Germination testing:

procedures and evaluation

Technical Information Sheet 13a

Germination tests are used by the

Millennium Seed Bank (MSB) for Box 1: How to make
two reasons: 1 litre of 1% agar
1. To monitor the viability of seed • Weigh out 10g of agar
collections. powder into a large jug
Figure 1: Alternative substrates: germination paper
2. To develop protocols for turning (left) and sand (right). and mix to a smooth paste
by adding 100ml of cold
seeds into plants. distilled water.
useful for large seeds) or germination
Initial viability is tested once collections paper. Agar is highly suitable as it is • Add 900ml of boiling water to
have been stored at -20°C for at least clear and so easy to see the seeds, the agar paste and stir well.
7 days, and ideally within 3 months of retains moisture, and can be combined • Gently heat the solution,
banking. Viability is then monitored at with chemical compounds such as stirring continuously until
least every 10 years. gibberellic acid (GA3). If using a medium boiling.
Germination testing is often the most other than agar, take care not to add
• Allow to cool to approximately
reliable way of assessing viability. It too much water (it should not pool on 50°C before pouring.
is important to monitor viability as the sand / paper) or to let it dry out.
1 litre of agar yields
non-viable seeds may not be apparent Germination containers approximately 33 x 9cm Petri
at other stages of processing. Optimal Choose a germination container (Petri dishes (30ml per plate)
germination conditions should allow dish, box, etc.) that allows light to
all viable seeds to germinate using the reach the seeds, and which is large To add gibberellic acid (GA3)
simplest method possible. For more enough to contain all the seeds without to agar:
information on choosing appropriate overcrowding. Seal containers inside • Add 5ml of stock solution
germination conditions, see Technical a plastic bag to prevent moisture loss (see below) to 445ml of
Information Sheet 13b. and reduce contamination. boiled agar (cooled to 50°C)
and pour plates as before.
Preparing for germination
testing Table 1: Recommended To prepare stock solutions
germination test numbers of GA3 (250mg/l)
Sample size
• Add 4.5g of GA3 to 200ml†
It is important to know the total number Collection Number cool deionised water.
of seeds available in the collection, Test size*
size* of tests**
including the portion expected to • Adjust pH to 6.5 using
be empty or infested. This portion sodium hydroxide /
> 1000 25 - 50 4 - 10 hydrochloric acid.
should be assessed after the cleaning
process using an x-ray or cut test, see • Filter the solutions using
> 500 25 2-4
Technical Information Sheet 14. You a sterile Nalgene filter and
syringe in a laminar flow
need to sow enough full seeds to allow > 250 10 2-4 cabinet.
proper analysis, without depleting an
important collection (see Table 1). The stock can be stored for
< 250 No test 0
3 months at 5°C in the dark.
Germination medium
* Excluding empty / infested portion †
To ensure final volume is not
Choose a germination medium such as (see Box 2 for over-sowing calculations) >200ml, start with 180ml and
1% agar (see Box 1), sand (particularly ** No more than 10% of the collection should be used adjust after setting pH.

Box 2: Over-sowing
When germination testing collections with empty or infested seed,
sow extra seed to compensate for the incompetent portion.

Number of
Number of full seeds full seeds in x-ray
needed for germination or cut test sample
Number of seeds to sow
sample x-ray or cut test sample
size

E.g. if 45 of 50 seeds full: 50 ÷ (45 / 50) = sow 56 seeds.

Figure 2: GA3 stock solutions at the MSB.

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Setting germination tests Place seeds on fresh substrate as
necessary (e.g. to control fungal
Prepare a germination test sheet contamination or remove inhibitors
for each collection (pg. 4). leached from the seed coat) noting this
Label each germination container on the test sheet each time.
and test sheet clearly, including; Chemical additives (such as GA3)
• date started should be renewed every four weeks,
• collection number as these degrade over time.
• species Figure 6: Rehydration over water.
Test duration
• number of seeds sown Rehydration can reduce risk of Continue tests until germination stops
• germination temperature imbibition damage, especially where or all seeds have germinated. If there
• any additional treatments a soaking treatment is applied (e.g. is no germination after 42 days, or
sanitisation). Suspend dry seed over germination has stopped for more
Sowing the seeds evenly in a grid can water in a sealed container for 24 than four weeks, decide whether to
reduce the spread of mould and allows hours before sowing (Fig. 6). continue the test (e.g. slow germination
easy inspection (Fig. 3). expected), apply a dormancy breaking
Sanitisation treatment (see Technical Information
Seeds can be sanitised by soaking Sheet 13b) or end the test. This
in a 0.5% sodium hypochlorite (NaOCl) decision may depend on the family,
solution, containing a 1% surfactant embryo size, seed health, etc. Seeds
(Tween 20) for 10 minutes and then of some wild species can take years
rinsing under running water for to germinate!
1 minute.

Figure 3: Sowing seeds in grid pattern.

Incubators
Suitable incubators for germination
have low energy, cool white fluorescent
tubes (Fig. 4). Incandescent lamps
should be avoided as they produce
too much far red light and heat.
Photoperiod should coincide with the
thermoperiod, with a cycle of 8/16
or 12/12 (light/dark) hours. Figure 7: Removal of germinated seedlings. Figure 9: Cut testing under a dissecting microscope.

Germination test monitoring Cut testing

Scoring It is important to dissect any seeds
Score germination weekly, removing that have not germinated at the end of
seedlings when the radicle is at least the test (Fig’s. 9 & 10). This will allow
2mm long (for very small seeds, proper evaluation and interpretation
shorter radicles are acceptable) of the result. Paying special attention
(Fig. 7). Record the date, days after to the embryo, record each seed as
sowing, and number germinated on the fresh, mouldy, empty, insect-infested
Figure 4: Incubator for germination tests. test sheet. Record abnormal seedlings or abnormal. Enter the results on the
(e.g. those with cotyledon growth only) germination test sheet (Fig. 11).
Wrap dark germination tests in separately (Fig. 8).
aluminium foil as below. Check for
germination in a dark room under 1 2 3 4
green light (Fig. 5).

Figure 10: Cut test examples of fresh (1), mouldy (2),

empty (3) and infested (4) Najas flexilis seeds.

Figure 8: Record weekly scoring data on the

Figure 5: Germination test wrapped in foil. germination test sheet.

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 reduced (e.g. from 10 to 5 years) when
Box 4: Recording data the first significant decline in viability
and databases is detected. Where possible, duplicate
short-lived collections to cryo-storage.
If you want to analyse or share
your germination data, it is Where no decline is shown after at
important that you record all raw least three retests, intervals can be
data in an appropriate format. extended (e.g. from 10 to 20 years).
Figure 11: Record cut test data on the
Use a data management system This can help to reduce staff costs
germination test sheet. with recognised seed bank data and conserve small seed collections.
standards such as BRAHMS
Germination test evaluation software (BRAHMS, 2015), Analyse retest data using a Z-test
which is capable of exporting (Ellis et al., 1985) or by probit analysis
Calculating percentage germination data in a standard format.
where possible. See Technical
and viability Information Sheet 01 in this series
Empty and insect-infested seeds are for further details on Probit analysis.
Conversely, if remaining seeds are
excluded from the calculations as they
‘mouldy’ on dissection, it can be
could never germinate. This fraction
assumed that all viable seeds have
(unlike viable/non-viable seeds) does
germinated and so germination cannot
not change over time and so does not
be improved. Low germination in this
need monitoring in a germination test.
case could suggest a low viability
Abnormal seedlings are recorded as
collection.
fresh but not germinated. To calculate
percentage germination and viability Statistical analysis for germination tests
use the following equations: To assess whether germination is
significantly less than viability, a
Germination (%) = G / X * 100
two-sample, one-sided binomial test
Viability (%) = (G+F+A) / X * 100 can be used (Fig. 12). If there is no
where: significant difference between viability
Figure 13: Dust containment hoods at the MSB.
G = number of seed germinated and germination (i.e. most or all viable
seeds have germinated) germination
X = number of seed sown
conditions are considered suitable.
(excluding empty and infested) Box 5: Health and safety
F = number of fresh seed As with other statistical tests, sample
size is important. Tests of less than • Scoring of tests should
A = number of abnormal seedlings 10 seeds are too small for meaningful be carried out in a dust
statistical analysis. containment hood (Fig. 13) to
Interpreting germination results
prevent inhalation of fungal
High viability collections should and bacterial spores.
achieve a germination result above Cut test non-germinated seed
• Take care when handling
85%, meeting the international Perform a binomial test on raw data
chemicals; safety glasses
regeneration standard (FAO, 2014). and disposable gloves
However, statistical analysis can should be worn.
also be used to make decisions on Most seeds Most seeds fresh
mouldy on cut test on cut test • Dispose of used scalpel
collections of any viability.
Viability is Viability is blades in a sharps container.
The most successful test (achieving not significantly significantly
different to greater than • Clean all surfaces after
highest germination with the simplest germination germination use with disinfectant.
method) can then be ‘accepted’ and
• Wash hands with biocide
those same conditions used for any
Germination Germination soap after working with
future testing. If a large proportion of requirements requirements
seeds.
seeds do not germinate but appear known not known
‘fresh’ at the end of a test, it is likely If viability = 0% Retest using • Treat all seeds as though
that the test conditions used were not then seeds are different poisonous.
likely dead conditions
optimum for that collection or that the • Wear a lab coat.
seeds were dormant. Figure 12: Statistical analysis of germination tests.

Monitoring collections over time

Box 3: Cut test reliability
Management decisions (e.g. to Acknowledgements
It is important to remember that collect again or regenerate) should
viability calculated from a cut R. Davies, A. Di Sacco & R. Newton,
be implemented if/when collection
test is only an estimate. Royal Botanic Gardens, Kew
Assessing viability across all quality drops to <85% of initial
tests and including controls viability. To begin with, viability should
should increase the reliability be monitored at least every 10 years
Further reading
of tests assessed in this way. (MSBP Seed Conservation Standards, Refer to Technical Information Sheet
2015). Test intervals should be 13b for references.

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Figure 14: Example of a blank germination test sheet. Include all relevant collection information on the test sheet including taxonomy, collection data
(location, date) and processing data (collection size, proportion of empty and infested seed).

Equipment specifications*

Description Model/Product Supplier

• Disposable Petri dishes SLS Select 90mm triple vent Scientific Laboratory Supplies
Plastic germination containers
• Box clear PS 174x115x60mm www.scientificlabs.co.uk

• Agar powder Fisher Bioreagents Fisher Scientific UK Ltd

• White seed germination blotter 2” circle, 3.25” circle www.fisher.co.uk
Germination substrates
and 9x6” rectangle (custom size) Anchor Paper Company Seed Solutions
• Sand (meets ISTA guidelines) www.anchorpaper.com/index.php/seed-solutions

Sigma-Aldrich Company Ltd.

Chemical additives to germination • Gibberellic acid BioReagent www.sigmaaldrich.com
substrates • Potassium nitrate 99.5+% for analysis Certified AR Fisher Scientific UK Ltd
www.fisher.co.uk

Germination incubators with LMS cooled incubators

LMS Ltd
appropriate lighting and temperature • 280 cyclic with timed lights, autodefrost
www.lms.ltd.uk
regimes • Series 1A available in various sizes

Containment hood for scoring Bigneat Ltd

Powder handling workstation with HEPA filtration
germination tests www.bigneat.com

• Nikon binocular stereo zoom microscope

Vision Engineering
Dissecting microscopes with Photonic cold lightsource with gooseneck
www.visioneng.com
• Lynx stereo microscope

Laboratory coat and disposable gloves Locally available

Permanent waterproof
Locally available
marker pens

Agar Scientific Ltd

Dissection kits Kit no. 2
www.agarscientific.com

Scientific Laboratory Supplies

• Distel/Trigene Advance Disinfectant 5L Green (1% solution made up) www.scientific-labs.com
Disinfectant
• Ethanol 99.8+% (GLC) 0.7897g/ml absolute duty free (70% solution made up)
(for bench and equipment) Sigma-Aldrich Company Ltd.
• Tween 20 or Polysorbate 20 nonionic detergent
www.sigmaaldrich.com

Disposable paper towel Locally available

Aluminium foil for dark germination Locally available

Surface sterilising solution Sodium hypochlorite and Tween in de-ionised water

*Please note that the above equipment is used by the Millennium Seed Bank and has been chosen carefully using our many years’ experience.
The list of suppliers is for guidance only and does not represent an endorsement by the Royal Botanic Gardens, Kew. The manufacturer’s instructions
must be followed when using any of the equipment referred to in this Information Sheet.

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