FERREIRA, D. A. Reações biocatalíticas usando células íntegras de Lens culinaris (Lentilha). 2012. 144 f. Tese (Doutorado em Química) - Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2012. / Submitted by José Jairo Viana de Sousa (jairo@ufc.br) on 2014-10-17T19:36:21Z No. of bitstreams: 1 2012_tese_daferreira.pdf: 4506710 bytes, checksum: 94cfd04a00906636065e44839b576c29 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa(jairo@ufc.br) on 2015-03-10T22:06:31Z (GMT) No. of bitstreams: 1 2012_tese_daferreira.pdf: 4506710 bytes, checksum: 94cfd04a00906636065e44839b576c29 (MD5) / Made available in DSpace on 2015-03-10T22:06:32Z (GMT). No. of bitstreams: 1 2012_tese_daferreira.pdf: 4506710 bytes, checksum: 94cfd04a00906636065e44839b576c29 (MD5) Previous issue date: 2012 / This paper describes the use of seeds of lentil (Lens culinaris) as intact plant system in biocatalytic reactions of reduction and hydrolysis. Initially, some plant sources were evaluated for their action in the biocatalytic reduction of aromatic ketone acetophenone. The plants that showed positive results in the reduction reaction underwent determination of their protein content by the methods of Lowry and Bradford. Lens culinaris was plant species revealed that most relevant biocatalytic activity, in agreement with their high protein content (1.33 mg / mL and 1.07 mg / mL according to Bradford and Lowry methods, respectively), and thus The plant was selected for further study. Subsequently, always using the model substrate acetophenone as the reaction parameters were optimized: concentration of biocatalyst; reaction kinetics; stirring speed the reaction medium. After selecting these parameters, the bioreduction reactions were extended to other derivatives of prochiral acetophenone, which were observed with bioconversions yields and enantiomeric excess (ee) ranging from low to high percentages. Generally, were observed steric and electronic influences due to the type and relative position of the various substituents on aromatic compounds. In the case of substrates containing two different functional groups susceptible to reduction, low chemoselectivity observed. Investigated also other carbonyl compounds such as aliphatic and aromatic ketones, aromatic aldehydes and aromatic nitro compounds. In addition investigated the possibility that hydrolytic action from compounds belonging functions esters, amides and nitriles. The yields of the reactions were calculated using Gas Chromatography coupled with Mass Spectrometry (GC-MS) and Nuclear Magnetic Resonance Hydrogen (1H NMR). The enantiomeric excess (ee) were determined by High Performance Liquid Chromatography (HPLC) equipped with chiral columns. / Este trabalho relata a utilização das sementes de lentilha (Lens culinaris) como sistema íntegro de planta em reações biocatalíticas de redução e hidrólise. Inicialmente, algumas fontes vegetais foram avaliadas quanto sua ação biocatalítica na redução da cetona aromática acetofenona. Os vegetais que revelaram resultados positivos na reação de redução foram submetidos à determinação do seu teor de proteínas através dos métodos de Lowry e Bradford. Lens culinaris foi à espécie vegetal que revelou atividade biocatalítica mais relevante, em acordo com seu alto teor de proteína (1,33 mg/mL e 1,07 mg/mL, segundo os métodos de Bradford e Lowry, respectivamente), e assim, foi o vegetal selecionado para estudos mais aprofundados. Posteriormente, utilizando sempre a acetofenona como substrato modelo foram otimizados os parâmetros reacionais: concentração do biocatalisador; cinética de reação; velocidade de agitação e meio reacional. Após seleção desses parâmetros, as reações de biorredução foram estendidas a outros derivados pró-quirais da acetofenona, onde foram observadas bioconversões com rendimentos e excessos enantioméricos (ee) que variaram de baixas a elevadas porcentagens. De modo geral, foram observadas influencias eletrônica e estérica devidas ao tipo e a posição relativa dos diferentes substituintes nos compostos aromáticos. No caso de substratos contendo dois grupos funcionais diferentes suscetíveis de redução, foi observada baixa quimiosseletividade. Foram investigados também outros compostos carbonilicos, como: cetonas alifáticas e aromáticas, aldeídos aromáticos e nitrocompostos aromáticos. Em adição foi investigado a possibilidade de ação hidrolítica a partir de compostos pertencente às funções ésteres, amidas e nitrilas. Os rendimentos das reações foram calculados utilizando Cromatografia Gasosa acoplada à Espectrometria de Massas (CG-EM) e Ressonância Magnética Nuclear de Hidrogênio (RMN 1H). Os excessos enantioméricos (ee) foram determinados através de Cromatografia Líquido de Alta Eficiência (CLAE) equipada com colunas quirais.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / This paper describes the use of seeds of lentil (Lens culinaris) as intact plant system in biocatalytic reactions of reduction and hydrolysis. Initially, some plant sources were evaluated for their action in the biocatalytic reduction of aromatic ketone acetophenone. The plants that showed positive results in the reduction reaction underwent determination of their protein content by the methods of Lowry and Bradford. Lens culinaris was plant species revealed that most relevant biocatalytic activity, in agreement with their high protein content (1.33 mg / mL and 1.07 mg / mL according to Bradford and Lowry methods, respectively), and thus The plant was selected for further study. Subsequently, always using the model substrate acetophenone as the reaction parameters were optimized: concentration of biocatalyst; reaction kinetics; stirring speed the reaction medium. After selecting these parameters, the bioreduction reactions were extended to other derivatives of prochiral acetophenone, which were observed with bioconversions yields and enantiomeric excess (ee) ranging from low to high percentages. Generally, were observed steric and electronic influences due to the type and relative position of the various substituents on aromatic compounds. In the case of substrates containing two different functional groups susceptible to reduction, low chemoselectivity observed. Investigated also other carbonyl compounds such as aliphatic and aromatic ketones, aromatic aldehydes and aromatic nitro compounds. In addition investigated the possibility that hydrolytic action from compounds belonging functions esters, amides and nitriles. The yields of the reactions were calculated using Gas Chromatography coupled with Mass Spectrometry (GC-MS) and Nuclear Magnetic Resonance Hydrogen (1H NMR). The enantiomeric excess (ee) were determined by High Performance Liquid Chromatography (HPLC) equipped with chiral columns. / Este trabalho relata a utilizaÃÃo das sementes de lentilha (Lens culinaris) como sistema Ãntegro de planta em reaÃÃes biocatalÃticas de reduÃÃo e hidrÃlise. Inicialmente, algumas fontes vegetais foram avaliadas quanto sua aÃÃo biocatalÃtica na reduÃÃo da cetona aromÃtica acetofenona. Os vegetais que revelaram resultados positivos na reaÃÃo de reduÃÃo foram submetidos à determinaÃÃo do seu teor de proteÃnas atravÃs dos mÃtodos de Lowry e Bradford. Lens culinaris foi à espÃcie vegetal que revelou atividade biocatalÃtica mais relevante, em acordo com seu alto teor de proteÃna (1,33 mg/mL e 1,07 mg/mL, segundo os mÃtodos de Bradford e Lowry, respectivamente), e assim, foi o vegetal selecionado para estudos mais aprofundados. Posteriormente, utilizando sempre a acetofenona como substrato modelo foram otimizados os parÃmetros reacionais: concentraÃÃo do biocatalisador; cinÃtica de reaÃÃo; velocidade de agitaÃÃo e meio reacional. ApÃs seleÃÃo desses parÃmetros, as reaÃÃes de biorreduÃÃo foram estendidas a outros derivados prÃ-quirais da acetofenona, onde foram observadas bioconversÃes com rendimentos e excessos enantiomÃricos (ee) que variaram de baixas a elevadas porcentagens. De modo geral, foram observadas influencias eletrÃnica e estÃrica devidas ao tipo e a posiÃÃo relativa dos diferentes substituintes nos compostos aromÃticos. No caso de substratos contendo dois grupos funcionais diferentes suscetÃveis de reduÃÃo, foi observada baixa quimiosseletividade. Foram investigados tambÃm outros compostos carbonilicos, como: cetonas alifÃticas e aromÃticas, aldeÃdos aromÃticos e nitrocompostos aromÃticos. Em adiÃÃo foi investigado a possibilidade de aÃÃo hidrolÃtica a partir de compostos pertencente Ãs funÃÃes Ãsteres, amidas e nitrilas. Os rendimentos das reaÃÃes foram calculados utilizando Cromatografia Gasosa acoplada à Espectrometria de Massas (CG-EM) e RessonÃncia MagnÃtica Nuclear de HidrogÃnio (RMN 1H). Os excessos enantiomÃricos (ee) foram determinados atravÃs de Cromatografia LÃquido de Alta EficiÃncia (CLAE) equipada com colunas quirais.

iv ABSTRACT GENETIC TRANSFORMATION OF LENTIL ( Lens culinaris M. cv.Sultan.1) WITH A TRANSCRIPTION FACTOR REGULATOR (MBF1c) AND ANALYSIS OF TRANSGENIC PLANTS KAM&Ccedil / I, Hamdi Ph.D., Biotechnology, Institute of Natural ad Applied Sciences Supervisor Prof. Dr. Meral Y&Uuml / CEL Co-Supervisor: Dr. Ufuk &Ccedil / elikkol AK&Ccedil / AY September 2011, 252 pages In this study, Agrobacterium mediated genetic transformation of lentil Sultan 1 cultivar with MBF1c and evaluation of transgenic plants was aimed. The study was initially based on optimized protocol with Agrobacterium tumefaciens KYRT1 strain and pTJK136 binary plasmid. Based on this protocol and transient marker gene expression in embryo apex, 15% stable transformation efficiency was aimed. However limited knowledge about pTJK136 and problem with curing KYRT1 leaded us to use Agrobacterium tumefaciens C58C1 strain and also to engineer an alternative binary plasmid / pPZP101. Hence, scope of this study became construction of a plant binary transformation vector and lentil transformation optimization with C58C1 strain.First plant transformation vector designed in this study was pPZP101ManA-MBF1c. Transformations with C58C1::pPZP101ManA-MBF1c were carried out with a reformulated co-cultivation media. Cotyledonary nodes were isolated from three days old lentil seedlings germinated with phytormone (BAP/TDZ) induction. Isolated nodes were either injured and pre-incubated in co-cultivation media or pre- incubated and then injured prior to transformation. Regeneration and necrosis behaviors of the transformed explants leaded us to the conclusion that explant preparation is the critical step of transformation. And data suggest that explants isolated from 2mg/l BAP, pre-incubated two days in co-cultivation media, injured and transformed performed significantly better scores for necrosis shoot regeneration and callus formation parameters. Transformed explants that survived in subsequent sub-cultures in mannose selection raised shoots. These shoots were grafted and regenerated into plantlets. The putative transgenic plantlets were screened for transgene with PCR. Initial amplification signals fainted and lost as grafts grew. In order to make a diagnosis of this fainting behavior the second plant transformation vector pPZP101ManA- GUSint-MBF1c was constructed and transient GUS expression analysis were made.

ABSTRACT GENETIC TRANSFORMATION OF LENTIL ( Lens culinaris M. cv.Sultan.1) WITH A TRANSCRIPTION FACTOR REGULATOR (MBF1c) AND ANALYSIS OF TRANSGENIC PLANTS KAM&Ccedil / I, Hamdi Ph.D., Biotechnology, Institute of Natural ad Applied Sciences Supervisor Prof. Dr. Meral Y&Uuml / CEL Co-Supervisor : Dr. Ufuk &Ccedil / elikkol AK&Ccedil / AY September 2011, 252 pages In this study, Agrobacterium mediated genetic transformation of lentil Sultan 1 cultivar with MBF1c and evaluation of transgenic plants was aimed. The study was initially based on optimized protocol with Agrobacterium tumefaciens KYRT1 strain and pTJK136 binary plasmid. Based on this protocol and transient marker gene expression in embryo apex, 15% stable transformation efficiency was aimed. However limited knowledge about pTJK136 and problem with curing KYRT1 leaded us to use Agrobacterium tumefaciens C58C1 strain and also to engineer an alternative binary plasmid / pPZP101. Hence, scope of this study became construction of a plant binary transformation vector and lentil transformation optimization with C58C1 strain. First plant transformation vector designed in this study was pPZP101ManA-MBF1c. Transformations with C58C1::pPZP101ManA-MBF1c were carried out with a reformulated co-cultivation media. Cotyledonary nodes were isolated from three days old lentil seedlings germinated with phytormone (BAP/TDZ) induction. Isolated nodes were either injured and pre-incubated in co-cultivation media or pre-incubated and then injured prior to transformation. Regeneration and necrosis behaviors of the transformed explants leaded us to the conclusion that explant preparation is the critical step of transformation. And data suggest that explants isolated from 2mg/l BAP, pre-incubated two days in co-cultivation media, injured and transformed performed significantly better scores for necrosis shoot regeneration and callus formation parameters. Transformed explants that survived in subsequent sub-cultures in mannose selection raised shoots. These shoots were grafted and regenerated into plantlets. The putative transgenic plantlets were screened for transgene with PCR. Initial amplification signals fainted and lost as grafts grew. In order to make a diagnosis of this fainting behavior the second plant transformation vector pPZP101ManA-GUSint-MBF1c was constructed and transient GUS expression analysis were made.

Lentils, one of the cool-season pulses, are consumed as a staple food in most developing countries. The demand for pulses in western societies is increasing because of its valuable nutritional quality and an increased awareness of health issues. Australia has a good reputation for producing clean low moisture legume products and could increase as market share of lentil production by identifying, developing and promoting good quality varieties.Lentils which are graded as good quality varieties must have a short and uniform cooking time, without 'hard to cook' seed, have the hull stay attached to the seed during cooking, and have a final acceptable taste, texture, flavour and appearance after cooking (Bhatty 1990). Cooking quality in this study is defined as the maximum force (N) that is required to compress the whole seed cooked product after cooking for a standard period of time. This study aims to develop an objective measurement to determine the cooking quality of lentils and thereby evaluates the relationships between lentil cooking quality and some of its physical and biochemical properties. Four cultivars used (Cassab, Digger, ILL 7180 and Matilda) were grown during 1999 at Mullewa and Pingaring, Western Australia. The relationship between the cooking quality of lentil and water absorption, seed size, seed coat thickness, phytic acid, mineral composition and initial moisture content was investigated.Texture measurement was carried out using the TA.XT2i meter as an alternative to the subjective method "Cooking time test". By comparing the cooking time determined by 'Cooking time test, 220 N was established and suggested as an optimal peak compression force to determine the adequate cooking time for lentils. Both methods showed that 35 minutes cooking time was adequate for red lentils (Cassab, Digger, and ILL 7180), and 45 minutes for green lentils ++ / (Matilda).Cooking significantly reduced the hardness of the seeds (R= - 0.752 to - 0.89) and promoted mineral leaching (P < 0.05). The interaction between environment and genotype had a significant effect on seed size, seed coat thickness, mineral composition (Phytic acid, Ne, K+, Mg2+, Ca2+ M+, Fe2+ and CU2+) and hardness (P< 0.05). Seed coat thickness did not correlated with this rate of water uptake and cooking quality. Growing environments had a greater influence on the cooking quality than genotypes. Lentils grown at Pingaring are generally had a higher in Phyti acid content, better mineral retention and were harder in texture than those grown at Mullewa.The results of this study implicated that the peak compression force (220 N) was identified as an indicator to determine the cooking time of lentil cultivars. Texture Profile Analysis (TPA) is a useful method to evaluate various texture characteristics (hardness, cohesiveness, chewiness, springiness, gumminess and adhesiveness) of lentil cultivars. Cooking quality of lentil is significantly affected by the effect of varieties and growing locations. However, not the various biochemical compositions (phytic acid and minerals) and the thickness of seed coat have no significant effect on the cooking quality of lentil.

Legumes being the most important crops worldwide are limited in terms of adaptability and productivity mainly by the abiotic stresses. In this study, the aim was to understand tolerance mechanisms of lentil cultivars under drought stress by physiological and biochemical analyses. This study was carried out with six Turkish Lentil cultivars (Seyran, Kafkas, Malazgirt, &Ccedil / agil, &Ccedil / ift&ccedil / i, &Ouml / zbek) subjected to drought stresses (10% and 15% PEG) and their physiological and biochemical properties were examined to select drought-tolerant and drought-sensitive cultivars. Drought stress was applied for 5 days to 7 days-grown lentil plants. 12-days old, stressed and control plant shoots and roots were analyzed in terms of physiological and biochemical parameters (length, fresh weight, ion leakage, proline, MDA and H

In this study, 14 days old lentil seedlings (Lens culinaris Medik cv. Sultan), which were subjected to 7 days of drought (20% PEG 6000), and salt (150 mM NaCl ) stress , were examined in a comparative manner for the effects of drought and salt stress treatments. In shoot and root tissues physiological parameters such as wet-dry weight, relative water content, root-shoot lengths, membrane electrolyte leakage, and lipid peroxidation in terms of malondialdehyde (MDA) were determined. H2O2 content, proline accumulation and chlorophyll fluorescence analysis were also performed. Changes in the activity of antioxidant enzymes such as superoxide dismutase (SOD: EC 1.15.1.1), catalase (CAT: EC 1.11.1.6) ascorbate peroxidase (APX: EC 1.11.1.11) and glutathione reductase (GR: EC 1.6.4.2) were observed upon stress treatments. In salt treated lentil seedlings, significant decreases in wet-dry weight, RWC, shoot-root length and chlorophyll fluorescence measurements indicated a sensitivity, when compared to drought treated plants. Higher MDA concentration and higher electrolyte leakage amounts are supported these results. APX, GR and proline seem to play important roles in antioxidant defense against salt stress for both tissues by removing reactive oxygen species and protecting macromolecules and membranes. GR and proline are also maintains the main protective mechanism against drought stress effects. SOD is active in drought stressed roots and salt stressed shoots, where the H2O2 contents are also observed to be increased.

In this study, the effects of different plant growth regulators on regeneration responses of various lentil explants through direct and indirect organogenesis and through somatic embryogenesis from calli and cell suspension cultures were investigated. Shoot regeneration was obtained in low frequencies from longitudinal embryonic axis explants and nodal buds of epicotyls, however whole plant regeneration was unsuccessful. Conditions provided for indirect organogenesis resulted only in swelling of hypocotyls and root directed ends of internodes and weak callus formation on leaves which were followed by tissue browning and necrosis. In somatic embryogenesis studies, the explants longitudinal embryonic axis and cotyledonary petioles produced soft and friable calli on MS media with Gamborg&rsquo / s vitamins supplemented with 0.75mg/L 2,4-D+0.5mg/L BA. The highest average number of embryos per explant, 12.36 was observed on media containing 0.75mg/L BA +0.5mg/L 2,4-D for cotyledonary petiole explants, whereas 3mg/L BA+1mg/L NAA was the only hormone combination that allowed embryo development to some extent, in both explants. Somatic callus failed to regenerate despite globular embryo formation and embryo development to some extent. Combination of sonication treatment with Agrobacterium transformation of three lentil explants / cotyledonary nodes, half cotyledons and cotyledonary nodes with intact shoots, had no effect on the improvement of transient gus gene expression on explants. Sonication treatment was also unable to form localized wounds on the petiole axils. The best gus gene expression on the axil region was obtained when cotyledonary nodes and KYRT1 strain were used in combination with vacuum infiltration and scalpel wounding of the axils. Gradual selection and repeated removal of regenerated shoots between selection cycles increased the number of gus expressing shoots significantly. The regenerated shoots were grafted on root stocks and whole plant regeneration was achieved in greenhouse conditions. By the use of the optimized Agrobacterium-mediated transformation protocol, 4 independent lines were obtained with 2.3% transformation efficiency. Southern blot analysis confirmed the integration of the gus gene into the genome of lentil plants. T0 plants were fertile and all plants showed Mendelian segregation of the gus gene in 3:1 ratio to their progenies except one line which carries three copies of the gene. Reverse transcription PCR has confirmed the expression of the genes in T0 and T1 generations. T0 plants and the following three generations strongly expressed gus gene uniformly in their tissues and the PCR amplifications of both gus and npt-II genes was successful through generations.

Salinity is the 2nd major limiting abiotic factor on plant growth. As a result of this, soil salinity greatly reduce the yield of the crop production by dual action on plants which are ionic toxicity and water deficit. Therefore, improvement of stress tolerance is greatly concerned. This study was performed to screen and select a salt-resistant and a salt-sensitive cultivar among 6 Turkish lentil cultivars (Lens culinaris M.) which are &Ccedil / agil, &Ccedil / ift&ccedil / i, Kafkas, Malazgirt, Seyran and &Ouml / zbek according to the physiological and biochemical properties. 12 days old lentil seedlings which were exposed to salt stress (100 mM NaCl and 150 mM NaCl) for 5 days as well as control groups analyzed physiologically by root-shoot fresh weights, and lengths / and biochemically by ion leakage, MDA, H

This study was carried out for understanding of antioxidant mechanisms of lentil under abiotic stress conditions. For this aim, 14 days old lentil seedlings (Lens culinaris Medik cv. Sultan-1) were subjected to drought (20% PEG 6000), and salt (150 mM NaCl ) stress for 6, 12 and 24 hours, for 3, 5 and 7 days. PCR conditions for Mn SOD, Cu/Zn SOD, chloroplastic/mitochondrial GR, CAT and chloroplast /stromal APX antioxidant enzymes were optimized. Then, total RNA was isolated from stressed and non-stressed plant roots and shoots. The gene expression levels of Mn SOD and Cu/Zn SOD were examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) technique. Arabidopsis 18S rRNA was used as internal control in multiplex PCR technique. Relative expression levels of Mn SOD were lower in shoots and roots under salt stress while no significant change was obtained under drought conditions in both tissues. Relative expression levels of Mn SOD were increased on 5th day of salt and drought applications in both shoots and roots. Relative expression levels of Cu/Zn SOD increased after 5th, and on 1st and 7th days of drough treatment in shoots and roots, respectively. On the other hand, expression levels of Cu/Zn SOD increased on 3rd and 5th days of salt treatment in shoot tissues. Although it is nearly impossible to understand the whole antioxidant mechanism of plants under environmental stresses, this study was the first step to learn about molecular background of antioxidant defence mechanisms in lentil.