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Tuber crops such as potato (Solanum tuberosum L.) can take up high levels of soil cadmium (Cd) which can be accumulated in their tubers. Thus, they can act as vehicles for transporting Cd to human body which can seriously threaten our health due to its high toxicity. In some circumstances, consumption of potato can contribute to more than 50 percent of human dietary Cd intake. In the present research two approaches were used to probe the potential for genetic improvement to contribute towards the goal of “minimisation of Cd level in potato” which is a novel food safety strategy: (1) assess the natural occurrence of variation in Cd accumulating potential among different potato cultivars already in cultivation in New Zealand, and (2) as a proof-of-concept study to generate potato plants with improved Cd resistance from a model experimental potato cultivar Iwa based on plant cell culture-selection (in vitro breeding approach). In the first approach, 10 New Zealand cultivars, namely Red Rascal (RR), Russet Burbank (RB), Fianna (F), Agria (A), Laura (L), Purple Heart (PH), Purple Passion (PP), Yukon Gold (YG), Moonlight (M) and Summer Delight (SD) were chosen randomly among over 30 cultivars grown for seed production in a block situated approximately one kilometre east of Lincoln, Canterbury within the 2011-2012 growing season. The tubers as well as the soil samples immediately surrounding the tubers were harvested and prepared for analytical analyses using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The results showed that the soil samples were low in Cd level (0.06 mg kg-1), compared with the national soil average (0.35 mg kg-1), while the tubers varied widely in Cd content from 0.04 to 0.34 mg kg-1 among different cultivars. Therefore, SD with the lowest mean Cd content (0.05 mg kg-1) and an Enrichment Factor (EF) of just below one showed promise as a potential cadmium safe cultivar (CSC). There might be of concern if L, YG, PP with the highest mean Cd contents (0.18-0.21 mg kg-1) are grown in soils with higher Cd levels. Potato tissue culture required for the second approach based on somaclonal variation was established from the model experimental genotype Iwa in the lab. Leaf and internode explants were used as the starting plant materials to initiate two morphologically distinct calli (type-A and -B). Upon morphological assessment and analysis of antioxidative enzymes such as peroxidase, it was revealed that they exhibited differential Cd sensitivity. The more Cd-resistant callus type (type-B) was chosen for in vitro selection using 18 different Cd treatments varying in Cd exposure timing and duration. Following shoot and root regeneration from these calli, 18 different new Iwa plant lines were obtained. After at least three months of sub-culturing of all 18 plant lines on Cd-free media, in vitro screening of the lines was carried out to identify the most promising plant lines as far as Cd resistance was concerned. After two rounds of in vitro growth screening under a low and high Cd levels, two lines including line 9 (L9) and line 11 (L11) were found to exhibit enhanced Cd resistance compared to control Iwa plants. Further studies of L9 and L11 compared to control Iwa plants including biochemical analysis of reactive oxygen species such as hydrogen peroxide, and transmission electron microscopic studies uncovered that L11 was more resistant to Cd than L9 and control plants. L11 plantlets had about 20 and 10 percent less H2O2 level than control and L9 plantlets, while antioxidative activities were four and two times higher in L11 compared with control and L9, respectively. Moreover, L11 seemed to exhibit a high rate of Cd compartmentalisation in the vacuoles and Cd binding to the cell walls in the roots, suggesting a potential to exclude or limit Cd translocation to other parts of the plant.

Somaclonal variation, to provide germplasm for crop improvement, must be screened, selected and characterized. Immature wheat, Triticum aestivum L., (PCYT-10) embryos (10-12 days old) were cultured on Hu rashige and Skoog (t1S) medium containing 0.5 mg L-1 6- furfurylaminopurine (kinet in) and 2, 3, or 4 mg L -1 of 1- methoxy-3,6- dichlorobenzoic acid (2,4-D). Dicamba, at 2 and 3 mg L -l and 0.2 mg L -1 2,4-D, produced 12.7%, 30.3%, and 28.2% of the somaclones, respectively. No plantlets were produced from other treatments. Variants were characterized by cytology, biochemistry and morphology. Somaclones showed significant differences in length and width of flag leaf, plant height, number of tillers, spike length, awn length, and number of seeds per main head when compared to parental controls for two sal fed recurrent generations. Number of spikelets per main head in the second generation showed no significant difference from controls. Stability and segregation of somaclones for measured traits indicated that genetic changes had occurred which could enhance wheat germplasm. Leaf isozymes of somaclones (SC 1 and sc 2 ) showed no variation in glutamine oxaloacetate transaminase (GOT) (E.C . 2. 6. 2.1. ), leucine aminopeptidase (LAP) (E.C. 3.4.11.1), or esterase (EST) (E.C. 3.4. 99) bands in 28% of the somaclones, 28% with light, and 44% missing a fast movind band. Approximately 30% of the normal group set no seeds. Mutants with the missing band were stable through the fourth-selfed generation, whereas, variants with the light EP band were still segregating . Plants with the missing EP band were morphologically normal compared to the parents except rachis internodes were longer than those of the parents. There was no correlation between the missing EP band and a missing chromosome in some mutants. The mutant may have been due to a point mutation, deletion, or activation of a repressor gene. Variants exhibited a wide range of protein density and missing or extra bands. Relative amounts of DNA per telophase nucleus were affected by inorganic salts and sucrose levels. Ploidy level increased with time within single-strength MS, but not within double MS medium. Cal li grown on the modified double MS medium exhibited a higher number of shoots than those grown on modified MS. Individual variants with desirable characteristics with high seed production, high protein levels, supernumerary spikelets, and larger flag leaves could be incorporated into a wheat improvement program.

Astilbe (Saxifragaceae) is a genus of herbaceous perennials widely cultivated for their ornamental value. The genus is considered taxonomically complex because of its geographic distribution, variation within species, and the lack of adequate morphological characters to delineate taxa. To date, an inclusive investigation of the genus has not been conducted. This study was undertaken to (a) develop a well-resolved phylogeny of the genus Astilbe using an expanded morphological data set and sequences from the plastid gene matK, (b) use single nucleotide polymorphisms to determine the lineages of cultivated varieties, and (c) successfully culture Astilbe in vitro and evaluate potential somaclonal variation of resulting Astilbe microshoots. Phylogenetic trees generated from a morphological character matrix of 28 character states divided Astilbe into three distinct clades. Relationships were well resolved among the taxa, though only a few branches had greater than 50% bootstrap support. There is evidence from the phylogeny that some described species may actually represent variation within populations of species. From our analysis I propose an Astilbe genus with 13 to 15 species and offer a key for distinguishing species and varieties. There was little matK sequence variation among taxa of Astilbe. Phylogeny of Astilbe generated from the maximum parsimony and maximum likelihood analysis of matK sequences resulted in a polytomy of seven Astilbe species, with relationships within the genus poorly resolved. A second phylogeny of 21 taxa of Astilbe was more informative, aligning cultivated varieties near species from which they were derived. The matK sequence variation for Astilbe taxa was aligned to reveal DNA polymorphisms. Closely related taxa retained polymorphisms at the same sites within the gene sequence. These polymorphic sites could potentially be utilized to confirm the lineage of popular cultivated Astilbe varieties. Propagation of Astilbe seedlings in tissue culture gave rise to various numbers of microshoots from each of 15 seedlings. Multivariate and cluster analysis of morphological characters from 138 plants derived from 15 seedlings revealed potential somaclonal variants. These variants were characterized by one or more of the following traits: dwarf habit, dark green leaves (high chlorophyll content), increased flowering, or larger plant size. Somaclonal variants with desirable phenotypes may be valuable for cultivar development. / Ph. D.

The objectives of this study were to develop an in vitro direct adventitious bud induction and an organogenic callus induction and shoot regeneration system via floral organ segments culture for bromeliads, moreover, explore the effect of auxin on plantlet elongation of Guzmania. And further, apply the above micropropagation system to physical and chemical methods to induce somaclonal variances of bromeliad plantlets in vitro for mutation breeding. The explant sources of bromeliads and the components of culture medium were studied to develop a micropropagation system for bromeliads. The results indicated that the 1/3MS basal medium supplemented with a combination of 1.0 mg l-1 BA + 0.5-1.0 mg l-1 NAA, or a combination of 3.0 mg l-1 BA + 0.5 mg l-1 NAA, showed the highest frequency of direct initiation of adventitious buds derived from shoot apex and lateral bud explants of Aechmea fulgens var. fulgens and Guzmania 'Focus'. The best results of adventitious buds induction of the both species were found in the lower lateral bud explants, at 47.5% and 35%, respectively. In addition, the adventitious buds began to form on day 16 after the G. 'Focus' decapitated plantlets had been cultured in medium supplemented with 3.0 mg l-1 BA + 0.5 mg l-1 NAA. However, this phenomenon did not occur in case of undecapitated explants, where only protruding nodules appeared. Petal- and ovary-derived calli of A. fasciata and G. 'Hilda' were induced on 1/2MS basal medium supplemented with 1.0-1.5 mg l-1 2,4-D in combination with 1.0 or 0.5 mg l-1 NAA. Organogenic calli were cultured on medium with 1.0 mg l-1 NAA and 0.5 mg l-1 TDZ could be induced to differentiate and regenerate the adventitious buds. Furthermore, the number of adventitious buds proliferating at the base of the plantlets derived from G. 'Hilda' floral organs, cultured in media with different concentrations of IAA, IBA, NAA, and 8-azaadenine, was only 1-2 adventitious buds individually. This result shows that auxin can indeed suppress cytokinin-effects. The influence on plantlet elongation was greatest in the treatments using 0.5 mg l-1 NAA and 1.0 mg l-1 NAA. After 4 months culture, plantlets grew to 5.73 and 5.62 cm in height, that was 2.22 and 1.95 cm higher than the control, respectively. Plantlets of A. fasciata hardened under the middle (50 £gmol m-2s-1) light intensity condition had a higher survival rate, 95%, than that hardened at a low light intensity (1 £gmol m-2s-1; 17.5%). The maximum number of newly developing roots, up to 4.15 per shoot, was also observed at the same light intensity treatment. During transplantation, plantlets growing in coir fiber showed the best results in terms of plant growth within 6 months ex vitro culture. The average length of the plantlets was 22.0 cm, and an average of 19.3 leaves per plantlet was achieved. When calli of G. 'Hilda' treated by sodium azide, the survival rate was 0%. The survival rate of decapitated plantlet explants treated with 0.5 mM sodium azide for 60 minutes was 51.3%, about half-lethal dose. In addition to the survival rates of decapitated plantlet explants of A. fasciata, G. 'Hilda', G. 'Cherry', G. 'Luna' and G. 'Focus' irradiated by £^-ray showed 74.2-100% with the exception of the G. 'Focus' irradiated by 15 Gy, which dropped to 45.0%. At present, mutant plantlets showed a great deal of chimeras in leaf and were transplanted to potting media.

The diploid woodland strawberry (Fragaria vesca) is a rapidly developing translational model for members of the family Rosaceae and other plants. This thesis represents some of the first forward genetics studies evaluating putative T-DNA insertional mutants in F. vesca. The observed phenotypes include alterations to floral development, anthocyanin pigmentation and leaf structure. The floral development mutant named green petal (gp) was not associated with the T-DNA insertions present. Based on similar phenotypes induced by mutation of transcription factors involved in floral development of Arabidopsis thaliana, we used a BLAST search of the F. vesca genome hybrid gene models to identify 30 candidate genes that may have caused the gp phenotype. Expression analysis of these genes revealed that it was due to a 37 bp deletion in a SEPALLATA3-like E-Class MADS box transcription factor. This mutation altered organ structure in the three inner whorls of the flower, affecting fertility and fruit development. The deletion was demonstrated to segregate with the mutant phenotype in a segregating population of 92 individuals, 22 of which had green petals. The anthocyanin biosynthesis mutant named white runner (wr) lacked red pigmentation in the stems and runners. The T-DNA insertion in this line was located in a highly repetitive LTR retrotransposon region, which complicated analysis. Segregation analysis of the wr lines revealed that the phenotype was unassociated with the T-DNA insertion as well. We used a targeted expression analysis of three critical structural genes in the flavonoid biosynthesis pathway that revealed a 20 bp deletion in the gene encoding flavanone 3-hydroxylase, an enzyme necessary for the production of flavonols, anthocyanins and proanthocyanidins. In an F2 segregating population, this deletion co-segregated with the phenotype. The third mutant line presented here displayed a curly leaf (cl) phenotype and was found to harbor a T-DNA insertion in a gene encoding a putative erythroblast macrophage attacher protein (EMP). Sequence and protein domain analysis indicated that FvEMP was related to the mammalian EMP protein that functions in cytoskeletal dynamics and red blood cell enucleation. Complementation analysis confirmed that introduction of the wild type FvEMP gene into the cl mutant plants restored wild type leaf phenotype. Further morphological analysis revealed additional pleiotropic effects of the mutation, including abnormalities in seed set and germination, pollen tube growth, adhesion of the abaxial epidermal layer to the mesophyll layer and reduced petiolule length. These phenotypes are consistent with actin binding and microtubule associated protein mutants in other plant species. Insertional mutagenesis is a critical molecular tool for model crop development. These studies highlight the precautions that must be taken when evaluating insertional mutants. These mutants are excellent tools for studying their respective disrupted gene function. The in depth molecular analysis of the mutants presented in this work was only possible because of the availability of the Fragaria vesca genome which was used extensively to identify T-DNA insertion sites and recover candidate gene sequences for expression analysis. / Ph. D.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-02-13T15:59:18Z No. of bitstreams: 1 2010 - ANDR?A PEREIRA DA FONSECA.pdf: 2103296 bytes, checksum: dca780e8029b629fbff3f70724ad309d (MD5) / Made available in DSpace on 2017-02-13T15:59:19Z (GMT). No. of bitstreams: 1 2010 - ANDR?A PEREIRA DA FONSECA.pdf: 2103296 bytes, checksum: dca780e8029b629fbff3f70724ad309d (MD5) Previous issue date: 2010-04-26 / CAPES / The culture of strawberry (Fragaria x ananassa Duch.) is an agricultural activity of great socio-economic importance. In recent years, its production has increased in Brazil due to the introduction of new cultivars and use of quality plants. The vegetative propagation by stolons, usual in the species of the genus Fragaria also provide a limited number of plants and can spread viral diseases. The production of healthy plants can be obtained by meristem culture. Been strawberry cultivars in vitro responsive, with this technique it is possible to obtain a greatest number of plants. However the increase in the number of subcultures may induce somaclonal variation. In order to expand the availability of healthy plants of strawberry with genetic identity guarantee, this study aimed to analyze, with the use of morphological and molecular markers, with precision, the number of subcultures that allows large scale multiplication of strawberry, without loss of genetic identity. Explants of the cultivars Aromas, Camarosa and Camino Real, at the Laboratory of Plant Tissue Culture, in a first step, was submitted to twelve cycles of subculture, and in the following year, explants of the same cultivars, from the same mother plants, were subcultured for three cycles. The acclimatization and evaluation of the plants in the field were conducted in a greenhouse at Horticulture Sector of the Instituto de Agronomia of UFRRJ. The experimental design was randomized blocks in a factorial scheme 3x2, with the three genotypes being a factor and the two subculture levels other factor. In the Laboratory of Molecular Genetics of the Instituto de Biologia of UFRRJ, extraction of DNA was proceeded from leaves of regenerated in vitro plantlets of the three cultivars submitted to three, five and twelve subcultures and of the mother plants that were not produced by tissue culture. During the in vitro multiplication, in two levels of subcultures, we observed a greater tendency of hyperhydricity in Camino Real cultivar and a higher frequency of callus production in ?Camarosa?. After the acclimatization, plants exposed to twelve subcultures showed a higher average height. However, the average root length was not significantly different between the two levels of subcultures. In the field evaluation, for other quantitative traits, there were no significant differences between the two levels of subcultures. Cultivars Camarosa and Camino Real showed the highest number of characteristics with a variation between the two levels of subcultures, with the greatest variations observed in plants subjected to three subcultures. Analysing the field qualitative characteristics, the variation observed between the third and 12th subcultures, was present only the frequency distribution of leaf brightness, length and width of terminal leaflet. / A cultura do morangueiro (Fragaria x ananassa Duch.) ? uma atividade agr?cola de grande import?ncia s?cio-econ?mica. Nos ?ltimos anos, o seu cultivo tem aumentado no Brasil devido ? introdu??o de novas cultivares, sendo a produ??o de mudas de qualidade um dos fatores que afetam a expans?o da produ??o. A propaga??o vegetativa de plantas do g?nero Fragaria al?m de fornecer um n?mero limitado de prop?gulos, pode disseminar doen?as vir?ticas e radiculares. A produ??o de mudas sadias pode ser obtida atrav?s da cultura de meristemas. Considerando que cultivares de morangueiro s?o responsivas a propaga??o in vitro, atrav?s desta t?cnica ? poss?vel obter o maior n?mero de plantas, aumentando o n?mero de subcultivos, contudo, este acr?scimo pode induzir a ocorr?ncia de varia??o somaclonal. A fim de ampliar a disponibilidade de mudas sadias de morangueiro e com garantia de identidade gen?tica, este trabalho teve como objetivo analisar, a partir do uso de marcadores morfol?gicos, o n?mero de subcultivos que permita a multiplica??o do morangueiro em larga escala, sem que ocorra a perda da identidade gen?tica dos clones submetidos a este processo. No Laborat?rio de Cultura de Tecidos Vegetais, explantes das cultivares Aromas, Camarosa e Camino Real, foram submetidos em uma primeira etapa a doze ciclos de subcultivos e, no ano seguinte, os explantes das mesmas cultivares provenientes das mesmas plantas matrizes foram subcultivados por tr?s ciclos. A aclimatiza??o e avalia??o das plantas a campo foram realizadas em estufa no Setor de Horticultura do Instituto de Agronomia da UFRRJ. O delineamento experimental foi em blocos casualizados em esquema fatorial 3x2, sendo um fator os tr?s gen?tipos e o outro, os dois n?veis de subcultivos. Durante a fase de multiplica??o in vitro, nos dois n?veis de subcultivos, foi observado um maior n?mero de frascos com brota??es com sintoma de hiperhidricidade na cultivar Camino Real e forma??o de calos na cultivar Camarosa. Ap?s a fase de aclimatiza??o foi observado que plantas submetidas a doze subcultivos apresentaram maior altura m?dia da parte a?rea, entretanto, para o comprimento m?dio da raiz n?o foi observada diferen?a significativa entre os dois n?veis de subcultivos. Na avalia??o a campo das demais caracter?sticas quantitativas, n?o foram observadas diferen?as significativas entre os dois n?veis de subcultivos. Com doze subcultivos in vitro de plantas de morangueiro, das cultivares Aromas, Camarosa e Camino Real, ? poss?vel obter maior n?mero mudas micropropagadas sem perda da estabilidade gen?tica. A cultivar Camarosa apresentou valores m?dios superiores para altura da parte a?rea ap?s a fase de aclimatiza??o e a campo e, massas fresca e seca da parte a?rea, quando submetida a doze ciclos de subcultivos. As cultivares Camarosa e Camino Real apresentaram maior n?mero de caracter?sticas quantitativas com varia??o entre os dois n?veis de subcultivos, sendo as maiores varia??es observadas em plantas submetidas a tr?s subcultivos. A cultivar Camino Real seguida da ?Camarosa? apresentaram um maior n?mero de caracter?sticas qualitativas com varia??o na distribui??o das frequ?ncias entre os dois n?veis de subcultivos. A cultivar Aromas apresentou uma maior estabilidade gen?tica em rela??o ?s caracter?sticas quantitativas e qualitativas.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The potato (Solanum tuberosum L.) is the fourth culture in economic importance in the world after the wheat, of the maize and the rice. Consequently, the research with this species can bring benefits for a considerable number of people. Tissue culture has contributed in the genetic improvement of the potato in the direction of attainment of free plants of virus and micropropagation of elite genotypes. However, plants regenerated from tissue culture can show variation in agronomic characteristics important. The induced variation by tissue culture is called somaclonal variation. The occurrence of somaclonal variation can be identified in vitro for parameters still auxiliary as the multiplication rate. In field, the use of morphologic markers can be used to identify somaclones. The work had as objective: To evaluate the behavior in vitro of cultivating them of potato Asterix and Macaca through the multiplication rate observed during a period of eight months. Identify variant somaclonais proceeding of the culture in vitro of them cultivate Asterix and Macaca through morphological markers.To evaluate the genotype effect, culture-age effect and the type of explant on the occurrence of somaclones. Multiplication rate was used in the attempt to identify the occurrence of somaclonal variation in cultivating of potato Asterix and Macaca with three sources of explantes (apex stems, stems and callus) and two ages of subculture (70 months-old clone and 12 months-new clone).The morphological characterization of the plants and basic tubercles was effected according to described methodology for COLLARES (2002) based in the minimum describers of the potato (Solanum tuberosum L.). The multiplication rat of cultivating Macaca was greater that of Asterix and the behavior of the multiplication rate of Macaca oscillated between extreme values different of the observed one in Asterix. The rate multiplication depends on cultivar.Through the morphological characterization it was possible to identify that to cultivate them Asterix and Macaca had presented different behavior how much to the stability of the morphologic characteristics when passing for culture in vitro. The explante derived from callus in such a way presented the biggest occurrence of somaclonais variants for the treatments that had included Asterix as Macaca. The age of subculture of 70 months and 12 months had not been adjusted for maintenance of disgnostic characteristics of them to cultivate Asterix and Macaca due to occurrence of somaclones. It was possible to identify variant somaclonais in the treatments that had included Asterix and Macaca through the minimum describers of the potato. / A batata (Solanum tuberosum L.) é a quarta cultura em importância econômica no mundo depois do trigo, do milho e do arroz. Conseqüentemente, a pesquisa com esta espécie pode trazer benefícios para um considerável número de pessoas. A cultura de tecidos tem contribuído no melhoramento genético da batata no sentido de obtenção de plantas livres de vírus e micropropagação de genótipos elite. No entanto, plantas regeneradas a partir de cultura de tecidos podem exibir variação em importantes caracteres agronômicos. A variação induzida por cultura de tecidos é chamada de variação somaclonal. A ocorrência de variação somaclonal pode ser identificada ainda in vitro por parâmetros auxiliares como a taxa de multiplicação. Em campo, o emprego de marcadores morfológicos pode ser usado para identificar somaclones. O trabalho teve como objetivos: avaliar o comportamento in vitro das cultivares Asterix e Macaca de batata através da taxa de multiplicação observada durante um período de oito meses. Identificar variantes somaclonais provenientes do cultivo in vitro das cultivares Asterix e Macaca através de marcadores morfológicos; avaliar o efeito do genótipo, do tempo de subcultivo e do tipo de explante sobre a ocorrência de variantes somaclonais. Foi empregada a taxa de multiplicação na tentativa de identificar a ocorrência de variação somaclonal na cultivares de batata Asterix e Macaca com três fontes de explantes (ápice caulinar, segmento nodal e calo) e dois tempos de subcultivo (70 meses-clone velho e 12 meses-clone novo). A caracterização morfológica das plantas e tubérculos básicos foi efetuada segundo metodologia descrita por COLLARES (2002) baseada nos descritores mínimos da batata. A taxa de multiplicação da cultivar Macaca foi maior que a de Asterix e o comportamento da de Macaca oscilou entre valores extremos diferente do observado em Asterix. A taxa de multiplicação in vitro depende da cultivar. Através da caracterização morfológica foi possível identificar que as cultivares Asterix e Macaca apresentaram comportamento diferente quanto à estabilidade dos caracteres morfológicos ao passarem por cultivo in vitro. O explante derivado de calo apresentou a maior ocorrência de variantes somaclonais, tanto para os tratamentos que incluíram Asterix como Macaca. Os tempos de subcultivo de 70 meses e 12 meses não foram adequados para manutenção dos caracteres diagnósticos das cultivares Asterix e Macaca devido à ocorrência de variantes somaclonais. Foi possível identificar variantes somaclonais nos tratamentos que incluíram Asterix e Macaca através dos descritores mínimos da batata.

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-01-10T14:27:33Z No. of bitstreams: 1 sirleiaparecidajulião.pdf: 1501615 bytes, checksum: c915a860117fdc2bf5cd1ba90fdab0b9 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-01-22T16:11:44Z (GMT) No. of bitstreams: 1 sirleiaparecidajulião.pdf: 1501615 bytes, checksum: c915a860117fdc2bf5cd1ba90fdab0b9 (MD5) / Made available in DSpace on 2018-01-22T16:11:44Z (GMT). No. of bitstreams: 1 sirleiaparecidajulião.pdf: 1501615 bytes, checksum: c915a860117fdc2bf5cd1ba90fdab0b9 (MD5) Previous issue date: 2017-08-11 / Lippia alba é uma espécie medicinal com ampla diversidade fenotípica, incluindo a composição do óleo essencial. A variação genética é provavelmente a principal causa dessa variação. A espécie foi descrita como um complexo poliploide com cinco números cromossômicos (2n=30, 38, 45, 60 e 90). Devido à importância econômica e à variação genética natural, essa espécie representa um excelente modelo em estudos sobre estabilidade genômica. Este trabalho teve como objetivo investigar a estabilidade genômica de 22 acessos naturais cultivados in vitro durante sete anos e em acessos poliploides sintéticos obtidos a partir da duplicação cromossômica de um acesso diploide natural usando colchicina. Para analisar a estabilidade do genoma de plantas cultivadas a longo prazo, foram analisadas quatro plantas (três mantidas in vitro e uma no campo) de 22 acessos. O tamanho do genoma foi verificado por citometria de fluxo e oito marcadores ISSR foram utilizados para verificara estabilidade em nível de sequencia de DNA. Para avaliar a estabilidade genômica após a poliploidização, onze plantas poliploides sintéticas, sendo 5 tetraploides (4X) e 6 mixoploides (2X / 4X) foram comparadas com a planta matriz diploide. As comparações foram baseadas no conteúdo de DNA, contagem cromossômica, marcadores moleculares ISSR e SSR e no perfil químico do óleo essencial. A comparação entre as plantas mantidas in vitro e as respectivas plantas mantidas no campo mostrou que 13 dos 22 acessos sofreram uma pequena redução no tamanho do genoma. Os marcadores ISSR detectaram polimorfismos na sequência de DNA variando de 1,61 a 33,87%. Somente três acessos não apresentaram bandas polimórficas. O número de bandas polimórficas entre os outros 19 acessos variou de um a 21. A análise da ploidia das plantas poliploides sintéticas realizada em folhas e raízes confirmou a estabilidade das plantas tetraploides. As plantas mixoploides apresentaram um único pico G1 correspondente a plantas triploides. A análise cromossômica revelou 60 cromossomos com 12 sítios de DNAr 45S nas plantas tetraploides e 9 sítios de DNAr 45S nos 45 cromossomos observados nas plantas triploides. Os marcadores ISSR mostraram polimorfismo entre a planta matriz diploide e as plantas poliploides sintéticas. A taxa de polimorfismo variou de 1,81 a 5,45% nas plantas tetraploides e de 43,63 a 56,36% nas plantas triploides. Alterações no tamanho dos alelos de microssatélites também foram detectadas. A planta matriz diploide apresentou dez alelos, três dos quais foram compartilhados com as plantas triploides e sete com as plantas tetraploides. Todas as plantas triploides apresentaram 13 alelos, sendo dez deles correspondentes a alelos novos. O número de alelos detectados nas plantas tetraploides variou de oito a 11 e o número de alelos novos como consequência da poliploidização variou de dois a quatro. O componente majoritário detectado no óleo essencial da planta matriz diploide e das plantas tetraploides foi o citral e nas plantas triploides foi o linalol. A instabilidade genômica detectada em L. alba após sete anos de cultura in vitro pode ser devido à consequência da instabilidade do genoma natural combinada com a cultura in vitro a longo prazo. Os efeitos da poliploidização que resultam em reorganização genômica e alterações fisiológicas podem explicar a variação observada nas plantas poliploides sintéticas. / Lippia alba is a medicinal species with a broad phenotypic diversity, including the essential oil composition. Genetic variation is probably the main cause of this variation. The species is a polyploid complex with five chromosome numbers (2n = 30, 38, 45, 60 and 90). Due to the economic importance and the natural genetic variation, this species represents an excellent model in studies on the genomic stability. This work aimed to investigate the genomic stability of 22 natural accessions grown in vitro for 7 years and in synthetic polyploid accessions obtained from the chromosome duplication of a natural diploid access using colchicine. To analyze the genome stability of long-term cultivated plants, four replicates (three maintained in vitro and one in the field) of 22 accessions were analyzed. The size of the genome was verified by flow cytometry and eight ISSR markers checked for stability in the DNA sequence. To evaluate the effect of polyploidization, eleven synthetic polyploid plants composed of 5 tetraploids (4X) and 6 mixoploids (2X/4X) were compared with the diploid parent plant. The comparisons were based on DNA content, chromosomal count, molecular markers ISSR and SSR, and chemical profile of the essential oil. The comparison between the plants maintained in vitro and the respective plants maintained in the field showed that 13 of the 22 accessions suffered a small reduction in the size of the genome. ISSR markers detected polymorphisms in the DNA sequence ranging from 1.61 to 33.87%. Only three accessions did not present polymorphic bands. The number of polymorphic bands among the other 19 accesses ranged from 1 to 21. Analysis of the ploidy of the synthetic polyploid plants carried out on leaves and roots confirmed the stability of the tetraploid plants. The mixoploid plants presented a single G1 peak corresponding to triploid plants. The chromosome analysis showed 60 chromosomes with twelve 45S rDNA sites in the synthetic tetraploids and nine 45S rDNA sites over the 45 chromosomes observed in the synthetics triploid plants. ISSR markers showed polymorphism between the diploid parent plant and synthetic polyploid plants. The polymorphism rate varied from 1.81 to 5.45% in the tetraploid plants and from 43.63 to 56.36% in the triploid plants. Changes in the size of the microsatellite alleles were also detected. The diploid parental plant presented 10 alleles, of which 3 were shared with the triploid plants and 7 with the tetraploid plants. All triploid plants had 13 alleles, 10 of which correspond to new alleles. The number of alleles detected in tetraploid plants ranged from 8 to 11 and the number of new alleles as a consequence of polyploidization ranged from 2 to 4. The major component detected in the essential oil of the parental diploid plant and the tetraploid plants was the citral and the triploid plants was the linalool. The genetic instability detected in L. alba after seven years of in vitro culture may be due to the consequence of natural genome instability combined with long-term in vitro culture. The effects of polyploidization that result in genomic reorganization and physiological changes may explain the variation observed in synthetic polyploid plants.

The genus Populus is ideally suited to investigate questions related to the interplay between an individual’s environmental history and its capacity to respond to external stimuli. In order to dissect the influence of individual history on subsequent plant responses, transcriptome level changes due to water deficit were assessed in clonal populations of Populus hybrids. Results indicate variation in the drought transcriptomes of genetically identical clones originating from different locations can be shaped by the individual history of the clone. Additionally, yearly variations in drought transcriptome patterns showed specific trends associated with a clonal population that were not related to an unknown influence at a location, nor with the biological source of cuttings. Despite these sources of transcriptome variation, a common shared response was identified across all populations. The findings hint at the influence of the environment and epigenetic factors in the dynamic regulation of transcriptome level responses in clonal individuals.