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methods of genetic purity testing

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Darya Khan Akbarzai

Date : 08/04/2019
CONTENT 2

 Introduction
 Definition and Objective of GP
 Morphological markers
 Chemical tests
 Biochemical markers
 Molecular markers
 Conclusion
 Future thrusts
 Seed Quality 3

It is a multiple trait which includes several components viz. purity (physical and
genetical), germination, viability, vigour, freedom from other crop seeds, weed seeds and
seed borne diseases, low seed moisture content and size. It is the degree of excellence in
regard to the referred seed characteristics that determines the seed quality
 Genetic purity ? 4

Genotypic purity is simply defined as true

to type plants / seeds conforming to the
characteristics of the variety as described
by the breeders.

Principle:
Genetic purity or genuiness of the cultivar
is tested by means of heritable characters
(morphological, physiological or chemical)
of seeds, seedlings or plants.
Causes of deterioration of genetic purity and its Maintenance
5
 Developmental  Improper / defective
Variation seed certification System
 Mechanical Mixtures  Control of seed source
 Mutations  Preceding crop
 Natural Crossing requirement
 Genetic drift  Periodic testing of
varieties for genetic
 Minor Genetic Variation purity
 Selective influence of  Isolation
Diseases
 Rouging of seed fields
 Techniques of the
 Seed certification
Breeder
 Grow out test
 Breakdown of male
sterility
Need for genetic purity testing 6

 To increase crop production at national

level.
 To increase farmers income and standard of
living.
 To make IPR (plant breeders right and plant
variety protection) part strong.
 For distinctiveness, uniformity and stability
(DUS) test.
 Quality control of grains for processing.
 Documentation of genetic resources.
DUS 7

 D: Distinctness – The variety should be clearly distinguishable

from any other existing variety at least for one character .
 U: Uniformity – The variety should be sufficiently uniform to
enable its description.
 S: Stability - The variety should be stable in its relevant
characteristics, that is, it must remain true to its initial
description even after repeated propagation.
Types of GP test 8

Testing of genetic purity of variety conveniently

divided into three gropes
 Laboratory
 Greenhouse
 Field plot tests and field inspection
Methods of GP test 9

Biochemical
Morphological
markers Molecular
/ Conventional Chemical test
(Proteins and markers (DNA)
grow out test
Isozymes)
 10

Morphological Methods
 Laboratory examination 11
 Laboratory examination is test of the varietal purity test which
carried out in a seed testing laboratory.
 The test is based on appearance of seed as morphological
character, seedlings or chemical test.
 The working simple consisted of 400 seed ( 4 rep of 100 seed
each) taken randomly from sub-sample.
 The morphological characters are examined with the aid of a
suitable magnification as length, breadth, thickness, size,
shape, surface, volume, roundness, texture etc.
 The color characteristics are examined under full daylight or
ultraviolet light.
 Chemical characteristics are examined after the treating the
seed with appropriate reagent and the reaction of each seed
is noted.
Field plots or grow-out test 12

 Morphological feature has been a major

component of cultivar identification in field testing
and observation on different qualitative and
quantitative characters are recorded from seeding
stage to maturity.
 Several character such as seedling, pigmentation,
stem colour and pubescence, length of internode,
growth habit, flowering type and colour and
maturity etc. are considered at all stages of plants
growth.
 Among them only reliable distinguishing characters
are taken into consideration
Morphological test 13

In field

 The seed sample is sown in the controlled

condition with the authentic sample.
 Genetic purity is determined on the basis of
observation made on the plant morphological
characters with reference to authentic sample.
 Genetic purity is always expressed in
percentage.
Grow out test (GOT) 14
 Characters
1. Highly heritability
2. Stable expression over a range of environments.
3. Easily discerned by visual observation.
 Sufficient spacing between rows and plants.
 Various samples of the same cultivar sown in succession
and standard samples are sown at suitable interval.
 Deviation from control sample counted.
 Mutual comparison between the samples to be tested
and the standard.
 Observations at full growing period.
15
 Procedures 16
 The minimum population required for taking the observations shall be
400 plants; however, it will also depend on the maximum permissible
off-type plants by following recommended cultural practices e.g.,
field preparation, size of the plot, etc
 Provide equal opportunity to each and every plant for full expression
of genetically controlled characters.
 Sow the various samples of the same variety/ cultivar in secession
and standard sample at suitable interval.
 Adjust of seed rate depending on germination % of individual sample
and subsequent thinning is not recommended.
 This test preferably conducted in area to which the variety is
recommended
 A minimum of 200 plants from control sample should be raised along
with test crop.
 The analyst employed for conducted ‘grow out test’ should possess
the basic as identified under seed rules, 1968.
 qualification
Size of working sample 17
Recommended row length, distances, spacing for some
important crops 18
 Methods for taking 19
observations
Grow-out test plots must be examined
throughout the growing season with emphasis on
the period from the flowering to ripening.
 All plants must be examined keeping in view the
distinguishing characters described for the
cultivars both in the test crop as well as the
control.
 While taking the observation, the plants showing
deviations in characters against the control
should be tagged and examined carefully at a
later stage to confirm whether they are off-types
or not.
Reject number for prescribed standards and sample size 20
Limitations of morphological 21
methods
 Environmental stress conditions often mask specific
morphological traits.
 Large amount of land required.
 Laborious
 Time consuming
 Unfavorable condition, i.e. disease and insect
infestation may limit GOT in field
 Morphological markers are becoming limited in relation
to rapid increase in number of varieties, hybrids and
transgenic.
Chemical Tests 22
 The field testing has to wait thought out the growing season until all traits
of the cultivar have been expressed.
 There is need for tests which can quickly establish the identity of a
cultivar.
 Quack tests so developed often rely on the reaction of a specific
chemical compound with some component of seed.
 Some chemical test are:
1. Phenol test 8. Ferrous sulphate test
2. Fluorescence test 9. 2,4-D soak test
3. Peroxidase test 10. coper sulphat-ammonia test
4. Potassium hydroxide (KOH)test 11. Gossypal test.
5. Potassium acid (HCI) test
6. Hydrogen peroxide (H2O2) test
7. Sodium hydroxide ( NaOH) rest
 Phenol test 23
A. Standard phenol test and B. Modified phenol test
Equipment and chemical
 Phenol (Carbolic acid) crystals, copper sulphate
(CuSO4), sodium carbonate (Na2CO3), Petri dishes, filter
paper, distilled water, amber collour bottle etc.
Procedure for standard phenol test
 Phenol solution ( 1.0%) is prepared by adding 5g phenol
crystals to 500 ml distilled water. Store the phenol solution
in an amber glass container in a cool place and do not
use phenol solution which is more than three months old.
 Place two layers of filter paper in a Pertidish and saturate
with distilled water.
Cont.. 24
 Pour off excess water and place the seed on the filter paper ( 50
seed per Pertidish of 9 cm diameter). Spread the seed uniformly
on the surface of moistened filter paper.
 Cover the Peridish and incubate the seed overnight ( 18-24 hrs) at
room temperature.
 Then add enough 1% phenol solution ( about 3-5 ml per petridish)
to moisten the filter paper.
 Close the pertridish and after 4 hours record the colour reaction
of seeds. The colour is observed as
1. Nill, no reaction
2. Light brown,
3. Brown,
4. Dark brown,
5. Balck
25
Cont… 26

B. Modified phenol test

 The procedure is similar to standard phenol
test but instead of distilled water the seeds
are soaked in 0.4% of copper sulphate
(CuSO4) or 0.6% sodium carbonate
(Na2CO3) soluation and incubated overnight
at room temperature.
 Than add 1% phenol solution. after 4 hours
the colour reaction is noted and observed.
 Based on the colour development the
varieties are categorized into different
groups.
Peroxidase test 27
Buttery and Buzzell (1968) separated soybean cultivar into two
groups based on the presence of either low or high seed coat
peroxidase activity.
Equipment and chemical: Guaiacols, hydrogen peroxide, test-tube
or other suitable container
Procedure:
 Place seed coats removed from the soybean seeds into a test
tube or suitable container
 Add 10 drops of 0-5% guaical solution to the test-tube
 After 10 minute add a drop of 0.1% hydrogen peroxide solution
 One minute after adding the hydrogen peroxide, record the
seed coat colour as peroxidase positive (high peroxidase activity)
indicated by a reddish –brown solution or peroxidase negative
(low or no peroxidase activity) indicated by colorless solution in
the test tube.
 Fluorescence test 28
Crops: Ryegrass, oat
Equipment: Fluorescence lamp, black paper, germinator etc.
Procedures:
 Place the seeds to be tested on a black background
 Evaluate the seeds for inflorescence under black light
tubes in a room from which all other sources of light are
excluded.
 Seeds are considered fluorescence of the lemma or pelea
fluoresce or appear light in colour, partially fluorescent
seeds should be considered fluorescent.
Seed are considered non- fluorescent if the lemma and
pelea do not fluoresce and appear dark in colour
29
Advantages of chemical tests 30

 They are quick.

 They require virtually no technical
expertise or training.
 Relatively inexpensive to conduct.
 No sophisticated equipment are
required.
 The test permits detection of percentage
admixture of other type.
 Its results are usually distinct and easily
interpretable.
 Biochemical and Molecular methods
31
Electrophoresis is a technique which separates mixture of
protein into district bands in a gel that has been placed
into an electric field.
Each variety has a specific banding pattern on the basis
of which admixture of other varieties, differing in banding
pattern could be detected.
Principle: the term of electrophoresis refers to the
migration of a charge particle under the influence of an
electric field. the movement of ions take place in a
suitable medium such as polyacrylamide gel. Which is
act as molecular sieve and cut down. This separation into
distinct bands is due to difference in
• Size (molecule weight); small partials migrate faster
than higher weight
Cont.. 32
There are various electrophoretic methods available:
 Acid-PAGE
 Alkaline- PAGE
 Sodium Dodecyl Sulphate Polyacryamide gel electrophoresis
for denatured protein (SDS-PAGE)
 Polyacryamide gel electrophoresis (PAGE)
Cont… 33

Steps
1. Sample preparation
2. Gel preparation
3. Electrophoresis
4. Staining

Equipment:
electrophoresis unites,
electricity supply unites,
micropipettes, gloves
Sample preparation 34
 De-coat single seed of cotton, crush and defat in 3 to 4 changes
for defatting solvent mixture. decant and dry and defatted meal
at room temperature.
 Or Heating the sample to 100 C to denature the protein.
 Or using liquid N for preparation of the simple.
Protein extraction:
Preparing the working protein extraction solution by mixing 4.25 ml of
stock protein extraction solution to 0.75 ml β-mercaptoethanol and
adding DW to 10 ml:
 2.5 ml 1 M Tris-HCl pH 6.8.
 0.5 ml of ddH20.
 1.0 g SDS.
 0.8 ml 0.1% Bromophenol Blue.
 4 ml 100% glycerol.
 2 ml 14.3 M β-mercaptoethanol (100% stock)
35
Cont…. 36

- Transfer the defatted single seed meal to

1.5 ml tub and add 0.3 ml solution.
- Leave the simple for 2 h at room
temperature and than store refrigerator
overnight.
- Heat in a boiling water bath for 10 min,
- cool the tubes and centrifuge at 15000
rpm for 10 min.
- Use the clear supernatant for
electrophorese.
 Gel preparation 37
 Clean and dry the gel plats and
assemble the caste as per the
instruction.
 A gel thickness of 1.0 to 1.5 mm is
generally used.
Separating gel:
- Tris Buffer ( B.1) pH 8.8: 12.0 ml
- Water: 7.4 ml
- 30% running gel acrylamide (B.11):
20.0 ml
- 5% APS ( B.4) : 0.4 ml
- 10% SDS ( B.2) : 0.4 ml
- Add 0.04 ml of TEMED just before using
Fill the caste till 3 to 4 cm fro the top and
overly DW and take 30-60 min to
prepared
Stacking gel 38
After the polymerization of the main gel, pour off the water layers
from the top and plot the cassette.
Pour the stacking gel mixture into it.
- Tris Buffer ( B.2) pH 6.8 : 1.50 ml
- Water: 6.00 ml
- 30% running gel acrylamide (B.10): 2.00 ml
- 5% APS ( B.4) : 0.4 ml
- 10% SDS ( B.2) : 0.10 ml
- Add 0.04 ml of TEMED just before using
Than set the comb and let the gel polymerize.
It will take 10-15 min and remove the comb, wish with tank buffer or
water
Electrophoresis 39
 Fix the gel cassette into the electrophoreses unit as
per design.
 Load 10 μl of clear seed extract to each well.
 conduct electrophoresis at 35 m A ( 20 sample) still
the sample migrates into the running gel.
 Subsequently, at 60 m A until the tracking dye reach
the bottom of gel.
 Fixing and staining the gel 40
 Remove the cassette from the unit and take out the gel
gently.
 Place it in a staining tray and incubate overnight in 15% TCA
solution.
 Wish throughoutly the excess SDS and pour sufficient staining
solution to cover the gel uniformly.
 Incubate for 6-18h, decant the stain and rinse with water.
Destaining
The in water
varieties are verifiedfor
onday
the or two
basis ofclears
bandingthe gel background
pattern
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑏𝑎𝑛𝑑
1. by measuring the Rm of band 𝑅𝑚 =
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡𝑟𝑎𝑐𝑘𝑖𝑛𝑔 𝑑𝑦𝑒

2. total number of bands

3. Presence or absent of specific band
4. Intensity of band
41
Advantages and limitations of Biochemical methods
42
 They are not affected by the environment.
 They are cost effective compared to other methods
and the turnaround time is relatively rapid.
 Multilocus analysis provide useful information for
verifying inbred and hybrid genotypes.
 Most are co-dominant and many loci express at all
stages of life cycle.
 An array of enzymatic analysis can be made using
small quantities of leaf and seed material.
 There are limited number of marker isozymes as
compared to molecular markers.
Molecular markers 43

DNA markers are defined as a fragment of DNA

revealing mutations/variations, which can be
used to detect polymorphism between different
genotypes or alleles of a gene for a particular
sequence of DNA in a population or gene pool.
 RAPD (Random Amplification of Polymorphic
DNA)
 SCAR (Sequence Characterized Amplified
Region)
 SSR (Simple Sequence Repeats)
 STS (Sequence Tagged Site) Different
molecular markers
Materials 44

CTAB buffer, centrifuge tubes, Mortar and Pestle,

Liquid Nitrogen, centrifuge, Absolute Ethanol (ice
cold), 70 % Ethanol (ice cold), 7.5 M Ammonium
Acetate 55o C water bath, Chloroform: Iso Amyl
Alcohol (24:1), Water (sterile), Agarose, 6x Loading
Buffer, 1x TBE solution, Agarose gel electrophoresis
system, Ethidium Bromide solution
Overview of (1) LEAF TISSUE
‘marker SAMPLING
genotyping’

(2) DNA EXTRACTION

(3) PCR

(4) GEL ELECTROPHORESIS

(5) MARKER ANALYSIS

Cont… 46
CTAB buffer 100ml

CI buffer: Chloroform: Iso Amyl Alcohol (24:1),

5x TBE buffer
54 g Tris base
27.5 g boric acid
20 ml of 0.5M EDTA (pH 8.0)
Make up to 1L with water.
To make a 0.5x working solution, do a 1:10 dilution of the concentrated stock

1% Agarose gel:
1 g Agarose dissolved in 100 ml TBE
Procedure 47
- Grind frozen plant material (100mg) in liquid Nitrogen
- In a 2 ml tube, add 800 uL of 1,5x CTAB and 1 ul of Beta-mercaptoethanol to the
ground leaf material and Incubate 1 hour at 60-65 degrees (C)
- Cool at Room Temp - Add 1 volume of Chloroform/Isoamylalkohol (24:1) mixture
- Centrifuge at 8000 rmp for 10 minutes
- Transfer supernatant to a new tube - Use wide pipette tips (cut point of tips if needed)
- Add 10 uL RNAse (10 mg/ml) - incubate at 37 degrees (C) for 30 minutes
- Add one volume 100% Isoponanol (pre-chilled at -20) - Allow sample to precepitate
for up to 20 minutes at -20
- Centrifuge: full speed, 10 minutes - Remove supernatant
- Add 1 ml 70% Ethanol - Centrifuge: full speed, 10 minutes - Remove Supernatant -
Repeat once
- Dry pellet in air or approx 20 minutes in vacuum centrifuge
-Dissolve pallet in 1x TE (10 mM Tris, pH8, 1 mM EDTA)
DNA extractions

Mortar and pestles

Porcelain grinding plates

LEAF SAMPLING
Wheat seedling tissue sampling in
Southern Queensland, Australia.

High throughput DNA extractions “Geno-Grinder”

DNA EXTRACTIONS
PCR-based DNA markers
 Generated by using Polymerase Chain Reaction
 Preferred markers due to technical simplicity and cost

PCR Buffer +
MgCl2 +
dNTPS +
Taq +
PCR
Primers +
DNA template

THERMAL CYCLING

GEL ELECTROPHORESIS
Agarose or Acrylamide gels
50
PCR 51
Agarose gel electrophoresis

http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/agardna.html

UV transilluminator
UV light
Acrylamide gel electrophoresis 1

UV transilluminator

UV light
Reading the band 54
Advantages and limitations of molecular techniques 55
 It has very large number of polymorphism development as
compared to the bio-chemical markers.
 Residual heterozygosity can be detected.
 It is reliable to all crops.
 Very fast method.
 Sophisticated instruments required.
 Very costly.
Examination of seedlings 56
Working sample: 400 seeds. ( or for ploidy test
initially 100 with a further 100 when initial
determination is inconclusive).
Determination: the seed are germinated in
replication of no more than 100. on a suitable
medium. When seedlings have reached a
suitable stage of development, they are
examined in whole or in part with or without
further treatment.
For determination of ploidy, root tip is excised and
processed for examination of chromosome
number.
 Cont..
57

Cereal
 Some varieties can be distinguished by
the colour of their coleoptiles, which
varies from purple to green and is
determined when the seedlings reach a
suitable stage of development.
Procedure (to intensify the colour): moisten
the filter paper (substrate) with a 1% solution
of NaCl or HCl; alternatively, place the
seedlings under UV light for 1–2 hours before
examination.
 cont… 58

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.
Procedure:
germinate 400 seeds in the dark at 20–30 °C;
after 5 days, place cotyledons in Petri dishes
containing alcohol (85–96%); place dishes on a
white surface; determine the colour of the
cotyledons after 4 hours.
 Cont.. 59

 Grasses
Ryegrass species can be identified using the fluorescence test
on seedlings. Root traces in the majority of Lolium multiform
cultivars exhibit fluorescence while in Lolium perenne they do
not.

Procedure:
 Place the seed on non-fluorescent white filter paper
moistened with distilled water for germination under the
prescribed conditions.
 After 14–18 days, when roots are well-developed, examine
seedlings under ultraviolet light from a lamp transmitting
360–370 nm radiations.
 Record the number of fluorescent seedlings and non-
fluorescent seedlings, as well as the number of normal
seedlings, for each repetition.
References 60

Agrawal, R.L. (2019). Seed technology (2nd Ed.).

New Delhi. CBS Publisher and distributor Pvt Ltd
Anirudha Kumar Sahu, I.S. Katageri, M.P. Jadhav
and Vamadevaiah, H.M. 2017. A Simple,
Rapid and Effective Protocol for Extraction of
Total Plant Proteins from Cotton Leaf.
Int.J.Curr.Microbiol.App.Sci. 6(12): 2968-2975
FAO (2019). SEEDS TOOLKIT Module 3: Seed
quality assurance.
Kharb, R.P.S and Punia, R.C. (2010) Practical
Manual on Seed Quality Testing. Hisar.
Department of Seed Science and T
echnology. CCSHAU.
 Thanks

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