1- Protocolo Aula Pratica i

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  • FISIOLOGIA VEGETAL 2012/2013

    BIOLOGIA; BIOTECNOLOGIA 2 ANO/2 SEMESTRE

    AULA PRTICA I

    DOCENTE: FILOMENA FONSECA

    UNIVERSIDADE DO ALGARVE

  • AULA PRTICA I

    Protocolos Fundamento terico do mtodo de Chardakov de determinao do potencial hdrico Mtodos e Instrumentos de medio do potencial hdrico, de presso e osmtico

  • mm (molal)

    Como determinar o potencial hdrico duma soluo

  • Molalidade ou Concentrao molal: a relao entre o nmero de mol de soluto (n1) e a massa do solvente (m2) em Kg

    (molalidade) m= n1/m2

    Exemplo: calcule a molalidade da seguinte soluo, em que dissolveu 4,25 g de NaNO3 em 2000g de H2O

    (Na= 23; N= 14; O=16)

    A massa molar de NaNO3 a soma das massas molares dos elementos:

    23 +14 +3 x16 = 85g ou seja, 85g/mol ou 85g mol-1

    Se temos 4,25g, temos 4,25g/ 85g mol-1 = 0,050 mol

    Aplicando a frmula da molalidade, temos:

    m= 0,050 mol /2 Kg = 0.025 molal

  • No caso da sacarose:

    mol de C12H22O11 = 342,3g

    De quanto preciso para fazer 500 ml de uma soluo aquosa de sacarose 1 m (1 molal)?

    1m = n de mol de sacarose / 0,5 Kg de gua = 0,5 mol de sacarose

    0,5 mol de sacarose pesa 342,3/0,5 = 171,15 g

    Ento: Preciso de 0,5 Kg de gua e de 171,15 g de sacarose

    Qual o potencial osmtico desta soluo, a T ambiente?

    s = -1m (mol/Kg) x 1 x 0,0831( litro. bar.K1.mol1) x (25+273) (K)= -24,76 bar ou -2,476 MPa

    1 atm = 1.01325 bar 1 atm = 0.101325 megapascals 1 bar = 0.1 megapascals

  • Na aula prtica vamos usar as seguines solues de sacarose:

    Molalidade Potencial hdrico (MPa) a T= 25 C 0.1m -0.2476

    0.3m - 0.7428

    0.5m - 1.2380

    0.7m - 1.7332

    0.9m - 2.2284

  • Fundamento terico do mtodo de Chardakov de Medio do Potencial Hdrico

    Os mtodos que vamos usar baseiam-se no facto de que um tecido vegetal colocado em contacto com uma soluo aquosa ir receber ou ceder gua a essa soluo, de acordo com o gradiente de potencial hdrico entre o tecido e a soluo.

    A observao do resultado feita aps ter sido atingido o equilbrio hdrico entre o tecido e a soluo. Considera-se que alteraes sofridas pelo tecido so s devidas movimentao da gua

  • Mtodos de Medio do Potencial Hdrico em tecidos vegetais

    1-Retirar todos os discos do mesmo tipo de folhas

    2- Aps 2 horas coram-se com azul de metileno as solues que contiveram os discos

    3- Introduz-se a gota corada na soluo testemunha

  • RESULTADOS ESPERADOS MTODO DE CHARDAKOV

    Se o potencial hdrico da soluo maior do que o das clulas dos discos (a soluo tem menos solutos do que os discos) a gua absorvida pelos discos e a soluo fica mais concentrada (> densidade do que a soluo testemunha)

  • RESULTADOS ESPERADOS MTODO DE CHARDAKOV

    Se o potencial hdrico da soluo menor do que o das clulas dos discos (a soluo tem mais solutos do que os discos) a gua cedida pelos discos e a soluo fica menos concentrada (< densidade do que a soluo testemunha)

    subir

  • RESULTADOS ESPERADOS MTODO DE CHARDAKOV

    Se o potencial hdrico da soluo igual ao das clulas dos discos, no h movimentao de gua para dentro ou fora dos discos e a soluo no sofre alterao de concentrao (= densidade que a soluo testemunha)

    pra

  • Clculo do teor relativo em gua

    Ao potencial hdrico encontrado para os tecidos foliares estudados ir corresponder um determinado teor relativo em gua.

    Como se determina?

    TRA = (Pf-Ps) / (Pt-Ps)

    Ou seja:

    Pesa-se uma amostra do material vegetal usado (Pf), que depois se coloca em gua destilada, no frigorfico. Espera-se pelo menos 2h (equilbrio) e pesa-se. Este peso corresponde ao peso trgido (Pt), ou seja, corresponde aos 100% de gua que o tecido consegue conter. De seguida coloca-se a amostra na estufa de secagem e pesa-se na aula seguinte. Este peso corresponde ao peso da amostra sem gua, peso seco (Ps).

    Assim: Pt-Ps= ao peso mximo de gua que o tecido consegue Pf-Ps= ao peso de gua que o tecido contem quando se mede o potencial hdrico Ex: TRA= (1.5g 0.5g) / (2.0-0.5g) = 0.67 ou 67%

  • OUTROS MTODOS

  • Retirar todos os discos do mesmo tipo de folhas usadas para o mtodo Chardakov

  • Se o peso dos discos aumentar depois do perodo de incubao com a soluo, isto significa que os discos receberam gua da soluo, logo tinham um portencial hdrico inferior ao da soluo.

    Os discos tero o mesmo potencial hdrico que a soluo que no lhes causar variao de peso

  • . Psychrometer (PSICRMETRO) -

    One psychrometric technique, known as isopiestic psychrometry, has been used extensively by John Boyer and coworkers (Boyer and Knipling 1965). Investigators make a measurement by placing a piece of tissue sealed inside a small chamber that contains a temperature sensor (in this case, a thermocouple) in contact with a small droplet of a standard solution of known solute concentration (known s and thus known w). If the tissue has a lower water potential than that of the droplet, water evaporates from the droplet, diffuses through the air, and is absorbed by the tissue. This slight evaporation of water cools the drop. The larger the difference in water potential between the tissue and the droplet, the higher the rate of water transfer and hence the cooler the droplet. If the standard solution has a lower water potential than that of the sample to be measured, water will diffuse from the tissue to the droplet, causing warming of the droplet. Measuring the change in temperature of the droplet for several s olutions of known w makes it possible to calculate the water potential of a solution for which the net movement of water between the droplet and the tissue would be zero signifying that the droplet and the tissue have the same water potential.

  • Psychrometers can be used to measure the water potentials of both excised and intact plant tissue. Moreover, the method can be used to measure the s of solutions. This can be particularly useful with plant tissues. For example, the w of a tissue is measured with a psychrometer, and then the tissue is crushed and the s value of the expressed cell sap is measured with the same instrument. By combining the two measurements, researchers can estimate the turgor pressure that existed in the cells before the tissue was crushed (p = w s).A major difficulty with this approach is the extreme sensitivity of the measurement to temperature fluctuations. For example, a change in temperature of 0.01C corresponds to a change in water potential of about 0.1 MPa. Thus, psychrometers must be operated under constant temperature conditions. For this reason, the method is used primarily in laboratory settings. There are many variations in psychrometric technique; interested readers should consult Brown and Van Haveren 1972, Slavik 1974, and Boyer 1995.

  • . Pressure chamber (CMARA DE PRESSO DE SCHOLANDER) -

  • A relatively quick method for estimating the water potential of large pieces of tissues, such as leaves and small shoots, is by use of the pressure chamber . This method was pioneered by Henry Dixon at Trinity College, Dublin, at the beginning of the twentieth century, but it did not come into widespread use until P. Scholander and coworkers at the Scripps Institution of Oceanography improved the instrument design and showed its practical use (Scholander et al. 1965). In this technique, the organ to be measured is excised from the plant and is partly sealed in a pressure chamber. Before excision, the water column in the xylem is under tension. When the water column is broken by excision of the organ (i.e., its tension is relieved allowing its p to rise to zero), water is pulled rapidly from the xylem into the surrounding living cells by osmosis. The cut surface consequently appears dull and dry. To make a measurement, the investigator pressurizes the chamber with compressed gas until the distribution of water between the living cells and the xylem conduits is returned to its initial, pre-excision, state. This can be detected visually by observing when the water returns to the open ends of the xylem conduits that can be seen in the cut surface. The pressure needed to bring the water back to its initial distribution is called the balance pressure and is readily detected by the change in the appearance of the cut surface, which becomes wet and shiny when this pressure is attained.

  • The pressure chamber is often described as a tool to measure the tension in the xylem. However, this is only strictly true for measurements made on a non-transpiring leaf or shoot (for example, one that has been previously enclosed in a plastic bag). When there is no transpiration, the water potential of the leaf cells and the water potential in the xylem will come into equilibrium. The balancing pressure measured on such a non-transpiring shoot is equal in magnitude but opposite in sign to the pressure in the xylem (p). Because the water potential of our non-transpiring leaf is equal to the water potential of the xylem, one can calculate the water potential of the leaf by adding together p and s of the xylem, provided one collects a sample of xylem sap for determination of s. Luckily s of the xylem is usually small (> 0.1 MPa) compared to typical midday tensions in the xylem (p of 1 to 2 MPa). Thus, correction for the s of the xylem sap is frequently omitted.

    Balancing pressure measurements of transpiring leaves are more difficult to interpret. The fact that water is flowing from the xylem to the leaf means that differences in water potential must exist. When the transpiring leaf