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No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) tese_leticia_neutzling_rickes.pdf: 1993198 bytes, checksum: 613414279ef93ce417b50eba90eee46c (MD5) Previous issue date: 2015-09-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / A fruticultura é um importante componente do agronegócio brasileiro, assumindo destaque cada vez maior no Rio do Grande do Sul, principalmente na região Sul deste Estado. Esta região se destaca por ser a maior detentora da produção de pêssegos do Brasil, porém ainda apresenta a menor produtividade média dos pomares, quando comparado a outros Estados. Isso se deve principalmente pelas condições edafoclimáticas da região, onde há presença de solos com horizonte „A‟ pouco profundo e com problema de drenagem, que dependendo do período do ano, podem sofrer situações de déficit hídrico em períodos críticos para a cultura, prejudicando o desenvolvimento e a produtividade da mesma. A restrição hídrica afeta diretamente o metabolismo das plantas, como por exemplo, a diminuição da taxa assimilatória líquida, o fechamento estomático, a utilização e partição de carboidratos e em nível molecular, a expressão de genes relacionados ao déficit hídrico. Desta forma, este trabalho visou caracterizar respostas diferenciais à nível fisiológico e molecular em plantas do gênero Prunus persica enxertadas sobre diferentes portaenxertos submetidas ao déficit hídrico, a fim de auxiliar na escolha de genótipos mais tolerantes à essa condição hídrica. O trabalho foi dividido em três experimentos, no primeiro, foram avaliados os parâmetros das trocas gasosas da cultivar copa Chimarrita enxertada sobre cinco diferentes portaenxertos (Tsukuba 1, Tsukuba 2, Tsukuba 3, Aldrighi 1 e Seleção UFPel 0402) submetidos ao déficit hídrico, com esquema fatorial: 2 x 8, sendo duas condições hídricas (controle e déficit hídrico) e oito dias de avaliação (0, 1º, 2º, 3º, 4º, 6º, 7º e 9º, sendo os dois últimos, período de recuperação: 24h e 72h, respectivamente). As diferentes combinações de portaenxertos com a cultivar copa Chimarrita, demonstraram comportamento fisiológico diferencial para a tolerância inicial ao déficit hídrico. A combinação „Chimarrita‟/Adrighi 1‟ apresentou-se com uma maior de tolerância inicial ao déficit hídrico. A redução da taxa assimilatória líquida perante o déficit hídrico não está relacionada principalmente à limitação estomática, sugerindo-se que ocorrem também limitações não estomáticas. No segundo experimento, objetivou-se analisar genes normalizadores para estudos de expressão gênica através da técnica de RT-qPCR em folhas de „Chimarrita‟, enxertadas sobre „Aldrighi 1‟ e „Tsukuba 2‟ submetidos ao déficit hídrico por um período de nove dias. Não houve influência das diferentes combinações „Chimarrita‟/portaenxertos para expressão dos genes normalizadores nas condições impostas do presente estudo, sendo os genes TUA e CYP2 considerados os mais adequados mediante as condições impostas. Por outro lado, os genes Ef-1α e ACT foram os menos indicados para as mesmas condições. No terceiro experimento, objetivou-se verificar através da técnica de RT-qPCR a expressão de sete genes-chave envolvidos no processo de resposta ao déficit hídrico em folhas de pessegueiro cultivar copa „Chimarrita‟ enxertadas sobre os portaenxertos „Aldrighi 1‟ e „Tsukuba 2‟. Os resultados demonstraram que houve indução de resposta diferencial da combinação „Chimarrita‟/portaenxertos em relação à expressão dos genes envolvidos no metabolismo do ajustamento osmótico, tais como, SDH, SIP1 GTL e P5SC. O gene S6PDH, apresentou expressão bastante pronunciada e similar para ambas as combinações copa/portaenxertos no quarto dia de estresse, evidenciando a participação e importância do sorbitol no ajustamento osmótico para uma possível tolerância ao déficit hídrico. No entanto, expressão dos genes relacionados ao metabolismo de sorbitol e de prolina, podem ser utilizados como marcadores moleculares para tolerância ao déficit hídrico em portaenxertos de Prunus persica. / Fruit growing is an important component of Brazilian agribusiness, assuming increasing prominence in the Rio Grande do Sul, mainly in the southern region of this state.This region stands out as the largest holder of the production of peaches in Brazil, but also has the lowest average productivity of the orchards when compared to other states. This is mainly the soil and climatic conditions of the region, where there is presence of soils with horizon 'A' superficial and with drainage problem, which depending on the time of year, may suffer water stress situations at critical periods for culture, impairing development and productivity of the same. Water restriction intake directly affects the metabolism of plants, such as the decrease in net assimilation rate, stomatal closure, use and carbohydrates partition and molecular level the expression of genes related to drought. Therefore, this study aimed to characterize differential responses to physiological and molecular level in the genus Prunus persica plants, grafted on different rootstocks subjected to water stress in order support choose the most tolerant to this condition water. The work was divided into three experiments, the first evaluated the parameters of gas exchange of the cultivar cup Chimarrita grafted on five different rootstocks (Tsukuba 1, Tsukuba 2, Tsukuba 3, Aldrighi 1 and Seleção UFPel 0402) submitted to water deficit, with factorial design: 2 x 8, two water conditions (control and water deficit ) and eight days of evaluations (0, 1, 2, 3, 4, 6, 7 and 9, the latter two, recovery time: 24 hours and 72 hours respectively). The different combinations of rootstocks with the cultivar cup Chimarrita demonstrated physiological performance differential for initial tolerance to water deficit. The combination 'Chimarrita'/Aldrighi 1 'presented with an initial greater tolerance to water deficit. The reduced net assimilation rate before the water deficit is not related mainly to stomatal limitation, suggesting that also occur not stomatal limitations. The second experiment aimed to analyze normalizing genes for gene expression studies by RT- qPCR technique in leaves 'Chimarrita' grafted on 'Aldrighi 1' and 'Tsukuba 2' and subjected to water stress for a period of nine days. There was no influence of different combinations 'Chimarrita'/rootstocks for normalizing gene expression of the imposed conditions of this study, and the TUA and CYP2 genes considered best suited by the conditions imposed. However, the EF-1α gene and the ACT less suitable for the same conditions. In the third experiment aimed to verify through RT- qPCR technique the expression of seven key genes involved in the response process to water deficit in peach leaves cultivar cup 'Chimarrita' grafted on rootstocks „Aldrighi 1' and „Tsukuba 2'. The results showed induction differential response of the combination 'Chimarrita'/rootstocks compared to the expression of genes involved in the metabolism of the osmotic adjustment, such as SDH, SIP1, GTL and P5SC. The S6PDH gene, presented quite pronounced and similar expression for both compounds canopy/rootstocks on the fourth day of stress, suggesting a participation and importance of sorbitol in osmotic adjustment for possible tolerance to water deficit. However, expression of genes related to the metabolism of sorbitol and proline, can be used as molecular markers for tolerance to water deficit in rootstocks of Prunus persica.

Maize is susceptible to biotic and abiotic stresses. The most important abiotic stresses in Africa are drought and low soil fertility. Aflatoxin contamination is a potential problem in areas facing drought and low soil fertility. Three studies were conducted to evaluate maize germplasm for tolerance to stress. In the first study, fifteen maize inbred lines crossed in a diallel were evaluated under drought, low N stress, and well-watered conditions at six locations in three countries to estimate general (GCA) and specific combining ability (SCA), investigate genotype x environment interaction, and estimate genetic diversity and its relationship with grain yield and heterosis. GCA effects were not significant for grain yield across environments. Lines with good GCA effect for grain yield were P501 and CML258 across stresses. Lines CML339, CML341, and SPLC7-F had good GCA effects for anthesis silking interval across stresses. Additive genetic effects were more important for grain yield under drought and well-watered conditions. Heterosis estimates were highest in stress environments. Clustering based on genetic distance calculated using marker data from AFLP, RFLP, and SSRs grouped lines according to origin. Genetic distance was positively correlated with grain yield and specific combining ability. In the second study, synthetic hybrids were evaluated at seven locations in three countries to estimate GCA and SCA effects under low N stress and optimal conditions and investigate genotype x environment interaction. GCA effects were significant for all traits across low N stress and optimal conditions. The highest yielding synthetic hybrids involved synthetics developed from stress tolerant lines. Synthetics 99SADVIA-# and SYNA00F2 had good GCA for grain yield across low N stress conditions. Heterosis was highly correlated with grain yield. Optimal environments explained more variation than stress environments. The third study evaluated the agronomic performance and aflatoxin accumulation of single and three-way cross white maize hybrids at five locations in Texas. Inbreds CML343, Tx601W, and Tx110 showed positive GCA effects for grain yield. Significant GCA effects for reduced aflatoxin concentration were observed in lines CML269, CML270, and CML78 across locations. Differences in performance between single and three-way crosses hybrids were dependent mostly on the inbred lines.

In order to improve our understanding on LEA proteins and their molecular functions in drought tolerance, the present work analyzes in the first place, the composition of LEA subproteomes from Arabidopsis seeds and maize embryos; second, three maize embryo LEA proteins from groups 1, 2, and 3 are analyzed in order to detect functional differences among them and finally, transgenic maize plants over-expressing group 5 “rab28” lea gene are characterized. The following results are presented: - Chapter 1. Proteomic approach to analyze the composition of LEA subproteomes from Arabidopsis seeds by mass spectrometry. The main objective was the development and isolation method to obtain enriched LEA populations from Arabidopsis seeds. LEA subproteomes were obtained using an extraction procedure that combines heat stability and acid solubility of LEA proteins. To identify the protein content, we followed two approaches: first, a classical 1D (SDSPAGE) gel-based procedure associated with MS analysis using an electrospray ionization source coupled on-line to liquid chromatography (LC-ESI-MSMS) and second, a gel-free protocol associated with an off-line HPLC and analysis via matrix assisted laser desorption/ionization (LC-MALDI-MSMS). - Chapter 2. Proteomic analysis of LEA proteins accumulated in maize mature seeds. Identification of LEA protein content by mass spectrometry and selection of three LEA proteins, Emb564, Rab17 and Mlg3, as representatives of groups 1, 2 and 3 for further study. Comparative functional analysis covering different aspects of maize Emb564, Rab17 and Mlg3 proteins, posttranslational modifications, subcellular localization and their properties in in-vitro and in- vivo assays. - Chapter 3. Characterization of transgenic maize plants expressing maize group 5 rab28 LEA gene under the ubiquitin promoter. Evaluation of Rab28 transcripts and protein levels, phenotype and stress tolerance traits of transgenic plants under drought stress. Investigation of the subcellular localization of transgenic and wild-type Rab28 protein using immunocytochemical approaches. / Las proteínas LEA, originalmente fueron descritas en las semillas de algodón; se acumulan en grandes cantidades en estructuras tolerantes a la desecación (semillas, polen) y en tejidos vegetativos sometidos a estrés abiótico, sequía, salinidad y frío. También se hallan en organismos anidrobióticos, en plantas de resurrección, algunos invertebrados y microorganismos. La presencia de proteínas LEA se correlaciona con la adquisición de tolerancia a la desecación. Desde un principio se les atribuyó un papel en las respuestas de las plantas en la adaptación al estrés (revisado en Bartels and Salamini 2001, Tunnacliffe 2007, Shih et al. 2010, Tunnacliffe 2010, Hand et al. 2011). Las proteínas LEA se clasifican en diversos grupos en función de dominios y secuencias de aminoácidos específicos (Wise 2010, Batagglia et al 2008, Bies-Ethève et al 2008). Los grupos 1, 2 y 3 son los más relevantes ya que abarcan la mayoría de las proteínas de la familia LEA. Una característica general de estas proteínas es su elevada hidrofilicidad, alto contenido de aminoácidos cargados y su falta de estructura en estado hidratado. A pesar de encontrarse mayoritariamente en forma de “random coil”, algunas adquieren un cierto grado de estructura durante la deshidratación o en la presencia de agentes promotores de α-hélices (Shih et al. 2010, Hand et al. 2011). A nivel celular se han hallado en todas las localizaciones, citosol, núcleo, nucleolo, mitocondria, cloroplasto, vacuola, retículo endoplásmico, peroxisoma y membrana plasmática, donde se supone ejercen su función protectora frente al estrés (Tunnacliffe and Wise 2007, Hundertmark and Hincha 2008). En relación a las modificaciones post-traduccionales, algunas se hallan fosforiladas (Jiang and Wang 2004; Plana et al. 1991, Heyen et al. 2002, Rohrig et al. 2006). Los efectos protectores de las varias proteínas LEA se han demostrado mediante ensayos in vitro y en aproximaciones transgénicas que han dado lugar a fenotipos resistentes a la sequía, sal y frío. Por lo general, se considera que estas proteínas contribuyen a la protección y a la estabilización de macromoléculas y estructuras celulares en las respuestas de adaptación al estrés en plantas; sin embargo, sus funciones específicas aún no han sido esclarecidas. A nivel molecular se ha propuesto que las funciones de las proteínas LEA pueden ser variadas: estabilización y renaturalización de proteínas, mantenimiento de membranas, en combinación, o no, con azúcares, tampones de hidratación (substitución de moléculas de agua), afinidad por iones y función antioxidante (Tunnacliffe and Wise 2007, Shih et al. 2010, Batagglia et al. 2008). Para finalizar, diremos que los objetivos principales de esta tesis consisten en ampliar los conocimientos sobre las proteínas LEA y sus funciones relativas a la tolerancia a la sequía. Los resultados están presentados en forma de capítulos.

Maize is susceptible to biotic and abiotic stresses. The most important abiotic stresses in Africa are drought and low soil fertility. Aflatoxin contamination is a potential problem in areas facing drought and low soil fertility. Three studies were conducted to evaluate maize germplasm for tolerance to stress. In the first study, fifteen maize inbred lines crossed in a diallel were evaluated under drought, low N stress, and well-watered conditions at six locations in three countries to estimate general (GCA) and specific combining ability (SCA), investigate genotype x environment interaction, and estimate genetic diversity and its relationship with grain yield and heterosis. GCA effects were not significant for grain yield across environments. Lines with good GCA effect for grain yield were P501 and CML258 across stresses. Lines CML339, CML341, and SPLC7-F had good GCA effects for anthesis silking interval across stresses. Additive genetic effects were more important for grain yield under drought and well-watered conditions. Heterosis estimates were highest in stress environments. Clustering based on genetic distance calculated using marker data from AFLP, RFLP, and SSRs grouped lines according to origin. Genetic distance was positively correlated with grain yield and specific combining ability. In the second study, synthetic hybrids were evaluated at seven locations in three countries to estimate GCA and SCA effects under low N stress and optimal conditions and investigate genotype x environment interaction. GCA effects were significant for all traits across low N stress and optimal conditions. The highest yielding synthetic hybrids involved synthetics developed from stress tolerant lines. Synthetics 99SADVIA-# and SYNA00F2 had good GCA for grain yield across low N stress conditions. Heterosis was highly correlated with grain yield. Optimal environments explained more variation than stress environments. The third study evaluated the agronomic performance and aflatoxin accumulation of single and three-way cross white maize hybrids at five locations in Texas. Inbreds CML343, Tx601W, and Tx110 showed positive GCA effects for grain yield. Significant GCA effects for reduced aflatoxin concentration were observed in lines CML269, CML270, and CML78 across locations. Differences in performance between single and three-way crosses hybrids were dependent mostly on the inbred lines.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Sutherlandia frutescens (L.) R. Br., also regarded as Lessertia frutescens, is a leguminous, perennial shrub indigenous to South Africa. Extracts prepared from the leaves have traditionally been used for the treatment of various diseases. Reports have also indicated that S. frutescens provides certain health benefits to cancer and HIV/AIDS patients. Analysis of extracts indicated the presence of several compounds (bitter triterpenoid glycosides, several flavonoids, amino acids, small amounts of saponins (no alkaloids though), asparagine, Larginine, canavanine, gamma-aminobutyric acid (GABA) and pinitol) which contribute to the medicinal properties of this plant. The first part of this study involved testing the effect of six treatments (light, dark, soaking of seeds, physical scarification, chemical scarification and flaming of seeds) on the in vitro germination of Sutherlandia seeds to elucidate the factors which control seed germination. Those treatments which removed the seed coat were most successful for germination with physical scarification being the most efficient method, resulting in 98.6% of the seeds germinating after 21 days. Although the organogenesis of Sutherlandia explants (cotyledons and hypocotyls) in vitro were investigated (results not included in this thesis), omitting plant growth regulators (PGR) in the cultivation medium was best for shoot multiplication. However, this PGR-free system successfully provided a continuous supply of plant material for further studies. It would be possible to successfully adopt it for commercial production of plants to assist with cultivation of Sutherlandia as a field crop. Another advantage of this system is spontaneous rooting with 85% of the in vitro microshoots rooting in PGR-free medium. These rooted plants were acclimated in the glasshouse using vented lids to harden off the shoots and this method resulted in 100% survival of plants. The second part of this study investigated the induction of hairy root cultures of S. frutescens using Agrobacterium-mediated transformation. The efficiency of three Agrobacterium strains (A4T, LBA9402 and C58C1) to transform different S. frutescens explants (cotyledons and hypocotyls) was analyzed. All three strains were equally efficient at inducing hairy roots in both hypocotyls and cotyledons. However, transformation of S. frutescens was dependent on the type of explant used with the hypocotyls being more efficiently transformed than the cotyledons. Overall the transformation of both the hypocotyl (93%) and cotyledon (47%) was highest when the strain A4T was used. Four hairy root clones were selected and their cultivation in a liquid system was optimized by investigating their growth in four different types of media (Gamborg B5 (Gamborg et al., 1968), White’s (White, 1934; White, 1954), MS (Murashige and Skoog, 1962) and half strength MS medium). All the growth of hairy root clones was best in the B5 and MS medium, with White’s medium being the least effective cultivation medium. Molecular analysis of hairy roots was used to prove the transgenic status of these four putative transgenic clones. This was achieved using polymerase chain reaction (PCR) amplification of rol A (320 bp), B (780 bp) and C (600 bp) genes to determine the presence of the TL-DNA in the plant genome. During Southern hybridization a radioactively labeled rol A probe was used to determine the copy number of the rol A gene. The three rol genes were present in all four hairy root clones. The third part of this study focused on the effect of three abiotic stress factors (nitrogen availability, salinity and drought) on the synthesis of four metabolites (gamma-aminobutyric acid (GABA), asparagine, arginine and canavanine). The effect of nitrogen availability on metabolite synthesis and the morphology was determined using in vitro shoot cultures as well as the hairy root clone C58C1-g. Nitrogen availability studies were conducted by cultivating the microshoots or root tips on modified MS medium. The MS medium contained either the normal amount of nitrogen (1.9 g L-1 KNO3 and 1.65 g L-1 NH4NO3) in the MS medium (1x nitrogen), half the normal nitrogen concentration in MS medium (0.5x nitrogen) or twice the normal nitrogen concentration in MS medium (2x nitrogen). The arginine and asparagine levels in the roots and shoots and the canavanine level in the shoots were directly correlated with the amount of nitrogen in the medium (as the nitrogen level increased, the metabolite levels increased). The GABA level in the shoots was inversely correlated with the amount of nitrogen in the medium. Several reasons may explain these metabolic changes including the assimilation of extra nitrogen into asparagine, canavanine and arginine in the shoots. The reduced GABA levels may indicate the preferential flux of the free GABA into other nitrogen assimilatory pathways such as protein synthesis as well as its rapid utilization to replenish the tricarboxilic acid cycle intermediates. The effect of water (induced by including 3% (w/v) PEG in the medium) and salt stress (induced by including either 50 or 100 mM NaCl in the medium) was only investigated in the shoot cultures as the root cultures lacked the synthesis of canavanine. Water stress did not significantly alter the metabolite levels, but resulted in a significant decrease in the growth (fresh weight and total shoot length) and the rooting response of these microshoots. Salt stress only resulted in a significant increase in arginine levels with increasing salinity and also caused a reduction in the rooting and growth response. Lowered plant vigour may be the first visual sign of water stress. Addition of NaCl may lead to ion toxicity and requires osmotic adjustment resulting in changes at the metabolic level concomitant to physiological growth changes. Finally, the anti-bacterial activity and the phytochemistry of transgenic root cultures and untransformed in vitro and ex vitro plant material was examined. Only the extracts prepared from the wild harvested leaf material exhibited moderate anti-bacterial activity (1.25 mg ml-1) against all the bacteria (Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis and Staphylococcus aureus) tested. Changes to the secondary metabolism of hairy roots were investigated using TLC and LC-MS analysis. Several of the compounds in the hairy root extracts were present in higher levels than in the control root extracts. Transformation also increased the complexity of the phytochemical pattern of the hairy roots, either due the synthesis of novel compounds or upregulated synthesis of existing metabolic pathways. The production of hairy roots and the establishment in a liquid system during this study was an important step towards upscaling these cultures to a bioreactor. In future these roots can assist in developing cultures which produce a high yield of the desired metabolites. / AFRIKAANSE OPSOMMING: Sutherlandia frutescens (L.) R. Br., ook bekend as Lessertia frutescens is ‘n peulagtige meerjarige struik, inheems tot Suid Afrika. Ekstrakte wat van die blare voorberei word, is tradisioneel gebruik vir die behandeling van verskeie siektes. Berigte het ook daarop gedui, dat S. frutescens sekere gesondheidsvoordele vir kanker en HIV/VIGS pasiënte inhou. ‘n Ontleding van die ekstrakte, dui op die teenwoordigheid van verskeie verbindings (bitter triterpenoïed glikosiede, verskeie flavonoïede, aminosure, klein hoeveelhede saponiene (alhoewel geen alkaloïede), asparagien, L-arginien, canavanien, gamma-aminobottersuur (GABS) en pinitol) wat tot die medisinale eienskappe van hierdie plant bydrae. Die eerste deel van die studie het die effek van ses behandelings (lig, donker, week van sade, fisiese skarifikasie, chemiese skarifikasie en die vlam van sade) op die in vitro ontkieming van Sutherlandia sade getoets met die doel om die faktore wat saadontkieming beheer, te identifiseer. Die beste behandeling vir saadontkieming was dié behandelings wat die saadhuid verwyder het. Die mees effektiewe metode van saadhuidverwydering was die fisiese skarifikasie van sade, wat gelei het tot ‘n 98.6% ontkieming van sade na 21 dae. Alhoewel in vitro organogenese van Sutherlandia eksplante (kotiel en hipokotiel) ondersoek was (resultate nie ingesluit in die tesis nie), was plant groei reguleerders (PGR) uitgesluit in die groeimedium om stingelvermeerdering te bevorder. Nie te min was die PGR-vrye sisteem suksesvol om ‘n voortdurende bron van plant material vir verder studies te verskaf. Dit sou egter moontlik wees om die PGR-vrye sisteem suksesvol te kon aanpas vir die kommersiële produksie van plante met die doel om Sutherlandia as ‘n landbougewas te bevorder. ‘n Verdere voordeel van dié sisteem, is die spontane wortelvorming, met 85% van die in vitro mikrostingels wat wortels in die PGR-vrye medium produseer het. Hierdie bewortelde plante was in die glashuis geakklimatiseer met behulp van geventileerde deksels (vir stingel afharding) en het tot ‘n 100% oorlewing gelei. Die tweede deel van die studie het die induksie van S. frutescens harige wortelkulture met behulp van Agrobacterium-bemiddelde transformasie ondersoek. Die effektiwiteit van drie Agrobacterium stamme (A4T, C58C1 en LBA9402) om verskillende S. frutescens eksplante (kotiel en hipokotiele) te transformeer, was geanaliseer. Al drie stamme was ewe effektief om harige wortels op beide hipokotiel en kotiele te induseer. S. frutescens transformasie blyk egter tog van die tipe eksplant afhanklik te wees, aangesien die hipokotiele meer effektief as die kotiele getransformeer kon word. Met inagneming van beide die hipokotiel (93%) en kotiel vii (47%), was transformasie optimaal met die gebruik van die A4T stam. Vier harige wortelklone was geselekteer en hulle produksie in ‘n vloeibare sisteem was geoptimiseer deur hulle groei in vier verskillende tipe media (Gamborg B5 (Gamborg et al., 1968), White’s (White, 1934; White, 1954), MS (Murashige and Skoog, 1962) en half-sterkte MS medium) te ondersoek. B5 en MS medium was beskou as die beste vir alle die harige wortelklone se groei, terwyl White’s medium die minste doeltreffende groeimedium was. Molekulêre analise van die harige wortels was gebruik ten einde die transgeniese status van die vier vermoedelike transgeniese klone te bewys. Dit was behaal deur polimerase kettingreaksie amplifisering (PKR) van die rol A, B en C gene ten einde die teenwoordigheid van die TL-DNS in die plant genoom aan te toon. Tydens Southern hibridisasie was ‘n radioaktief gemerkte peiler gebruik om die aantal rol A geen kopieë te bepaal. Die drie rol gene was teenwoordig in al vier harige wortelklone. Die derde deel van die studie het gefokus op die effek van drie abiotiese stress faktore (stikstof beskikbaarheid, sout- en droogte stres) op die produksie van vier metaboliete (GABS, asparagien, canavanien en arginien). Die effek van stikstof beskikbaarheid op die metaboliet produksie asook die morfologie was bestudeer deur gebruik te maak van in vitro mikrostingels asook die harige wortel kloon C58C1-g. Stikstof beskikbaarheidstudies was uitgevoer deur die mikrostingels of wortelpunte in ‘n gewysigde MS medium te groei. Die MS medium was aangepas om die normale hoeveelheid stikstof (1.9 g L-1 KNO3 en 1.65 g L-1 NH4NO3) in MS medium (1x stikstof), of die helfte van die normale stikstof konsentrasie (0.5x stikstof) of twee keer die normale stikstof konsentrasie in MS medium (2x stikstof) te bevat. Die arginien en asparagien vlakke in die wortels en stingels, asook die canavanien vlak in die stingels was positief gekorreleerd aan die stikstof konsentrasie in die medium. Die GABS vlak in die stingels was egter omgekeerd eweredig aan die stikstof konsentrasie in die medium. Verskeie redes kan aangevoer word om die metaboliet veranderinge te verduidelik, insluitende die assimilasie van addisionele stikstof in asparagien, canavanien en arginien in die stingels. Die verlaagde GABS vlakke kan dui op die voorkeur van vrye GABS vloei na ander stikstofassimilerende metaboliese paaie soos proteïen sintese, asook die snelle benutting van GABS ten einde die Trikarboksielsuursiklus intermediêre produkte aan te vul. Die effek van droogte (geïnduseer deur die byvoeging van 3% (m/v) PEG tot die medium) en sout stres (geïnduseer deur 50 of 100 mM NaCl byvoeging tot die medium) was slegs in die stingel kulture ondersoek weens die afwesigheid van canavanien produksie in die wortel kulture. Water stres het nie ‘n betekenisvolle verandering in die metaboliet vlakke meegebring nie, maar dit het wel tot ‘n beduidende afname in groei (vars massa en totale stingel lengte) en bewortelingsreaksie in die mikrostingels gelei. Sout stres het slegs tot ‘n betekenisvolle viii toename in arginien vlakke asook ‘n afname in die wortelvorming en groeireaksie tydens die toenemende sout vlakke gelei. ‘n Verlaging in plant groeikragtigheid mag ‘n eerste visuele teken van water stres wees. Die toevoeging van NaCl tot die medium kan tot ioontoksisiteit lei en plante reageer deur middel van osmotiese aanpassing wat tot veranderinge in die metaboliet vlakke asook veranderinge in fisiologiese groei, lei. Die finale deel van die studie het die anti-bakteriële aktiwiteit en die fitochemie van die transgeniese wortel kulture asook die ongetransformeerde in vitro en ex vitro plant materiaal ondersoek. Slegs die ekstrakte verkry vanaf blaar materiaal geoes uit die natuur, het matige anti-bakteriële aktiwiteit (1.25 mg ml-1) teen al die bakterië (Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis en Staphylococcus aureus) wat ondersoek is, getoon. Aanpassings in die sekondêre metabolisme van die harige wortels is deur middel van dunlaag chromatografie (DLC) en vloeibare chromatografie-massa spektroskopiese (VC-MS) analise ondersoek. Verskeie verbindings was in hoër vlakke in die harige wortels teenwoordig, as in die kontrole wortel ekstrakte. Transformasie het ook die kompleksiteit van die harige wortels se fitochemiese patroon verhoog, moontlik weens die produksie van nuwe verbindings of weens die opregulasie van bestaande metaboliese paaie. Die produksie van harige wortels en die vestiging daarvan in ‘n vloeibare sisteem tydens hierdie studie word beskou as ‘n belangrike stap na die opskalering van die kulture na bioreaktore. Hierdie wortels kan toekomstig tot die ontwikkeling van kulture met ‘n hoë produksie van gewenste metaboliete lei.

All plants have a protection against the surrounding environment called a cuticle coating. When this cuticle coating is constructed it is believed that the family of protein called lipid transfer proteins (LTPs) is involved. The LTPs are small and cysteine rich. In Marchantia polymorpha the groups of LTPs called LTPd and LTPg can be found. 8 and 4 in each group respectively. In the genes of LTPd there is an intron placed downstream of the start codon. Firstly, a sequence database search was performed and LTPd2 and LTPd3 were chosen for further experiments in this study. Secondly, a control that the intron was present in the samples were done by preforming a PCR reaction of cDNA from isolated RNA taken from untreated Marchantia polymorpha. A gel electrophoresis of the product was also performed. Lastly, the amount of alternative splicing in LTPd2 and LTPd3 from Marchantia polymorpha after treated with cold and dehydration were studied using quantitative PCR. For the qPCR MpACT and the exon of respective gene were used as references. The ΔCt values and the expression fold (2ΔΔCt) calculated from the qPCR results showed that most of the transcript with introns preserved were upregulated after subjected to stress. Only the intron in MpLTPd2 and MpLTPd3 with MpACT as reference showed a small downregulation after the cold treatment. The intron in MpLTPd3 with MpLTPd3s exon as reference didn’t show any difference. None of the intron transcript in any of the genes on the other hand showed any significant difference in the alternative splicing. This could be because of small sample groups when the test was performed. In conclusion, there were no significant difference in intron expression between treated and control samples. Therefore, nothing can be said about the change in alternative splicing in MpLTPds after cold and dehydration treatments.

Doctor of Philosophy / Department of Agronomy / S.V. Krishna Jagadish / Sorghum (Sorghum bicolor (L.) Moench) is one of the hardiest crop to abiotic stresses compared with other grain crops. However early stage chilling, terminal heat and drought stress are three most damaging abiotic stresses that have limited sorghum productivity in the US Great plains and other locations having similar environmental conditions. Three studies were conducted with an overall goal aimed at increasing grain sorghum’s resilience to harsh climatic conditions. In the first study, four promising chilling stress tolerant sorghum advanced breeding lines, a known early stage chilling tolerant Chinese landrace (Shan Qui Red - SQR) and a susceptible US elite cultivar (RTx430) as checks were assessed for chilling tolerance during emergence and early growth under field and controlled environments. Aerial phenotyping using unmanned aircraft systems (UAS) fitted with multispectral camera was used to capture reflectance-based vegetation indices (NDVI and NDRE) in field experiments. Some advanced breeding lines with superior agronomic background also recorded significantly better emergence, seedling growth and vigor compared to SQR under chilling conditions. Aerial phenotyping indices from images taken between 30 and 60 days after emergence were consistently correlated with destructive measurements under early plantings, indicating their effectiveness in differentiating chilling responses. Second study was conducted to understand physiological mechanisms inducing heat stress resilience in sorghum during flowering. A diverse set of sorghum inbreds and selected hybrids were tested under greenhouse, growth chamber facilities and field conditions. A highly conserved early-morning-flowering mechanism was observed across all the inbreds and hybrids, with the peak anthesis wherein >90% of florets completed flowering within 30 min after dawn. The conserved response was consistent even under drought stress and heat stress exposure imposed at different times of the day. Our findings report a novel heat escaping early-morning-flowering mechanism effectively employed by sorghum to minimize heat stress impact at anthesis. Another experiment with sequential increase in daytime temperature treatments suggest heat stress induced loss in pollen viability to be a key factor resulting in reduced seed-set and grain yield. The findings suggest heat stress could have a greater impact on post-pollen germination processes such as fertilization, embryo formation and development. We identified a heat tolerant genotype “Macia” which appears to be a promising donor for developing improved heat tolerant sorghum hybrids. In the third study, a bi-parental recombinant inbred lines (RILs) mapping population developed from elite post flowering drought susceptible cultivar (RTx430) and a known drought tolerant cultivar (SC35) were evaluated under wide spectrum of environments and moisture conditions. Several novel and major QTL for grain yield, panicle neck diameter, effective quantum yield of photosystem II and chlorophyll content were identified. The genomic regions and the candidate genes within these regions can potentially help in improving source and sink dynamics in sorghum under diverse environments. The findings from these studies will complement ongoing efforts in developing future sorghum with enhanced resilience to different abiotic stresses that continue to limit sorghum productivity.

Made available in DSpace on 2014-06-11T19:33:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-04-28Bitstream added on 2014-06-13T21:06:43Z : No. of bitstreams: 1 carlin_sd_dr_jabo.pdf: 560339 bytes, checksum: 8b2e1669f83b62325f0519f3baf373d4 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / No presente trabalho foi verificado o ajuste osmótico no crescimento inicial de cana-de-açúcar, cv. IAC91-5155, sob efeito da interação dos estresses hídrico e ácido no solo. Para tanto, foram quantificados os teores de três solutos compatíveis, prolina, glicina betaína e trealose. Também foi estudada a atividade da nitrato redutase e os teores de clorofilas. O estudo de todas estas variáveis teve por objetivo propor a utilização destes compostos como indicadores fisiológicos de aclimatação da cana-deaçúcar sob efeito da interação de ambos os estresses. O experimento foi realizado em casa de vegetação com 29,7±4,3oC e 75±10 UR%. Foram utilizados três tratamentos de disponibilidade hídrica (capacidade de campo, CC): controle (70%), estresse moderado (55%) e estresse severo (40%) e três tratamentos de acidez no solo (saturação por bases, V%): controle (55%), média acidez (33%) e alta acidez (23%). O delineamento experimental utilizado foi o de blocos casualizados em esquema fatorial 3x3, com quatro repetições. Após 60 dias sob estresses foram determinados os teores dos solutos compatíveis, a atividade da nitrato redutase e os teores das clorofilas. Os resultados mostram que houve acúmulo dos três solutos compatíveis evidenciando que ocorreu ajuste osmótico. Prolina e glicina betaína são indicadores fisiológicos do efeito da interação de ambos os estresses enquanto trealose é indicadora apenas para o estresse hídrico. Também, a atividade da nitrato redutase pode ser utilizada como indicador do estresse hídrico e os teores das clorofilas a, b e total para os estresses hídrico ou ácido. A massa de matéria seca da parte aérea, o número de folhas e a área foliar das plantas jovens de cana-de-açúcar, ajustadas osmoticamente, são reduzidos pelos estresses. Os efeitos interativos de ambos... / In this present work was verified the osmotic adjustment in the initial plant growth sugarcane cv. IAC91-5155 under interaction effect of water and acid stress in the soil. For this, were quantified three compatible solutes content, as follows: proline, glycine betaine and trehalose. It was verified also the nitrate reductase activity as well the chlorophyll content. The study of all parameters aimed to propose the use components as physiological indicators to acclimation of sugarcane plants under interaction the both stress. The experiment was carried out in a greenhouse under 29.7±4.3oC and 75±10%. Three water availability treatments (Field Capacity – FC%): control (70%), moderate stress (55%), and extreme stress (40%) and three soil acidification treatment (Base Saturation – BS%): control (55%), average acidity (33%), and high acidity (23%) were used. The experimental design used was the randomized blocks in a 3x3 factorial scheme and four replicates. After sixty days under stress were determined compatible solutes, nitrate reductase and chlorophyll content. Our results showed that there was the accumulation of three compatible solutes exhibiting osmotic adjustment. Glycine betaine and proline are both physiologic indicators of stresses interaction effect whereas trehalose indicates only water stress. The nitrate reductase activity can be used as water stress indicator and chlorophylls a, b and total are indicators to water or acid stress. The dry mass of shoot part, leaves number and leaf area are significantly reduced by stress in young sugarcane plants adjusted osmotically. The interaction of stresses cause decrease of dry mass while water stress reduces leaves number and leaf area.

Made available in DSpace on 2015-03-26T13:42:03Z (GMT). No. of bitstreams: 1 texto completo.pdf: 289132 bytes, checksum: 74e110bc985b149f74592a1fcdd77125 (MD5) Previous issue date: 2007-08-15 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Aluminum (Al) toxicity is one of the major constraints for agriculture on acid soils, which occupy large regions of the world s agricultural area. At low pH values associated with these soils Al3+ is solubilized into soil solution and is toxic to plants inhibiting root growth and crop yield. Cultivars genetically adapted to acid soils may offer an environmental compatible solution. The aims of this work were (1) to estimate parameters and genetics gains in three cycles of divergent selection to aluminum tolerance and (2) to identify changes in allelic frequencies at five loci near a QTL in chromosome 5 of maize that explain 13% of the Al tolerance. The phenotypic index employed was relative seminal root length (CLR) obtained from nutrient solution. Simple sequence repeats (SSR) were used to detect linkage disequilibrium between marker and QTL. An expressive genetic gain of 49,15% was observed from base population to first generation. This evidence is characteristic from traits that have oligogenic inheritance. Shifts in allelic frequencies in 4 loci in first generation and in all loci, in second generation were exclusively due to effects of genetic drift. / A toxicidade ao alumínio (Al) é um dos maiores problemas para a agricultura em solos ácidos, que ocupam grandes áreas agricultáveis no mundo. Em condições de baixo pH associado a estes solos, o Al3+ é solubilizado e se torna tóxico para as plantas, inibindo o crescimento de suas raízes e comprometendo a produtividade das culturas. Genótipos adaptados aos solos ácidos podem oferecer uma solução sustentável a este problema. Os objetivos deste trabalho foram (1) estimar parâmetros e ganhos genéticos obtidos ao longo de três ciclos de seleção divergente para a tolerância e (2) identificar alterações nas freqüências alélicas em cinco locos próximos a um QTL no cromossomo 5 do milho que explica 13% da tolerância ao Al. O índice fenotípico empregado, foi o Crescimento Liquido Relativo (CLR) obtido a partir do cultivo em solução nutritiva. Marcadores microssatélites (SSR) foram utilizados para detectar desequilíbrio de ligação entre as marcas e o QTL. Ocorreu um ganho genético expressivo da população base à primeira geração de 49,15%, diminuindo drasticamente para os ciclos posteriores. Tal evidência é patente de caráter de herança oligogênica. Variações nas freqüências alélicas em 4 locos, na primeira geração, e em todos os locos, na segunda geração, foram explicadas exclusivamente pela deriva genética.

Submitted by Renata Bruna dos Santos Coscolin null (rbscoscolin@fca.unesp.br) on 2017-01-16T22:27:20Z No. of bitstreams: 1 definitivo tese 8_VF_Completa.pdf: 3379003 bytes, checksum: 659bf515460d03427a658914feaa471a (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-01-19T18:33:58Z (GMT) No. of bitstreams: 1 coscolim_rbs_dr_bot.pdf: 3379003 bytes, checksum: 659bf515460d03427a658914feaa471a (MD5) / Made available in DSpace on 2017-01-19T18:33:58Z (GMT). No. of bitstreams: 1 coscolim_rbs_dr_bot.pdf: 3379003 bytes, checksum: 659bf515460d03427a658914feaa471a (MD5) Previous issue date: 2016-11-17 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O amendoim é uma cultura de interesse econômico para o estado de São Paulo, principalmente para renovação de canaviais e pastagens na região oeste do estado. Nesta região, os crescentes custos de produção do amendoim acompanhados por baixo rendimento por área devido à suscetibilidade às variações climáticas são influências negativas e contribuem para desestimular a produção dessa cultura. Nesse âmbito, a associação com fungos micorrízicos arbusculares (FMAs) e suplementação com bioestimulante a base de extrato solúvel de algas (Ascophyllum nodosum) (ESA) podem promover melhorias no crescimento e desenvolvimento da planta, além de atenuarem os efeitos negativos provocados pela deficiência hídrica. As plantas foram cultivadas em estufa agrícola, com monitoramento das relações hídricas além do estudo de trocas gasosas, análise de crescimento, análises microbiológicas e de produção, bem como das respostas metabólicas das plantas em função dos tratamentos. A presente pesquisa valida os benefícios para a cultura do amendoim quando em associação com os FMAs e / ou com a suplementação com o ESA, pois além de incrementarem o crescimento e taxa de assimilação líquida de carbono nas plantas em condições hidratadas, mantiveram o status hídrico das plantas e proveram também o acionamento de enzimas do complexo antioxidativo nas plantas em condições de deficiência hídrica moderada e severa. / Water is essential element for plant development and its absence or deficiency induces physiological changes with severe consequences for productivity. Peanut is a culture of economic interest to the State of São Paulo especially in the western region. In this region, the increasing of production costs in peanuts is associate by low yield due to susceptibility to climate variations, such as dry seasons. They are negative influences and do not contribute to production in that crop. In this context, the association with arbuscular mycorrhizal fungi (AMF) and supplementation with biostimulant based on soluble algal extract (Ascophyllum nodosum) (ESA) can promote improvements in plant growth and development, as well as attenuate the negative effects caused by water deficiency. The plants grown in a greenhouse with monitoring of water relations in addition to the study of gas exchange parameters, growth analysis, microbiological analysis, production, and metabolic responses of plants in the treatments. The present study validates the benefits for peanut cultivation with fungus and/or seaweed extract supplementation. Plants under hydrated conditions had better performance in growth and carbon assimilation rate and under conditions of moderate and severe water deficiency.of, they maintained water status of the plants and also provided the activation of enzymes of the antioxidative complex. / CNPq: 141167/2014-9