid cell frequency ranged from 48.6% to 59.9%. Tetraploid cell frequency ranged from 11.9% to 25.3%. Several families of anther-derived monoploid clones of S. phureja were analyzed for differences among clones within a family and among families. Significant differences were found in both categories. Finally, unstained protoplasts of monoploid S. phureja clone AM3 were sorted based on forward angle light scatter (FALS) and autofluorescence. Fractions selected for low FALS and weak autofluorescence appeared to be selectively enriched for monoploid protoplasts. / Master of Science

Mammalian estrogens and estrogen-like compounds known as xeno-estrogens are being found in and excreted into the environment in ever increasing amounts. The xeno-estrogen DDE has been found at high concentrations of 1-5 mg/kg of soil (Aislabie et. al, 1997). These estrogens and xeno-estrogens are having a devastating effect on animal-life, yet little is known or understood on the effects of estrogens on plant-life. Thus it is important to determine what effects (if any) estrogens may have on plants. Other research has shown that estrogen has an effect on plants grown in vitro (Janeczko and Skoczowski, 2005). This research aims to help increase the amount of information on what effects estrogens may have on plants. In this study, the effects of mammalian estrogens (17-β-estradiol, estrone and estriol) on the growth and tuberization of potato plants (Solanum tuberosum L. cv 'Iwa') grown in liquid tissue culture medium are presented. It was found that at even 0.1 mg/L of estrogen, root growth of the plants was diminished and at 10 mg/L of estrogen, plant deformity was apparent and callus growth induced. Acid phosphatase activity of the plants was increased with the addition of 0.1 mg/L and 1 mg/L of estrogen but then decreased with the addition of 10 mg/L of estrogen. Tuber production was slightly reduced in plants treated with estrogen compared to the control.

A publication by the National Academy of Sciences, USA (1979) outlined some of the research need for a great variety of economically important woody species whose remaining genetic resources need urgently to be collected and conserved. A viable regeneration system was established via tissue and cell suspension culture for Albizia falcataria and A. lebbeck, two important wood yielding leguminous tree species. The culture medium was standardized after several trials to obtain callus from the leaflet explants of these two tree species. The optimum use of casein hydrolysate (w/v) and coconut milk (v/v) in addition to 6-Benzylaminopurine and Indole-3-butyric acid could induce morphogenesis and somatic embryogenesis in the cultured tissue. This reports the first observation on somatic embryogenesis ofA. lebbeck using leaflets as the explants. Scanning Electron Microscopy and histological studies were done on the different stages plant development following standard techniques. Embryogenesis in suspension culture followed regeneration of plantlets in A. lebbeck. In A.falcaaria the regenerative process followed via organogenesis from the shoot buds developed on the leaf explants. After hardening the regenerated plants were transferred to the greenhouse. Some of the trees grew more than 25 feet tall within a few months outside the greenhouse. Karyotype of the three leguminous trees Albizia lebbeck, A. falcataria, and Dalbergia sissoo was analyzed. In D. sissoo, various chromosomal anomalies were observed in the cultured tissue. The abnormality indices and ploidy level varied with the age and the frequency of the subculture. In the aged culture the regenerative potential declined but was reinstated to some extent with the addition of two complex growth factors, coconut milk and casein hydrolysate. Seed anatomy of 26 species of 4 leguminous genera was studied with SEM. The main distinguishing anatomical features observed in the seed sections were uniseriate or multiseriate epidermis, epidermal projections, and number of rows and nature of columns of hypodermal layer, especially the nature of endosperm. Three species of Dalbergia, Acacia and Cassia and two species of Albizia are difficult to distinguish externally even with seed coat study under SEM, but this study with cross sections provided enough characteristic features to distinguish one from the other.

Muitos estudos têm sido direcionados para gerar melhorias na cultura de cana-de-açúcar, sendo uma das alternativas o uso de técnicas biotecnológicas, como a embriogênese somática (ES). O objetivo deste projeto foi estudar a multiplicação celular na ES de cana-de-açúcar da variedade SP80-3280. Culturas embriogênicas (CE) e não-embriogênicas (CNE) foram monitoradas e parâmetros bioquímicos foram analisados. Foi observado que CE e CNE apresentaram crescimento máximo aos 24 dias de cultivo, sendo que as CE apresentaram maior incremento em matéria fresca. Dos carboidratos analisados, a sacarose foi predominante em CE, enquanto a glicose predominou em CNE. Os conteúdos de glicose e frutose variaram simultaneamente em CE. As concentrações de amido, poliaminas (Pas) totais e a razão entre PAs [Razão PA= Put/(Spd+Spm)] apresentaram maiores valores nas CNE, sendo a espermidina a poliamina predominante em CE e putrescina em CNE. O conteúdo de proteínas totais foi significativamente maior em CE, em todas as fases de crescimento. / Many studies have been directed to generate improvements in the sugarcane cultures, one of the alternative use of biotechnology techniques such as somatic embryogenesis (SE). The objective of this project was to study the cell multiplication in SE of sugarcane, variety SP80-3280. Embryogenic cultures (EC) and non-embryogenic (NEC) were monitored and biochemical parameters were analyzed. Was observed that EC and NEC showed maximum growth to 24 days of culture, and the EC showed greater increase in fresh weight. Of the carbohydrates tested, sucrose was predominant in EC, while glucose was predominant in the NEC. The contents of glucose and fructose varied simultaneously in EC. The concentrations of starch, polyamines (PAs) and the ratio of total PAs [Ratio PA = Put / (Spd + Spm)] showed higher values in the NEC, and the spermidine is predominant polyamine to EC and putrescine is predominant in NEC. The content of total protein was significantly higher in CE, at all stages of growth.

Thesis (MTech (Horticulture))--Cape Peninsula University of Technology, 2018. / Drosanthemum micans and Drosanthemum hallii are endangered succulent shrubs of horticultural and medicinal value. They are restricted to the Succulent Karroo, which is one of the world’s biodiversity hotspots. The species risk extinction from illegal over-harvesting for water-wise gardens, erosion by occasional flush floods from ephemeral rivers, competition from alien invasive species, overgrazing and clearing of land for agriculture and human settlement. Although seeds and cuttings may be used in propagating these species, they often require seasonal collection and planting and cuttings struggle to establish, hence the need for in-vitro propagation as an alternative solution. Thus, the main objective of the study was to develop a method for rapid in-vitro shoot and root multiplication and acclimatization of D. micans and D. hallii. To initiate shoot formation, disinfected leaf and stem nodal explants were cultured in Murashige and Skoog (1962) media supplemented with different rates (0, 10, 20 or 30μM) of 2-isopentyladenine, 6-Benzyladenine and kinetin for D. hallii or 2-isopentyladenine, 6-Benzyladenine and Thiadiazuron for D. micans. Shoots from explants were rooted in varying rates (0, 10, 20 or 30μM) of IAA for root initiation. Three media, which were used in previous studies, were tested for acclimatization of rooted explants in i) vermiculite, ii) sand (50%): vermiculite (50%) or iii) sand (75%): perlite (25%). For quantitative evaluation of plant stress, chlorophyll fluorescence index (Fv/Fm) was measured as a proxy for plant stressf stress. It emerged that stem nodal explants of D. hallii tend to produce multiple shoots whilst leaf explants tended to produce callus when cultured in full-strength Murashige and Skoog (1962). Shoot multiplication was optimal in both D. hallii and D. micans at 10 μM of kinetin. Root formation in both D. hallii and D. micans only occurred when shoots were transferred to a full-strength Murashige and Skoog (1962) media without any phytohormones added. The intensity of tissue browning increased at higher levels of cytokinins, suggesting an interaction of plant growth regulators with exudates from explants. Different acclimatization media tested showed no significant differences in the level of stress (Fv/Fm). It is recommended that Murashige and Skoog (1962) media with10 μM kinetin be used for shoot development and multiplication, followed by transfer of the shoots to fresh full-strength Murashige and Skoog (1962) media without hormones for root development. Acclimatization of the rooted explants was possible in one of the following media: i) vermiculite, ii) sand (50%): vermiculite (50%) or iii) sand (75%): perlite (25%) and in a misted greenhouse (ca. 60% RH), with gradual weekly reductions in humidity by 10% over 2 weeks.