Practical 3 & 4 – Germination, Mineral Nutrition and Hormone Response
Introduction
The following experiments will run over three weeks as outlined in the Table below. This includes an
interim week between the practicals. The results from all the experiments will be submitted at the end
of practical 4.
WEEK A WEEK B WEEK C
3.1 The role of light and hormones in prepare measure submit
germination of light sensitive seeds
3.2 Hormones and growth analysis measure measure complete
/submit
3.3 Hormones and apical dominance complete submit
3.4 Commercial use of hormones complete submit
3.5 Description of deficiency symptoms for measure measure complete
selected mineral nutrients /submit
Week B Interim measurements
After Practical 3 each group must arrange with the Teaching Assistant to make interim measurements of
the following:
After two days: Germination of light sensitive seeds (this experiment will be discontinued after two days).
You must submit your results to the Teaching Assistant. Data from all of the groups will be available
from Myelearning. You must download the data, carry out a statistical analysis and write a scientific
abstract.
After one week: Hormones and growth analysis. Harvest the plants from treatments 2&3 and place in the
oven. Use the same procedure as for treatment 1 (including the measurements of leaf area) and clearly
label all material with your group number.
After one week: Mineral nutrition. Interim observations of nutrient deficiency are necessary to distinguish
between the different treatments.
In your own time: Commercial use of hormones. You will need to compile this information before the next
practical.
3.1 The role of light and hormones in germination of light sensitive seeds
Seeds of lettuce (Lactuca sativa; variety Grand Rapids) are dormant when freshly shed. This dormancy
can be broken when seeds are hydrated (imbibition) and exposed to light. This light affect is mediated
by the pigment-protein phytochrome, which is synthesized in the mature dormant seed as the red
absorbing form (Pr). The Pr form undergoes a conversion to the far-red absorbing form (Pfr) upon
exposure to red light. Conversion of Pr to Pfr by exposure to red light promotes germination. Certain
hormones also affect seed germination by reducing or eliminating the light requirement.
Week A:
Place a piece of filter paper in each of five small petri dishes. Identify treatments by labelling the top of
the petri dishes in a way which will not block light from the seeds. Spread 20 seeds over the filter paper
in each petri dish. Add sufficient distilled H2O to moisten the filter paper in each petri dish, except in
treatment 3 where GA3 solution is added instead. Seal the edges of all the petri dishes with plastic wrap
to prevent desiccation of developing seedlings. For treatment 2 & 3, wrap dishes with 2 layers of
aluminum foil. Treatments 4 & 5 will be placed in boxes with coloured cellophane filters that block the
transmittance of light at specific wavelengths. All work must be done in the dark room using a green
save light.
Approx. 20 seeds will be added to each.
Treatment Description
1 White light
2 Dark (control)
3 Dark + 50 ppm GA3
4 Far-red light
5 Red light
Week B & C:
After two days remove the lids and count the number of germinated seeds in each dish. It is important
that you do not disturb other student’s seeds or expose them to light at this time. Record the
percentage germination and submit the data to the Teaching Assistant. The data from all the groups will
be distributed on Myelearning. Each person must carry out a statistical analysis of the data (using
ANOVA), and write an abstract on the effects of gibberellic acid and light on germination of the lettuce
seeds.
Germination %
Treatment Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Mean
Results from ANOVA
Your abstract should follow the format used in scientific journals. The general outline is given in the
template below (only 1 or 2 short statements are needed for each heading).
Introduction (or rationale for the study):
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Materials and Methods:
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Results:
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Discussion:
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Conclusion:
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3.2 Hormones and growth analysis
Here we use classical growth analysis to investigate the impact of hormones on plant development. We
will use many of the formulas discussed in the Lectures:
Absolute growth rate = (dW/dT) = (W2-W1) / (T2- T1) [g day-1]
Relative growth rate = (dW/dT) x (1/W) = (W2-W1) / (T2- T1) x (1/W1) [g g-1 day-1] %
Net Assimilation Rate
-1 -2
NAR = (dW/dT) x (1/leaf area) = (W2-W1) / (T2- T1) x (1/ leaf area) [g day m ]
Leaf Area Ratio LAR = (leaf area / plant weight (W))
[m-2 g-1 ]
Leaf Area Index = leaf area / ground area [m-2 m-2 ratio units]
Specific Leaf Area
SLA = (leaf area / leaf weight) [m-2 g-1 ]
Root: Shoot ratio
(root weight / shoot weight) [g : g ]
Week A:
Investigate the effects of gibberellic acid (GA) spray on the growth of bodi (Vigna unguiculata) seedlings.
You are provided with one-week-old bodi seedlings growing in seedling trays. Each group should select
three small trays containing seedlings. Randomly assign one of the following treatments to each tray:
1. Initial 2. Control 3. GA treated
For ‘3 GA treated’, plants are to be sprayed with 100ppm gibberellic acid (GA 3) to wet all leaves (ask your
demonstrator for help with this). Plants are to be left unsprayed in treatments 1 and 2.
Treatment 1, this will be used to estimate the biomass of the plants at the start of the experiment and
provide a reference for the growth that will occur in the two other treatments. Carefully cut the stem of
the plant at the soil level:
• Divide each plant into (a) stem plus petioles (b) leaf.
• For leaf area index, measure the total leaf area per plant. To estimate leaf area, draw outlines of
the leaves on graph paper then cut out the shapes and weigh them. A separate section of graph
paper of known area is also weighed to give a conversion factor for calculating leaf area. You
will also need an estimate of the ground area occupied by each plant- this is approx. 10 cm 2.
• For biomass (dry weight), combine the 5 plant samples and place stems and leaves separately
into labelled paper bags and dry at 80°C to constant mass (you need only 2 paper bags). Dry
mass will be taken after two weeks (note: constant mass will be achieved after 2-3 days, but the
material can stay in the oven until the next Lab session).
Week B:
Repeat all the above measurements for treatments 2 and 3 after one week of growth. Theses will be
kept in the oven until Week C.
Week C:
Weigh all samples from week A and Week B.
Leaf area conversion factor
Week A Week B
Mass (g) of 100 cm2 graph paper
Area conversion factor (cm2 per g paper)
Ground area (cm2) occupied per plant (avg. row × column spacing between plants)
Table 3.1 Results for treatment 1. Initial
Variable Bodi (initial)
Leaf area (cm2 per plant)
Leaf mass (g per plant)
Stem mass (g per plant)
Total above ground mass
(g per plant)
Table 3.2 Results for Control and GA treated plants
Control Bodi GA treated Bodi
Leaf area (cm2 per plant)
Leaf mass (g per plant)
Stem mass (g per plant)
Total above ground mass
(g per plant)
Table 3.3 Using the mean values above, calculate growth indices for your data (give units).
Measurements made at both times (weeks A & B) are used for calculation of AGR, RGR and NAR. Use
data only from week B to calculate the other indices.
Growth index Bodi
Control GA treated
Total mass
Leaf area
AGR
RGR
NAR
LAR
LAI
SLA
Give short statements to describe and/or explain the effects of the gibberellic acid treatment on each of
the following indices (from results given in Table 3.3)
Absolute growth rate
Relative growth rate
Net assimilation rate
Leaf area ratio
Leaf area index
Specific leaf area
Summarize the major effects of the GA treatment on growth of the plant species.
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3.3 Hormones and apical dominance
Tomato (Solanum lycopersicum) seedlings were decapitated (by cutting just below the apical bud) and
the hormone Auxin was applied to the cut stem as indole-3-acetic acid (IAA) in a paste. For control
plants an inert paste was applied to the cut surface without auxin. This experiment was set up on three
occasions: 4, 7 and 10 days ago. Make observations on the growth of axillary buds (if any) in these
plants.
Table 3.4 Length (mm) of the topmost axillary bud
Treatment applied 4 Treatment applied 7 Treatment applied 10
days ago days ago days ago
Replication
Paste IAA-Paste Paste IAA-Paste Paste IAA-Paste
alone alone alone
1
2
Mean
Give an physiological explanation for the difference you observed between the control and treated
plants:
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3.4. Commercial use of hormones Week
B&C:
Obtain details on the use of a plant growth regulator in any of the following:
• Control of fruit ripening in bananas or tomatoes
• Induction of flowering in pineapples
• Stimulation of rooting in cuttings
• Weed control
Select only one example and including product names, active ingredients, concentrations used and
application techniques (you can obtain information from: internet sources, visits to garden shops,
conversations with farmers, etc.).
Give details of the commercial use of a hormone/plant growth regulator.
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3.5 Description of deficiency symptoms for selected mineral nutrients
Week A, B & C:
A mineral is defined as essential if normal growth and reproduction cannot be carried out in its absence.
Plants usually respond to the deficiency of an essential mineral by characteristic visual symptoms and
stunted growth. Such symptoms are of interest because they shed light on the necessary function of the
element in the plant and because they are used by agriculturists to determine how and when to fertilize
their crops. A trained observer can predict the mineral deficiency of a given soil simply by examining the
conditions of the plants growing in it.
In this experiment you will record the development of symptoms in plants growing under several
mineral deficient conditions with coded labels. At the end of three weeks you will attempt to determine
which deficiency exists in each set of plants.
The roots of three healthy sand-grown seedlings were rinsed and placed into one of ten containers, each
of which was filled with one of ten aerated experimental solutions. Only the distilled water and the
complete medium treatments are labelled. The other five treatments (- Ca, - Mg, - N, - P, - Fe, and –
micronutrients) are coded.
Stock solution to add for each treatment (ml)
Stock solution Complete Ca Mg N P Fe Micro H2O
0.5M Ca(NO3)2 5 0 5 0 5 5 5 0
0.5M KNO3 5 5 5 0 5 5 5 0
0.5M MgSO4 2 2 0 2 2 2 2 0
0.5M KH2PO4 1 1 1 1 0 1 1 0
FeNaEDTA 1 1 1 1 1 0 1 0
Microelements 1 1 1 1 1 1 0 0
0.5M NaNO3 0 5 0 0 0 0 0 0
0.5M MgCl2 0 0 0 0 0 0 0 0
0.5M Na2SO4 0 0 2 0 0 0 0 0
0.5M NaH2PO4 0 0 0 0 0 0 0 0
0.5M CaCl4 0 0 0 5 0 0 0 0
0.5M KCl 0 0 0 5 1 0 0 0
For the next three weeks, make weekly observations of plant height, leaf number and visual appearance
(avoid touching the plants as this can cause damage). A list of typical deficiency symptoms is included
below for your guidance (Table 3.1). Symptoms to look for include:
Colour changes and their location; Dead spots and their location; Peculiarities of venation; other
pertinent information
In the tables below make your suggestion for which mineral is absent from each solution, indicate which
symptoms led you to make your choices. Be specific:
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Does the presence of a symptom on young and old leaves give you any information as to the mobility of
a each mineral? Cite specific examples from your data to support your answer.
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Person
……………………………………………………………………… Date………………………………………………………………….
Suggested
Observations mineral absent
Complete
1 minerals
Distilled water
8 (no minerals)
Person
Suggested
Observations mineral absent
Complete
1 minerals
Distilled water
8 (no minerals)
Table 3.1 Typical nutrient deficiency symptoms seen in plants (treatments included here are in bold)
Mineral Deficiency symptoms
Calcium Newer or upper leaves affected; young leaves of terminal bud typically hooked,
(Ca) later dying back at tips and margins. Growth characterized by a cut-out
appearance; stalk dies at the terminal bud; growth is restricted.
Magnesium Older or lower leaves affected; mottled or chlorotic leaves; leaves may
(Mg) redden; sometimes with necrotic spots; tips and margins turned upwards;
stalk slender; interveinal chlorosis with veins remaining green.
Nitrogen Older or lower leaves affected; plant light green; lower leaves yellow, drying to
(N) light brown colour; stalks short and slender if deficiency at later stage of growth;
a reddish or purple colour may occur along the veins.
Phosphorus Older or lower leaves affected; plant dark green, often developing red and
(P) purple colours; stalks short and slender if deficiency occurs in later stages of
growth; necrotic areas may develop on leaves, petioles and fruit.
Iron Newer or upper leaves affected; young leaves chlorotic, may become almost
(Fe) white; principal veins typically green; stalks short and slender.
Micronutrients Usually a delayed response with symptoms most evident in the youngest leaves.
Typical symptoms are: Manganese – spots of dead tissue on leaf, smallest veins
tend to remain green giving a reticulated pattern. Copper – young leaves
permanently wilted without spotting or marked chlorosis, twig or stalk below tip
unable to stand erect in later stages. Zinc – rapidly enlarging necrotic areas
between veins, leaves thick, shortened internodes. Boron – young leaves of
terminal bud light green at base, with final breakdown here, leaves become
twisted, stalk dies back at terminal bud. Molybdenum – required for nitrogen
metabolism, so symptoms resemble nitrogen deficiency.
Potassium Older or lower leaves affected; mottled or chlorotic leaves with spots of dead
tissue usually at tips and between veins; also seen on leaf margins; stalks
slender; plants stunted.
Sulphur Newer or upper leaves affected; young leaves with veins and tissue between
veins light green; growth continues normally; apical leaves appear quite yellow
to white.
