Orientador: Carmen Silvia Fernandes Boaro / Banca: João Domingos Rodrigues / Banca: Giuseppina Pace P. Lima / Banca: Marcos Roberto Furlan / Banca: Antonio Natal Gonçalves / Resumo: Pertencente à família Meliaceae, a espécie Trichilia catigua A. Juss é conhecida popularmente como catigua, cataguá, argelim-rosa e mangalto-catingam. Sua casca apresenta propriedades adstringente, inseticida, purgativa, tônica, bactericida, antiinflamatória e antidepressiva. Com o objetivo de propagar a espécie T. catigua foram desenvolvidos experimentos testando o enraizamento de estacas e a micropropagação com explantes de matrizes e sementes. Os experimentos de enraizamento de estacas foram realizados na primavera 2004, verão 2004/2005, outono, inverno e primavera 2005 e primavera 2006. Em todos os experimentos, estacas com aproximadamente 15 cm de comprimento foram coletadas de árvores adultas e preparadas da parte apical e mediana dos ramos. A seguir, foram submetidas aos reguladores vegetais IBA (ácido indolbutírico), NAA (ácido naftalenoacético) e IAA (ácido 3-indolacético), variando as dosagens. Para a avaliação dos experimentos determinou-se a percentagem de estacas enraizadas, não enraizadas e mortas e quando enraizadas, seu comprimento e diâmetro. No experimento primavera de 2004 foram testadas as concentrações de 1000 e 2000 mg L-1 dos reguladores vegetais IBA, NAA e IAA. As avaliações aos 90 dias após sua instalação revelaram maiores percentagens de enraizamento e iguais a 33,33, 25,00, 22,91 e 23,43 % para estacas submetidas a IBA 1000, 2000 mg L-1 e NAA 1000 e 2000 mg L-1, respectivamente. No verão 2004/2005, outono, inverno e primavera 2005 os experimentos foram conduzidos com as concentrações dos reguladores IBA, NAA e IAA iguais a 1000, 2000 e 3000 mg L-1 e as avaliações foram realizadas após 120 dias. Não houve enraizamento no outono e inverno. A análise conjunta dos resultados obtidos na primavera e no verão mostrou percentagem de enraizamento superior na primavera. A maior percentagem de enraizamento, igual a 19,17%... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Trichilia catigua A. Juss from the Meliaceae family is popularly known as catigua, cataguá, argelim-rose and mangalto-catingam. Its bark has astringent, insecticide, purgativa, tonic, bactericide, anti-inflammatory and antidepressant properties. With the aim of propagate T. catigua, experiments of rooting with stem cuttings and of micropropagation with explants of trees and seeds were carried out. In all the rooting experiments the stem cuttings with approximately 15 cm of length were collected from adult trees and prepared from the apical and intermediate parts. The cuttings were immersed in the vegetable regulators IBA (Indole-3- butyric acid), ANA (Naphthalene acetic acid) and IAA (Indole-3 acetic acid). The rooted stem cutting and not rooted stem cutting percentage and, when rooted, the length and diameter of roots, were evaluated. In the experiment spring 2004 the concentrations of 1000 and 2000 mg L-1 of IBA, ANA and IAA were tested, with evaluations 90 days after installation. The highest rooting percentage were 33,33, 25,00, 22,91 and 23,43% for IBA 1000, 2000 mg L-1 and ANA 1000 and 2000 mg L-1, respectively. In the summer of 2004/2005, autumn, winter and spring of 2005 IBA, ANA and AIA, with concentration of 1000, 2000 and 3000 mg L-1, were tested. The evaluation was carried out at 120 days. No rooting was observed in autumn and winter. The analysis of data from summer and spring showed higher rooting percentage in spring. The highest rooting percentage was obtained with IBA 3000 mg L-1 (19,17%). In the spring 2006 IBA (1000, 2000, 3000, 4000 and 5000 mg L-1) and ANA (1000, 2000, 3000 mg L-1) were tested. The highest rooting percentage (41,67%) was obtained with IBA 5000 mg L-1. In the in vitro cultivation, explantes obtained from trees were submitted to asepsis treatments with HgCl2, CaOCl2 and NaOCl and inoculated in Murashige & Skoog culture medium (MS) with 25%... (Complete abstract click electronic access below) / Doutor

O experimento foi conduzido com o objetivo de avaliar a influência da interação entre o nitrato de amônio e nitrato de potássio sobre as respostas morfogênicas de embriões somáticos de pupunheiras in vitro a fim de otimizar o protocolo de micropropagação da espécie. As concentrações utilizadas foram 0, 825, 1650, 2475 e 3300 mg L-1 de nitrato e amônio e 0, 950 , 1900, 2850 e 3800 mg L-1 de nitrato de potássio, combinadas entre si, perfazendo um total de 25 tratamentos. Os tratamentos foram preparados a partir da solução completa de Murashige e Skoog, devidamente modificado para as proporções desses íons no suprimento de nitrogênio. Utilizaram-se duzentos e cinqüenta embriões somáticos com características morfológicas homogêneas, isentos de raízes e folhas, obtidos a partir de microplantas mantidas em sala de crescimento com temperatura e luminosidade controladas. Foram aferidos dados do comprimento da parte aérea e da raiz, o número de raízes, brotações e folhas, a porcentagem de ramificação da raiz, a porcentagem de raízes finas, medianas e grossas, em quatro períodos de cultura, a cada 60 dias, totalizando 240 dias de cultivo. Ao final desse período, avaliou-se também o teor de proteínas totais solúveis, o teor de clorofila pelo índice SPAD e os teores de macro e micronutrientes nas microplantas. Utilizou-se delineamento estatístico inteiramente casualizado e os dados foram submetidos ao teste de Bartlett a 5% e análise de variância (ANOVA) a 1% e 5% de probabilidade de erro ou foi elaborada uma matriz de correlação de Pearson a 1% e 5% de probabilidade de erro, conforme o caso. Os resultados permitiram inferir que aos 240 dias de cultivo as microplantas passam a investir mais em formação de parte aérea e aumentam a porcentagem de raízes finas, e funcionais. Os tratamentos que melhor favoreceram a formação de proteínas foram aqueles com concentração de 2475 mg L-1 de NH4NO3 ou com 2850 mg L-1 de KNO3. Os maiores índices SPAD ocorreram nos tratamentos com até 1650 mg L-1 de NH4NO3 combinado com até 1900 mg L-1 de KNO3. Diferentes combinações dos sais de NH4NO3 e KNO3 podem favorecer a absorção de cada nutriente. Conclui-se que os resultados obtidos no trabalho podem contribuir com a melhoria do protocolo de micropropagação de B. gasipaes, na medida em que permitem estabelecer o melhor tratamento para maximização de uma resposta específica desejada para cada momento do processo de cultivo dos embriões somáticos de pupunheiras, como enraizamento, crescimento da parte aérea, formação de proteínas e formação de brotações, facilitando dessa forma a otimização da produção de microplantas com características desejáveis e, conseqüentemente, sua aclimatização e desenvolvimento ex vitro. / This study aimed to evaluate the influence of the interaction between ammonium nitrate and potassium nitrate on the morphogenetic responses of peach palm somatic embryos in vitro cultivated, to optimize the micropropagation protocol for this species. The concentrations used were 0, 825, 1650, 2475 and 3300 mg L-1 of ammonium nitrate and 0, 950 , 1900, 2850 and 3800 mg L-1 of potassium nitrate, in all possible associations, totalizing 25 treatments. The treatments were prepared with the complete solution of Murashige and Skoog, with modified proportions of these ions in relation to the nitrogen supply. Two hundred and fifty somatic embryos with homogeneous morphological characteristics, without roots and leaves, obtained from microplants from a controlled temperature and luminosity room, were used in the experiment. Every 60 days, during 240 days, the length of shoot and root, the number of roots, propagules and leaves and the root architecture were measured. At the end of 240 days of cultivation, it was also analyzed the total soluble proteins, the foliar chlorophyll by a chlorophyll meter equipment (SPAD-502) and the macronutrients and micronutrients concentrations in the microplants. The experiment was conducted in a randomized design. The data were subjected to Bartlett´s test at 5% and variance analysis (ANOVA) at 1% and 5% of probability of error, or it was made a correlation matrix at 1% and 5% of probability of error, according to each analysis. The results showed that at 240 days of cultivation the microplants spent more energy building the shoot part and raised the percentage of thin, functional root. The best treatments for proteins formation were those with 2475 mg L-1 of NH4NO3 or with 2850 mg L-1 of KNO3. The highest SPAD index occurred in the treatments with at most 1650 mg L-1 of NH4NO3 associated with at most 1900 mg L-1 of KNO3. Different associations of NH4NO3 e KNO3 may favor the absorption of each nutrient. We conclude that the results obtained may contribute to the optimization of the B. gasipaes micropropagation protocol, as it is possible to establish the best treatment for maximization of a specific answer for each moment of the somatic embryos cultivations process, such as rooting, shoot growth, propagules and protein formation, and thus increase the optimization of microplants production with wanted characteristics and hence its acclimatization and ex vitro development.

O cultivo in vitro de plantas possibilita o controle de fatores ambientais e nutricionais, facilitando o estudo da interação planta-bactéria e a interpretação de eventuais alterações morfofisiológicas nas microplantas, decorrentes dessa interação. Entretanto, a presença de bactérias endofíticas na micropropagação é quase sempre caracterizada como contaminação microbiana prejudicial, sendo prontamente eliminada com o uso de quimioterápicos. Essa abordagem desconsidera os efeitos benéficos que bactérias endofíticas podem trazer ao desenvolvimento vegetal, aniquilando a possibilidade de se explorar esse potencial no ambiente in vitro. Em geral, devido a sua baixa culturabilidade, as comunidades bacterianas endofíticas requerem o uso de técnicas independentes de cultivo para seu estudo. Neste contexto, o presente trabalho teve como objetivo principal comprovar a presença de uma comunidade bacteriana endofítica em microplantas de abacaxizeiro (Ananas comosus (L.) Merrill) cv. IAC Gomo-de-mel consideradas axênicas e os efeitos dessa comunidade na morfofisiologia vegetal após antibioticoterapia. Para tanto, técnicas de PCR-DGGE, PCR-ARISA, clonagem, sequenciamento, análises estruturais e ultraestruturais foram utilizadas. Os resultados evidenciaram que existe uma comunidade bacteriana endofítica composta por membros de Alfa, Beta, gama -proteobactérias, actinobactérias e cianobactérias, que coloniza as microplantas. Os resultados convergentes de PCR-DGGE e PCR-ARISA mostraram que essa comunidade apresentou alteração na sua estrutura e diversidade de acordo com seu nicho de colonização e com o período de cultivo da planta hospedeira, sem renovação de meio de cultura. A ação da antibioticoterapia influenciou a estrutura da comunidade bacteriana, promovendo impactos diferenciados em cada grupo bacteriano avaliado. A antibioticoterapia não promoveu a total eliminação das bactérias endofíticas. Contudo, os tratamentos ocasionaram alterações na comunidade bacteriana, resultando na redução do crescimento de raízes e outras alterações histológicas no sistema radicular das microplantas. As análises ultraestruturais comprovaram a presença de bactérias endofíticas colonizando intracelularmente o mesofilo foliar e o córtex de raízes, mesmo após a antibioticoterapia. Dessa forma, conclui-se que em microplantas de abacaxizeiro assintomáticas existe uma comunidade bacteriana endofítica intracelular, rompendo o principal paradigma da cultura de tecidos vegetais, no qual microplantas são obrigatoriamente axênicas. / The in vitro culture of plants by the control of environmental and nutritional factors, allows the study of the plant-bacteria interaction and the interpretation of possible morphophysiological changes in the microplants, from such interaction. However, the presence of endophytic bacteria in micropropagation is often characterized as harmful microbial contamination, which is promptly eliminated by the use of chemotherapeutics. This approach does not take into account the beneficial effects that endophytic bacteria can bring to the plant development, and annihilates the possibility of exploiting this potential in the in vitro environment. In general, due to its low culturability, endophytic bacterial communities require the use of culture-independent techniques for their study. In this context, this work aimed to prove the presence of bacterial endophytes in pineapple (Ananas comosus (L.) Merrill) cv. IAC Gomo-de-mel microplants considered axenics, and the effects of these in plant morphophysiology after antibiotictherapy. For this, PCR-DGGE, ARISA-PCR, cloning, sequencing, structural and ultrastructural analysis were used. The results showed that there is an endophytic bacterial community, composed of members of , , -proteobacteria, actinobacteria and cyanobacteria, which colonizes the microplants. The converging results of PCR-DGGE and ARISA-PCR showed that this community changed in its structure and diversity according to its niche of colonization and the period of cultivation of the host plant, without renewal of culture medium. The action of antibiotics influenced the bacterial community structure, promoting differential impacts on each bacterial group evaluated. Antibiotictherapy did not promote the total elimination of endophytic bacteria. However, the treatments caused changes in bacterial community, resulting in reduced growth of roots and other histological changes in the root system of the microplants. The ultrastructural analysis confirmed the presence of endophytic bacteria colonizing the intracellularly the leaf mesophyll and the cortex of roots, even after antibiotictherapy. Thus, it is concluded that in asymptomatic pineapple microplants there is an intracellular endophytic bacterial community, breaking the main paradigm of plant tissue culture, in which axenic microplants are mandatory.

O cultivo in vitro de plantas possibilita o controle de fatores ambientais e nutricionais, facilitando o estudo da interação planta-bactéria e a interpretação de eventuais alterações morfofisiológicas nas microplantas, decorrentes dessa interação. Entretanto, a presença de bactérias endofíticas na micropropagação é quase sempre caracterizada como contaminação microbiana prejudicial, sendo prontamente eliminada com o uso de quimioterápicos. Essa abordagem desconsidera os efeitos benéficos que bactérias endofíticas podem trazer ao desenvolvimento vegetal, aniquilando a possibilidade de se explorar esse potencial no ambiente in vitro. Em geral, devido a sua baixa culturabilidade, as comunidades bacterianas endofíticas requerem o uso de técnicas independentes de cultivo para seu estudo. Neste contexto, o presente trabalho teve como objetivo principal comprovar a presença de uma comunidade bacteriana endofítica em microplantas de abacaxizeiro (Ananas comosus (L.) Merrill) cv. IAC Gomo-de-mel consideradas axênicas e os efeitos dessa comunidade na morfofisiologia vegetal após antibioticoterapia. Para tanto, técnicas de PCR-DGGE, PCR-ARISA, clonagem, sequenciamento, análises estruturais e ultraestruturais foram utilizadas. Os resultados evidenciaram que existe uma comunidade bacteriana endofítica composta por membros de Alfa, Beta, gama -proteobactérias, actinobactérias e cianobactérias, que coloniza as microplantas. Os resultados convergentes de PCR-DGGE e PCR-ARISA mostraram que essa comunidade apresentou alteração na sua estrutura e diversidade de acordo com seu nicho de colonização e com o período de cultivo da planta hospedeira, sem renovação de meio de cultura. A ação da antibioticoterapia influenciou a estrutura da comunidade bacteriana, promovendo impactos diferenciados em cada grupo bacteriano avaliado. A antibioticoterapia não promoveu a total eliminação das bactérias endofíticas. Contudo, os tratamentos ocasionaram alterações na comunidade bacteriana, resultando na redução do crescimento de raízes e outras alterações histológicas no sistema radicular das microplantas. As análises ultraestruturais comprovaram a presença de bactérias endofíticas colonizando intracelularmente o mesofilo foliar e o córtex de raízes, mesmo após a antibioticoterapia. Dessa forma, conclui-se que em microplantas de abacaxizeiro assintomáticas existe uma comunidade bacteriana endofítica intracelular, rompendo o principal paradigma da cultura de tecidos vegetais, no qual microplantas são obrigatoriamente axênicas. / The in vitro culture of plants by the control of environmental and nutritional factors, allows the study of the plant-bacteria interaction and the interpretation of possible morphophysiological changes in the microplants, from such interaction. However, the presence of endophytic bacteria in micropropagation is often characterized as harmful microbial contamination, which is promptly eliminated by the use of chemotherapeutics. This approach does not take into account the beneficial effects that endophytic bacteria can bring to the plant development, and annihilates the possibility of exploiting this potential in the in vitro environment. In general, due to its low culturability, endophytic bacterial communities require the use of culture-independent techniques for their study. In this context, this work aimed to prove the presence of bacterial endophytes in pineapple (Ananas comosus (L.) Merrill) cv. IAC Gomo-de-mel microplants considered axenics, and the effects of these in plant morphophysiology after antibiotictherapy. For this, PCR-DGGE, ARISA-PCR, cloning, sequencing, structural and ultrastructural analysis were used. The results showed that there is an endophytic bacterial community, composed of members of , , -proteobacteria, actinobacteria and cyanobacteria, which colonizes the microplants. The converging results of PCR-DGGE and ARISA-PCR showed that this community changed in its structure and diversity according to its niche of colonization and the period of cultivation of the host plant, without renewal of culture medium. The action of antibiotics influenced the bacterial community structure, promoting differential impacts on each bacterial group evaluated. Antibiotictherapy did not promote the total elimination of endophytic bacteria. However, the treatments caused changes in bacterial community, resulting in reduced growth of roots and other histological changes in the root system of the microplants. The ultrastructural analysis confirmed the presence of endophytic bacteria colonizing the intracellularly the leaf mesophyll and the cortex of roots, even after antibiotictherapy. Thus, it is concluded that in asymptomatic pineapple microplants there is an intracellular endophytic bacterial community, breaking the main paradigm of plant tissue culture, in which axenic microplants are mandatory.

Dentre as poucas espécies de Eucalyptus que apresentam aptidão ao cultivo em regiões de baixas temperaturas e a geadas frequentes, destacam-se genótipos de Eucalyptus benthamii que representam opções para futuros plantios florestais em diferentes regiões brasileiras. Porém, existem poucas informações quanto a obtenção de mudas clonais, e se focarmos as espécies aconselhadas para o plantio em condições subtropicais, tal carência é ainda maior, principalmente ao considerar os fatores endógenos e exógenos para o enraizamento adventício. Baseado no exposto, o presente trabalho teve como objetivo geral avaliar aspectos morfofisiológicos da clonagem de genótipos de Eucalyptus benthamii por meio das técnicas de miniestaquia e micropropagação. Para tanto, o trabalho foi dividido em quatro estudos básicos. O primeiro estudo (Capítulo 2) baseou-se na avaliação da morfofisiologia de um minijardim clonal em relação a diferentes concentrações de Zn e B ao longo de sucessivas coletas de brotações. O segundo estudo (Capítulo 3) foi baseado em avaliar o enraizamento de miniestacas quanto a diferentes concentrações de Zn e B e aplicação de AIB ao longo de sucessivas coletas de brotações. O terceiro estudo (Capítulo 4) baseou-se em avaliar a dinâmica de enraizamento de miniestacas quanto a diferentes concentrações de AIB, tempo ótimo de permanência de miniestacas enraizadas em casa de vegetação e a origem da conexão vascular da raiz emitida. Por fim, o quarto estudo (Capítulo 5) foi baseado no desenvolvimento e estabelecimento de protocolo para a micropropagação visando a produção de microcepas para formar um microjardim clonal. Em termos gerais, a sobrevivência das minicepas, produção de miniestacas por metro quadrado ao ano e os teores foliares de macro e micronutrientes variaram significativamente em relação as concentrações de Zn e B, apresentando diferentes respostas ao longo das coletas de brotações no minijardim clonal. Os teores foliares de carboidratos solúveis não estruturais variaram significativamente de acordo com as coletas de brotações e solução nutritiva, sendo que o aumento das concentrações de Zn e B na solução nutritiva induziu a redução dos teores de carboidratos solúveis não estruturais. A porcentagem de enraizamento dos materiais clonais foi baixa, sendo considerados de difícil propagação pela miniestaquia. A presença de Zn e B na solução nutritiva (concentrações variando de 1,0 a 2,0 mg L-1) associadas a presença de AIB induziram os maiores índices de enraizamento. A aplicação de AIB na concentração de 2.000 mg L-1 favoreceu a indução de raízes, e o intervalo de 35 a 42 dias foi o mais indicado para a permanência das miniestacas enraizadas em casa de vegetação. De acordo com as análises histológicas da rizogênese foi verificado que a raiz adventícia apresentou conexão direta com o câmbio vascular. A multiplicação in vitro de gemas axilares dependeu do clone, meio de cultura e concentração de regulador de crescimento e, o alongamento de brotações dependeu do clone e regulador de crescimento. O protocolo de micropropagação foi eficiente para a produção de microplantas de Eucalyptus benthamii as quais podem ser usadas para a formação de um microjardim clonal. / Few Eucalyptus species present adaptation for cultivation in regions subject to low temperatures and frequent frosts, and Eucalyptus benthamii genotypes may represent options for future forest plantations in different regions of Brazil, in view of its excellent silvicultural performance in these conditions. However, there is little information on obtaining clones, 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 adventitious rooting. Based on these information, the present work was aimed the conducting of studies on morphophysiological aspects during the cloning of Eucalyptus benthamii through of the mini-cuttings and micropropagation techniques. Therefore, the work was divided into four basic studies. The first study (Chapter 2) was based in evaluate the morphophysiology of a clonal mini-garden regarding to Zn and B concentrations during successive shoot collections. The second study (Chapter 3) was based in evaluate the induction of adventitious rooting in mini-cuttings regarding to genotype, Zn and B concentrations, shoot collections and IBA application. The third study (Chapter 4) was based in evaluate the adventitious rooting percentage of selected genotypes regarding the IBA concentration, optimal time of permanence of rooted mini-cuttings in a greenhouse and the origin of the vascular connection. Finally, the fourth study (Chapter 5) was based in develop a method for cloning of selected genotypes through micropropagation technique for the formation of a clonal micro-garden. In overall terms, mini-stumps survival, mini-cuttings production per square meter per year and foliar content of macro and micronutrients varied significantly in relation to treatments, presenting different responses according to shoots collection of the clonal mini-garden. The content of soluble carbohydrates non-structural of leaves varied regarding the shoots collection and nutrient solution. The increasing of the Zn and B concentrations in the nutrient solution induced reduction of the total content of soluble carbohydrates non-structural of leaves. The adventitious rooting percentage was low, and the genotypes were considered difficult to propagation by mini-cuttings technique. The ministumps fertigated with nutrient solutions containing Zn and B (concentrations of 1.0 at 2.0 mg L-1) associated with the IBA application presented the greater adventitious rooting percentage. The IBA application in the concentration of 2,000 mg L-1 resulted in the greater speed of rooting and rooting percentage, and the interval of 35 to 42 days was the most suitable for the permanence of mini-cuttings rooted in a greenhouse. According to the histological analysis of rhizogenesis was verified that the adventitious root presented direct connection to the vascular cambium. The in vitro multiplication of axillary buds depends of the genetic material, culture medium and concentration of plant growth regulator and, the shoots elongation depends of the genetic material and plant growth regulator. The micropropagation protocol was efficient for the microplants production of Eucalyptus benthamii and can be used to form a clonal microgarden.

Thesis submitted in fulfilment of the requirements for the degree Master of Technology: Horticultural Sciences in the Faculty of Applied Sciences at the CAPE PENINSULA UNIVERSITY OF TECHNOLOGY Supervisor: Dr L Kambizi Co-supervisor: Dr NP Makunga Cape Town December 2013 / Agathosma betulina (Berg.) Pillans, previously known as Barosma betulina, is a member of the Rutaceae family, and indigenous to the fynbos botanical biome of the Western Cape of South Africa. It is commonly known as buchu. Extracts as well as powdered leaves have traditionally been used for the treatment of various ailments. The increase in the international demand for A. betulina for health as well as food and beverage benefits, have raised concerns over exploitation of wild populations and the lack of horticultural information necessitates this study to evaluate the propagation of this economical important species. The main objective of this study was to establish a simple and highly productive micropropagation protocol for A. betulina through experimenting with nodal explants. Testing of the effect of various treatments (physical scarification, chemical scarification, GA, stratification, smoke and combinations thereof) on the in vitro germination of A. betulina seeds was done to elucidate the factors which control seed germination. The study revealed that the physical scarification and smoke-induced germination had a significant effect on germination percentages. In terms of germination rate, the radical generally started to appear after approximately 10 days in the physical scarification with smoke treatment. Initial decontamination methods with the exposure of various concentrations of NaOCl gave fatal results, however 1.5% NaOCl had more phenolic reactions rather than fungal or bacterial contamination. Interestingly, contamination rates of explants were influenced by the stage of maturity of the explant material. This plant material was used to test different strengths of regeneration media, to ensure that the explants receive ample nutrients. Results made exhibited that ½ MS was the best strength for growing A. betulina nodal explants. Compared comparison between in vitro derived explants and ex vitro collected explants showed that the ex vitro derived explants had significant results, but the explants lost vigour soon after the initial exponential growth leading to the explants dying off. Furthermore, ex vitro decontaminated plant material was not economically viable to continue with. Seedlings derived from germinated seeds appeared to be the preferred method of propagation as this spent the least time in culture and produced a stable plant with an established root system, which is essential during the hardening off process after in vitro growth. When exposing nodal explants to phytohormone 2,4-D it responds best to dosages 0.5mg Lˉ¹ and 1mg Lˉ¹. Phytohormone BA was very effective in producing soft friable callus. The best results were shown when 0.5mg Lˉ¹ BA was applied to ½ MS media. For both shoot length and multiple shoot production, a combination of phytohormones BA-NAA (1: 0.5mgLˉ¹) had the most significant results. Interestingly, a higher phytohormone concentration of NAA is necessary to develop multiple adventitious roots. The effect of 3mg Lˉ¹ was significant in that it resulted in multiple adventitious roots, but fewer calli was observed in this treatment. Micropropagation becomes valuable as little attention between subcultures is needed; making it less labour intensive compared to conventional nursery propagation systems where weeding watering and spraying of plants are labour intensive. In the traditional world of medicine, more so in Southern Africa, extracts are prepared by adding boiling water to the plant material; however commercial ethanol is used as an extractant. Establishment of the essential oil quality of the in vitro cultures post exposure to various treatments was done. Analysis of essential oils from A. betulina resulted in the identification of twenty one compounds. The results showed qualitative as well as quantitative differences amongst the samples used in the study. The highest relative concentration of limonene was observed in the callus of nodal explants after it was exposed to 0.5mg lˉ¹ NAA. No pulegone was found in this treatment making it ideal for limonene production. This suggests that liquid culture with the same treatment may produce more calli making it ideal for the production of limonene.

Research into the micropropagation of black wattle in South Africa is important for two reasons. Firstly micropropagation technology allows breeders to select and propagate mature tissue, which in turn allows them to better capture selected traits. Secondly, tissue culture may control the highly invasive nature of black wattle. If triploid black wattle can be developed, foresters will then have to rely on clonal propagation to supply material for their growing operations. This research was part of the Institute for Commercial Forestry’s Acacia mearnsii vegetative propagation programme. The main focus of this research was to overcome various problems associated with direct organogenesis of ex vitro material. The shoot apex region was used as the explant in all studies because this region is thought to harbour relatively few internal microbial contaminants and is of sufficient size to withstand stresses associated with micropropagation. The initial research was focussed on the screening of sterilants, searching for a viable alternative to mercuric chloride. Surface sterilisation is integral to any micropropagation technique. This process should do the least amount of plant damage, whilst reducing microbial contamination to an acceptable level. Explants were cultured on Murashige and Skoog (MS) medium supplemented with 2.0 mg L-1 BA and monitored for signs of contamination and shooting. Household bleach proved an excellent alternative to mercuric chloride because it did significantly less damage to the explants than mercuric chloride and is handled easily. There was no significant effect of sterilant exposure time on explant decontamination levels, whilst the shortest exposure time resulted in significantly higher levels of shoot development than the other two times tested. The results of this initial research was developed into a protocol and utilised in subsequent investigations. Due to a considerable variation in the success of the developed surface sterilisation protocol according to different times of the year, a further investigation into the effects of season and mother plant material on shoot apex culture of Acacia mearnsii was undertaken. The success of any tissue culture technique depends on a large array of ex vitro and in vitro variables. The objective of this research was to determine the ii effect of two ex vitro variables, season and mother plant, on shoot apex culture of Acacia mearnsii. Explants from individual mother plants were cultured on MS medium supplemented with 2.0 mg L-1 BA during four separate seasons and monitored for signs of contamination and shooting. Spring was found to be the best harvesting season because spring explants showed significantly higher decontaminated explant levels and shooting levels than explants harvested in the other three seasons. The effect of mother plant selection on the performance of Acacia mearnsii explants during shoot apex culture was also found to be significant, especially with regard to shooting levels. Finally factors influencing shoot elongation of A. mearnsii during shoot apex culture were investigated. In the past, induction of shoot elongation during micropropagation of A. mearnsii was attained through the addition of plant growth regulators and other supplements to the basal culture medium. However, some micropropagation methods in other species have utilised red light as a means of promoting shoot elongation. The objective of this study was to test the effects of an alternative basal medium, red light and differing concentrations of chemical additions to the culture medium on shoot elongation of Acacia mearnsii during shoot apex culture. Four independent experiments were undertaken comparing: shoot elongation on Woody Plant Medium (WPM) to the MS basal medium control; shoot elongation under a red cellophane box compared to control culture light conditions; shoot elongation on media supplemented with various concentrations of GA3 to the un-supplemented control and shoot elongation on media supplemented with combinations of BA and IBA compared to a control. Although no significant effects were observed, many trends were noted. The results indicated that there was no advantage to using WPM instead of MS medium when attempting to elongate shoots, rejuvenated through shoot apex culture of A. mearnsii, whilst the effect of GA3 showed a negative trend. The effects of red light and some BA and IBA combinations showed positive trends on the elongation of initiated shoots. This research successfully addressed some of the problems associated with micropropagation of A. mearnsii. Shoot apex culture shows promise and further research into this technique should be considered. A viable surface sterilant alternative to mercuric chloride was successfully identified. This alternative is not only iii safer to use but shows a large reduction in phytotoxic effects. The effects of season and mother plant on shoot apex culture was successfully investigated, resulting in a better understanding of mother plant influences on tissue culture as well as the identification of an optimum season for explant selection. Finally two possible shoot elongation promoters were identified for further research and a more affordable alternative to red light sources and screens was identified. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.

Dentre as poucas espécies de Eucalyptus que apresentam aptidão ao cultivo em regiões de baixas temperaturas e a geadas frequentes, destacam-se genótipos de Eucalyptus benthamii que representam opções para futuros plantios florestais em diferentes regiões brasileiras. Porém, existem poucas informações quanto a obtenção de mudas clonais, e se focarmos as espécies aconselhadas para o plantio em condições subtropicais, tal carência é ainda maior, principalmente ao considerar os fatores endógenos e exógenos para o enraizamento adventício. Baseado no exposto, o presente trabalho teve como objetivo geral avaliar aspectos morfofisiológicos da clonagem de genótipos de Eucalyptus benthamii por meio das técnicas de miniestaquia e micropropagação. Para tanto, o trabalho foi dividido em quatro estudos básicos. O primeiro estudo (Capítulo 2) baseou-se na avaliação da morfofisiologia de um minijardim clonal em relação a diferentes concentrações de Zn e B ao longo de sucessivas coletas de brotações. O segundo estudo (Capítulo 3) foi baseado em avaliar o enraizamento de miniestacas quanto a diferentes concentrações de Zn e B e aplicação de AIB ao longo de sucessivas coletas de brotações. O terceiro estudo (Capítulo 4) baseou-se em avaliar a dinâmica de enraizamento de miniestacas quanto a diferentes concentrações de AIB, tempo ótimo de permanência de miniestacas enraizadas em casa de vegetação e a origem da conexão vascular da raiz emitida. Por fim, o quarto estudo (Capítulo 5) foi baseado no desenvolvimento e estabelecimento de protocolo para a micropropagação visando a produção de microcepas para formar um microjardim clonal. Em termos gerais, a sobrevivência das minicepas, produção de miniestacas por metro quadrado ao ano e os teores foliares de macro e micronutrientes variaram significativamente em relação as concentrações de Zn e B, apresentando diferentes respostas ao longo das coletas de brotações no minijardim clonal. Os teores foliares de carboidratos solúveis não estruturais variaram significativamente de acordo com as coletas de brotações e solução nutritiva, sendo que o aumento das concentrações de Zn e B na solução nutritiva induziu a redução dos teores de carboidratos solúveis não estruturais. A porcentagem de enraizamento dos materiais clonais foi baixa, sendo considerados de difícil propagação pela miniestaquia. A presença de Zn e B na solução nutritiva (concentrações variando de 1,0 a 2,0 mg L-1) associadas a presença de AIB induziram os maiores índices de enraizamento. A aplicação de AIB na concentração de 2.000 mg L-1 favoreceu a indução de raízes, e o intervalo de 35 a 42 dias foi o mais indicado para a permanência das miniestacas enraizadas em casa de vegetação. De acordo com as análises histológicas da rizogênese foi verificado que a raiz adventícia apresentou conexão direta com o câmbio vascular. A multiplicação in vitro de gemas axilares dependeu do clone, meio de cultura e concentração de regulador de crescimento e, o alongamento de brotações dependeu do clone e regulador de crescimento. O protocolo de micropropagação foi eficiente para a produção de microplantas de Eucalyptus benthamii as quais podem ser usadas para a formação de um microjardim clonal. / Few Eucalyptus species present adaptation for cultivation in regions subject to low temperatures and frequent frosts, and Eucalyptus benthamii genotypes may represent options for future forest plantations in different regions of Brazil, in view of its excellent silvicultural performance in these conditions. However, there is little information on obtaining clones, 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 adventitious rooting. Based on these information, the present work was aimed the conducting of studies on morphophysiological aspects during the cloning of Eucalyptus benthamii through of the mini-cuttings and micropropagation techniques. Therefore, the work was divided into four basic studies. The first study (Chapter 2) was based in evaluate the morphophysiology of a clonal mini-garden regarding to Zn and B concentrations during successive shoot collections. The second study (Chapter 3) was based in evaluate the induction of adventitious rooting in mini-cuttings regarding to genotype, Zn and B concentrations, shoot collections and IBA application. The third study (Chapter 4) was based in evaluate the adventitious rooting percentage of selected genotypes regarding the IBA concentration, optimal time of permanence of rooted mini-cuttings in a greenhouse and the origin of the vascular connection. Finally, the fourth study (Chapter 5) was based in develop a method for cloning of selected genotypes through micropropagation technique for the formation of a clonal micro-garden. In overall terms, mini-stumps survival, mini-cuttings production per square meter per year and foliar content of macro and micronutrients varied significantly in relation to treatments, presenting different responses according to shoots collection of the clonal mini-garden. The content of soluble carbohydrates non-structural of leaves varied regarding the shoots collection and nutrient solution. The increasing of the Zn and B concentrations in the nutrient solution induced reduction of the total content of soluble carbohydrates non-structural of leaves. The adventitious rooting percentage was low, and the genotypes were considered difficult to propagation by mini-cuttings technique. The ministumps fertigated with nutrient solutions containing Zn and B (concentrations of 1.0 at 2.0 mg L-1) associated with the IBA application presented the greater adventitious rooting percentage. The IBA application in the concentration of 2,000 mg L-1 resulted in the greater speed of rooting and rooting percentage, and the interval of 35 to 42 days was the most suitable for the permanence of mini-cuttings rooted in a greenhouse. According to the histological analysis of rhizogenesis was verified that the adventitious root presented direct connection to the vascular cambium. The in vitro multiplication of axillary buds depends of the genetic material, culture medium and concentration of plant growth regulator and, the shoots elongation depends of the genetic material and plant growth regulator. The micropropagation protocol was efficient for the microplants production of Eucalyptus benthamii and can be used to form a clonal microgarden.