?rea de concentra??o: Biotecnologia e melhoramento gen?tico florestal. / Incluir a Companhia Energ?tica de Minas Gerais S.A. (CEMIG), Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM) e Funda??o de Amparo ? Pesquisa do Estado de Minas Gerais (FAPEMIG) como ag?ncias financiadoras. / Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-12-18T17:15:46Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) lilian_almeida_brito.pdf: 1387334 bytes, checksum: b11b197308560b439026c5ce3e4ce1fc (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2018-01-03T17:04:10Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) lilian_almeida_brito.pdf: 1387334 bytes, checksum: b11b197308560b439026c5ce3e4ce1fc (MD5) / Made available in DSpace on 2018-01-03T17:04:10Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) lilian_almeida_brito.pdf: 1387334 bytes, checksum: b11b197308560b439026c5ce3e4ce1fc (MD5) Previous issue date: 2017 / Herbicidas com baixo poder de sor??o podem deixar res?duos no ambiente pass?veis de contamina??o em cursos h?dricos. A passagem dos res?duos para os cursos h?dricos pode ser diminu?da ou mesmo anulada por camadas vegetadas, notadamente, em matas ciliares. Contudo, pouca informa??o existe sob os efeitos de herbicidas em esp?cies florestais, principalmente na fase inicial de desenvolvimento. Com esse prop?sito avaliou-se a sensibilidade das esp?cies Senegalia polyphylla (DC.) Britton & Rose (monjoleiro) e Senna macranthera (DC. Ex collad.) H.S. Irwin & Barnaby (fedegoso) a res?duos dos herbicidas ametryn, atrazine, hexazinone e 2,4-D. Para o primeiro cap?tulo adotaram-se doses correspondentes a 0; 12,5; 25 e 50% daquela comercialmente recomendada de ametryn, atrazine e hexazinone em esquema fatorial 3 x 4 x 2 (herbicidas x doses x esp?cies) com quatro repeti??es, conduzidos em blocos ao acaso em casa de vegeta??o com temperatura controlada. Avaliou-se emerg?ncia, ?ndice de Velocidade de Emerg?ncia (IVE), taxa de mortalidade, massa fresca e seca, altura, di?metro, n?mero de folhas, al?m de vari?veis fisiol?gicas obtidas com aux?lio de um analisador a gases no infravermelho (IRGA). No segundo cap?tulo foram instalados dois experimentos paralelos (um por esp?cie) delineados inteiramente ao acaso em esquema fatorial 2 x 4, correspondentes a duas fontes de 2,4-D (produto comercial DMA? ou auxina Sigma-Aldrich? P.A.) e quatro concentra??es (0; 0,1; 1 e 10 mg.L-1 de 2,4-D), com quatro repeti??es e oito r?plicas cada. As esp?cies foram avaliadas quanto ? germina??o, ?ndice de Velocidade de Germina??o (IVG), contamina??o, di?metro, massa fresca e seca. Como resultado, de maneira geral o monjoleiro se mostrou mais sens?vel aos herbicidas, todavia, demonstrou potencial fitorremediador para atrazine. A compara??o entre os tipos de 2,4-D revela que o herbicida n?o pode ser utilizado como regulador de crescimento em laborat?rio, potencialmente devido aos aditivos que apresenta. Por fim, os resultados apontam que o uso recorrente de herbicidas lixivi?veis pr?ximos a fragmentos florestais podem afetar a perpetua??o de esp?cies nativas por efeito delet?rio silencioso. Novas pesquisas precisam ser realizadas com outras esp?cies nativas a fim de conhecer os efeitos dessas e de outras mol?culas ao ecossistema em geral. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Ci?ncia Florestal, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2017. / Herbicides with low sorption capacity can leave residues without an environment liable to contamination in water courses. Passage from the residues to the watercourses can be reduced or even negated by layers vegetated, notably in riparian forests. However, scarce information exists about the effects of herbicides on forest species, especially in the early stages of development. For this purpose the sensitivity of the species Senegalia polyphylla (DC.) Britton & Rose (monjoleiro) and Senna macranthera (DC. Ex collad.) H.S. Irwin & Barnaby (fedegoso) to residues of the herbicides ametryn, atrazine, hexazinone and 2,4-D were evaluated. For the first chapter doses corresponding to 0, 12.5, 25 and 50% of that commercially recommended for ametryn, atrazine and hexazinone were adopted in a 3 x 4 x 2 factorial arrangement (herbicides x doses x species) with four replicates, conducted in randomized blocks under controlled temperature greenhouse. It was evaluated the emergence, Emergency Speed Index (IVE), mortality rate, fresh and dry mass, height, diameter, number of leaves, and physiological variables obtained with the aid of an infrared gas analyzer (IRGA). In the second chapter were installed two parallel experiments (one per species) completely randomly delineated in a 2 x 4 factorial scheme, corresponding to two sources of 2,4-D (commercial product DMA? or auxin Sigma-Aldrich? P.A.) and four concentrations (0; 0,1; 1 e 10 mg.L-1 of 2,4-D), with four repletitons and eight replicates each. The species were evaluated for germination, Germination Speed Index (IVG), contamination, diameter, fresh and dry mass. As a result, in general the monjoleiro showed to be more sensitive to herbicides, however, it demonstrated a phytoremediation potential for atrazine. The comparison between the 2,4-D types reveals that herbicide cannot be used as a growth regulator in the laboratory, potentially due to the additives it presents. Finally, the results indicate that the recurrent use of leachable herbicides close to forest fragments can affect the perpetuation of native species by a silent deleterious effect. New research is done for other native species to know the effects of other molecules on the ecosystem in general.

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

The development of the science of micropropagation from the very initial concept of totipotency to the modern day advancement and sophistication has been affected by a wide range of problems such as hyperhydricity, shoot-tip necrosis and somaclonal variation. These problems are largely the result of the obvious fact of trying to grow plants in an environment that is different from the one plants are used to naturally. The extent of these problems ranges from minor technical inconvenience to significant economic loss. Characterization and control of micropropagation problems has been one of the priorities of plant tissue culture research due to the enormous contribution of this discipline for plant production, improvement and conservation. The prevalence and severity of these tissue culture problems varies widely among plant species. The rationale of this research project was therefore, to identify plant species most affected by the problems studied, characterize the problem and find mechanism(s) to control or minimize the damage caused by the problem. The literatures reviewed provide sufficient background information for the experimental chapters. Due to the different nature of the problems and variation in the plant species they affect, the model plant, the methodologies used and parameters analysed were also different. The findings of these investigations, in their own different way, addressed certain problems that individually and collectively pose difficulties to the micropropagation industry. The difference in the content of the experimental chapters is therefore the result of the broader objective of the research project to tackle such difficulties. The success and failure of tissue culture system greatly depends on the choice of PGR’s. This choice can be made based on comparative study of their biological activity. Some promising reports on the role of topolins in micropropagation led to the idea of testing these cytokinins for their potential in tissue culture. As a prerequisite to subsequent investigations, the biological activity of some selected topolins and BA derivatives was tested using the soybean callus bioassay. The activity of the cytokinins tested varied significantly. The results demonstrated that the structure of a cytokinin dictates its activity. Modifications of side-chain improved the activity of oT but had no effect on pT. The presence of the methyl group had an enhancing effect on cytokinin activity of topolins or at least it did not reduce it. BA derivatives BA9THP (conjugated at N9 position), 3FBA and 2Cl6(3OHBA)R (halogenated derivatives) also showed good cytokinin activity and hold good promise for future research. In an attempt to alleviate hyperhydricity in Aloe polyphylla and optimize the micropropagation protocol, meta-topolin and its derivatives were tested at various concentrations together with BA and zeatin. Of all the cytokinins tested mT produced the best results in terms of shoot and root growth. Five μM was found to be the optimum concentration at which complete control of hyperhydricity was achieved without compromising shoot and root growth. Plantlets rooted in a multiplication media. BA generally had a negative effect on growth and development both in vitro and ex vitro. Acclimatization of plantlets was achieved easily by initially transferring plantlets to a mist house (for three weeks) followed by transfer to the greenhouse. The type of cytokinin also had an effect on ex vitro growth with BA-treated plants producing the lowest shoot and root biomass. Various experiments were conducted to characterize and control factors affecting STN in Harpagophytum procumbens. Media type and strength, PGR, carbon sources, sub-culturing, calcium and boron were tested. Results indicated that all of the tissue culture components tested affected STN. From the different media types tested, half strength was MS found to be the preferred medium. Increasing cytokinin concentration increased the incidence of STN and the problem was aggravated by the addition of auxin to the multiplication medium. Optimum shoot multiplication was achieved by omitting auxin and using the cytokinin mTR. Plantlets produced basal callus which interfered with rooting. The quantity of this basal callus was minimum when mTR was used. Sub-culturing plantlets onto fresh medium every two weeks helped minimize STN. Off all the sugars tested 3% sucrose was optimum. Other sugars either aggravated STN or inhibited growth when compared at equi-molar concentration. Increasing the concentration of either Ca or B prevented the development of necrotic shoots. When the concentration of both elements is increased simultaneously negative effects on both growth and STN were observed. Using 6 mM Ca in half strength MS medium was optimum. B was toxic at higher concentrations. Plantlets rooted readily in half strength cytokinin-free MS media supplemented with 2.5 μM IAA. Rooted plantlets produced using the optimized protocol were acclimatized successfully by transferring directly to a greenhouse in a 1:1 ratio of sand and soil mixture. The effect of meta-toplins on micropropagation and somaclonal variation of banana was investigated. Tissue cultured explants of cultivars ‘Williams’ and ‘Grand Naine’ were cultured in MS media containing the cytokinins BA, mT, MemT, MemTR and mTR at various concentrations. Results of the investigation revealed that superior multiplication and lower abnormality index was recorded from the mTR and mT treatments at 22.2 μM concentration. These treatments, however, had an inhibitory effect on rooting. The effect of these treatments (22.2 μM mT and mTR) in comparison with equi-molar concentration of BA on somaclonal variation of ‘Williams’ banana was tested using RAPD-PCR at the 7th multiplication cycle. No significant difference was found between the treatments. It should however be highlighted that cultures were initially maintained for three multiplication cycles in media containing BA. The inherent stability and initial effect of BA could have influenced the results. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

The focus for the first part of the thesis is on the systematics of species belonging to Silene subgenus Silene. Phylogenetic relationships are inferred from DNA sequences from both the plastid (the rps16 intron) and the nuclear (ITS, intron of the RPB2 gene) genomes. Silene section Rigidulae is shown to be non-monophyletic in its previous circumscription, but instead consisting of six separate clades, each correlated to the geographical distribution of the included species. The taxonomic consequences for each clade are discussed. One of the clades is recognized as a new section and described as Silene sect. Arenosae sect. nov. The morphological descriptions of the species are formalized using a novel implementation of the Prometheus Description Model. Two proposals are included in the thesis, one to reject the name Silene polyphylla L., which is a senior synonym to S. portensis L. Silene linearis Decne. is proposed for conservation against the rarely used S. linearis Sweet. Silene antirrhina, a weedy American annual, is strongly supported as sister to the Hawaiian endemic species of Silene, suggesting an American origin for these. Two of the endemics have evolved woodiness after introduction to Hawaii. In the second part of the thesis we use four nuclear DNA regions, (introns from RPA2, RPB2, RPD2a, RPD2b), and the chloroplast psbE-petG spacer. A framework is developed to evaluate different phylogenetic explanations for conflicting gene trees, where divergence times are used to discriminate among inter- and intralineage processes. The incongruences observed regarding the relationships among the three major lineages of Heliosperma are best explained by homoploid hybridization. The pattern regarding the origin of Heliosperma itself is more complicated and is likely to include several reticulate events. Two lineages have probably been involved in the origin of Heliosperma, one leading to Viscaria and Atocion and the other to Eudianthe and/or Petrocoptis.

Micropropagation of Aloe polyphylla, an endangered species with a high ornamental and medicinal value, is an important part of its conservation. However, the in vitro culture was hindered by the phenomenon of hyperhydricity. The research reported in this thesis was undertaken for two reasons. Firstly, to understand the role of various culture factors involved in the process of hyperhydricity in A. polyphylla and to identify the in vitro conditions, under which this disorder can be prevented. Secondly, we conducted an investigation into the underlying mechanisms of this phenomenon by probing if it was mediated through internal cytokinins. Ammonium (NH4 +) ions, applied cytokinins (CKs) and CK concentrations were tested in multifactorial combinations and significantly influenced the regeneration rate and occurrence of hyperhydricity. Shoots were grown on media with different NH4 + concentrations (10.3, 20.6 and 61.8 mM) and supplemented with BA, zeatin or TDZ at 0, 5 or 15 ìM. Elevating the levels of NH4 +, in the absence of CKs, could not induce hyperhydricity. Similarly, very low hyperhydricity was observed when CKs were added to media containing low NH4 + (10.3 mM). However, in the presence of higher NH4 + concentrations, CKs increased hyperhydricity in a concentrationdependant manner, suggesting that they were capable of inducing this syndrome only when other factors in the culture system were not optimised. High numbers of healthy looking shoots were produced on media with low NH4 + and low BA or zeatin (5 ìM). The use of TDZ resulted in the formation of buds, which did not develop into shoots. In view of the fact that NH4 + was supplied in the form of NH4NO3, it was difficult to determine if NH4 + or nitrate (NO3 -) ions were associated with the increase in hyperhydricity. We further examined the role of nitrogen (N) supplied as inorganic NH4 + or NO3 -, or organic glutamine. The omission of total N from the culture medium resulted in low multiplication and hindered shoot growth. Ammonium as the sole source of N depressed shoot regeneration and growth and escalated the frequency of hyperhydricity to ca. 50%. When NO3 - was used as the sole N source, shoots of fine quality were produced and hyperhydricity was completely eliminated. Overall, the MS N mix was superior to any single N source for multiplication and growth of shoots, suggesting a synergistic effect between NH4 + and NO3 - on shoot regeneration. Furthermore, not only the absolute amount of N, but also the relative amounts of NH4 + and NO3 - influenced the multiplication rate, frequency of hyperhydricity and shoot quality. The highest regeneration was obtained with NH4 + : NO3 - ratios (mM) of 20 : 40, 30 : 30 and 40 : 20. Decreasing the ratio of NH4 + : NO3 - lowered the occurrence of hyperhydricity. The potential of glutamine as the sole source of N was also demonstrated, since its application resulted in the production of good quality shoots and almost no hyperhydricity. Shoot explants grown in static liquid media became hyperhydric and lost their ability to regenerate. The type of gelling agent used to solidify the medium affected greatly hyperhydricity and shoot multiplication. Gelrite resulted in a significantly lower multiplication rate and four times higher hyperhydricity (64.7%) compared to when agar was used. Gelrite was further selected to test the hypothesis if hyperhydricity can be overcome by decreasing the relative matric potential of the media, and respectively the availability of water, as represented by increasing gelrite concentrations. Satisfactory reduction in hyperhydricity was achieved only at 16 g l-1 gelrite, however the regeneration also decreased. The nature of the gelling agent is therefore essential for the successful control of this phenomenon. It appears that a crucial prerequisite for the reduction of hyperhydricity in tissue cultures of A. polyphylla is the gaseous exchange between the in vitro atmosphere and the outside environment. In ventilated cultures, achieved by using a modified lid with a hole (d = 7 mm) covered with polyester or cotton mesh, hyperhydricity was completely eliminated, irrespectively of the type of gelling agent. Ventilation was further advantageous for the in vitro regenerants by increasing their leaf chlorophyll content as well as epicuticular wax deposition, the last one being indicative of the development of the water loss regulation mechanisms of explants. The increased culture ventilation, however, was negatively correlated with the regeneration rate and shoot growth. Endogenous CKs were measured in in vitro regenerants after an eight-week cycle to examine whether the hyperhydricity-inducing effect of exogenous CKs and gelling agents is associated with changes in the endogenous CK content. The content of endogenous CKs, determined by HPLC-mass spectrometry, in the shoots grown on CK-free media comprised isopentenyladenine-, trans-zeatin- and cis-zeatin-type CKs. The application of exogenous CKs resulted in an increase in the CK content of the shoots. Following application of zeatin, dihydrozeatin-type CKs were also detected in the newly-formed shoots. Application of BA to the media led to a transition from isoprenoid CKs to aromatic CKs in the shoots. Shoots grown on gelrite media contained higher levels of endogenous CKs compared to those on agar media. Total CK content of hyperhydric shoots was higher than that of normal shoots grown on the same medium. We suggest that the ability of exogenous CKs and gelrite to induce hyperhydricity in shoots of Aloe polyphylla is at least partially due to up-regulation of endogenous CK levels. However, hyperhydricity is a multifactor process in which different factors intervene. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.