Imagens multiespectrais são utilizadas em diferentes aplicações, que vão desde sensoriamento remoto a processos médicos. No caso de imagens multiespectrais oriundas de microscopia confocal de varredura à laser (Confocal Laser Scanning Microscopy-CLSM), a extração da informação se inicia pela conversão das assinaturas espectrais, em uma imagem RGB. Esta imagem é a referência para a seleção da região de interesse, da qual se obtém a assinatura espectral média, originada do arquivo multiespectral (LSM). Mesmo utilizando um padrão muito bem estabelecido de conversão, alguns pontos devem ser considerados: i) o processo de conversão reduz a informação, a uma ordem de 10-145%; ii) a cor é uma experiência sensorial, subjetiva e pessoal, interferindo na seleção da região de interesse e; iii) a assinatura é obtida pela média espectral, da região de interesse, selecionada manualmente.Assim, esta tese de doutorado propõem um método de mapeamento e visualização das informações de imagens multiespectrais, combinando um algoritmo de agrupamento não supervisionado(kmeans) e um algoritmo que define uma paleta de cores coerentes com a informação espectral das regiões mapeadas. Aplicou-se o método em três casos de estudos de tecidos vegetais: i) no pré-tratamento de paredes celulares da cana-de-açúcar; ii) na plasticidade foliar do Jacaranda caroba e; iii) no uso de assinaturas espectrais na classificação de plantas do Cerrado. Os resultados demonstraram que o método é bastante robusto, permitindo de forma inovadora a: visualização, análise e comparação de imagens multiespectrais qualitativa e quantitativamente, e que seu uso é viável em qualquer área de pesquisa que utilize imagens multiespectrais. / Multispectral images are used in different applications, ranging from remote sensing images to medical images. In the case of multispectral images derived from confocal laser scanning microscopy (CLSM), the extraction of information begins with the conversion of spectral signatures in an RGB image. This is the reference for selecting the region of interest, from which it gets the average spectral signature, originated from multispectral file (LSM). Even using a very well established pattern of conversion, some points should be considered: i) the conversion process reduces the information on the order of 10-145%; ii) the color is a sensory experience, subjective and personal, interfering in the selection of the interest region and; the signature is obtained by the spectral average, from interest region which is selected manually. Thus, this doctoral thesis proposes a method of mapping and visualization of multispectral imaging information, combining an unsupervised clustering algorithm (kmeans) and an algorithm that defines a consistent color palette with the spectral information of mapped regions. The proposed method was applied in three cases plant tissue studies: i) in the pre-treating the cell walls of sugarcane; ii) in the leaf plasticity of Jacaranda caroba; iii) in the use of spectral signatures in the Cerrado plant classification. The results showed that the proposed method is quite robust. It presents innovation to the visualization and analysis of multispectral images and makes possible a qualitative and quantitative comparison of a group of multispectral images. Besides that, its use is feasible in any area of research, which are using multispectral images.

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.

Capillary electrophoresis (CE) is an analytical chemistry approach that allows for the efficient separation by charge of diverse classes of compounds for analysis, including secondary metabolites. The goal of this work was to optimize a buffer system for plant tissue analysis using micellar electrokinetic chromatography (MEKC), and by doing so to understand the role of buffer components in the performance of this form of capillary electrophoresis. In this experiment we implemented a factorial design to optimize buffer composition for separating plant tissue and secondary metabolites. The results of this experiment will be used to optimize a universal buffer for MEKC analysis that can be used on any variety of plant tissues. To determine the feasibility of this, a diverse set of plant secondary metabolite chemical standards in solution were tested as well as Helianthus annuus tissue to confirm the separation in a real biological sample. The results of this optimization yield insights into the utility of buffer components like electrolyte and pH for MEKC separation.

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.

Atualmente a procura por produtos sustentáveis têm-se mostrado cada vez mais frequente e promissora. Em espécies de importância comercial, procura-se obter a maior produtividade possível dentro de um curto espaço de tempo aliado à preservação do meio ambiente. Dentro disso, a transformação genética de plantas se mostra uma alternativa atrativa para a geração de variedades de cana-de-açúcar que gerem produtos de maneira mais eficaz. O sucesso da transformação genética está diretamente associada a cultura de tecidos de plantas que precisa ser adequada a cada genótipo e situação de cultivo, sendo a luminosidade um dos principais fatores para a produção de plantas vigorosas. Outro fator importante é a seleção das plantas transgênicas, que precisam ser submetidas a uma quantidade de agente seletivo suficiente para identificar as plantas modificadas geneticamente. Em cana-de-açúcar, a identificação de plantas transgênicas por PCR e a definição do número de cópias é um procedimento de difícil execução e muito oneroso. Isto se dá pois no processo transformação via biolística, a inserção de genes é aleatória, produzindo plantas com variados números de cópias. Em consideração a estes fatores envolvidos na eficiência de obtenção de plantas transgênicas de cana-de-açúcar, os objetivos deste trabalho foram o aperfeiçoamento do protocolo de cultura de tecidos, transformação genética da variedade SP803280 com os genes xth, AtDdm1, como também, definir genes de referência para a quantificação do número de cópias dos genes xth e AtDdm1 inseridos na variedade SP803280 e do gene neo na RB835089, análise de ploidia e tamanho de genoma dos eventos transgênicos comparado com as plantas controle. No estudo a respeito da melhor qualidade de luz durante o cultivo in vitro na fase de regeneração de plantas, tem-se que a luz branca e a junção das luzes LED e branca se mostraram melhores para regeneração e desenvolvimento das plantas enquanto que para plântulas, as luzes LED e branca separadamente foram mais efetivas no crescimento. Para a seleção das plantas uma concentração de geneticina entre 40 e 50 mgL-1 é recomendada. As taxas de sucesso nas transformações genéticas para o gene xth variaram entre 2,5 a 18,3% dependendo do experimento e para AtDdm1 foi de 2,2% em um bombardeamento.Não houveram alterações de ploidia e tamanho do genoma nos transgênicos das duas variedades em relação à planta selvagem. Os genes p4h e prr foram identificados como os melhores para a quantificação relativa de genes inseridos por PCR em tempo real na variedade SP803280 enquanto que para a RB835089 aprt e prr se mostraram mais eficazes. A análise do número de cópias inseridas em eventos transgênicos por PCR em tempo real foi possível através das duas metodologias de cálculo testados por este trabalho, com resultados que concordam com uma tendência nesta determinação de maneira simples e rápida. / Currently the demand for sustainable products has been shown to be frequent and promising. In species of commercial importance, there is an effort to obtain the highest possible productivity in a short time, along with the environment preservation. In this context, genetic transformation of plants appears as an attractive alternative for the development of sugarcane varieties able to generate products in a more effective way. The genetic transformation success is directly associated to plant tissue culture that requires specific condition for each genotype and cultivation process, in which, luminosity is one of the main factors that determines the production of vigorous plants. Another important factor is the selection of transgenic plants, that occur by exposing plants to a sufficient amount of selective agent in order to identify only genetic modified plants. In sugarcane, identification of transgenic plants by PCR and the definition of copy numbers is a difficult procedure to implement and usually is very costly. It is because in the process of genetic transformation by biolistic, the insertion of genes occurs randomly and also produce plants with varied copy numbers. In consideration of these factors directly involved in the efficiency to obtain sugarcane transgenic plants the objectives of this study were the improvement of a tissue culture protocol, the genetic transformation of the variety SP803280 with xth and AtDdm1 genes. Also, the studies include the definition of reference genes for determining the number of copies inserted of xth and AtDdm1 genes into the variety SP803280 and neo gene in RB835089, ploidy analysis and genome size of the transgenic events compared to control plants. In the study related to the best light quality for in vitro plant regeneration, white light and the combination of LED and white lights proved to be better for plants regeneration and development while for seedlings, LED and white light separately were more effective for growth. In order to obtain selection of transgenic plants, geneticin concentration between 40 and 50 mg L-1 is recommended. Success rates in xth genetic transformation ranged from 2.5 to 18.3% depending on the experiment, and for AtDdm1 was only 2.2% in just one biolistic bombardment. There were no changes in ploidy and genome size in transgenic events related to their wild type plant. The genes p4h and prr were defined to be the best for determining the copy number of transgenic events by real time PCR in SP803280 variety, while for RB835089, the genes aprt and prr were the most effective. The analysis of the number of inserted copies was possible using the two calculation methodologies tested by this work, with results that agree with a tendency in a simple and fast quantification methodology.