Sampling and analytical procedures for purity testing and detection of spurious seeds

Seed sampling
It is required to obtain a sample of required quantity representing the seed lot in true sense.
Samples are derived from different portions of seed lot; these samples are mixed.
From this composite sample smaller portion of required quantity is obtained in such a way
that
even after reduction it represent the seed lot.
Equipment
 Trier
 Plastic tubs
 Bags
 Balance
 Seed divider
 Sticker
 Label

Fig.- Seed divider

Fig.- Trier
Primary sample:
- It is a small quantity of seed taken from one point of the processed lot.
- The seed lot is arranged to approach conveniently up to individual container.
- Primary samples are taken from different portions and depth by inserting the stick
trier with the closed slot diagonally in the seed bag or container up to desirable depth
with minimum damage to seed.
- The flow of seed is facilitated in the tube by opening and closing of the slot.
- Finally, the slot is closed, the trier is withdrawn and collected sample is transferred to
a container.
Composite sample:
Primary samples drawn from different places of a lot are mixed and the mixture is known as
composite sample.
The size of composite sample should be 10 times more than the required submitted sample.

Submitted sample:
The required quantity of seed which, is sent to seed testing lab is known as submitted sample.
To prepare a submitted sample, the composite sample is mixed thoroughly and reduced up to
required quantity with the help of seed divider or by repeated halving method.

Sampling in seed testing lab:

The submitted sample received in seed testing lab is registered and designated by a code
number.
Submitted sample is tested for determination of seeds of other crop, weed, objectionable
weeds, objectionable diseases and other distinguishing varieties by number.
Three working samples of the submitted sample, which passes the seed certification standard
by number are prepared. Each working sample consists of at least 2500 seeds.

Preparation of a working sample

 Use of seed divider:
- The number of the sample is mentioned on the bag.
- The divider and its pans are cleaned properly.
- The valve of hopper in Boerner divider is closed.
- The pans placed at both the spout and the valve of Boerner divider is opened whereas,
hopper of soil divider containing seed is put on ports and ducts.
- The seed is poured on rotated spinner of Gamet type divider.
- The divided samples from both the spouts are collected.
- The seed of one collecting pan is kept in a container and the seed of other pan is again
divided with the help of a divider to get the required quantity of working sample.

Repeated halving method:

The seed is poured on a clean smooth surface and shaped as a mound after thorough mixing.
Mound is divided into two halves, each half is again halved, each portion is again halved
giving total 8 portions.
Alternate portions are combined i.e. 1st and 3rd of first row and 2nd and 4th of second row.
The remaining portion is kept in a pan and the process is repeated to obtain required size of
the working sample.
Random cup method:
- Six to eight small cups of equal size and shape are arranged at random on a tray.
- The seed is poured uniformly over the tray.
- The seeds, which fall into the cups are collected as working sample.
- This method is useful for the crops with small seed size but not for chaff and round
seeds.

Spoon method:
The seeds are poured evenly in one direction over the tray. If required, seed can be poured
second time in opposite direction. Shaking of the tray is avoided, small quantity of seeds are
collected with the help of spatula from minimum 5 random places to make a working sample
of required quantity.

Distribution of seed lot:

Submitted samples and working samples of different crops:

Physical Purity analysis by number

To identify and determine the seeds of species other than the seed crop present in submitted
sample and constituting the seed lot by number. Seed crop refers to all the botanical varieties
and cultivars of the species considered for seed production.
 Apparatus:
- Physical purity work board, forceps, cups and specimen of objectionable weed seed
(OWS).

Procedure:
The sample is spread uniformly over the physical purity work board and the following
categories of seeds are sorted out with the help of forceps by visual observation and separated
in different cups.
• Other crop seed
• Total weed seed
• Objectionable weed seed
Seed Standards:

Physical Purity analysis by weight

To determine the physical purity of a seed lot by weight of the other matter than the crop seed
present in the working sample. The working sample is separated into pure seed, other seed
and inert matter. The physical purity percentage is determined by weight.

Pure seed:
It is the seed of an allotted crop produced by a grower, which includes all botanical varieties
and cultivars of that particular species. The seed of allotted crop is considered as pure even if
it consists of immature, shrivelled, diseased, germinated, under sized seed or piece of seed
unit larger than half of the original size provided it can be authentically be identified as of
that crop.
Other seed:
Seed of any other crop or weed mixed in the sample.
Inert matter:
All the matter present in the sample not defined as seed e.g., Plant parts other than seed Soil
particles insect Nematode galls.
Seed of the allotted crop with half of its original size or more than half damaged part Any
appendages of seed not defined as part of the seed (glume of wheat) are considered as inert
matter.
Procedure:
The working sample of desired weight is prepared and distributed evenly over the physical
purity work board.
Pure seed, other seed and inert matter are sorted out with the help of spatula and forceps and
kept in different cups. Separation is done without use of any pressure and magnification
except some grasses. During physical purity analysis the seed of any crop from family
Leguminosae and Cruciferae without seed coat is considered as inert matter.

Multiple seed unit

- one fertile floret is attached with sterile or fertile floret, in grasses are weighed
separately without breaking the attachment and reported in percentage.
- Insects are considered as inert matter.
- Insect damaged seed is examined thoroughly and when the extent of damage is more
than half of the seed then the seed and insect both are considered as inert matter.

Husk less seed

- Husk less seed from the sample of rice and sunflower are removed and counted to
work out the percentage of husk less seed.
- The maximum permissible limit for husk less seed is 2%.

Use of seed blower: It is used to remove the light weight inert

matter from the seeds of grasses or vice-versa. Working sample is kept at the lower portion of
the tube and the required uniform upward flow of air is regulated up to prescribed period of
time. Lighter matter is separated from the sample by air flow and settled down in the partition
provided in the tube of the blower. The tube is removed and the inert matter is collected.
Purity%:

General Procedures
The I.S.T.A. Rules (Para. 2) prescribe the various techniques to be used in sampling to
provide a representative sample from a lot.
Briefly the Rules prescribe:
1. The rate of sampling, i.e. how many bags need to be sampled or how many samples
should be taken from a bulk specified as the bulk sample.
2. The type of sampler to be used.
3. The minimum weights of a sample to be submitted for analysis. which vary with the
size of the seed being sampled—referred to as submitted sample.
4. The minimum weight of seed to be examined for a purity analysis, again depending
on the size of the seed—the working sample. The working sample should be
approximately 2,500—3.000 seeds —the new I.S.T.A. tables are based on 2,300 for
chaffy grasses and 2,750 for other seeds.
5. The sampling methods which may be used to take the "Submitted Sample" from the
"Bulk Sample" and in the laboratory, to take the ' 'Working Sample" from the
"Submitted Sample".
The methods recommended for dividing a sample are as follows:
(a) An efficient mechanical divider, such as the Boerner or Gamet, one of which is
available in seed testing laboratories.
(b) The random cups method.
The I-S.T. A. Rules (para 2.3.3) prescribe the sampling intensities for bulk and bagged lots of
seed as follows:
Bulk Sampling
When sampling seed lots stored in bulk (heaps, bins, wagons, etc.) or streams of seeds during
processing operations the following sampling intensity should be regarded as a minimum
requirement for obtaining the "bulk sample".
(a) up to 500 kg. at least 5 individual samples except for very small lots (50 kg.) where a
smaller - number of samples is sufficient, but not less than, 3 samples need be taken.
(b) of 501—3,000 kg one individual sample for each 300 kg., but •not less than 5 individual
samples.
(c) of 3,001—20,000 kg one individual sample for each 500 kg, but not less than 10
individual samples.
For seed in bulk the individual samples should be distributed at random all over the bulk and
the samples drawn from varying depths.

FIG.—Bag triers can be inserted carefully between the threads of gunny bags without
breaking them when sampling.

Sampling Records
Information submitted with the sample should include the following:
 Date sampled
 Who sampled seed
 Name and address of sender
 Kind and hybrid or variety
 Origin or class of seed
 Sample designation or lot number
 Quantity of seed in lot
 Kind of tests desired: Germination, Purity or Moisture.
Using Spectroscopy to Detect Spurious Seeds

Seed fraud of spurious seeds are umbrella terms for seeds of sub-par quality, seeds that are
mislabelled – intentionally or not, and contaminant seeds that contain weeds or any invasive
species that higher levels than allowed by regulation.

Near Infrared Spectroscopy

Near infrared spectroscopy involves irradiating the sample of interest with near infrared light
and recording which frequencies of light are absorbed.

When analysing thin samples, near infrared spectroscopy is normally performed in a

transmission geometry, where light is passed through the sample, and the detector receives
the light that is transmitted without being absorbed.

When it comes to the analysis of seeds, it is often more convenient to design the spectrometer
in a reflective geometry so that the detector collects the light that has been reflected from the
surface of the seed. An infrared spectrum, regardless of the specific geometry used, contains
information on the chemical bonds in a sample.

Seed Identification

Near infrared spectroscopy is useful for seed classification on both single and bulk
collections of seeds. As this spectroscopic method can be used on bulk or single seeds,
impurity fractions can be calculated upon batch seed collection, and the uniformity can thus
be analysed.

With regards to quality control, near infrared spectroscopy can be used to distinguish real
seeds from fake ones, specific chemical species, including certain oils and isoflavones, can
also be screened for in the near infrared spectrum. In addition, near infrared spectroscopy is
highly sensitive to water and can hence be used to assess moisture levels.

In order to assess the phenotype profile of a seed, near infrared spectroscopy can evaluate the
difference in the protein, oil content and fibre structure. Near infrared spectroscopy is
sensitive to the combined chemical makeup of the compounds present in the oil; therefore,
any changes in this composition can be a sign of modification of the seed type or adulteration
of the final product.

Chemometrics

One challenge in using near infrared spectroscopy to assess seed profiles is the complexity of
the information that has been obtained.

A single infrared spectrum from a single molecule can have a number of peaks. Substances
like oils, however, are complex chemical mixtures and will therefore produce a spectrum that
is a superposition of the spectra of all of the present chemical species. The intensity of each
molecule's spectral features will be scaled proportionally to the concentration.
However, analytical methods can be employed to gain meaningful information from mixtures
like those present in seeds or their extracted products, in spite of the spectral complexity of
mixtures.

The solution to the problem lies in chemometrics – a powerful tool for extracting information
from complex multivariate datasets which has been used with great success for distinguishing
between seed types.

Chemometrics methods can be sensitive to even slight changes between samples and help
identify whether or not the chemical profile of a particular seed is within expected ranges.

References:

 [Link]
 [Link]
 [Link]
 Dhirendra Khare, Plant Breeding and Genetics, JNKVV, Jabalpur
 Chapter 5, [Link]
 [Link]