Lecture No.

15 & 16
VARIETAL IDENTIFICATION
1. Grow - Out Test

Objective
To determine the genetic purity status of a given seed lot of the notified
the extent to which the sample in cultivar/hybrid and
question conforms to the
prescribed standards.
Field of applicability

Grow-out Test is the official measure for

controlling the genetic purity of the seed lot. It
serves as a pre-control as well
'post-control' test for avoiding genetic
as a

According to the official regulations in ndia, it is prerequisite for seed contaminations.

certification of
hybrids of certain species such as cotton, castor, musk melon and
brinjal
The test is required to be conducted for
checking the sellers label with
purity status of the seed lot under the proVisions of the seeds Act 1966. In respect genetic
to

test can also be used as a measure to

addition, grow-out
judge the efficacy of the certification agency or the
inspector,

Sampling: The samples for "Grow-out test shall be

drawn
for other seed quality tests in accordance with simultaneously with the samples
the prescribed sampling procedures.
Size of submitted
sample: The size of submitted
samples shall vary according to the species
as exemplified in this Table.

Recommended size of submitted sample for Grow-out Test

For maize, cotton,

1,000 g groundnut, soybcan and species of other genera
with seeds of similar size
For sorghum, wheat, paddy and
500 g species of other genera with seeds of
similar size
250 g Beta and species of other genera with seeds ofSimilar size
100 g For bajra, jute and species of all other genera
250 tubers/ planting
stakes roots/ corms Seed potato, sweet potato and other vegetatively propagating crops.

Size of working
sample
The working sample for grow out test shall be obtained through subsequent mixing and
dividing of the submitted sample in accordance with the prescribed
procedure for seed
Sampling. The minimum population required for taking the observations shall be 400
plants;
however, it will also depend on the maximum permissible off type
plants prescribed for the
species under consideration in the Indian Minimum seed Certification standards.
The number
of seeds required for raising the crop to obtain the required number of plants shall
e
germination percentage of the seed
depend on
sample and hence seed rate should be adjusted
accordingly
Number of plants required per sample for grow out test

Maximum permissible off

Minimum Number of plants required
types (%) genetic Purity (%)
per sample
0.10 99.9 4,000
0.20 99.8 2,000
0.30 99.7 1,350
0.50
99.5 800
1.00 and above 99.0 and below 400

Procedures

To achieve the accuracy and reproducibility of the out test

grow results; the procedures
provided hereunder must be followed:

Location of the grow out test

Grow out test shall be conducted in

specified areas recommended for the cultivar/hybrid or in
off-season nurseries.
Standard sample

The standard sample of a cultivar (control) is the official standard against which all other
samples of the seed of the cultivar will be
judged. The standard sample must not differ
significantly any character and be obtained from the
in
originating plant breeder/breeding
institute and be stored under controlled and
temperature humidity conditions so as to use it
cach year to sow control
plots for cultivars under test. Further quantities of sample must be
obtained from the originating plant breeder as and when
required. A comparison must be
made between the two lots of the standard
sample before changing from one standard sample
to another.

Method of raising the erop

Standard and recommended
agronomic/cultural practices such as field preparation, size of the
plot, row length, distance between the rows, distance between the
plants, irrigation and
fertilization, ete., in respect of the specific crop shall be followed both for the
question and its control (standard sample). The sample in
germination percentage
of the sample (s) in
question and the standard sample must be determined to adjust the seed rate. The
should be done sowing
by dibbling or small plot drill. Seed drill must be
carefully checked to ensure
its cleanliness. Subsequent thinning is not recommended. The samples of the
must be sown in
same cultivars
succession and the
standard samples are sown at suitable intervals. (one
standard sample for
every ten sample to be tested). The size of the plot, row length and
spacing shall differ according to the crop. Recommended
variables are provided in Table mentioned below specification for the above
which can suitably be
considered essential. modified if
 Recommended roW length, distances, spacing for some important crops

Row Plant to Row to Plot to

S.No Crop length (m) plant (cm) row (cm) plot (cm)

Wheat, barley, oats 6 2 25 50

6 10 45 90
Pea, Cowpea
Chickpea, green gram, 10 30 60
3
black gram
10 25 60 90
4 Maize
10 45 45
Hybrid cotton

6 Paddy:
Early to medium 6 15 20 45

6 25 30 60
Late and very late

6 10 60 90
Pearl millet

6 10 45 60
Sorghum

should be grown in two replicates to guard against

failure in one part of the
The field plots
variations.
field and to reduce environmental and soil fertility

Methods for taking observations

season with emphasis on the
Grow-out test plots must be examined throughout the growing
must be examined keeping in view the
period from the flowering to ripening. All plants
both in the test crop as well as the
distinguishing characters described for the cultivars
deviations in characters against the
control. While taking the observation, the plants showing
to confirm whether they are
control should be tagged and examined carefully at a later stage
the off-type plants found should be
off-types or not. The number of the total plants and
record

Calculation and interpretation of the results

Percentage of other cultivars, species or aberrant found must be calculated up to first decimal
place. While interpreting the results, tolerances should be applied by using the reject number
sample size as provided in
for prescribed standards with reference to Table
Reject number for prescribed standards and sample size

Reject numbers for samplesize of

800 400
99.5 (1 in 200) 8
*

99.0 (1 in 100) 16 8
95.0 (5 in 100) 48 24
90.0 (10 in 100) 88 44
85.0 (15 in 100) 128 64
1ndicates that the sample size is small
too for a valid test.
Reporting of results

The results of the grow-out test shall be reported as

percentage of other species,
cultivars off-type plants.
or

If the sample is found to be a cultivar other than stated by the sender, the results shall
be reported as such.
If plants of other cultivars are more than 15 percent, the report shall state that the
sample consists of mixture of different cultivars.
If nothing worthy of special comments is found, the report shall state that the results
of the grow-0ut test of the sample in question revealed nothing to indicate that the
name of the cultivar or species stated by the sender is incorrect.

2. Electrophoresis (Biochemical test)

It is the 1latest method of cultivar identification based on protein banding and isoenzyme
activity. Here single seeds are defatted and extracted for protein and esterases. The extracted
proteins or esterases are separated by polyacrylamide gel electrophoresis. Based on the
banding pattern of protein and esterase's the varieties can be differentiated and identified.

Electrophoresis for proteins and enzymes: Seeds, seedlings or mature leaves, etc. of a crop
plant have a specific mix of proteins which are not only crop specific but also variety specific

genotype specific). electrophoresis in a suitable medium separates the mixture of

The
leaves into distinct bands. Each variety (or
proteins extracted from seeds, seedlings or mature
the basis of which admixtures of other
genotype) thus has a specific "banding pattern" on
detected. This is done by comparing the
varieties, differing in "banding pattern" could be
the standard banding pattern of that variety. The
banding pattern of analyzed sample with
used for determining the genetic purity of seed
electrophoresis is now being increasingly
samples.
'electrophoresis' refers to migration of a charged particle under the
the
Principle: The term
of ions takes place in a suitable medium, such as
influence of an electric field. The
movement

a molecular sieve and

cut down convection currents and
polyacrylamide gel, which acts as
renain as sharp zones With maximum resolution.
diffusion, so that the separated components
The separation into distinct bands
is due to,
variOus proteins. Particles
molecules (molecular weight) of
1. Differences in the size of
faster than those with higher weights.
with smaller molecular weights migrate
Differences in charge. The molecules with the higher charge migrate faster than those
2
with a lower charge.
at any pi otner than their point, they migrate in
isoelectric
Since proteins carry a net charge
an electric field, the rate of
which depends on the charge density (that is, the rate of charge to
mass of the molecule). Proteins with higher charge density will migrate faster, thus resulting
in differential rates of movement of proteins when a mixture of different proteins is subiected
to an electric field. By altering the gel pore size (using polymers at different concentrations)
and the charge on the protein molecule (by changing the pH of the system) a high degree of
molecules in a mixture.
resolution can be achieved for separation of protein
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