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FAO/AfricaSeeds	
  Valida1on	
  Workshop	
  
Seeds	
  Toolkit	
  
Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
SEEDS	
  TOOLKIT	
  
MODULE	
  3:	
  
Seed	
  Quality	
  Control	
  and	
  
Cer;fica;on	
  
FAO/AfricaSeeds	
  Valida1on	
  Workshop	
  
Seeds	
  Toolkit	
  
Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Seed testing
WHY SEED TESTING IS IMPORTANT
q  To predict performance of seed in the field
q  To determine its value for planting.
For farmers, seed testing:
ü  guarantees that seeds meet minimum quality standards in terms
of physical purity and germination percentage;
ü  minimizes the risk of crop failure; and
ü  avoids problems resulting from use of seed contaminated with
noxious weeds, seed infested with disease or insects, and seed
having a low viability level.	
  
3	
  
Ø Standard	
  tes*ng	
  procedures.	
  	
  
Ø Consistency	
   and	
   uniformity	
   of	
   	
   the	
  
evalua*on	
   methods	
   and	
   test	
   results	
   are	
  
(ISTA	
  1924)	
  
FAO/AfricaSeeds	
  Valida1on	
  Workshop	
  
Seeds	
  Toolkit	
  
Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Role of seed-testing laboratory
A seed laboratory does not improve the
quality of seeds produced
Technical sections of the laboratory
ü Analytical purity
ü Varietal purity
ü Germination
ü Seed health
Ø  Calibration and maintenance of Equipment
Ø  Laboratory working documents
FAO/AfricaSeeds	
  Valida1on	
  Workshop	
  
Seeds	
  Toolkit	
  
Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Procedures for laboratory seed testing
Code	
  
Test	
  sheet	
  
	
  
	
  
	
  
q Submitted sample reception
q Sample registration
Ø  Registra*on	
  number	
  (code)	
  
Ø  Recep*on	
  date	
  
Ø  Species,	
  variety	
  and	
  seed	
  class	
  
Ø  Requested	
  test(s)	
  
FAO/AfricaSeeds	
  Valida1on	
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Seeds	
  Toolkit	
  
Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Physical purity analysis
The working sample is divided into three
components:
q  Pure seed
q  Other seeds
q  Inert matter
Apparatus:
Purity board –Hand lenses and binocular microscopes - Seed
blowers - Sieves –Analytical balance, forceps and fine needles.
Objectives:
Ø Determine percentage composition by weight of the sample being
tested (and by inference the composition of the seed lot).
Ø Identify the various species of seeds and inert particles constituting
the sample.
FAO/AfricaSeeds	
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Seeds	
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Abidjan,	
  Côte	
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14-­‐18	
  November,	
  2016	
  
Physical purity analysis
Procedure:
ü  Identify the seed.
ü  Determine the correct weight of the working sample (≥ 2 500 seeds with a
max. w 1 000 g).
ü  Analysis on one working sample or two subsamples of at least half this
weight, each independently drawn.
ü  Weigh the working sample (or each subsample) in grams to the minimum
number of decimal places necessary to calculate the percentage of its
component parts to one decimal place.
Weight	
  of	
  working	
  
sample	
  or	
  subsample	
  (g)!
Minimum	
  number	
  
of	
  decimal	
  places!
Example!
<	
  1.000! 4! 0.7056!
1.000–9.999! 3! 7.056!
10.00–99.99! 2! 70.56!
100.0–999.9! 1! 705.6!
≥	
  1	
  000! 0! 7	
  056!
FAO/AfricaSeeds	
  Valida1on	
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Seeds	
  Toolkit	
  
Abidjan,	
  Côte	
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14-­‐18	
  November,	
  2016	
  
Physical purity analysis
Laboratory	
  reports	
  of	
  purity	
  analysis	
  
Results are expressed as a percentage with two decimals.
Components! Weight	
  
(g)!
Percentage! Example	
  (rice)!
Weight	
  (g)! %!
Pure	
  seed	
   X! (X	
  ×	
  100)	
  ÷	
  W! X	
  =	
  68.88! 98.4!
Other	
  seeds	
   Y! (Y	
  ×	
  100)	
  ÷	
  W! Y	
  =	
  0.14! 0.2!
Inert	
  ma_er	
   Z! (Z	
  ×	
  100)	
  ÷	
  W! Z	
  =	
  0.98! 1.4!
Total	
  	
   W! 100! W	
  =	
  70! 100.0!
FAO/AfricaSeeds	
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Seeds	
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Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Determination of other seeds by number
“Other seeds” – i.e. species specified by the applicant (besides
those tested) – may refer to:
Ø  a general group (e.g. all other species);
Ø  one category of seeds (e.g. species registered as noxious);
Ø  a specific species (e.g. Elytrigia repens).
In international trade, this test is particularly useful for
determining the presence of seeds of noxious or undesirable
species	
  
ü  Results	
  expressed	
  as	
  the	
  number	
  of	
  seeds	
  found	
  
ü  If	
  difficulty	
  for	
  species	
  ,	
  genus	
  name	
  only	
  is	
  reported
FAO/AfricaSeeds	
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Abidjan,	
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14-­‐18	
  November,	
  2016	
  
Other seeds by number
Procedure:
Ø  Obtain a working sample of a prescribed
Ø  If a species indicated by the applicant is difficult to identify, use a
minimum of one-fifth of the ISTA prescribed working sample
weight.
Ø  Examine the working sample for seeds of all other species as
requested by the applicant.
Ø  Count the number of seeds found of each species.
Ø  Keep seeds of the other species found and
store for reference until sample disposal.
FAO/AfricaSeeds	
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Seeds	
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Abidjan,	
  Côte	
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14-­‐18	
  November,	
  2016	
  
Other seeds by number
Laboratory	
  reports	
  
ü  Actual weight of seed examined to the minimum number of
decimal places.
ü  Scientific name and number of seeds of each species sought and
found.
ü  Genus name only if the seed characteristics are insufficient for
precise identification (e.g. Astragalus sp.).
The determination of other seeds is reported using one of
the tests:
q  Complete test –
q  Limited test –
q  Reduced test –
q  Reduced-limited test –
FAO/AfricaSeeds	
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Seeds	
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Abidjan,	
  Côte	
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14-­‐18	
  November,	
  2016	
  
Germination test
Germination is the emergence and development of the seedling
to a stage at which the appearance of its essential structures
indicates whether it can develop further into a satisfactory plant
under favourable conditions in the field.
Objective: Determine the germination potential of a seed lot
FAO/AfricaSeeds	
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Abidjan,	
  Côte	
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14-­‐18	
  November,	
  2016	
  
Essen*al	
  seedling	
  structures	
  
Depending on the species tested, the structures essential for
development into a satisfactory plant are:
ü  Root system (primary root; in certain cases seminal roots)
ü  Shoot axis (hypocotyl; epicotyl; in certain Poaceae mesocotyl;
terminal bud)
ü  Cotyledons (one or more)
ü  Coleoptile (in all Poaceae)
The 50 % rule to evaluate
cotyledons and primary leaves	
  
Germination test
FAO/AfricaSeeds	
  Valida1on	
  Workshop	
  
Seeds	
  Toolkit	
  
Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Procedure (TP):
ü  Four replicates of 100 seeds
ü  Split replicates of 50 seeds (or even 25, if seed-borne pathogens or saprophytes
present.
ü  Place Petri dishes in the germination apparatus;
ü  record the number of seeds set and the date.
ü  Make two counts of seedlings. Schedule the first and final counts according to the
ISTA Rules.
ü  Keep the seed moist throughout the test period.
Growing media:
§  Top of paper (TP) –
§  Between paper (BP) –
§  Pleated paper (PP) –
§  Top of sand (TS), top of organic growing medium
(TO) –
§  Sand (S), organic growing medium (O) –
Germination (%) = Number of normal seedlings ×100
Number of seeds set for germination	
  
Germination test
FAO/AfricaSeeds	
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Abidjan,	
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14-­‐18	
  November,	
  2016	
  
Germination test
Seedlings are evaluated and categorized as follows:
q Normal seedlings:
ü Intact - With slight defects –With secondary infection
q Abnormal seedlings :
ü Damaged –Deformed or unbalanced –Decayed
FAO/AfricaSeeds	
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Seeds	
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Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Germination test
Ungerminated seeds Classification:
ü  Hard
ü  Fresh
ü  Dead
ü  Other	
  
Ø  The result expressed as % by number of normal and abnormal seedlings and hard, fresh
and dead seeds.
Ø  The percentages are rounded to the nearest whole number.
Ø  Example, 75.00 and 75.25 are rounded to 75%; 75.50 and 75.75 are rounded to 76%.
Ø  The sum of the percentages of normal, abnormal and ungerminated seeds must be 100.
FAO/AfricaSeeds	
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Seeds	
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Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Tetrazolium test for viability
ü  Tetrazolium solution is an indicator and produces a substance called formazan in living cells.
ü  A viable seed shows staining in all those tissues whose viability is necessary for normal seedling
development.
ü  Seeds can then be classified into viable and non-viable seed classes
Objective: Make a rapid assessment of seed viability and seed vigour in the
following circumstances:
ü  Seeds need to be sown shortly after harvest.
ü  Seeds have deep dormancy or show slow germination.
ü  A very quick estimate of germination potential is required
ü  A solution is required to problems encountered in a germination test
Apparatus:
Petri dishes, filter paper, magnifying glass, dropper and
bottle, solution of tetrazolium, dissecting needles, forceps.	
  
FAO/AfricaSeeds	
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Seeds	
  Toolkit	
  
Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Procedure:
ü  Draw four replicates of 100 pure seeds at random,
ü  Mix the pure seed fraction thoroughly
ü  Soak seeds in water overnight to soften the embryo and endosperm and activate the enzyme
system.
ü  Make a cut or completely remove the seed-coat (depending on the species) – to expose the
embryo and facilitate contact with the tetrazolium solution.
ü  Immerse the prepared seeds or embryos in tetrazolium salt solution.
ü  Avoid exposure to direct light, as it would cause a reduction of the tetrazolium salt.
ü  Refer to the ISTA Rules for optimum temperatures and staining times.
ü  Wash seeds repeatedly with distilled water.
ü  Examine seeds under a magnifying glass.
Tetrazolium test for viability
Seeds are classified as follows:
ü  Living – red or purple embryo.
ü  Dead – no colour in the embryo, even when red colour develops on other parts, the seed is still
classified as dead.
Viable seed (%) = Number of living seeds ×100
Number of seeds set for viability test	
  
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  November,	
  2016	
  
Objectives
ü  Assess the extent of seed lot deterioration and/or physical damage occurred in
storage.
ü  Distinguish important differences in physiological potential among seed lots of
commercial value (in particular those of similar germination percentage).
There are several definitions of seed vigour, including:
ü  Seed properties that determine the potential for rapid uniform emergence and
development of normal seedlings under a wide range of field conditions (AOSA, 1983).
ü  Sum total of properties of seeds that determine the activity and performance of seed
lots of acceptable germination in a wide range of environments (ISTA, 1995)
Seed Vigour tests
Ø Seed vigour is not a single measurable property, but
Ø A concept describing several seed performance characteristics including:
ü rate and uniformity of seed germination and seedling growth;
ü ability of seeds to emerge under unfavourable environmental conditions; and
ü performance after storage, particularly retention of the ability to germinate
FAO/AfricaSeeds	
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Seeds	
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Abidjan,	
  Côte	
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14-­‐18	
  November,	
  2016	
  
Seed Vigour tests
Ø  It is possible to identify lots more likely to perform well under non-optimal environmental
conditions.
For example, two seed lots may have similar germination potential (> 90%), but
significant differences in seed vigour	
  
Seed	
  lot	
   Germina;on	
  (%)	
   Seedling	
  emergence	
  (%)	
  
	
  	
   	
  	
  
Field	
  1	
  (near	
  ideal	
  
condi;ons)	
  
Field	
  2	
  (unfavourable	
  
condi;ons)	
  
A	
  (high	
  vigour)	
   90	
  	
   88	
  	
   75	
  
B	
  (low	
  vigour)	
   90	
   87	
  
50	
  
Seed vigour tests allows to obtain information about the planting value in a
wide range of environments and the storage potential of the seed lot.
FAO/AfricaSeeds	
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Seeds	
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Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Seed Vigour tests
q Since seed vigour describes several characteristics associated with seed
performance
q  There are several vigour test methods as described in detail in the AOSA
(2002) and ISTA (1995) handbooks.
Ø Stress tests – cold, accelerated ageing and controlled
deterioration tests
Ø Biochemical tests – conductivity and tetrazolium tests
Ø Seedling growth and evaluation tests.	
  
Cold test
q  The cold test developed in the US for maize but also used for other crops
q  The cold test places the seed under the influence of two stress factors:
Ø  low temperature; and
Ø  presence of pathogens.
To guarantee the presence of certain pathogens, soil from the previous year’s plot is
used as substrate.
FAO/AfricaSeeds	
  Valida1on	
  Workshop	
  
Seeds	
  Toolkit	
  
Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Seed Vigour tests
Cold test
Modified cold test methods exist with a mixture of soil and sand, or only sand.
In such cases, the seed is exposed only to low temperature and the effect of pathogens is
completely ignored.
Apparatus
Procedure
Many different cold test procedures have
been developed range of containers from
plastic boxes and trays to rolled towels	
  
ü  Calculate the germination percentage by counting the
number of normal seedlings (as in the germination test).
ü  The higher the germination percentage, the greater the
vigour..
FAO/AfricaSeeds	
  Valida1on	
  Workshop	
  
Seeds	
  Toolkit	
  
Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Seed Vigour tests
Accelerated ageing test
Ø  The accelerated ageing test was developed initially to determine storage
potential.
Ø  Used to predict the seedling field emergence potential of seed lots (soybean)
Ø  The ageing process at high temperature (40–45 °C) and high relative humidity
(around 95%) in the ageing chamber for 72 hours.
Ø  Seeds are then subjected to a germination test.
Ø  Seed lots that show high germination in the accelerated ageing test have
high vigour and are expected to maintain high viability during ambient
storage. Apparatus: Accelerated ageing chamber, equipment for
germination test, plastic accelerated ageing boxes with wire
mesh, distilled water..
Procedure:
ü  Use two boxes with 42 g of seed (100 seeds) in each.
ü  Place seeds on a dry wire mesh (screen)
ü  Place in the ageing chamber at 41 ± 0.3 °C for 72 hours.
ü  Conduct a standard germination test using four replications
of 50 seeds each.
FAO/AfricaSeeds	
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Abidjan,	
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14-­‐18	
  November,	
  2016	
  
Seed Vigour tests
Conductivity	
  test
Ø The electrical conductivity (EC) test is based on the
assumption that cell membranes disintegrate during seed
deterioration.
Ø The test is used for garden pea (Pisum sativum).
Ø Less vigorous or more deteriorated seeds have a slower rate
of cell membrane repair during seed water uptake for
germination, and therefore release greater amounts of solutes
to the external environment
Ø Electrolyte leakage is an indication of vigour.
Ø Low conductivity indicates low electrolyte leakage and thus
high vigour;
Ø High conductivity indicates low vigour
FAO/AfricaSeeds	
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Seeds	
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Abidjan,	
  Côte	
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14-­‐18	
  November,	
  2016	
  
Seed Vigour tests
Conductivity test
Apparatus: Conductivity meter, beaker, 0.1% mercuric chloride, distilled
water, seed sample, wash bottle, tissue paper.
Procedure:
ü  Use 4 replicates of 50 seeds and weigh each replicate to two decimal
places (0.01 g).
ü  Fill four containers of the same size with 250 ml of deionized water,
cover to prevent contamination and maintain at 20 ± 2 °C for 24 h.
ü  Put the seeds to soak in the containers and re-cover to avoid pollution and evaporation. Place in a
germinator at 20 ± 2 °C for 24 h.
ü  Use two other containers containing only de-ionized water as control for each test run.
ü  At the end of the incubation period, stir the seeds and measure the electrical specific conductivity – either
between seeds or after the seeds have been removed from the water.
ü  Measure the conductivity of the control containers and subtract the mean value from the readings for the
seed samples. Between readings, rinse the dip cell in deionized water
Conductivity (µS cm-1 g-1)=Conductivity reading - Mean value of the conductivity of the control
Weight of replicate (g)	
  
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  2016	
  
Seed Vigour tests
Tetrazolium test
To obtain a rapid general estimation of seed viability,
To determine seed lot vigour, the procedure is as for the viability test,
but classification is more precise:
ü  High vigour – staining uniform and even, tissue firm and bright.
ü  Medium vigour – embryo completely stained or embryonic axis stained in dicots. Extremities
may be unstained, while some areas may be more or less stained.
ü  Low vigour – large areas of non-essential structures unstained. Only one root may be
stained (monocots) or the extreme tip of radicle unstained (dicots). Tissue is milky and over-
stained.
Ø  For reliable results, an experienced
tetrazolium analyst must evaluate the test
and the method must be accurately followed.
Ø  The test is widely adopted for cereal crops
and used successfully with field pea. It is
also applied to soybean, cotton, corn, and
large-seeded legumes.	
  
FAO/AfricaSeeds	
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Seeds	
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Abidjan,	
  Côte	
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14-­‐18	
  November,	
  2016	
  
Seed Vigour tests
Seedling growth and evaluation tests
Ø Several tests based on seedling performance are used to
evaluate seed vigour:
ü  First count of germination test.
ü  Speed of germination – one of the oldest manifestations of seed
vigour. Rapid germination is important, because it usually
corresponds to rapid seedling emergence in the field.
ü  Uniform seedling emergence – evaluation of seedling length or
seedling dry weight.
ü  Rate of primary root emergence
Ø  Tests are simple to perform and do not usually require special equipment.
Ø  Seedling tests are useful for evaluating seed vigour in species where no
other seed vigour tests.
FAO/AfricaSeeds	
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Seeds	
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Abidjan,	
  Côte	
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14-­‐18	
  November,	
  2016	
  
Determination of moisture content
Ø  Moisture content is crucial for preserving quality of
stored seed and maintaining viability.
Ø  The moisture content of a sample is expressed as a
percentage of the weight of the original sample.
Ø  The ISTA Rules differentiate between direct and
indirect methods to determine moisture content.
Ø  Direct methods involve oven drying, desiccation and
other physicochemical approaches.
Ø  Note that some species require grinding before testing.
Objectives:
ü  Determine the appropriate drying conditions for optimum
preservation during storage.
ü  Verify whether seed complies with the maximum moisture content
specified in the seed regulations.
FAO/AfricaSeeds	
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Abidjan,	
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14-­‐18	
  November,	
  2016	
  
Determination of moisture content
Constant temperature oven method
Ø  A direct method, reduces oxidation, decomposition and loss of other
volatile substances,
Ø  Ensures the removal of as much moisture as possible.
Ø  The temperature (high/low) depends on the species. Pre-drying may be
necessaryApparatus: Grinding mill, electrically heated oven, containers, thermometer,
desiccator, suitable desiccant (e.g. silica gel), analytical balance, sieves, cutting tool.
Procedure:
ü  Use 4 replicates of 50 seeds and weigh each replicate to two decimal
places (0.01 g).
ü  Distribute the working sample evenly over the surface of the container.
ü  Weigh the container and its cover before and after filling.
ü  Quickly place the container on top of or next to its cover, in an oven.
ü  At the end of the prescribed period, cover the container, place in a
desiccator (containing desiccant) and leave to cool at ambient
temperature.
ü  Once cooled, weigh the container with its cover and contents.
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  Toolkit	
  
Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Moisture meter method
ü  An indirect method, any type of moisture meter can be used, as long it meets the calibration and determination
requirements.
ü  The moisture content measured is calibrated against the moisture content obtained using the oven method.
ü  Meters are very practical and particularly useful when a rapid result is required,
ü  Example, when seed arrives at the processing plant after harvest and it is necessary to decide whether further
drying is required.
Determination of moisture content
Constant temperature oven method
MC(%) = Loss of weight x 100 = M2 –M3 x 100
Initial weight M2 – M1	
  
M1 = weight (g) of container and cover
M2 = weight (g) of container, cover and contents before drying
M3 = weight (g) of container, cover and contents after drying
Percentage by weight calculated to three decimal places for each replicate
Species! Grinding	
  
/	
  cugng!
Method	
  to	
  
be	
  used!
Drying	
  at	
  high	
  
temperature	
  (h)! Pre-­‐drying	
  requirement!
Red	
  beet	
  	
  
Beta	
  vulgaris	
  L.! No! High! 1! -­‐!
Cowpea	
  	
  
Vigna	
  unguiculata	
  (L.)	
  Walp.! Coarse! High! 1! ≤	
  17%	
  moisture	
  content!
Groundnuts	
  	
  
Arachis	
  hypogaea	
  L.! Cut! Low! –! ≤	
  17%	
  moisture	
  content	
  !
Maize	
  	
  
Zea	
  mays	
  L.! Fine! High! 4! ≤	
  17%	
  moisture	
  content	
  !
Rice	
  	
  
Oryza	
  sa;va	
  L.! Fine! High! 2! ≤	
  13%	
  moisture	
  content	
  !
Sorghum	
  	
  
Sorghum	
  bicolour	
  (L.)	
  Moench! Fine! High! 2! ≤	
  17%	
  moisture	
  content	
  !
Wheat	
  	
  
Tri;cum	
  aes;vum	
  L.! Fine! High! 2! ≤	
  17%	
  moisture	
  content	
  !
FAO/AfricaSeeds	
  Valida1on	
  Workshop	
  
Seeds	
  Toolkit	
  
Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Seed health testing
Ø Seed pathology – the study of seed-borne diseases
Ø Seed health – the presence or absence of disease-causing
organisms and animal pests.
Objectives:
ü  Prescribe seed treatment.
ü  Enable seed certification.
ü  Determine the need for plant
quarantine.
ü  Conserve seeds in gene banks.
Methods
There are various ways of detecting the presence or
absence of seed-borne organisms:
1.  Inspection of dry seed (described in detail below)
2.  Seed washing for suspension (described in detail
below)
3.  Whole embryo count method
4.  Blotter method (described in detail below)
5.  Agar plate method
6.  Water agar plate method
7.  Freezing method
8.  Seedling symptom method
9.  Serological test
10. Growing on test
11. Enzyme-linked immunosorbent assay (ELISA)
12. Molecular biology methods (PCR)
13. Field trials
14. Inspection of seed crops
FAO/AfricaSeeds	
  Valida1on	
  Workshop	
  
Seeds	
  Toolkit	
  
Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Seed health testing
Inspection of dry seed
The seed sample is examined with the naked eye or hand lens stereo-
microscope;
The presence of fungi, if any, is observed and recorded.
ü Fruiting structures of fungi are visible as acervuli or pycnidia;
ü Whole or broken ergot sclerotia are mixed with the seed.
ü Spores or spore masses of fungi are visible on the seed surface (rusts,
smuts, downy mildews, spores of other fungi – e.g. Drechslera,
Alternaria, Nigrospora, Curvularia).
ü Nematode galls are present.
FAO/AfricaSeeds	
  Valida1on	
  Workshop	
  
Seeds	
  Toolkit	
  
Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Seed health testing
Washing test
Seeds are washed and the suspension is examined.
This test is used principally to detect fungi whose inoculum is present on the seed surface –
e.g. teliospores of bunts and smuts, oospores of downy mildew, chlamydospores of
Protomyces macrosporus, rust of sugar beet (Uromyces betae) and safflower (Puccinia
calcitrapae).
This method is used to test for pathogens in various
crops:
Ø  Soybean – downy mildew
Ø  Pearl millet – downy mildew, smut
Ø  Wheat – bunt, dwarf bunt, karnal bunt
Ø  Rice – bunt	
  
Blotter method
ü  This is an incubation method (seeds are placed on moist blotters) with seeds incubated for
7 days at 22 °C under alternating cycles of light and dark.
ü  After incubation, the fungi developed are observed under a stereo-binocular microscope
and identified based on the habit characteristics and morphology of the spores. Fruiting
bodies are observed under a compound microscope. Normally 400 seeds are tested in the
blotter method. It is recommended that one analyst examines 200 seeds and another
analyst examines the other 200.c
This method is the most commonly used in seed health testing
to detect pathogenic and saprophytic fungi on almost all crops.
FAO/AfricaSeeds	
  Valida1on	
  Workshop	
  
Seeds	
  Toolkit	
  
Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Seed health testing
Selection of pathogen testing method
The analyst needs to know the exact location of the
pathogen (i.e. in/on individual seeds or among the
seed as separate bodies), and whether there is one
or more types of inoculum.
Some crops require several tests to obtain the full picture of
seed-borne pathogens. Example, for wheat:
Ø dry inspection for ergot;
Ø washing test for Tilletia spp.;
Ø embryo count method for loose smut;
Ø blotter and deep-freezing blotter method for
Alternari spp., Bipolaris sorokiniana, Fusarium spp.
and Stagonospora nodorum; and
Ø agar plate method for quick screening for B.
sorokiniana.
FAO/AfricaSeeds	
  Valida1on	
  Workshop	
  
Seeds	
  Toolkit	
  
Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Varietal purity testing
Ø  Varietal or species purity is tested in the laboratory, in the greenhouse, and in field
plots.
Ø  Variety identification in the laboratory is not standard practice, because most
species have insufficient morphological features for accurate identification.
Ø  The best way to ensure varietal purity is field inspection and post-control plots.
q  Traditional varietal purity tests in the laboratory (e.g. visual examination of seeds and
seedlings and chemical tests) are the first step towards identifying a variety or narrowing
down the range of possibilities.
q  The morphological and physiological characteristics of seeds, seedlings and plants are
examined.
q  In all tests, authentic samples of the species/variety should be available for comparison.
Species
Laboratory
only (g)
Field plot and
laboratory (g)
Glycine, Lupinus, Phaseolus, Pisum, Vicia, Zea
and species of other genera with seeds of similar
size
1 000 2 000
Avena, Hordeum, Secale, Triticum and species of
other genera with seeds of similar size
500 1 000
Beta and species of other genera with seeds of
similar size
250 500
All smaller-seeded species 100 250
FAO/AfricaSeeds	
  Valida1on	
  Workshop	
  
Seeds	
  Toolkit	
  
Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Varietal purity testing
Examination of seeds
Ø  It	
  is	
  possible	
  to	
  differen*ate	
  between	
  cul*vars	
  on	
  the	
  basis	
  of	
  colour,	
  
morphological	
  characters	
  and	
  chemical	
  characteris*cs.	
  	
  
Ø  Various	
  tes*ng	
  methods	
  are	
  available.	
  
Ø  Identifying characters include:
ü  Wheat – colour, shape and size of caryopsis; frequency and length of bristles at the
end of the caryopsis (brush).
ü  Rice – shape, size and colour of grain; size and colour of caryopsis
ü  Barley – shape of grain, base of lemma, colour, hairs in the ventral crease, opening
of the ventral crease, rachilla hairs, dentation of the lateral dorsal nerves, wrinkling
of the lemma and palea, shape and hairiness of the lodicules.
ü  Oat – grain colour (white, yellowish grey or black).
ü  Pea – colour, size and shape.
FAO/AfricaSeeds	
  Valida1on	
  Workshop	
  
Seeds	
  Toolkit	
  
Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Varietal purity testing
Examination of seeds
It	
  is	
  possible	
  to	
  differen*ate	
  between	
  cul*vars	
  on	
  the	
  basis	
  of	
  colour,	
  morphological	
  characters	
  
and	
  chemical	
  characteris*cs.	
  Various	
  tes*ng	
  methods	
  are	
  available.	
  
Wheat varieties develop a
characteristic brown colour varying
from pale to very dark under the
phenol test
Chemical tests
Various chemical tests are available to differentiate between varieties of several species:
ü  Phenol test – distinguishes between cultivars of wheat, barley, oat and ryegrass.
ü  Peroxidase test – separates soybean cultivars into high and low seed-coat
peroxidase activity.
ü  Cooper sulphate-ammonia test – separates yellow sweet clover (Melilotus officinalis)
from white sweet clover (M. alba).
ü  Sodium hydroxide (NaOH) test – distinguishes white wheat from red wheat.
FAO/AfricaSeeds	
  Valida1on	
  Workshop	
  
Seeds	
  Toolkit	
  
Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Varietal purity testing
Examination of seeds
Fluorescence test
Ø  The sample is examined under ultraviolet light and the fluorescence is observed.
Ø  This test is useful for differentiating between species of ryegrass and between varieties of oats.
Ø  Identifying characters include:
ü  Oat - lemma and palea of the seed. A sample of 75 g of pure seed is placed on a black work
surface and examined under ultraviolet light for off-type or variant fluorescence.
ü  Ryegrass - roots of seedlings. The test is used to differentiate between annual (Lolium
multiflorum) and perennial (Lolium perenne)
Electrophoresis
Ø  Electricity is used to separate molecules (protein or DNA) based on their charge and/or size.
Ø  Specific stains are observed and the pattern is compared with known standards.
Ø  ISTA recommends the following standard techniques for certain crops:
ü  Polyacrylamide gel electrophoresis (PAGE) – barley (Hordeum), pea (Pisum) and
ryegrass (Lolium).
ü  Isoelectric focusing (IEF) - maize (Zea mays).
FAO/AfricaSeeds	
  Valida1on	
  Workshop	
  
Seeds	
  Toolkit	
  
Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Varietal purity testing
Examination of seedlings
Cereal
Some varieties can be distinguished by the colour of their coleoptiles, which varies from
purple to green
Determined when the seedlings reach a suitable stage of development.
Brassica
White-fleshed cultivars are distinguished from yellow-fleshed cultivars by the colour of the
cotyledons of germs in turnip:
lemon for white-fleshed cultivars and orange for yellow-fleshed cultivars.
Lolium
Ryegrass species can be identified using the fluorescence test on seedlings.
Root traces in the majority of Lolium multiflorum cultivars exhibit fluorescence while in Lolium
perenne they do not
FAO/AfricaSeeds	
  Valida1on	
  Workshop	
  
Seeds	
  Toolkit	
  
Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Storage of samples
3	
  
Do not delay testing
Ø Moisture content can dramatically increase or decrease
during storage, depending on the temperature and humidity
of the storage room.
Ø Dormancy may be alteredü After analysis, the samples should be kept for ≥ 1 year to
allow for audit retesting.
ü Samples must be stored in a room with controlled
temperature and humidity and protected against insects and
rodents to minimize changes in seed quality.
ü Samples should be placed on appropriate shelves in a clear
and orderly manner.
FAO/AfricaSeeds	
  Valida1on	
  Workshop	
  
Seeds	
  Toolkit	
  
Abidjan,	
  Côte	
  d’Ivoire	
  
14-­‐18	
  November,	
  2016	
  
Working Group Discussion
3	
  

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Day 3 - Module 3: Seed Quality Control - Session 2

  • 1. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   SEEDS  TOOLKIT   MODULE  3:   Seed  Quality  Control  and   Cer;fica;on  
  • 2. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Seed testing WHY SEED TESTING IS IMPORTANT q  To predict performance of seed in the field q  To determine its value for planting. For farmers, seed testing: ü  guarantees that seeds meet minimum quality standards in terms of physical purity and germination percentage; ü  minimizes the risk of crop failure; and ü  avoids problems resulting from use of seed contaminated with noxious weeds, seed infested with disease or insects, and seed having a low viability level.   3   Ø Standard  tes*ng  procedures.     Ø Consistency   and   uniformity   of     the   evalua*on   methods   and   test   results   are   (ISTA  1924)  
  • 3. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Role of seed-testing laboratory A seed laboratory does not improve the quality of seeds produced Technical sections of the laboratory ü Analytical purity ü Varietal purity ü Germination ü Seed health Ø  Calibration and maintenance of Equipment Ø  Laboratory working documents
  • 4. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Procedures for laboratory seed testing Code   Test  sheet         q Submitted sample reception q Sample registration Ø  Registra*on  number  (code)   Ø  Recep*on  date   Ø  Species,  variety  and  seed  class   Ø  Requested  test(s)  
  • 5. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Physical purity analysis The working sample is divided into three components: q  Pure seed q  Other seeds q  Inert matter Apparatus: Purity board –Hand lenses and binocular microscopes - Seed blowers - Sieves –Analytical balance, forceps and fine needles. Objectives: Ø Determine percentage composition by weight of the sample being tested (and by inference the composition of the seed lot). Ø Identify the various species of seeds and inert particles constituting the sample.
  • 6. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Physical purity analysis Procedure: ü  Identify the seed. ü  Determine the correct weight of the working sample (≥ 2 500 seeds with a max. w 1 000 g). ü  Analysis on one working sample or two subsamples of at least half this weight, each independently drawn. ü  Weigh the working sample (or each subsample) in grams to the minimum number of decimal places necessary to calculate the percentage of its component parts to one decimal place. Weight  of  working   sample  or  subsample  (g)! Minimum  number   of  decimal  places! Example! <  1.000! 4! 0.7056! 1.000–9.999! 3! 7.056! 10.00–99.99! 2! 70.56! 100.0–999.9! 1! 705.6! ≥  1  000! 0! 7  056!
  • 7. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Physical purity analysis Laboratory  reports  of  purity  analysis   Results are expressed as a percentage with two decimals. Components! Weight   (g)! Percentage! Example  (rice)! Weight  (g)! %! Pure  seed   X! (X  ×  100)  ÷  W! X  =  68.88! 98.4! Other  seeds   Y! (Y  ×  100)  ÷  W! Y  =  0.14! 0.2! Inert  ma_er   Z! (Z  ×  100)  ÷  W! Z  =  0.98! 1.4! Total     W! 100! W  =  70! 100.0!
  • 8. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Determination of other seeds by number “Other seeds” – i.e. species specified by the applicant (besides those tested) – may refer to: Ø  a general group (e.g. all other species); Ø  one category of seeds (e.g. species registered as noxious); Ø  a specific species (e.g. Elytrigia repens). In international trade, this test is particularly useful for determining the presence of seeds of noxious or undesirable species   ü  Results  expressed  as  the  number  of  seeds  found   ü  If  difficulty  for  species  ,  genus  name  only  is  reported
  • 9. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Other seeds by number Procedure: Ø  Obtain a working sample of a prescribed Ø  If a species indicated by the applicant is difficult to identify, use a minimum of one-fifth of the ISTA prescribed working sample weight. Ø  Examine the working sample for seeds of all other species as requested by the applicant. Ø  Count the number of seeds found of each species. Ø  Keep seeds of the other species found and store for reference until sample disposal.
  • 10. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Other seeds by number Laboratory  reports   ü  Actual weight of seed examined to the minimum number of decimal places. ü  Scientific name and number of seeds of each species sought and found. ü  Genus name only if the seed characteristics are insufficient for precise identification (e.g. Astragalus sp.). The determination of other seeds is reported using one of the tests: q  Complete test – q  Limited test – q  Reduced test – q  Reduced-limited test –
  • 11. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Germination test Germination is the emergence and development of the seedling to a stage at which the appearance of its essential structures indicates whether it can develop further into a satisfactory plant under favourable conditions in the field. Objective: Determine the germination potential of a seed lot
  • 12. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Essen*al  seedling  structures   Depending on the species tested, the structures essential for development into a satisfactory plant are: ü  Root system (primary root; in certain cases seminal roots) ü  Shoot axis (hypocotyl; epicotyl; in certain Poaceae mesocotyl; terminal bud) ü  Cotyledons (one or more) ü  Coleoptile (in all Poaceae) The 50 % rule to evaluate cotyledons and primary leaves   Germination test
  • 13. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Procedure (TP): ü  Four replicates of 100 seeds ü  Split replicates of 50 seeds (or even 25, if seed-borne pathogens or saprophytes present. ü  Place Petri dishes in the germination apparatus; ü  record the number of seeds set and the date. ü  Make two counts of seedlings. Schedule the first and final counts according to the ISTA Rules. ü  Keep the seed moist throughout the test period. Growing media: §  Top of paper (TP) – §  Between paper (BP) – §  Pleated paper (PP) – §  Top of sand (TS), top of organic growing medium (TO) – §  Sand (S), organic growing medium (O) – Germination (%) = Number of normal seedlings ×100 Number of seeds set for germination   Germination test
  • 14. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Germination test Seedlings are evaluated and categorized as follows: q Normal seedlings: ü Intact - With slight defects –With secondary infection q Abnormal seedlings : ü Damaged –Deformed or unbalanced –Decayed
  • 15. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Germination test Ungerminated seeds Classification: ü  Hard ü  Fresh ü  Dead ü  Other   Ø  The result expressed as % by number of normal and abnormal seedlings and hard, fresh and dead seeds. Ø  The percentages are rounded to the nearest whole number. Ø  Example, 75.00 and 75.25 are rounded to 75%; 75.50 and 75.75 are rounded to 76%. Ø  The sum of the percentages of normal, abnormal and ungerminated seeds must be 100.
  • 16. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Tetrazolium test for viability ü  Tetrazolium solution is an indicator and produces a substance called formazan in living cells. ü  A viable seed shows staining in all those tissues whose viability is necessary for normal seedling development. ü  Seeds can then be classified into viable and non-viable seed classes Objective: Make a rapid assessment of seed viability and seed vigour in the following circumstances: ü  Seeds need to be sown shortly after harvest. ü  Seeds have deep dormancy or show slow germination. ü  A very quick estimate of germination potential is required ü  A solution is required to problems encountered in a germination test Apparatus: Petri dishes, filter paper, magnifying glass, dropper and bottle, solution of tetrazolium, dissecting needles, forceps.  
  • 17. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Procedure: ü  Draw four replicates of 100 pure seeds at random, ü  Mix the pure seed fraction thoroughly ü  Soak seeds in water overnight to soften the embryo and endosperm and activate the enzyme system. ü  Make a cut or completely remove the seed-coat (depending on the species) – to expose the embryo and facilitate contact with the tetrazolium solution. ü  Immerse the prepared seeds or embryos in tetrazolium salt solution. ü  Avoid exposure to direct light, as it would cause a reduction of the tetrazolium salt. ü  Refer to the ISTA Rules for optimum temperatures and staining times. ü  Wash seeds repeatedly with distilled water. ü  Examine seeds under a magnifying glass. Tetrazolium test for viability Seeds are classified as follows: ü  Living – red or purple embryo. ü  Dead – no colour in the embryo, even when red colour develops on other parts, the seed is still classified as dead. Viable seed (%) = Number of living seeds ×100 Number of seeds set for viability test  
  • 18. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Objectives ü  Assess the extent of seed lot deterioration and/or physical damage occurred in storage. ü  Distinguish important differences in physiological potential among seed lots of commercial value (in particular those of similar germination percentage). There are several definitions of seed vigour, including: ü  Seed properties that determine the potential for rapid uniform emergence and development of normal seedlings under a wide range of field conditions (AOSA, 1983). ü  Sum total of properties of seeds that determine the activity and performance of seed lots of acceptable germination in a wide range of environments (ISTA, 1995) Seed Vigour tests Ø Seed vigour is not a single measurable property, but Ø A concept describing several seed performance characteristics including: ü rate and uniformity of seed germination and seedling growth; ü ability of seeds to emerge under unfavourable environmental conditions; and ü performance after storage, particularly retention of the ability to germinate
  • 19. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Seed Vigour tests Ø  It is possible to identify lots more likely to perform well under non-optimal environmental conditions. For example, two seed lots may have similar germination potential (> 90%), but significant differences in seed vigour   Seed  lot   Germina;on  (%)   Seedling  emergence  (%)           Field  1  (near  ideal   condi;ons)   Field  2  (unfavourable   condi;ons)   A  (high  vigour)   90     88     75   B  (low  vigour)   90   87   50   Seed vigour tests allows to obtain information about the planting value in a wide range of environments and the storage potential of the seed lot.
  • 20. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Seed Vigour tests q Since seed vigour describes several characteristics associated with seed performance q  There are several vigour test methods as described in detail in the AOSA (2002) and ISTA (1995) handbooks. Ø Stress tests – cold, accelerated ageing and controlled deterioration tests Ø Biochemical tests – conductivity and tetrazolium tests Ø Seedling growth and evaluation tests.   Cold test q  The cold test developed in the US for maize but also used for other crops q  The cold test places the seed under the influence of two stress factors: Ø  low temperature; and Ø  presence of pathogens. To guarantee the presence of certain pathogens, soil from the previous year’s plot is used as substrate.
  • 21. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Seed Vigour tests Cold test Modified cold test methods exist with a mixture of soil and sand, or only sand. In such cases, the seed is exposed only to low temperature and the effect of pathogens is completely ignored. Apparatus Procedure Many different cold test procedures have been developed range of containers from plastic boxes and trays to rolled towels   ü  Calculate the germination percentage by counting the number of normal seedlings (as in the germination test). ü  The higher the germination percentage, the greater the vigour..
  • 22. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Seed Vigour tests Accelerated ageing test Ø  The accelerated ageing test was developed initially to determine storage potential. Ø  Used to predict the seedling field emergence potential of seed lots (soybean) Ø  The ageing process at high temperature (40–45 °C) and high relative humidity (around 95%) in the ageing chamber for 72 hours. Ø  Seeds are then subjected to a germination test. Ø  Seed lots that show high germination in the accelerated ageing test have high vigour and are expected to maintain high viability during ambient storage. Apparatus: Accelerated ageing chamber, equipment for germination test, plastic accelerated ageing boxes with wire mesh, distilled water.. Procedure: ü  Use two boxes with 42 g of seed (100 seeds) in each. ü  Place seeds on a dry wire mesh (screen) ü  Place in the ageing chamber at 41 ± 0.3 °C for 72 hours. ü  Conduct a standard germination test using four replications of 50 seeds each.
  • 23. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Seed Vigour tests Conductivity  test Ø The electrical conductivity (EC) test is based on the assumption that cell membranes disintegrate during seed deterioration. Ø The test is used for garden pea (Pisum sativum). Ø Less vigorous or more deteriorated seeds have a slower rate of cell membrane repair during seed water uptake for germination, and therefore release greater amounts of solutes to the external environment Ø Electrolyte leakage is an indication of vigour. Ø Low conductivity indicates low electrolyte leakage and thus high vigour; Ø High conductivity indicates low vigour
  • 24. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Seed Vigour tests Conductivity test Apparatus: Conductivity meter, beaker, 0.1% mercuric chloride, distilled water, seed sample, wash bottle, tissue paper. Procedure: ü  Use 4 replicates of 50 seeds and weigh each replicate to two decimal places (0.01 g). ü  Fill four containers of the same size with 250 ml of deionized water, cover to prevent contamination and maintain at 20 ± 2 °C for 24 h. ü  Put the seeds to soak in the containers and re-cover to avoid pollution and evaporation. Place in a germinator at 20 ± 2 °C for 24 h. ü  Use two other containers containing only de-ionized water as control for each test run. ü  At the end of the incubation period, stir the seeds and measure the electrical specific conductivity – either between seeds or after the seeds have been removed from the water. ü  Measure the conductivity of the control containers and subtract the mean value from the readings for the seed samples. Between readings, rinse the dip cell in deionized water Conductivity (µS cm-1 g-1)=Conductivity reading - Mean value of the conductivity of the control Weight of replicate (g)  
  • 25. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Seed Vigour tests Tetrazolium test To obtain a rapid general estimation of seed viability, To determine seed lot vigour, the procedure is as for the viability test, but classification is more precise: ü  High vigour – staining uniform and even, tissue firm and bright. ü  Medium vigour – embryo completely stained or embryonic axis stained in dicots. Extremities may be unstained, while some areas may be more or less stained. ü  Low vigour – large areas of non-essential structures unstained. Only one root may be stained (monocots) or the extreme tip of radicle unstained (dicots). Tissue is milky and over- stained. Ø  For reliable results, an experienced tetrazolium analyst must evaluate the test and the method must be accurately followed. Ø  The test is widely adopted for cereal crops and used successfully with field pea. It is also applied to soybean, cotton, corn, and large-seeded legumes.  
  • 26. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Seed Vigour tests Seedling growth and evaluation tests Ø Several tests based on seedling performance are used to evaluate seed vigour: ü  First count of germination test. ü  Speed of germination – one of the oldest manifestations of seed vigour. Rapid germination is important, because it usually corresponds to rapid seedling emergence in the field. ü  Uniform seedling emergence – evaluation of seedling length or seedling dry weight. ü  Rate of primary root emergence Ø  Tests are simple to perform and do not usually require special equipment. Ø  Seedling tests are useful for evaluating seed vigour in species where no other seed vigour tests.
  • 27. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Determination of moisture content Ø  Moisture content is crucial for preserving quality of stored seed and maintaining viability. Ø  The moisture content of a sample is expressed as a percentage of the weight of the original sample. Ø  The ISTA Rules differentiate between direct and indirect methods to determine moisture content. Ø  Direct methods involve oven drying, desiccation and other physicochemical approaches. Ø  Note that some species require grinding before testing. Objectives: ü  Determine the appropriate drying conditions for optimum preservation during storage. ü  Verify whether seed complies with the maximum moisture content specified in the seed regulations.
  • 28. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Determination of moisture content Constant temperature oven method Ø  A direct method, reduces oxidation, decomposition and loss of other volatile substances, Ø  Ensures the removal of as much moisture as possible. Ø  The temperature (high/low) depends on the species. Pre-drying may be necessaryApparatus: Grinding mill, electrically heated oven, containers, thermometer, desiccator, suitable desiccant (e.g. silica gel), analytical balance, sieves, cutting tool. Procedure: ü  Use 4 replicates of 50 seeds and weigh each replicate to two decimal places (0.01 g). ü  Distribute the working sample evenly over the surface of the container. ü  Weigh the container and its cover before and after filling. ü  Quickly place the container on top of or next to its cover, in an oven. ü  At the end of the prescribed period, cover the container, place in a desiccator (containing desiccant) and leave to cool at ambient temperature. ü  Once cooled, weigh the container with its cover and contents.
  • 29. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Moisture meter method ü  An indirect method, any type of moisture meter can be used, as long it meets the calibration and determination requirements. ü  The moisture content measured is calibrated against the moisture content obtained using the oven method. ü  Meters are very practical and particularly useful when a rapid result is required, ü  Example, when seed arrives at the processing plant after harvest and it is necessary to decide whether further drying is required. Determination of moisture content Constant temperature oven method MC(%) = Loss of weight x 100 = M2 –M3 x 100 Initial weight M2 – M1   M1 = weight (g) of container and cover M2 = weight (g) of container, cover and contents before drying M3 = weight (g) of container, cover and contents after drying Percentage by weight calculated to three decimal places for each replicate Species! Grinding   /  cugng! Method  to   be  used! Drying  at  high   temperature  (h)! Pre-­‐drying  requirement! Red  beet     Beta  vulgaris  L.! No! High! 1! -­‐! Cowpea     Vigna  unguiculata  (L.)  Walp.! Coarse! High! 1! ≤  17%  moisture  content! Groundnuts     Arachis  hypogaea  L.! Cut! Low! –! ≤  17%  moisture  content  ! Maize     Zea  mays  L.! Fine! High! 4! ≤  17%  moisture  content  ! Rice     Oryza  sa;va  L.! Fine! High! 2! ≤  13%  moisture  content  ! Sorghum     Sorghum  bicolour  (L.)  Moench! Fine! High! 2! ≤  17%  moisture  content  ! Wheat     Tri;cum  aes;vum  L.! Fine! High! 2! ≤  17%  moisture  content  !
  • 30. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Seed health testing Ø Seed pathology – the study of seed-borne diseases Ø Seed health – the presence or absence of disease-causing organisms and animal pests. Objectives: ü  Prescribe seed treatment. ü  Enable seed certification. ü  Determine the need for plant quarantine. ü  Conserve seeds in gene banks. Methods There are various ways of detecting the presence or absence of seed-borne organisms: 1.  Inspection of dry seed (described in detail below) 2.  Seed washing for suspension (described in detail below) 3.  Whole embryo count method 4.  Blotter method (described in detail below) 5.  Agar plate method 6.  Water agar plate method 7.  Freezing method 8.  Seedling symptom method 9.  Serological test 10. Growing on test 11. Enzyme-linked immunosorbent assay (ELISA) 12. Molecular biology methods (PCR) 13. Field trials 14. Inspection of seed crops
  • 31. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Seed health testing Inspection of dry seed The seed sample is examined with the naked eye or hand lens stereo- microscope; The presence of fungi, if any, is observed and recorded. ü Fruiting structures of fungi are visible as acervuli or pycnidia; ü Whole or broken ergot sclerotia are mixed with the seed. ü Spores or spore masses of fungi are visible on the seed surface (rusts, smuts, downy mildews, spores of other fungi – e.g. Drechslera, Alternaria, Nigrospora, Curvularia). ü Nematode galls are present.
  • 32. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Seed health testing Washing test Seeds are washed and the suspension is examined. This test is used principally to detect fungi whose inoculum is present on the seed surface – e.g. teliospores of bunts and smuts, oospores of downy mildew, chlamydospores of Protomyces macrosporus, rust of sugar beet (Uromyces betae) and safflower (Puccinia calcitrapae). This method is used to test for pathogens in various crops: Ø  Soybean – downy mildew Ø  Pearl millet – downy mildew, smut Ø  Wheat – bunt, dwarf bunt, karnal bunt Ø  Rice – bunt   Blotter method ü  This is an incubation method (seeds are placed on moist blotters) with seeds incubated for 7 days at 22 °C under alternating cycles of light and dark. ü  After incubation, the fungi developed are observed under a stereo-binocular microscope and identified based on the habit characteristics and morphology of the spores. Fruiting bodies are observed under a compound microscope. Normally 400 seeds are tested in the blotter method. It is recommended that one analyst examines 200 seeds and another analyst examines the other 200.c This method is the most commonly used in seed health testing to detect pathogenic and saprophytic fungi on almost all crops.
  • 33. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Seed health testing Selection of pathogen testing method The analyst needs to know the exact location of the pathogen (i.e. in/on individual seeds or among the seed as separate bodies), and whether there is one or more types of inoculum. Some crops require several tests to obtain the full picture of seed-borne pathogens. Example, for wheat: Ø dry inspection for ergot; Ø washing test for Tilletia spp.; Ø embryo count method for loose smut; Ø blotter and deep-freezing blotter method for Alternari spp., Bipolaris sorokiniana, Fusarium spp. and Stagonospora nodorum; and Ø agar plate method for quick screening for B. sorokiniana.
  • 34. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Varietal purity testing Ø  Varietal or species purity is tested in the laboratory, in the greenhouse, and in field plots. Ø  Variety identification in the laboratory is not standard practice, because most species have insufficient morphological features for accurate identification. Ø  The best way to ensure varietal purity is field inspection and post-control plots. q  Traditional varietal purity tests in the laboratory (e.g. visual examination of seeds and seedlings and chemical tests) are the first step towards identifying a variety or narrowing down the range of possibilities. q  The morphological and physiological characteristics of seeds, seedlings and plants are examined. q  In all tests, authentic samples of the species/variety should be available for comparison. Species Laboratory only (g) Field plot and laboratory (g) Glycine, Lupinus, Phaseolus, Pisum, Vicia, Zea and species of other genera with seeds of similar size 1 000 2 000 Avena, Hordeum, Secale, Triticum and species of other genera with seeds of similar size 500 1 000 Beta and species of other genera with seeds of similar size 250 500 All smaller-seeded species 100 250
  • 35. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Varietal purity testing Examination of seeds Ø  It  is  possible  to  differen*ate  between  cul*vars  on  the  basis  of  colour,   morphological  characters  and  chemical  characteris*cs.     Ø  Various  tes*ng  methods  are  available.   Ø  Identifying characters include: ü  Wheat – colour, shape and size of caryopsis; frequency and length of bristles at the end of the caryopsis (brush). ü  Rice – shape, size and colour of grain; size and colour of caryopsis ü  Barley – shape of grain, base of lemma, colour, hairs in the ventral crease, opening of the ventral crease, rachilla hairs, dentation of the lateral dorsal nerves, wrinkling of the lemma and palea, shape and hairiness of the lodicules. ü  Oat – grain colour (white, yellowish grey or black). ü  Pea – colour, size and shape.
  • 36. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Varietal purity testing Examination of seeds It  is  possible  to  differen*ate  between  cul*vars  on  the  basis  of  colour,  morphological  characters   and  chemical  characteris*cs.  Various  tes*ng  methods  are  available.   Wheat varieties develop a characteristic brown colour varying from pale to very dark under the phenol test Chemical tests Various chemical tests are available to differentiate between varieties of several species: ü  Phenol test – distinguishes between cultivars of wheat, barley, oat and ryegrass. ü  Peroxidase test – separates soybean cultivars into high and low seed-coat peroxidase activity. ü  Cooper sulphate-ammonia test – separates yellow sweet clover (Melilotus officinalis) from white sweet clover (M. alba). ü  Sodium hydroxide (NaOH) test – distinguishes white wheat from red wheat.
  • 37. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Varietal purity testing Examination of seeds Fluorescence test Ø  The sample is examined under ultraviolet light and the fluorescence is observed. Ø  This test is useful for differentiating between species of ryegrass and between varieties of oats. Ø  Identifying characters include: ü  Oat - lemma and palea of the seed. A sample of 75 g of pure seed is placed on a black work surface and examined under ultraviolet light for off-type or variant fluorescence. ü  Ryegrass - roots of seedlings. The test is used to differentiate between annual (Lolium multiflorum) and perennial (Lolium perenne) Electrophoresis Ø  Electricity is used to separate molecules (protein or DNA) based on their charge and/or size. Ø  Specific stains are observed and the pattern is compared with known standards. Ø  ISTA recommends the following standard techniques for certain crops: ü  Polyacrylamide gel electrophoresis (PAGE) – barley (Hordeum), pea (Pisum) and ryegrass (Lolium). ü  Isoelectric focusing (IEF) - maize (Zea mays).
  • 38. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Varietal purity testing Examination of seedlings Cereal Some varieties can be distinguished by the colour of their coleoptiles, which varies from purple to green Determined when the seedlings reach a suitable stage of development. Brassica White-fleshed cultivars are distinguished from yellow-fleshed cultivars by the colour of the cotyledons of germs in turnip: lemon for white-fleshed cultivars and orange for yellow-fleshed cultivars. Lolium Ryegrass species can be identified using the fluorescence test on seedlings. Root traces in the majority of Lolium multiflorum cultivars exhibit fluorescence while in Lolium perenne they do not
  • 39. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Storage of samples 3   Do not delay testing Ø Moisture content can dramatically increase or decrease during storage, depending on the temperature and humidity of the storage room. Ø Dormancy may be alteredü After analysis, the samples should be kept for ≥ 1 year to allow for audit retesting. ü Samples must be stored in a room with controlled temperature and humidity and protected against insects and rodents to minimize changes in seed quality. ü Samples should be placed on appropriate shelves in a clear and orderly manner.
  • 40. FAO/AfricaSeeds  Valida1on  Workshop   Seeds  Toolkit   Abidjan,  Côte  d’Ivoire   14-­‐18  November,  2016   Working Group Discussion 3