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 25oC
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)(µg/ml) = 20A645 + 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)µg (chl a + b)(µg/ml) × (10ml)
=
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. That’s 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, there’s 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.