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Lab 5: Hormonal Control of Leaf Senescence

Objectives
1. To study the role of cytokinins in leaf senescence
Materials
Plant, 3 cork borer, paper towel, deionized water, 5 petri dishes, N6-
benzyladenine (BAP), screw-top test tube, aluminium foil, gooseneck lamp,
freezer, ethanol, spectrophotometer
Procedure:
1. The plant was prepared to be used in this experiment
2. By using 3 cork borer, 35 40 discs were cut from two primary leaves. The
primary leaves are removed from the plant and layed topside down on
several layers of paper towel. Cork borer was pressed down firmly and
evenly on the desired are of the leaves.
3. Then, it was placed into 400ml beaker containing about 200ml of
deionized water
4. 5 petri dishes are obtained and numbered from 1 -5. 15ml of deionized
water are added to each of dishes 1 and 2. 15 ml of N6-
benzyladenine(BAP) solution at 1.3 10-4 M, 1.3 10-5 M or 1.3
10-6 M are added into dish 3,4,5 respectively
5. 5 leaf discs are transferred by using a spatula or forceps from the beaker
to Kimwipe tissue. 5 dry discs are blotted gently with tissues, and then
being measure and recorded in Table 1. After being weighted, 5 discs are
placed adaxial side up on the solution in dish number 1
6. 5 dry disc are selected and 5 discs are weighed for each of the other
dishes in the number 2-5
7. Another 5 leaf discs are selected, dried and weighed and then being
dropped into an empty screw-top test tube labelled 6 initial. The tube is
closed, wrapped with aluminium foil and stored in freezer until we are able
to harvest other leaf disc from the petri dishes
8. Then the discs in the petri dishes are incubated about 25 oC
9. The discs are examined after 2 days of incubation and any visible
differences will be observed. Then incubation process is being continued
as before

The second week of the plant hormone lab


10.After 7 days of incubation, the discs are harvested. 5 test tube are labelled
with number 1-5 and transferred from each dish into the corresponding
tube. Tube number 6 that contain 5 disc that were stored in freezer is
retrieved
11.10ml of 80% ethanol are added into the discs in each of the 6 tubes and
capped with marble
12.Tubes are placed in 75-78% water bath for 35 minutes to extract the
chlorophyll from the leaf disc.
13.After 35 minutes, the tubes are removed from the bath and allowed to cool
14.Forceps is used to remove the leaf disc from the tubes and it is discared.
The volume of each extract with 10-ml graduated cylinder are checked
and 80% ethanol is added to restore the volumes to 10ml
15.Each pigment extract are measure and recorded in the table. The
absorbance at 645 nm and 663 nm. The spectrophotometer are calibrated
on blank composed of 80% ethanol
16.For each extract, calculate combined concentrations of chlorophyll a and n
being calculated according to the formula:
(chl a + b)(g/ml) = 20A 645 + 8A663
17.For each extract, the final mass of chlorophyll a and b per mg of initial
fresh mass are calculated by using the combined mass of the 5 discs that
was measured in previous week and the fact that the alcohol extract 10-ml
volumes. Thus:
( chl a+b)g (chla+ b)(g /ml)(10 ml)
=
fresh mass mg mass of 5 disc( mg)
18.For extracts 1-5, final amount of chlorophyll per fresh mass as a
percentage of the initial amount is calculated, i.e., by dividing (chl a +
b)/fresh mass for each extract by the dividing (chl a + b)/fresh mass of
extract 6
Discussions
Senescence is the final stage of development of leaf in plant. Plant undergo
senescence so that it can recycle the nutrient to other parts of the plant. For
examples, nitrogen is used for the synthesis of storage protein in stem that will
support the growth of plant. Thats the reason why senescence is frequently
occur at lower parts of plant leaves. Senescence is part of physiological,
biochemical process in plant. Therefore, it involves the changes in cell structures,
metabolism indeed it also controlled by gene in plant. This gene will promote the
cell to activate cell death programme thus it activate a self-destruction of leaf.
Leaf senescence is not destructive process in plant but it is very significant in
plant growth. The purpose is for recycle of nutrient. Usually it only occur at the
bottom part of leaves. Usually bottom parts of leaves in covered from getting
sunlight. Therefore it cannot undergo the photosynthesis process effectively. It
will be waste of nutrient to supply it to the leaf that cannot undergo the
photosynthesis process efficiently. Therefore in will undergo programmed cell
death and the nutrient can be transported to the upper parts of plant where this
part receive huge amount of sunlight can able to carry out photosynthesis
process effectively. Senescence process starts when the chlorophyll of leaf is
degenerated. The it will be followed with degeneration of protein, nucleus, and
other organelles. It explains why senescence leaf is undergo necrosis which
indicates the tissue of plant is dead. Therefore, in this experiment the indication
that have been used to measure the leaf senescence is the presence of
chlorophyll in each plant. To measure the concentration of chlorophyll in solution
spectrophotometer has been used in this experiment. Spectrophotometer is an
instrument that have been used to measure the concentration of solutes (in this
experiment is chlorophyll) by measuring the amount of light that have been
absorbed. If the spectrophotometer reading is high, it indicates that the amount
of chlorophyll is high and the process if leaf senescence is slow and vice versa.
Leaf senescence is actually influenced by internal and external factors. The
external factors is such as light intensity whereas internal factors is such as
concentration of hormone. Therefore, in this experiment light factors and
concentration of hormone are used as independent variable to study how these
factors will influence the process of leaf senescence. Light played as a major
factor that contributing leaf senescence. With the presence of light, plant will be
able to carry out the photosynthesis process and producing their product which is
oxygen and glucose. These product can be used in the process of respiration.
Respiration is the process where plant convert the complex molecule to become
simpler and this process will release the energy. The cell inside of the plant will
be able to survive therefore it slow down the senescence process. However, the
absence of light will initiate the senescence process in leaves. It is because the
photosynthetic rate will drops below a certain threshold which is may be at or
near the compensation plant in plant. At this point, leaf will be no longer
contributes fixed carbon to the rest of the plant. Therefore, plant will be unable
to produce oxygen and glucose for respiration process. Respiration process
cannot occur and cell inside of the plant unable to gain energy therefore it
chlorophyll, nucleus, protein and other organelle will begin to degenerate and
senescence process will occur faster. It explains why in this experiment plant that
exposed to light have highest concentration of chlorophyll among all where plant
that not exposed to light shows least concentration of chlorophyll.
However, theres an internal factor that influence the leaf senescence which is
hormone. It was observed that when we applied a N6-benzyladenine (BAP) it
gives a significant reading towards the concentration of chlorophyll. In this
experiment, when the leaf discs are not exposed to light it will gives a least
reading of chlorophyll. However, when we applied BAP eventhought under dark
condition it will gives a higher number of chlorophyll which is in dark condition
only without the presence of hormone is 0.386. Under dark condition with the
hormone with different concentration which are BAP 1.3 10-4 M, 1.3 10-
5
M and 1.3 10-6 M will gives a reading 0.6337, 0.845 and 0.635 respectively.
BAP is type of cytokinin hormone. The function of cytokinin are promote cell
division in plant, promote cell differentiation, maintaining cell meristem and the
most significant in this experiment is delay the leaf senescence. Supposedly, the
higher the concentration of cytokinin the senescence process should be slower.
However, in this experiment 1.3 10-5 M shows the highest concentration of

chlorophyll compared to 1.3 10-4 M. error might be occurred in this


experiment because only very small amount of BAP is enough to influence the
senescence process in leaf. However, the most significance in this experiment is
when we apply cytokinin under dark condition, it will increase the senescence
process drastically. It shows that cytokinin hormone will delay the senescence
process in leaf.

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