A mist reactor was used to study plant growth and development under various environmental conditions towards the production of healthy plantlets ready for soil transplant in one step from inoculation. In addition, a 3D type of cultivation via surface attachment of explants to vertically hanging strips inside the mist reactor was also investigated to maximize productivity with minimal footprint. Using carrot as the model species, pre-embryogenic cell suspensions were successfully spray-inoculated onto hanging poly-L-lysine (PLL)-coated nylon mesh to which they then attached and remained for several weeks while they developed into rooted plantlets. To study single step micropropagation from shoot explants to fully acclimatized plantlets, Artemisia annua was used as the model species. Nodal cuttings of A. annua were inoculated onto PLL-coated mesh strips by briefly immersing the strips in the suspension of nodal cuttings. Investigation of medium, phytohormones, CO2, ventilation level and humidity ensued resulting in selection of a preferred final process that reduced physiological aberrations like hyperhydricity and was time efficient. The nodal cuttings that attached to the strips were first misted with half strength shooting medium for 7 days to develop new shoots. Then the new shoots were misted with the rooting medium supplemented with NAA for 12 days to develop roots. Rooted plantlets were acclimatized in the same rooting medium for 9 days to acquire fully functional stomata prior to planting into soil. Taken together this study suggested that fully developed plantlets ready for planting into soil could be obtained in a single step in a bioreactor from embryogenic cells or from nodal explants.

Made available in DSpace on 2017-07-25T19:31:00Z (GMT). No. of bitstreams: 1 Marina Oliveira.pdf: 1283767 bytes, checksum: 902cc2416d1d157460b534a7f6b62ed8 (MD5) Previous issue date: 2016-02-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The expansion of cultivated blueberry fields in Brazil is directly linked to the provision of quality seedlings to the market, which may result in the increase of production. The plant micropropagation technique enables the production and provides high quality plants, physiologically viable and healthy and free from viruses in a short space of time. Woody plants presente difficulty in the in vitro rooting, in this context the development of a more efficient system of rooting results in plants with higher physiological quality and reduction of losses during the acclimatization phase. In the first experimente the treatments consisted in 8 doses of BIOBRAS 16 (0; 0,1; 0,2; 0,3; 0,4; 0,5; 0,6; 0,7 mg L-1) added to the culture medium at the time of preparation. The following characteristics were evaluated: number of corns, callus diameter, number of roots, the total root length, total length, number of shoots, length of shoots, number of leaves and fresh mass. The lowest concentration of BIOBRAS 16 used (0,1 mg L-1) presented a higher percentage of rooting, root length and number of leaves, but also stimulated the development of calluses. The dose of 0,4 mg L-1 stimulated the formation of shoots and length thereof. There was no significant difference for other parameters evaluated. In the second experiment the treatments consisted in diferent doses of BIOBRAS 16 (0,1; 0,3; 0,5 mg L-1) associated to different doses of indole acetic acid (1; 3; 5 L-1 μM) added to the culture medium during preparation. The parameters evaluated were the same as the experiment 1. The use of BIOBRAS 16 associated with indolacetic acid showed a positive effect for the development of in vitro characteristics of blueberry. For rooting, the concentration of 0,3 mg L-1 BIOBRAS 16 + 5 μM AIA showed a higher percentage. Concentrations of 0,5 mg L-1 BIOBRAS 16 + 5 μM of AIA induced the formation and callus diameter, undesirable feature when the goal is rooting. For the parameters number of shoots and number of leaves, the best result was the concentration of 0,1 mg L-1 BIOBRAS 16 + 5 μM of AIA. / A expansão da área cultivada de mirtileiro no Brasil está diretamente vinculada a oferta de mudas de qualidade ao mercado, que poderá resultar em aumento da produção. A técnica da micropropagação de plantas viabiliza a produção e fornece plantas de elevada qualidade, fisiologicamente viáveis, sadias e isentas de vírus emcurto espaço de tempo. Plantas lenhosas apresentam dificuldade de enraizamento in vitro, nesse contexto o desenvolvimento de um sistema de enraizamento mais eficiente resulta em mudas com maior qualidade fisiológica e diminuição de perdas durante a fase de aclimatização. No primeiro experimento os tratamentos consistiram em 8 doses de BIOBRAS 16 (0; 0,1; 0,2; 0,3; 0,4; 0,5; 0,6; 0,7 mg L-1) adicionados no meio de cultura no momento do preparo. As seguintes características foram avaliadas: número de calos, diâmetro do calo, número de raízes, comprimento total de raízes, comprimento total, número de brotações, comprimento de brotações, número de folhas e massa fresca. A menor concentração de BIOBRAS 16 utilizada (0,1 mg L-1) apresentou maior percentual de enraizamento (%), comprimento de raízes (cm) e número de folhas, porém também estimulou o desenvolvimento de calos. A dose de 0,4 mg L-1 estimulou a formação de brotações e o comprimento das mesmas. Não houve diferença significativa para os demais parâmetros avaliados. No segundo experimento os tratamentos consistiram em diferentes doses de BIOBRAS 16® (0,1; 0,3; 0,5 mg L-1) associadas a diferentes doses de ácido incolacético (1; 3; 5 μM L-1) adicionados ao meio de cultura no momento do preparo. As características avaliadas foram número de calos,diâmetro do calo, número de raízes, comprimento total de raízes, comprimento total, número de brotações, comprimento de brotações, número de folhas e massa fresca. A utilização de BIOBRAS 16 associado a ácido indolacético mostrou efeito positivo para características de desenvolvimento in vitro de mirtileiro. Para enraizamento, as concentrações de 0,3 mg L-1 de BIOBRAS 16 + 5 μM de AIA apresentaram maior percentual. As concentrações de 0,5 mg L-1 de BIOBRAS 16® + 5 μM de AIA induziram a formação e diâmetro de calos, característica indesejável quando o objetivo é o enraizamento. Para as características número de brotações e número de folhas, o melhor resultado foi da concentração de 0,1 mg L-1 de BIOBRAS 16 + 5 μM de AIA.

As atividades que envolvem a floricultura vêm se destacando positivamente no cenário econômico do agronegócio brasileiro. Dentre as espécies de plantas utilizadas para este fim ressaltam-se as espécies pertencentes à família botânica Asteraceae, principalmente as do gênero Chrysanthemum. A espécie C. cinerariaefolium Vis. (Piretro da Dalmácia) é mundialmente cultivada visando à produção de aleloquímicos que apresentam atividades de inseticidas naturais (piretrinas). No Brasil chegou a ser produzida para esse fim em larga escala, no município de Taquara-RS na década de 30 a 40. Recentemente o interesse de seu cultivo tem aumentado como uma alternativa natural de manejo de pragas como planta ornamental, principalmente para jardins. Contudo, o fato de não produzir sementes férteis limita sua capacidade de propagação massal. Visando aperfeiçoar sua micropropagação foram realizados dois experimentos. No experimento 1 foi avaliado aos 30 dias de cultivo a influência de cinco concentrações de hipoclorito de sódio (10, 20, 30, 40 e 50% do produto comercial Mazzarolo) e cinco tempos de permanência na solução durante a desinfestação dos explantes (5, 10, 15, 20, 25 min.). Ao todo foram isolados 125 meristemas totalizando 25 tratamentos com cinco repetições. Os explantes isolados foram inoculados no meio de isolamento, o qual foi constituído de MS + 1,0 mg.L-1 de BAP + 0,1 mg.L-1 de GA3 + 0,01 mg.L-1 ANA + 30 g.L-1 de Sacarose em 7,0 g.L-1 de Agar. No experimento 2 foi avaliado a influência das diferentes concentrações de hipoclorito e dois tempos de permanência na solução desinfetante sobre a porcentagem de necrose, estruturas amorfas, plântulas formadas e comprimento de brotos. As plântulas regeneradas do experimento 1 foram inoculadas no meio de multiplicação que constou do MS + 0,4 mg.L-1 de Thiamina HCl + 100 mg.L-1 de Mio-Inositol + 0,05 mg.L-1 de ANA + 1,0 mg.L-1 de BAP + 40 g.L-1 de Sacarose e 7,0 g.L-1 de Agar. O pH dos meios foi ajustado em 5,9 antes da autoclavagem. Os resultados obtidos no experimento 1 indicaram que quanto maior o tempo de permanência na solução, maior a porcentagem de necrose. Porém, quanto menor a permanência maior a taxa de contaminação microbiana. No experimento 2, observou-se que a porcentagem de necrose é maior na concentração de 50%. Não houve influência dos fatores analisados na produção de estruturas amorfas. Para o número de brotos/explante e comprimento de brotos, o melhor desempenho se deu na solução 30%. Porém, a taxa de brotos por explante diminuiu à medida que aumentou o tempo de permanência na solução. Com relação ao comprimento dos brotos, os melhores resultados foram observados até o tempo de 15 minutos de permanência. Assim conclui-se que C. cinerariaefolium Vis. responde positivamente ao cultivo in vitro. A taxa de necrose aumenta à medida que aumenta a concentração de hipoclorito. Por outro lado, a taxa de contaminação aumenta à medida que diminui a concentração da solução desinfetante. A concentração de hipoclorito de sódio e o tempo de permanência na solução desinfetante afetam o número e o comprimento de brotos produzidos nos subcultivos. / The activities that involve floriculture have distinguished positively in the economic scenario of the Brazilian agribusiness. Among the plant species used for that purpose it can be distinguished the species belonging to the botanical family Asteraceae, mainly from the genus Chrysanthemum. The species C. cinerariaefolium Vis. is worldwide cultivated in order to produce the alelochemicals, which have activities of natural insecticides (pyretrine). In Brazil it was produced in large scale, in the Taquara-RS County in the decade of 30 to 40. Recently the interest on its cultivation has increased as natural alternative of pest management as ornamental plant, mainly in yards. However, the fact of no seed is produced by the plant that limits its capacity of massal production. In order to optimize its micropropagation two experiments were developed. In the experiment 1 it was evaluated 30 days after cultivation the influence of five concentrations of sodium hipochlorine (10, 20, 30, 40 and 50% of the commercial product Mazzarolo), and five time lengths of the explants permanence in the disinfecting solution (5, 10, 15, 20 and 25 min.). It was isolated 125 meristems, 25 treatments with five replications. The isolated explants were inoculated in isolation media, that was constituted of MS + 1.0 mg.mL-1 of BAP + 0.1 mg.L-1 of GA3 + 0.01 mg.L-1 ANA + 30 g.L- 1 of sucrose in 7.0 g.L-1 of Agar. In the experiment 2 it was evaluated the influence of different concentrations of hipochlorine and two length of time in disinfection solution measuring the percentage of necrosis, amorphous structures, seedlings formed and length of the sprouts. The regenerated seedlings from experiment 1 were inoculate in a medium of multiplication that was of MS + 0.4 mg.L-1 of Thiamine HCl + 100 mg.L-1 of Mio-Inositol 0.05 mg.L-1 of ANA + 1.0 mg.L-1 of BAP + 40 g.L-1 of sucrose and 7.0 g.L-1 of Agar. The pH of the medium was adjusted to 5.9 before the autoclaving process. The results obtained in the experiment 1 indicated that the higher the time length in the solution, the higher is the percentage of necrosis. However, the higher is the permanence of higher rate of microbial contamination. In the experiment 2; it was observed that the percentage of necrosis is higher at the concentration of 50%. There was no influence of the analyzed factors in the yield of amorphous structures. For the number of sprouts/explants and length of sprouts, the better performance was in the solution of 30%. However, the rate of sprouts per explants diminished as the length of time in the solution increased. The best results for sprout length were obtained up to 15 minutes of permanence. Therefore, it can be concluded that C. cinerariaefolium Vis. responds positively to in vitro culture. The rate of necrosis increases as the hipochlorine concentration is higher. On the other hand, the contamination rate increases as the disinfection solution diminishes. The concentration o hipochlorine sodium as the length of time of permanence in the initial disinfecting solution used during the explants disinfection affects the number and the sprout length produced during the subculture.

Made available in DSpace on 2016-04-28T14:57:34Z (GMT). No. of bitstreams: 1 2008 - Everaldo Hans Studt Klein.pdf: 5165097 bytes, checksum: ccfbfbc6baec84bc829dad886555adf9 (MD5) Previous issue date: 2008-07-15 / It was conducted a survey of the major diseases that occur in orchids in the State of Rio de Janeiro, through collection of plants in various localities in the period April 2006 to June 2008. Fifty-three plants had their diseases and pathogens identified, of which 35.9% were infected by fungi, being 17% by Fusarium oxysporum, 13.2% by Colletotrichum gloeosporioides, 1,9% by Botrytis sp., 1,9% by Puccinia sp. and 1.9% by Phyllosticta capitalensis. 51% of the plants were infected by virus with simple and double infections of Cymbidium mosaic virus - CymMV and Odontoglossum ringspot virus - ORSV. 1.9% were infected by the bacterium Pectobacterium carotovorum subsp. carotovorum and 3.8% by nematodes of the genus Aphelenchoides. The nematodes Aphelenchoides fragariae and A. ritzemabosi were identified infecting hybrid orchids, which is the first report of parasitism of these nematodes in orchid and of A. ritzemabosi parasiting Asplenium nidus L. in Brazil. It also made the first record of the occurrence of Cymbidium mosaic virus - CymMV infecting Bamboo orchid Arundina bambusifolia Lindl. in the State of Rio de Janeiro. Based on the identification of pathogens of that survey, it was developed a diagnostic kit for the main diseases of orchids. Aiming to test antimycotic properties of three substances in culture medium for cultivation of orchids, using as test- plants Sophronitis cernua Lindl. and Epidendrum ellipticum Grah. Of the substances tested, only powdered cinnamon (Cinnamomum zeylanicum J. Presl), at the dose of 6 grams / liter positively influenced the germination of seeds of Epidendrum ellipticum Graham, and the addition of clove (Syzygium aromaticum) at doses of 3, 5 and 6 grams / liter caused the inhibition of in vitro germination of seeds of Epidendrum ellipticum Graham. and Sophronitis cernua (Lindl.) Hook. The addition of commercial garlic (Allium sativum L.) powder in doses of 6 and 10 grams / litre, also inhibited in vitro germination of seeds of Epidendrum ellipticum Graham. / Foi realizado levantamento das principais doen?as que ocorrem em orqu?deas no Estado do Rio de Janeiro, atrav?s de coleta de plantas em v?rias Localidades, no per?odo de abril de 2006 a junho de 2008. Cinq?enta e tr?s plantas tiveram suas doen?as e pat?genos identificados, dos quais 35,9% das plantas estavam infectadas por fungos, sendo 17% por Fusarium oxysporum, 13,2% por Colletotrichum gloeosporioides, 1,9 % por Botrytis sp., 1,9% por Puccinia sp. e 1,9% por Phyllosticta capitalensis. 51% das plantas estavam infectadas por v?rus com infec??es simples e duplas de Cymbidium mosaic virus - CymMV e Odontoglossum ringspot virus - ORSV. 1,9 % estavam infectadas pela bact?ria Pectobacterium carotovorum subsp. carotovorum e 3,8 % por nemat?ides do g?nero Aphelenchoides. Foram identificados os nemat?ides Aphelenchoides fragariae e A. ritzemabosi infectando orqu?deas h?bridas, sendo esse o primeiro relato de parasitismo desses nemat?ides em orqu?dea e de A. ritzemabosi em Asplenium nidus L., no Brasil. Foi feito tamb?m o primeiro registro da ocorr?ncia Cymbidium mosaic virus CymMV infectando plantas da esp?cie Arundina bambusifolia Lindl. no Estado do Rio de Janeiro. Com base na identifica??o dos pat?genos desse levantamento, desenvolveu-se um kit diagn?stico para os principais fitopat?genos de orqu?dea. Objetivou-se testar tr?s subst?ncias de propriedades fungist?ticas em meio de cultivo de propaga??o de orqu?deas, utilizando-se como plantas-teste Sophronitis cernua Lindl. e Epidendrum ellipticum Grah.. Das subst?ncias testadas, apenas canela em p? (Cinnamomum zeylanicum J.Presl), na dosagem de 6 gramas / litro influenciou positivamente a germina??o de sementes de Epidendrum ellipticum Graham, sendo que o acr?scimo de cravo-da-?ndia (Syzygium aromaticum) comercial picado nas doses de 3, 5 e 6 g/l causou a inibi??o da germina??o in vitro das sementes de Epidendrum ellipticum Graham. e Sophronitis cernua (Lindl.) Hook. O acr?scimo de alho (Allium sativum L.) em p? comercial nas doses de 6 e 10 g/l, tamb?m inibiu a germina??o in vitro das sementes de Epidendrum ellipticum Graham.

Made available in DSpace on 2017-07-10T17:37:07Z (GMT). No. of bitstreams: 1 Luciani_Marcia_Scherer.pdf: 497371 bytes, checksum: 27b02ebdf1f1cef92c900c635b6a2a91 (MD5) Previous issue date: 2007-02-14 / Secretaria de Estado da Educação do Paraná / Citronella of Java (Cymbopogon winterianus Jowitt.) is an aromatic plant of great economical value, it is also well known in all over the world due to repellent characteristics to its essential oil in citronellal. The propagation in vitro is a good method to multiply citronella, due to the fact that the division of the citronella roots ordinarily used facilitates the cutting contamination. However, there are reports that citronella does not present good results in the propagation in vitro when collected in determined seasonal conditions. Therefore, the aim of this paper was to evaluate the effect of the seasonally about the propagation in vitro of citronella of Java, being used different concentration of IBA and BAP, as well as evaluating the income of the essential oil for each season. The explants were collected in each season of the year and inoculated in MS in addition to combination of IBA and BAP. At the end of 30 days the following variants had been evaluated: Height (cm), number of shootings, formation of callus, oxidation and contamination. In the realized conditions, was observed a good multiplication rate during the summer, being just necessary the addition of 1,0 mg/L-1 of BAP in the medium culture, commonly to this it did not have oxidation in this season. The height only presented significant result in the summer. It had formation of callus in the base of the explants either in the autumn or in the winter and in the summer, but in the summer it had a concomitant increase in the number of shootings. The oxidation was higher in the spring. (100% of oxidation). The contamination did not present significant results in any season, therefore, independent from seasonal condition. The extraction of the essential oil was accomplished in each season of the year by in Clevenger appliance. The great income of the essential oil was gotten in the winter with 1,37% of oil, when the plants were totally bloomy. Although the normal conditions of bloom of citronella in the south region is during the spring and in the beginning of the summer. The spring and the summer had 0, 77 and 0,76% income of essential oil, respective. In the autumn presented a lesser income, with 0,57% of essential oil / A citronela de Java (Cymbopogon winterianus Jowitt.) é uma planta aromática de grande valor econômico e também muito conhecida no mundo inteiro devido a sua propriedade repelente atribuída ao seu óleo essencial, rico em citronelal. A propagação in vitro é uma boa forma de multiplicar a citronela, visto que a divisão de touceiras comumente empregada facilita a contaminação das mudas. No entanto, existem relatos de que a citronela não apresenta bons resultados na propagação in vitro quando coletada em determinadas condições sazonais. Sendo assim, o objetivo deste trabalho foi avaliar o efeito da sazonalidade sobre a propagação in vitro da citronela de Java, utilizando diferentes concentrações de IBA e BAP, bem como avaliar o rendimento do óleo essencial para cada estação. Os explantes foram coletados em cada uma das estações do ano e inoculados em meio MS acrescido de combinações de IBA e BAP. Ao final de 30 dias foram avaliadas as variáveis: altura (cm), número de brotações, formação de calo, oxidação e contaminação de explantes. Nas condições realizadas, foi observada uma boa taxa de multiplicação durante o verão, sendo necessária a adição somente de 1,0 mg L-1 de BAP ao meio de cultura, concomitante a isto não houve oxidação nesta estação. A altura dos explantes somente apresentou resultados significativos no inverno. Tanto no outono, como no inverno e no verão houve formação de calo na base dos explantes, sendo que no verão houve também o aumento no número de brotações por explante. A oxidação se deu de forma mais intensa na primavera (100% de oxidação). Para a contaminação de explantes não houve resultados significativos para nenhuma estação, sendo, portanto, independente das condições sazonais. A extração do óleo essencial foi realizada em cada estação do ano utilizando um aparelho de Clevenger. O maior rendimento do óleo essencial foi obtido no inverno, com média 1,37% de óleo, sendo este o momento em que as plantas matrizes se encontravam plenamente floridas. No entanto, as condições normais de florescimento da citronela na região sul são durante a primavera e início do verão. Na primavera e verão o rendimento do óleo essencial foi de 0,77 e 0,76% respectivamente. No outono foi constatado o menor rendimento, com 0,57% de óleo essencial

Poucas espécies de Eucalyptus apresentam aptidão ao cultivo em regiões de baixas temperaturas e geadas frequentes, limitando seu cultivo em algumas regiões do Brasil. Genótipos de Eucalyptus globulus podem representar opções futuras para plantios florestais, tendo em vista o seu ótimo desempenho silvicultural nessas condições. No entanto, informações quanto à obtenção de mudas clonais são escassas, e se focarmos as espécies recomendadas para plantio em condições subtropicais, tal carência é ainda maior, principalmente em termos endógenos e exógenos de enraizamento de propágulos. Esta espécie se destaca também, pelo grande interesse no setor florestal, sobretudo em relação às características de sua madeira para obtenção de celulose, porém, é considerada recalcitrante ao enraizamento de estacas, especialmente quando envolve material adulto, dificultando o aproveitamento dos benefícios da clonagem. Por essa razão, o presente trabalho teve como objetivo principal avaliar a técnica de micropropagação de clones de E. globulus, visando a reversão à juvenilidade, aumentando os índices de enraizamento e, consequentemente a produção de mudas clonais. Para tanto, os clones (USP 01, USP 24 e USP 33) foram estudados e apresentaram estabelecimento satisfatório, onde avaliou-se: (a) a multiplicação in vitro, testando-se três meios de cultura (WPM, JADS e MS) suplementados com concentrações do regulador de crescimento BAP (0; 0,50 e 1,0 mg L-1) combinado com ANA (0; 0,05 e 0,10 mg L-1); (b) o alongamento in vitro, testando-se o meio de cultura WPM suplementado com concentrações de BAP (0; 0,05 e 0,10 mg L-1) combinadas com ANA (0; 0,1; 0,2 e 0,3 mg L-1); (c) e o enraizamento in vitro, em que brotações alongadas in vitro dos clones foram coletadas e transferidas para meio de cultura WPM, suplementado com 0 e 0,05 mg L-1 de BAP combinado com 0; 0,1; 0,5 e 1,0 mg L-1 de AIB. Os resultados revelaram comportamentos diferenciados dos clones quanto à multiplicação e alongamento in vitro. As taxas de multiplicação in vitro dos clones variaram ao longo dos 10 subcultivos, com uma estabilização a partir do 6º subcultivo para todos os clones avaliados, sendo a maior taxa de multiplicação (número médio de 13 gemas) na concentração de 0,7 mg L-1 de BAP e 0,05 mg L-1 de ANA no meio de cultura WPM. O meio de cultura WPM avaliado no alongamento in vitro, contribuiu favoravelmente para o alongamento das brotações, assim como ANA na concentração de 0,3 mg L-1 combinando com 0,1 mg L-1 de BAP. Portanto, o protocolo desenvolvido mostrou-se eficiente para micropropagação via proliferação de gemas axilares dos clones avaliados, porém, em relação ao enraizamento constatou-se apenas uma raiz emitida na concentração de 0,5 mg L-1 de AIB no clone 01. De forma geral, em razão dos resultados observados, conclui-se que apesar de mostrar-se viável, a micropropagação não promoveu rejuvenescimento significativo dos clones de E. globulus, uma vez que não foi observado enraizamento expressivo das microestacas para as características avaliadas. / Few Eucalyptus species present adaptation for cultivation in regions with low temperatures and frequent frosts, limiting their cultivation in some regions of Brazil. E. globulus genotypes may to represent future options for forest plantations, in view of its optimal silvicultural performance in these conditions. However, information about obtaining clonal seedlings are scarce, and considering the species recommended for planting in subtropical conditions, this lack of information is even greater, mainly when considering the endogenous and exogenous factors for the rooting. This species is distinguished by great interest in forestry, particularly in relation to characteristics of its wood to obtain cellulose, however, it is considered recalcitrant to rooting, especially when it involves adult material, making it difficult to utilize the cloning benefits. Therefore, this study aimed to evaluate the micropropagation technique of E. globulus clones, in order to revert to juvenility, increasing the rate of rooting and consequently the clonal production. For this, the clones (USP 01, USP 24 and USP 33) were studied and showed satisfactory in vitro culture establishment, where it was evaluated: (a) the in vitro multiplication, testing three culture media (WPM, JADS and MS) supplemented with concentrations plant growth regulator BAP (0; 0,50 and 1,0 mg L-1) combined with NAA (0; 0,05 and 0,10 mg L-1); (b) the in vitro elongation, testing the WPM medium culture supplemented with BAP concentrations (0; 0,05 and 0,10 mg L-1) combined with NAA (0; 0,1; 0,2 and 0,3 mg L-1); and (c) in vitro rooting, where elongated shoots in vitro of the clones were collected and transferred to WPM, supplemented with 0 and 0,05 mg L-1 BAP combined with 0; 0,1, 0,5 and 1,0 mg L-1 IBA. The results showed different behaviors of clones as in vitro multiplication and elongation. Rates of clones in vitro multiplication varied over the 10 subcultures, with the stabilization from the sixth subculture for all clones, the largest rate multiplication (average number of 13 buds) in the concentration of 0,7 mg L-1 BAP and 0,05 mg L-1 NAA in WPM. The culture media WPM evaluated in vitro elongation, contributed positively to the shoots elongation, with 0,3 mg L-1 NAA and 0,1 mg L-1 BAP. Therefore, with the micropropagation protocol using axillary buds proliferation of these clones showed be well developed , however, in relation to rooting, only one root was emitted at 0,5 mg L-1 IBA in clone 01. In general, it is possible concluded that despite viable, the micropropagation did not promote significant rejuvenation of E. globulus clones, due to the not expressive rooting for the microcutting evaluated.

No abstract available. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

An efficient micropropagation protocol for large scale production of hazelnut plants is required for consistent supply of elite germplasm to support the growing industry in Ontario. The main focus of current research was to develop a bioreactor based micropropagation protocol for hazelnut multiplication. An integrated approach was developed to increase the multiplication rate by optimizing the nutrient medium supplements and culture technology using a temporary immersion bioreactor system (TIS). As compared to conventional semi-solid and liquid based culture system, the Liquid LabTM temporary immersion bioreactor system (LIS) showed a significant enhancement in the number of shoots (3.3 shoots/explant), shoot height, leaf area and chlorophyll content in micropropagated plantlets. Antioxidant supplements such as ascorbic acid and melatonin along with iron (460 µM FeEDDHA) significantly increased the shoot proliferation (5.5 shoots/explant). However, a much higher shoot proliferation (10.9 shoots/explant) was observed with the addition of aspirin (10 µM) in the presence of BA (17.6 µM). Among several hazelnut cultivars, HF-16 had the highest multiplication rate followed by Geneva and Epsilon. Medium supplemented with 10 µM IBA was the best for rooting of microshoots of HF-16 and the plantlets acclimatized in the green house with 80% survival rate. / Ontario Ministry of Agriculture and Food and Rural Affairs

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.

Romulea is a genus with numerous attractive and endangered species with horticultural potential. This genus in the Iridaceae has its centre of diversity in the winter-rainfall zone of South Africa. This thesis uses ecophysiological and biotechnological techniques to investigate the physiology behind the propagation of some species in this genus. The ecophysiological techniques of soil sampling and analysis and germination physiology were used to determine the natural and ex vitro growth and development requirements of these plants, while biotechnological techniques are used to determine the in vitro growth and development requirements of these plants and to increase the rate of multiplication and development. Soil sampling and analysis revealed that R. monadelpha and R. sabulosa, two of the most attractive species in the genus, grow in nutrient poor 1:1 mixture of clay and sandy loam soil with an N:P:K ratio of 1.000:0.017:0.189 with abundant calcium. To investigate the physical properties of the seeds, imbibition rate, moisture content and viability of seeds were determined. The seed coat and micropylar regions were examined using scanning electron microscopy. To test for suitable stimuli for germination, the effect of temperature and light, cold and warm stratification, acid and sand paper scarification, plant growth promoting substances, deficiency of nitrogen, phosphorous and potassium, and different light spectra (phytochromes) on germination were examined. An initial germination experiment showed germination above 65% for R. diversiformis, R. leipoldtii, R. minutiflora and R. flava seeds placed at 15°C; while seeds of other species placed at 15°C all had germination percentages lower than 30%. More extensive germination experiments revealed that R. diversiformis and R. rosea seed germinate best at 10°C, R. flava seed germinates best when cold stratified (5°C) for 21 days and R. monadelpha germinates best at 15°C in the dark. Seeds of R. diversiformis, R. flava, R. leipoldtii, R. minutiflora, R. monadelpha and R. sabulosa seem to all exhibit non-deep endogenous morphophysiological dormancy while seeds of R. camerooniana and R. rosea appear to have deep endogenous morphophysiological dormancy. The suitability of various explant types and media supplementations for culture initiation was examined for various species of Romulea. Both embryos and seedling hypocotyls can be used for R. flava, R. leipoldtii and R. minutiflora in vitro shoot culture initiation. R. sabulosa shoot cultures can only be initiated by using embryos as explants, because of the lack of seed germination in this species. Shoot cultures of R. diversiformis, R. camerooniana and R. rosea could not be initiated due to the lack of an in vitro explant shooting response. Shoot cultures can be initiated on media supplemented with 2.3 to 23.2 M kinetin for all species that showed an in vitro response. The most suitable concentration depended on the species used. Some cultures appeared embryogenic, but this was shown not to be the case. A medium supplemented with 2.5 M mTR is most suitable for R. sabulosa shoot multiplication. BA caused vitrification of shoots in all the experiments in which it was included and is not a suitable cytokinin for the micropropagation of these species. The effect of various physical and chemical parameters on in vitro corm formation and ex vitro acclimatization and growth was examined. Low temperature significantly increased corm formation in R. minutiflora and R. sabulosa. A two step corm formation protocol involving placing corms at either 10 or 20°C for a few months and then transferring these cultures to 15°C should be used for R. sabulosa. When paclobutrazol and ABA were added to the medium on which R. minutiflora shoots were placed, the shoots developed corms at 25°C. This temperature totally inhibits corm formation when these growth retardants are not present. BA inhibited corm formation in R. leipoldtii. Corms can be commercialized as propagation units for winter-rainfall areas with minimum temperatures below 5°C during winter. Although an incident of in vitro flowering was observed during these experiments, these results could not be repeated. Although none of the corms or plantlets planted ex vitro in the greenhouse survived, a small viability and an ex vitro acclimatization experiment shows that the corms produced in vitro are viable. One embryo of the attractive R. sabulosa, produces 2.1 ± 0.7 SE shoots after 2 months; subsequently placing these shoots on a medium supplemented with 2.5 μM mTR for a further 2 months multiplies this value by 5.5 ± 1.3 SE. Each of these shoots can then be induced to produce a corm after 6 months. This means that 1 embryo can produce about 12 corms after 10 months or about 65 corms after 12 months (if shoots are subcultured to medium supplemented with 2.5 μM mTR for another 2 months). Embryo rescue can enable wider crosses within this genus. These results can be used for further horticultural development of species in this genus and their hybrids and variants. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.