Tree Seed Quality Testing
Tree Domestication Course –Seed Physiology lab exercise 17th to 22nd November 2003
Summary of activities
Author: Lucy Mwaura
World Agroforestry Centre (ICRAF) P.O .Box 30677, Nairobi.
Learning Objectives
Participants will perform a practical on purity analysis on sample of Leucaena trichandra and Tephrosia
vogelii seed. They will be expected to give the percentage of pure seed in the sample and fill the relevant
information in a given form.
Handouts on viability testing using the x-raying and the tetrazolium staining tests will be issued to the
trainees. They will also be shown on-going germination tests of Moringa oleifera, Calliandra calothyrsus,
Gliricidia sepium and Sesbania sesban to demonstrate different sowing media for use in such tests.
Tree Seed Purity analysis (30 minutes)
It is very important to use only pure tree seed for all sowing activities so that the maximum number of
seedlings of a desired tree species are obtained. Seed purity may also affect the price of a given seed lot.
Thus it is of great importance that anybody using tree seed should be able to assess the purity of a seed lot.
Pure seed should not contain any impurities including foreign material (inert matter), pieces of broken seed,
twigs, leaf parts or seed of any other species. Participants will be provided with impure seed of Leucaena
trichandra and Tephrosia vogelii, they will be expected to sort/clean it of all foreign matter and calculate
the level of purity of the seedlot that will be provided.
Viability assessment (notes will be provided)
Viability assessment can be carried out in various ways. The best of these is the germination test, which is
nearest expression of the true viability of a seedlot, but some seeds take many days and even months to
germinate. For this reason, quick viability tests should be carried out. The tests provide an estimate of the
seed viability percentage that can be used for various purposes e.g. paying for seed, estimating seed needs
for planting activity.
Due to the time constraint, Participants will be provided with notes on how to perform two types of quick
viability testing (x-raying and Tetrazolium salt staining.
Germination testing Media and test analysis (30 minutes)
Germination testing is important for determining what quantity of seed may be needed for a given purpose
an also to provide information about conditions necessary to cause germination in seeds of different tree
species.
The tests can be carried out in different media. The medium should be inert, porous, and not heavy as
compared to the size of the seed being tested. Proper sowing should also be carried out, not too shallow or
too deep. The ISTA rules should be adhered to whereby four replicates of hundred seeds each is picked at
random from working sample to represent the seedlot. The demonstration will be set up on four kinds of
media as used in the seed lab for germination of Moringa oleifera, Calliandra calothyrsus, Gliricidia
sepium and Sesbania sesban
Further reading
FAO FORESTRY PAPER 20/2 1985. “A guide to Forest Seed Handling.” By William R.C for DFSC.
Albrecht J (1993) (Ed) “Tree seed Handbook of Kenya.”
International Rules for Seed Testing, Rules (1996). (Volume24, Supplement, Rules 1996) “Seed Science
and Technology.”
“Guide to handling of Tropical and subtropical Forest seed” by Lars Schmidt.
TREE DOMESTICATION COURSE
TREE SEED PHYSIOLOGY LABORATORY
Purity analysis
Tree seed samples can contain impurities such as weeds, seeds of other trees species
detached seed structures, leaf particles and other materials. Purity analysis is a test done
to determine the percentage of pure seed in a given sample from an impure seedlot.
Subsequent tests are made from the pure seed component.
Objectives
The objective of the purity analysis is to determine
 The percentage composition by weight of the sample being tested and by
inference the composition of the seedlot.

The identity of the various species of seed and inert particles constituting
the sample.
Pure Seed
Refers to the species under consideration and in addition to mature, undamaged seed
includes: undersized, shriveled, immature and germinated seed provided they can be
identified as the species under consideration and, pieces resulting from breakages that are
more than one-half their original size.
Other Seed
Includes seed of all species except that under test and innert matter
Inert Matter
Consists of pieces of broken or damaged seed less than half the original size, wings of
coniferous species, leguminous and coniferous seed with the seedcoat entirely removed
and other matter such as fragments of leaves, twigs, stones, soil, etc.
PRACTICAL EXERCISE ON PURITY ANALYSIS
Apparatus
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Seed of a selected species (In this case we shall use either Leucaena trichandra or
Tephrosia vogelii)
Weighing balance (with a capacity of up to 1000g)
Petri dishes.
Plain paper/work board
Forceps and Spatulas
Magnifying glasses
Procedure
The working sample containing all the impurities is weighed and the pure seed is
removed and weighed separately (in grams).
Weigh about 30 grams from the working sample.
Calculation
The percentage of pure seed is calculated as follows.
Purity % = Weight of pure seed x 100
Total weight of original sample
Fill in the relevant information in the form provided and give your recommendations.
Seed purity analysis report form.
Lab No……………………
ICRAF Acc No……………………
Species…………………….
Provenance…………………………
Collector…………………..
Date of test………………..
Working sample weight………………
Category
Results
Weight in grams
Percentage (%)
Pure seed
Other seeds
Inert matter
Purity of the seedlot (%)……………………
List and Percentage of seed of other species (%)
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List of other impurities
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Remarks and other observations if any……………………………………………….
…………………………………………………………………………………………
Date………………….
Analysed and compiled by…………………….
TREE DOMESTICATION COURSE
Tree Seed physiology Laboratory
Germination testing media
These are substrates that are used for germination testing in the laboratory. Suitable
media should have the following characteristics:
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Non-toxic to the germinating seedlings.
Free of fungi and other micro-organisms should be sterilised at 1030C for 24hrs
for sand)
Should porous in texture to enable adequate aeration and moisture for the
germinating seeds.
.
Choice of media
Choice of media on which the seeds are placed depends on:
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The species (For large seeds, sand is recommended)
The working condition (The space available in the incubators)
The experience of the worker.
Types of media used in the Laboratory.
 Filter paper (Usually for small seeds e.g. Sesbania sesban.
 Sand (For large seeds e.g. Moringa oleifera
 Vermiculite (Medium sized seeds e.g Gliricidia sepium
Demonstration and analysis of completed germination test

See and analyse results of the already germinated seeds of Moringa oleifera in
sand, Calliandra calothyrsus in soft tissue, Gliricidia sepium in vermiculite and
Sesbania sesban on filter paper.
Tetrazolium Viability test
Salt Preparation
Solution –1 Dissolve 9.078gms in KH2 PO4 in 1000mls
Solution –2 Dissolve9.472gms in Na2HPO4 in 1000mls or dissolve 11.876gms
Na2HPO4 x 2H2O in 1000mls water.
Mix two parts of solution 1 with three parts of solution 2 and check the PH which must
be between 6.5 –7.5.
Dissolve the correct amount of tetrazolium salt (either chloride or bromide) in this buffer
to obtain the right concentration, e.g.1g salt per 100mls buffer gives a 1% solution.
`
Objectives
The objectives of these biochemical tests are:
1. To make a quick estimate of the viability of seed samples in general and those
showing dormany in particular. This mainly includes species of hardwoods and
confers which germinate slowly by regular germination methods.
2. In the case of particular samples which at the end of a germination test reveal a high
percentage of dormant seeds, to determine the viability of individual dormant seeds or
the viability of a working sample.
Principle
In the topographical tetrazolium test a colourless solution of 2,3,5-triphenyl
tetrazolium chloride or bromide is used as an indicator to reveal the processes, which take
place within living cells .The indicator is imbibed by the seed. Within the tissues it
interacts with the reduction processes of living cells and accepts hydrogen from the
dehydrogenases. By hydrogenation of the 2,3,5-triphenyl tetrazolium chloride a red,
stable and non-diffusible substance, triphenyl forazan, is produced in living cells. This
makes it possible to distinguish the red-colored living parts of the seeds from the
colourless dead ones
In addition to completely stained viable seeds and completely unstained nondiffusable seeds partially stained seeds may occur. Varying proportions of necrotic tissue
are found in different zones of these partially stained seeds. The position and size of the
necrotic areas, and not necessarily the intensity of the colour, determine whether such
seeds are classified as viable or non-viable. However, colour differences along with tissue
soundness are to be considered decisive mainly to the extent that they permit recognition
and location of sound, weak or dead tissue.
Procedure
Working sample
A full test is carried out on four replicates of 100 seeds drawn at random from the pure
seed fraction of a purity test. Test may be carried out also on individual seeds that are
found dormant at the end of a germination test. The seeds are soaked in water for about
20hours, and then the seed coat is punctured to facilitate entry of the 1% aqueous solution
tetrazolium (TZ) then they are immersed in the dark for 48 hours
Seed preparation and treatment
1
2
3
The seeds are soaked in water for about 20hours.
Bisect the seeds longitudinally through the embryo with a razor blade to facilitate
entry of the 1% aqueous solution of tetrazolium (TZ).
Immerse seeds in the dark for 48 hours.
Evaluation of the results
1
Immerse seeds in plenty of running water to remove excess stain.
2
Soak the seeds in lacto phenol solution for 1-2 hrs (200mls phenol, 400mls lactic
acid, 200mls glycerine, 200mls water to make one litre) Phenol preparation
(dissolve 0.5-1.0gms of phenol detached crystals in 100mls.use ethyl alcohol if
the crystals do not dissolve).
4
Use low power binocular microscopy to evaluate the staining pattern.
Viable tissues stain bright red. Pink and very dark red stains are indicative
of dead tissue.
Classification
Completely stained and viable seeds
Completely unstained seeds that is non-viable
Partially stained seeds.
Advantages and disadvantages
Advantages
It’s a quick method for testing viability of a seedlot.
It’s useful for hardwood and conifer seeds that take longer time to germinate.
Disadvantages
There is danger of overstaining, and this may portray wrong evaluation.
This biochemical test requires skilled staff to evaluate the results.
The test is limited to certain species due to difficulties in staining.
There is lack of uniform interpretation of staining and difficulty in interpreting the
significance of different degrees of staining
Recommendation
The method is not practical due to many disadvantages and so is not recommended for
routine seed quality testing.
Seed X-Ray Viability Test
What is seed x-ray test?
It’s a diagnostic method of tree seed analysis which permits the detection of
empty seeds, mechanical damage and abnormally developed internal seed
structures measurements of thickness of the seedcoat and assessment of the seed
viability when combined with a contrast agent (special staining techniques)
Why carry out the X-ray test?
Objectives
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To provide a quick, non-destructive method of differentiating between filled,
empty insect and physically damaged seeds from the morphological
characteristics evident on an x-radiography.
To create a permanent photographic record of the proportions of filled, empty,
and insect physically damaged seeds in a sample
.
Apparatus
The following apparatus is necessary: X-ray Machine
Developer, film or paper
X-Ray film or paper.
Holder for film
Holder for seed.
Procedure
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The test is performed on four replicates of 100 seeds each drawn at
random from the pure seed fraction.
Load the film/paper on holder
Spread the seeds evenly on top of the film.
Place lead letters or other x-ray opaque marking devices on the film/paper
to identify the sample.
View the set-up on the screen then make the exposure. (Note that
individual X-ray machines will require different exposure time and
voltage settings to produce the best image and also vary for different
species).
Develop the film/paper in the darkroom.
Evaluation of Results
Seeds are classified according to the internal anatomy revealed by the radiography
as follows
 -Filled fruit/seed: Fruit/seed containing all tissues essential for
germination
 -Empty fruit/seed: fruit/seed containing less than 50% seed tissue.
 -Insect-damaged fruit/seed: Fruit/seed containing insect, insect larvae
frass, or showing other evidence of insect damage affecting the ability of
the seed to germinate.
 -Physically damaged fruit/seed: Filled Fruit/seed with coat outline cracked
or damaged.
Reporting results
Results are expressed as percentages of filled, empty, insect damaged, and
physically damaged.(see form) provided)
Advantages and Disadvantages
Advantages.
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-Helps the analyst to make a quick judgment on the status of the seedlots.
-The test is non-destructive.
Disadvantages
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-Equipments are expensive.
-Can be a health hazard to the worker due to x-rays
Tree Seed Physiology Lab Analysis
X-ray Viability test Results form.
Species…………………………..
Test No………………………..
Provenance……………………..
ICRAF No……………………..
Date of collection……………….
Storage condition………………..
Type of paper/film………………
Exposure time………………….
Evaluation
Filled seed(%)
Empty seed(%)
Insect damaged(%)
Physically damaged(%)
Remarks:……………………………………………………………………………………
.
………………………………………………………………………………………………
………………………………………………………………………………………………
Date………………….
Compiled by…………….