José Donizeti Alves

Otimização dos parâmetros de bombardeamento de partículas para a transformação genética de linhagens brasileiras de milho

The objective of this work was to develop a genetic transformation system for tropical maize genotypes via particle bombardment of immature zygotic embryos. Particle bombardment was carried out using a genetic construct with bar and uidA genes under control of CaMV35S promoter. The best conditions to transform maize tropical inbred lines L3 and L1345 were obtained when immature embryos were cultivated, prior to the bombardment, in higher osmolarity during 4 hours and bombarded at an acceleration helium gas pressure of 1,100 psi, two shots per plate, and a microcarrier flying distance of 6.6 cm. Transformation frequencies obtained using these conditions ranged from 0.9 to 2.31%. Integration of foreign genes into the genome of maize plants was confirmed by Southern blot analysis as well as bar and uidA gene expressions. The maize genetic transformation protocol developed in this work will possibly improve the efficiency to produce new transgenic tropical maize lines expressing desirable agronomic characteristics.

Flooding tolerance and cell wall alterations in maize mesocotyl during hypoxia

Abstract€Œ€This research aimed to characterize the tolerance to flooding and alterations in pectic andhemicellulose fractions from mesocotyl of maize tolerant to flooding when submitted to hypoxia.In order to characterize tolerance seeds from maize cultivars Saracura BRS-4154 and BR€107 tolerantand sensitive to low oxygen levels, respectively, were set to germinate. Plantlet survival was evaluatedduring five days after having been submitted to hypoxia. After fractionation with ammonium oxalate0.5% (w/v) and KOH 2M and 4M, Saracura BRS-4154 cell wall was obtained from mesocotyl seg-ments with different damage intensities caused by oxygen deficiency exposure. The cell wall fractionswere analyzed by gel filtration and gas chromatography, and also by Infrared Spectrum with FourrierTransformation (FTIR). The hypoxia period lasting three days or longer caused cell lysis and in ad-vanced stages plant death. The gelic profile from pectic, hemicellulose 2M and 4M fractions fromsamples with translucid and constriction zone showed the appearance of low molecular weight com-pounds, similar to glucose. The main neutral sugars in pectic and hemicellulose fractions were arabi-nose, xilose and mannose. The FTIR spectrum showed a gradual decrease in pectic substances frommesocotyl with normal to translucid and constriction appearance respectively.Index terms: pectic substances, hemicellulose.Tolerância ao alagamento e alteracies de parede celular em mesocotilo de milho durante hipoxiaResumo€Œ€Objetivou-se caracterizar a tolerância ao alagamento e alteracies nas fracies pOcticas ehemicelulosicas de mesocotilos de milho submetidos a hipoxia. Sementes de milho cultivar SaracuraBRS-4154 e BR€107, tolerante e sensivel a hipoxia, respectivamente, foram submetidas a germinacaoe a sobrevivŒncia das plântulas em condicies de hipoxia, foi avaliada durante cinco dias. O€material deparede celular, obtido de segmentos de mesocotilos da cultivar Saracura BRS-4154 com diferentesintensidades de danos causados por hipoxia, foi submetido a fracionamento com oxalato de amonio0,5% e KOH 2M e€4M. As€fracies de parede celular obtidas foram analisadas por cromatografia emgel, cromatografia gasosa e espectro de infravermelho com transformacao dos dados pela sOrie deFourrier (FTIR). Periodos de hipoxia superiores a trŒs dias causaram a lise celular (aparŒncia translœcida),e, em estAEdios mais avancados, a morte das plantas. O€perfil gOlico das fracies pOcticas, hemicelulose2M e 4M das amostras de mesocotilos translœcidos e com constricao apresentaram compostos de bai-xos pesos moleculares semelhantes a glicose. Os€principais acœcares neutros nas fracies pOcticas ehemicelulosicas foram arabinose, xilose e manose. O€espectro de FTIR mostrou um decrOscimo gradu-al nas substâncias pOcticas do mesocotilo com aparŒncia normal, para translœcido e constricao respec-tivamente.Termos para indexacao: substâncias pOcticas, hemicelulose.

Tolerance of Sesbania virgata plants to flooding

Sesbania virgata (Cav.) Pers. is a bush, pioneer and occurs naturally in Brazil. It belongs to the Fabaceae family and it is indicated for recovery of degraded areas because of its rusticity and capacity to tolerate flooding. The present research was carried out to investigate the ability of S. virgata plants to adapt to flooding conditions. Plants containing six expanded leaves were placed in masonry tanks and were subjected to the following conditions: control (well watered), soil-waterlogging (water to the setup level of 1 cm above the soil surface – roots and parts of the stems flooded) and complete submergence (whole plant flooded). The evaluations were conducted on the day of the stress induction and after 7, 14, 21, 28, 35, 42, 49 and 56 days of the treatment. After 15 days of return to normoxic environment, plant survival was assessed. Growth (height, dry mass of shoots and roots), vigour, carbohydrate content and the activity of enzymes involved in anaerobic metabolism (lactate dehydrogenase, pyruvate decarboxylase and alcohol dehydrogenase) were also evaluated. Our results suggested that sesbania plants are tolerant to flooding, because they can survive being submerged for 56 days. The reasons for this tolerance include the accumulation and use of carbohydrates in the leaves and roots, maintenance of growth and the activation of anaerobic metabolism, particularly in steps catalysed by the enzymes pyruvate decarboxylase and alcohol dehydrogenase.

Purification of polyphenoloxidase from coffee fruits

Polyphenoloxidase (PPO) was purified from coffee beans at the pinhead stage. Purification was carried out by precipitation of contaminating proteins with 30% (NH4)2SO4, dialysis of the supernatant and chromatography on phenyl sepharose and DEAE columns. A purification factor of 364 was obtained. Two bands (29 and 64 kDa) of the purified PPO were detected after SDS-PAGE which presented activity in partially denaturing SDS-PAGE and gave a positive response when probed in Western blots with an apple 27 kDa PPO antibody. It is suggested that the 29 kDa band was a cleavage product of the 64 kDa PPO. The N-terminal sequencing of the 29 kDa protein did not show similarity with others PPOs. Other reports have shown that the 64 kDa is a pre-protein which is converted to a 45 kDa mature form by action of a chloroplastid protease. The 45 kDa was not detected in pinhead fruits but usually detected in the leaves and endosperm of coffee. It is suggested that the appearence of the 45 kDa form might be controlled by the developmental stage of the fruit. Chlorogenic acid was the preferred substrate for the purified PPO.

Caracterização morfofisiológica de clones precoces e tardiosde batata visando à adaptação a condições tropicais

Os objetivos do trabalho foram avaliar os processos de crescimento, de tuberizacao e de particao de materia seca em tuberculos de clones de batata precoces e tardios ao longo do ciclo vegetativo. Avaliou-se, ainda, a viabilidade do uso desses caracteres morfofisiologicos para a selecao de clones mais adaptados as condicoes tropicais. O trabalho foi desenvolvido em Lavras (MG), na safra das aguas (agosto a dezembro de 2005). Os clones tardios foram mais produtivos que os precoces, devido ao crescimento mais vigoroso, a maior longevidade da area foliar fotossinteticamente ativa, a maior taxa de tuberizacao e ao periodo mais longo para enchimento de tuberculos. Os teores de clorofila a, b e total, a area foliar, a duracao da area foliar, a taxa de crescimento relativo e a taxa assimilatoria liquida para os tuberculos foram relevantes na diferenciacao dos clones tardios e precoces. Sugere-se como estrategia possivel para aumentar a produtividade da cultura da batata em regioes tropicais, a selecao de clones com ciclo vegetativo mais longo.

Aspectos fisiológicos do desenvolvimento de mudas de café cultivadas sob telas de diferentes colorações

Abstract – The objective of this work was to evaluate the physiological aspects of the development of coffee seedlings grown under colored screens with different spectral characteristics. Seedlings of Catucai Amarelo 2SL, in the stage known as “orelha de onca”, were arranged in a randomized block design, with five replicates, under structures individually covered with blue, white, gray, black or red screens with 50% shade. Four months after, evaluations were done for seedling growth, pigment content of the leaves, total soluble sugars and starch contents of the leaves and roots. The red screen was the most effective in promoting growth in four out of the seven studied traits: plant height, leaf area and leaf dry weight and total dry matter. For the other characteristics, there was no difference among the screens. The pigment analysis showed that, except for the gray screen, the other ones did not differ for this trait. In leaves, the red screen promoted higher levels of carbohydrates and starch. At the root, carbohydrate contents were higher under the red and black screens. Among the five screen colors, the red one was the most efficient in the production of coffee seedlings with higher vigor and quality, with outstanding carbohydrate contents and biomass.Index terms:

Cell wall biochemistry of sapodilla (Manilkara zapota) submitted to 1-methylcyclopropene

Sapoti (Manilkara zapota) e um fruto climaterico que amadurece rapidamente apos a colheita e os estudos acerca da extensao de sua vida util pos-colheita sao principalmente restritos ao uso de baixa temperatura e atmosfera modificada. Este trabalho avaliou a influencia do 1-metilciclopropeno (1-MCP) sobre o amolecimento e bioquimica da parede celular durante o amadurecimento de sapoti, assim como o seu potencial de conservacao pos-colheita. Sapotis foram tratados com 1-MCP, um antagonista do etileno, a 300 nL L-1 por 12 h e, em seguida, armazenados sob atmosfera modificada a 25oC por 23 d. O 1-MCP retardou significativamente o amadurecimento de sapoti por 11 d, via diminuicao da atividade das enzimas hidroliticas da parede celular e, portanto, os frutos tratados com 1-MCP exibiram menor solubilizacao dos poliuronideos, hemicelulose e acucares neutros livres quando comparados com os frutos-controle. O 1-MCP retardou o amaciamento do sapoti, indicando que esse processo e dependente da producao e percepcao de etileno.

Stomatal behavior and components of the antioxidative system in coffee plants under water stress

Coffee (Coffea arabica) plants show a positive relationship between stomatal closure and formation and accumulation of H2O2. However, for coffee plants under water restriction such relationship has never been studied. The objective of the present study was evaluate the stomatal movement and the antioxidant capacity of coffee seedlings under different water regimes. Eight months old coffee seedlings of cv. Catuai IAC 99 were submitted to field capacity, gradual and total suspension of irrigation during a period of 21 days. Evaluations of leaf water potential (Ψw) were performed in the beginning of the morning, and stomatal resistance, transpiration rate and vapor pressure deficit were determined at 10 am and 5 pm. All biochemical and enzymatic determinations were performed in leaves collected at 5 pm. Evaluations and samplings were performed at three days intervals. There was no variation in Ψw during the evaluated period for plants in field capacity. However, an expressive decrease of Ψw following day 12, reaching values near -2.5 MPa at the end of the experiment was observed for plants submitted to gradual suspension of irrigation. For plants submitted to total suspension of irrigation, Ψw decreases after the sixth day, reaching -2.5 MPa at day 15. The decay of Ψw in plants submitted to gradual and total suspension of irrigation reflected in increased stomatal resistance and in a decreased transpiration rate leading to an increase in hydrogen peroxide formation and, on final stages, increase in lipid peroxidation. As a conclusion, an increase in the activity of antioxidant enzymes as well as in the levels of ascorbate and dehydroascorbate was observed, which act in the detoxification of free radicals formed as result of the water stress.

Características fisiológicas e anatômicas de plantas de sibipiruna submetidas à hipoxia

Plants in their natural environment are subject to adverse conditions, and the stress periods can be short or long. Some species develop mechanisms that make them bear the adverse conditions, allowing their survival. The aim of this study was to evaluate the anatomic and physiological responses in the initial development of Sibipiruna plants submitted to hypoxia. Plants with three to four leaves fully expanded were subjected to three treatments: plants without flooding, flooded on roots and totally flooded. Evaluations were carried out in the starch, total soluble sugars and reducing sugars levels in the leaves and roots collected every five days, for 30 days. Anatomical characteristics were evaluated 15 days after the beginning of each treatment. The largest reduction in starch level in leaves and in roots was observed in plants completely flooded. For total soluble sugars and reducing sugars were observed behavior similar to that achieved in starch However, the plants which had flooded roots, in some periods of assessment, had levels, mainly in the roots, higher than the plants without flooding. These plants also showed higher stomatal density than the other treatments. For the thickness of the cortex and the cylinder vascular roots, a reduction was observed for the flooded treatment, which completely flooded plants had a higher apoplastic barrier imposed by thickening in phi, which is characterized by the formation of septa in cells near the endodermis.

Efeito da aplicação de zinco via foliar na síntese de triptofano, aminoácidos e proteínas solúveis em mudas de cafeeiro

An experiment was conducted aiming to evaluate the effect of the application of Zn on tryptophan synthesis, total amino acids and total soluble proteins of leaves of coffee seedlings (Coffea arabica L.). The seedlings were grown in nutrient solution without Zn for a four-month period. After that time treatments were applied. The plants were sprayed with 0.6 % ZnSO4.7H2O in three different regions (basal: 6 pairs of lower leaves; apical: 6 pairs of upper leaves; and plagiotropic branches). The plants were harvested at times: 0 (control), 12, 24, 48 or 72 h after application of Zn. The leaves were then separated into lots taken from the basal as well as from the plagiotropic branches. These were analysed for tryptophan, concentration, total amino acids and total soluble proteins. From the results obtained a reduction in the tryptophan concentration was observed following Zn application together with an increase in the concentration of total amino acids and total soluble proteins