1. Assignment on Varietal IdentificationMethods and their Importance
SUBJECT: GPB – 221 Principles of Seed Technology (2+1)
Submitted by – Submitted to -
Bhavya Singh Mr. Girish Tantuway
18230AGC009
B.Sc. (hons) Ag. 2nd year 4th semester

2. VARIETAL IDENTIFICATION METHODS
& THEIR IMPORTANCE

3. WHAT IS VARIETY ?
In agriculture , variety can be described as a taxonomic category that ranks below subspecies
(where present) or species, its members differing from others of the same subspecies or species
in minor but permanent or heritable characteristics.
Within a species there is possibility of presence of a wide range of different types of plant
present. Farmers and growers need plants with particular characteristics and that are adapted to
their environment and their cultivation practices. A plant variety represents a more precisely
defined group of plants, selected from within a species, with a common set of characteristics.
Examples are the varieties of strawberries, apples, corn, wheat, cotton, etc. ‘Variety,’ in this
sense, is thus a generic word including races, strains, and clones.

4. WHAT IS VARIETAL IDENTIFICATION ?
• Varietal Identification test can be described as process that is used in
identifying and separating different varieties of crop present in the
same species /subspecies/lowest taxonomic division.
• The different morphological, molecular, biochemical and chemical
procedures are present which serve as tools for varietal identification.
Latest technologies like molecular and other test give more precise and
quicker results in comparison to the older methods.

5. Need for Varietal Identification
 To have identical type of seed lots (variety) for a particular field.
 The identical seed lots will lead to uniformity in crop production.
 Varietal Identification is a prerequisite for assurance of seed quality and therefore
yields.
 Usage of better and advanced methods not depending on morphological and
phenotypic characters for identifying a variety so that its not affected by environmental
fluctuations and rather are permanent criteria for identification.
 To bring about increase in crop production at national level by having identified high
yielding varieties possessing uniformity.
 It is a necessary step to increase farmer’s income and standards of living.
 For quality control of grains for processing.
 For documentation of genetic resources.

6. METHODS OF VARIETAL IDENTIFICATION
• - DUS test - Seedling examination
• - Grow Out Test - Greenhouse examination
• - Seed morphology test - Germination test
Morphological
method
• - Phenol test - 2,4-D Auxin test
• - Modified phenol test - Ferrous sulphate beach test
• - Potassium hydroxide(KOH) test - Alkaloid test
• - Sodium hydroxide test (NaOH) - Iodine test
• - Peroxidase test - Gibberellin soaking test
Chemical
method
• - Electrophoresis
• - Polymerase Chain Reaction ( PCR)
• - Molecular Markers
Molecular
method
Biochemical
method

7. 1. MORPHOLOGICAL METHODS
1. DUS Test
DUS stands for Distinctness, Uniformity, Stability.
• Distinctness - The variety should be clearly distinguishable from any other existing variety at least for one character.
• Uniformity - The variety should be sufficiently uniform to enable its description.
• Stability - The variety should be stable in its relevant characteristics, that is, it must remain true to its initial
description even after repeated propagation.
A DUS test is usually conducted in the field or in a glasshouse over two successive growing seasons. During this period
a number of mainly morphological characteristics are recorded both on the new (or candidate) variety and on similar
varieties in what is known as “Common Knowledge”. Differences, if existing, are established by observation and
measurement using internationally agreed protocols.
It is conducted in the trial field and also in the laboratory in accordance with guidelines and requirements of
International Union for the Protection of New Varieties of plants (UPOV). A variety is considered in common
knowledge which is granted rights and is a registered variety.

8. Some characteristics have to be analysed for DUS test and these characteristics are classified as
bellow :
• Qualitative characteristics – It is expressed in discontinuous states which are self -explanatory and independently
meaningful. All states are important for describing the full range of characteristics and each and every form of expression
can be described by a single state and are not influenced by environmental factors.For example- panicle and awns in rice
(expression states – present or absent ) , anthocyanin colouration of collar in leaf of rice (expression states – present or absent)
, types of grains in corn ( expression states- flint, dent like , sweet , pop , waxy etc.)
• Quantitative characteristics – the expression will cover the full range of variation starting from one extreme to the other
extreme and expressions can be recorded at a one dimensional, continuous or discrete, linear scale.The range of expression is
divided into number of states for description. For example- intensity of anthocyanin colouration in leaf sheath of rice and in
stem of corn (expression states – very weak, weak, medium and strong ) ,
• Pseudo-qualitative characteristics- In this the range of expression is at least partly continuous but varies in more than one
dimension. It cannot be adequately described by just defining two ends of a linear range. Each individual state of expression
needs to be identified to adequately describe the range of the characteristic. For example – spikelet: colour of stigma in rice (
expression states :white, light green, light purple, yellow , purple) , main colour of top of grain in ear of a corn ( expression
states : white , yellow white , yellow, yellow orange, purple)

9. • Examining Distinctness (D)
The candidate and common knowledge varieties are compared based on: consistently different and clearly distinguishing
characters. The methods for examining include observation, measurement and using statistical methods. For measured
Characteristics: we need to establish a minimum allowable distance between the varieties. For observational characteristics –it
depends on the types of characteristics (qualitative , quantitative or pseudo – qualitative). For example – length of longest awn
in the rice panicle ( expression states: very short, short, medium, long, very long) , anthocyanin colouration of leaf sheath in
rice ( absent or present : QL)
• Examining Uniformity (U)
The variation in the expression of relevant characteristics forms the basis for the assessment of Uniformity. Particular features
of propagation of the variety must be considered when examining U are : Truly self-pollinated or Mainly self-pollinated ;
Inbred lines; Vegetative ;Cross pollinated or Mainly cross pollinated ;Synthetic varieties ;Hybrid varieties.
• Examining Stability (S)
Difficult to examine in comparison to Distinctness and Uniformity for the DUS test. Stability is assessed indirectly
through Uniformity: if a variety is uniform then it is considered generally as stable.If there is a doubt, then special testis
done i.e. compare the new and old material stocked by the applicant. If a variety is not stable –its material will be
definitely refused from granting of rights. Hybrids can be examined by other test or indirectly through their parents.

10. 2. Grow Out Test
Objective of Grow Out Test - To determine the genetic purity status of a given seed lot of the notified cultivar /
hybrid and the extent to which the sample in question conforms to the prescribed standards.
Field of applicability- It serves as a pre-control as well as a ‘post-control’ test for avoiding genetic
contaminations..In addition the grow-out test can also be used as a measure to judge the efficacy of the
certification agency or the inspector.
Sampling - The samples for ‘Grow-out test shall be drawn simultaneously with the samples for other seed
quality tests in accordance with the prescribed sampling procedures.
Size of submitted sample - The size of submitted samples shall vary according to the species.
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
number of seeds required for raising the crop to obtain the required number of plants shall depend on the
germination percentage of the seed sample and hence seed rate should be adjusted accordingly.
Procedure of GOT includes the following steps : location for GOT ; setting standard sample ; method of raising
the crop ; methods for taking observations ; calculation and interpretation of results ; reporting of results.

12. 3. Seed Morphology Test
These are based on morphological characters which are examined with the help of suitable and required
magnification. For ex.- size and shape of grain, base of lemma, vertical crease hairs, rachilla hairs, deviation of
lateral dorsal nerves etc. The colour characteristics are examined under full day light or light of limited
spectrum e.g. ultraviolet light.Scanning with electron microscope is used for the purpose of studying
differences in seed coat surface and its inner structure have also been used in some species.
4. Examination of seedlings
Many useful varietal identification tests have been performed on seedlings. Such tests are useful
because they may yield more information than observations of the un-germinated seed and do not
require as much time as field grow-out tests.

13. Limitations of morphological methods -
 The environmental stress conditions often mask or cover up specific morphological traits.
 Large amount of land requirement is a major drawback
 It is very laborious procedure.
 It consumes a lot of time.
 Presence of the unfavourable condition, i.e. disease and insect infestation may limit Grow Out
Test in the field.
 Morphological markers are becoming limited in relation to rapid increase in number of varieties,
hybrids and transgenics.

14. 2. CHEMICAL METHODS
• There are various laboratory or chemical tests which have additional benefits for varietal identification. These chemical
tests are very quick, easy to do, reproducible and can be conducted throughout the year under controlled conditions.
• The chemical tests reveal differences of colour among the seeds. Study of phenotypic characters along with chemical and
biochemical techniques have additional benefits for producing more authentic result. In these chemical tests, the chemical
agents react with the seed and help in varietal identification. Some of the major chemical methods are :-
- Phenol test - Alkaloid test
- Modified Phenol test - Iodine test
- Potassium Hydroxide (KOH) test - Gibberellin soaking test
- Sodium Hydroxide (NaOH) test - 2,4 – D Auxin test
- Ferrous Sulphate bleach test - Peroxidase test

15. 1. Phenol test
The Standardized phenol test for varietal purity testing as suggested by walls (1965) was followed. This test is highly
specific for varieties. Phenol reaction is monogenically controlled response. An enzyme polyphenol oxidase (PPO) or
tyrosinase present in seed coat is responsible for the oxidation of externally supplied phenol into quinones and their further
polymerization yield melanin like pigments which result in development of brown colouration in seeds. Coloured reaction
serves as a basis for grouping intensity of the colour received and is graded on a scale of 0-9 where 0 is negative or no
change gradual positive intensification of colour from light brown to deep black proceeds. It is applied mostly on wheat ,
rye , sorghum, foxtail millet, mustard.
2. Modified phenol test
It is similar to phenol test but in this 2 samples are taken and one is soaked in copper sulphate solution and other in sodium
carbonate solution. . Based on the colour development in both the tests groups were made and in CuSO4 soaked seed
groups were classified in to three groups dark brown, brown and reddish brown colour , and for Na2CO3 soaked seeds
were classified in to three groups brown, dark brown and strong brown.

16. 3. Potassium Hydroxide (KOH) test
Hundred seeds in three replications were soaked in five per cent KOH solution for 2 hrs at room temperature.
Changes in colour of seeds is observed after 1 hr. Based on the colour intensity of the seed, the genotypes were
classified into three group’s viz., dark brown, reddish brown and light brown. The duration for test varies
according to seeds taken and their seed coat and ;are observed for the presence of dark pigmented tannic acid
and light pigmented testa.
4. Sodium hydroxide (NaOH) test
100 seeds in three replications are soaked in 5% NaOH solution f hr at room temperature. Changes in colour of
seeds is observed after 1 hr. Based on colour intensity of the seed, the genotypes are classified. It is used for
several cereal crops.
5. Peroxidase test
Under this test, 60 seed are soaked in water for 24 h after that 15 seeds are incubated in 2.5 ml of guaiacol
solution (0.05%) for 20 mins.. 2ml of guaiacol was taken out and 0.2 ml of H2O2 (0.1%) was added. Itwas
quantified by DU 64 spectrophotometer at 480 nm. The reading was taken after 2 mins of adding H2O2 in
guaiacol. The whole test was carried out at 25 degree celsius on basis of transmittance percent. The results and
duration of test varies with each crop species. Positive or high peroxidase activity shows reddish brown

17. 6. 2,4 D Auxin test - The effect of 2, 4-D test at 5ppm concentration on seedling was studied. Seedlings
were evaluated after 7days and ten seedling were selected at random and seedling length (shoot
length + root length) was measured in Centimetres. The results and duration of procedure varies with
each crop. Based on the variations achieved conclusions are drawn out.
7. Ferrous sulphate test - Seeds are soaked in 1.5% solution of ferrous sulphate for 4 hrs under ambient
conditions. Excess moisture is removed from seeds & are evaluated for colour change & grouped as
dark grey streaks , brown streaks, brown spots on husk of cereal seeds.
8. Alkaloid test - In lupinous species presence or absence of alkaloid is a diagnostic feature. Seeds are
soaked in water for 24 hrs. Thin slice of seeds are cut & placed on a glass plate over a white surface.
Subsequently 1-2 drops of Lugol’s solution is added. An appearance of a distinct brown red precipitate
indicates presence of alkaloid.
9. Iodine test - It is mainly used to differentiate millet seeds from weed seeds. After soaking seeds in
5% iodine solution for 5-7 mins , the weed seeds develop brown colour , whereas millet seeds remain
same or change into a light green colour.
10. Gibberellins soaking test- Seedlings are raised in germination paper towels moistened in the GA
solution of 100 ppm. After 7-10 days seedling growth is observed and cultivars are grouped on the
basis of response to root and shoot elongation as high and low.

18. Advantages of chemical methods :-
Chemical tests prove to be better choice for varietal identification over the morphological tests due to following
reasons:-
 They are quick and give faster results.
 They require virtually lesser technical expertise or training for conducting out the tests.
 Relatively inexpensive to conduct and so can be used for larger scale operations.
 No sophisticated equipments are required and thus can be practised by more people.
 The test permits detection of percentage admixture of other type.
 Its results are usually distinct and easily interpretable

19. MOLECULAR METHODS
The molecular systems, which are based on variety specific protein and DNA profiles (fingerprints), respectively, have
shown their potential for rapid varietal identification in several crops. Some molecular methods most commonly used are
:-
 Electrophoresis
 PCR
 Molecular markers
1. Electrophoresis
• Electrophoresis may be defined as the migration of the charged particle through a solution under the influence of an
external electrical field. Same works as its principle also Ions that are suspended between two electrodes tends to travel
towards the electrodes that bears opposite charges. It is one of the latest method of cultivar identification based on
banding of protein and activity of isoenzyme. Here single seeds are defatted and extracted for protein and esterases.
The extracted proteins or esterases are separated by gel electrophoresis . Based on the banding pattern of protein and
esterase's the varieties can be differentiated and identified. The gel used may be Agarose gel or PAGE (Poly acrylamide
Gel Electrophoresis) depending upon the pore size needed.
• The common types of electrophoresis used mainly for varietal identification are : Gel electrophoresis done agar or agarose gel ;
Polyacrylamide gel electrophoresis (PAGE) ; Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) ; Starch
gel electrophoresis

20. • Seeds, seedlings or mature leaves etc. of a crop plant have specific mix of proteins which are crop specific & also variety
specific (genotype specific). The electrophoresis in a suitable medium separates the mixture of proteins extracted from
seeds, seedlings or mature leaves into distinct bands. Each variety has a specific "banding pattern" on the basis of which
admixtures of other varieties, differing in "banding pattern" is detected. This is done by comparing the banding pattern of
analysed sample with the standard banding pattern of that variety. The separation into distinct bands is due to,
1. differences in the size of molecules (molecular weight) of various proteins. Particles with smaller molecular weights
migrate faster than those with higher weights. 2. differences in charge.
This results in differential rates of movement of proteins when a mixture of different proteins is subjected to an electric
field. By altering gel pore size and charge on protein a high degree of resolution can be achieved for separation of proteins
in a mixture.
• Seeds, seedlings or mature leaves etc. of a crop plant have specific mix of proteins which are crop specific & also variety
specific (genotype specific). The electrophoresis in a suitable medium separates the mixture of proteins extracted from
seeds, seedlings or mature leaves into distinct bands. Each variety has a specific "banding pattern" on the basis of which
admixtures of other varieties, differing in "banding pattern" is detected. This is done by comparing the banding pattern of
analysed sample with the standard banding pattern of that variety. The separation into distinct bands is due to,
1. differences in the size of molecules (molecular weight) of various proteins. Particles with smaller molecular weights
migrate faster than those with higher weights. 2. differences in charge.
This results in differential rates of movement of proteins when a mixture of different proteins is subjected to an electric
field. By altering gel pore size and charge on protein a high degree of resolution can be achieved for separation of proteins
in a mixture.

21. 2. Polymerase Chain Reaction (PCR)
PCR was originally developed in 1983 by the American biochemist and Nobel Laureate Kary Mullis. Polymerase
chain reaction (PCR) is a common laboratory technique used to make many copies of a particular region of DNA.
PCR combines the principles of complementary nucleic acid hybridization with those of nucleic acid replication
that are applied repeatedly through numerous cycles. It results in the exponential production of the specific
target DNA/RNA sequences by a factor of 10^7 within a relatively short period.
• The essential components used in PCR are Taq polymerase, PCR primers.
• The DNA polymerase typically used in PCR is called Taq polymerase, isolated from Thermus aquaticus. Its
DNA polymerase is very heat stable and this heat-stability makes Taq polymerase ideal for PCR.
• Taq polymerase can only make DNA if it's given a primer, a short sequence of nucleotides that provides a
starting point for DNA synthesis. PCR primers are short pieces of single-stranded DNA, usually
around 20 nucleotides in length. They are given sequences that will make them bind to opposite strands of the
template DNA, just at the edges of the region to be copied. The primers bind to the template by
complementary base pairing.

22. The key ingredients of a PCR reaction are Taq polymerase, primers, template DNA, and nucleotides (DNA building blocks). The
ingredients are assembled in a tube, along with cofactors needed by the enzyme, and are put through repeated cycles of heating
and cooling that allow DNA to be synthesized. The results of a PCR reaction are usually visualized using gel
electrophoresis.
The basic steps are:
1. Denaturation (96 °C): Heat the reaction strongly to separate, or denature, the DNA strands. This provides single-stranded
template for the next step.
2. Annealing (55 - 65°C): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded
template DNA.
3. Extension (72 °C): Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA.

23. PCR is remarkably effective at identifying species, varieties, or individuals by genetic fingerprinting. It is
simply to amplify nucleotide sequences that are specific to species, variety, or individual. The coding sequences
correspond to the genes and are translated into proteins. The noncoding sequences, which are therefore not
translated, represent a large proportion of eukaryotic genomic DNA (up to 98%). Coding sequences are of little
relevance in terms of identification.
The noncoding sequences are very polymorphous between species as between individuals of the same species.
They thus present a large choice of genetic markers that make it possible to establish identification tests which
are highly discriminating.Among these markers are minisatellites (or VNTR, variable number of tandem
repeats) and microsatellites (or STR, short tandem repeats). VNTRs and STRs are repetitive polymorphisms
composed of sequences that are repeated in tandem.
The amplification products are then either analysed by electrophoresis or undergo fragment analysis using a
capillary sequencer. The variety of amplification products obtained leads to footprints that are specific
individuals
Two techniques that are relevant are the Random Amplification of Polymorphic DNA (RAPD) and the
Amplification of Fragment Length Polymorphism (AFLP). RAPD is a PCR for varietal identification that uses
pairs of random primers of reduced size (about 10 base pairs). Amplification of Fragment Length Polymorphism
(AFLP) is a much more efficient method. It consists hydrolising the genomic DNA with 1 or better 2 restriction
endonucleases. Then, we proceed with the ligation of adapters (defined sequences of DNA of about 15
nucleotides) at the level of the generated cohesive ends by restriction enzymes. Finally, the product of the
ligation is amplified by PCR with a pair of primers that hybridizes at the level of the adapters.

24. 3. Molecular markers
These are heritable DNA sequences, phenotypically neutral, developmentally and environmentally stable and identified by techniques
such as Southern hybridization or PCR (Polymerase Chain Reaction). Molecular techniques have been applied to plant cultivar
identification in the past decade by developing molecular markers that detect differences in DNA sequences between different cultivars.
Highly specific marker profiles commonly known as DNA fingerprinting can be developed for each cultivar and used for its identification.
Different types of molecular markers are being used currently which are detected by Southern Hybridization :
• RFLPs -Restriction Fragment Length Polymorphisms
• VNTRs – Variable Number of Tandem Repeats)
Detected by PCR based methods:
• RAPD - Randomly Amplified Polymorphic DNA
• AFLP – Amplification Fragment Length Polymorphism
• CAPS – Cleaved Amplified Polymorphic Site, SSR – Simple Sequence Repeats (microsatellites)
• SNP - Single Nucleotide Polymorphisms
• SCAR - Sequence Characterized Amplified Region
• SSR - Simple Sequence Repeats
• STS - Sequence Tagged Site

25. The best molecular markers are those that distinguish multiple alleles per locus (i.e. are highly polymorphic) and are co-
dominant (each allele can be observed). Among the molecular markers, microsatellites also called Simple Sequence
Repeat (SSR) markers are most suitable because of the, highly polymorphic nature, co-dominant inheritance
(determination of homozygous and heterozygous states of diploid organisms), frequent occurrence in genome, easy
access (availability), easy and fast assay, high reproducibility and easy exchange of data between laboratories.
General methodology for molecular markers:
 DNA extraction
 PCR amplification using nucleotide primer
 Initial Denaturation
 Repeated Cycles
 Denaturation
 Annealing
 Extention
 Final Extention
• Electrophoretic run and identification of PCR amplified product

26. Advantages of molecular methods:-
 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.
 It gives precise and quick results.
 Time saving procedure
Limitations of molecular methods:-
 Sophisticated instruments required.
 Very costly.
 Needs expert staff for handling and conducting the experiment.
• Not possessed by all institutes in developing countries.

27. BIOCHEMICAL METHODS
• Biochemical is term associated to biochemistry. It refers to chemical substances present in a living organism or
chemical processes occurring in a living organism. For example- proteins, isozymes, enzymes , carbohydrates
etc. The biochemicals are generally present in distinct forms in the various varieties in different forms and thus it
helps in identifying the particular varieties by detecting the presence or absence of the particular biochemical.
The presence or absence is detected using various techniques.
• Electrophoretic analysis of proteins and isoenzyme offers an efficient and cost effective method towards cultivar
identification and varietal purity tests in seeds lots.The analysis of the seed proteins and isozymes by (SDS-
PAGE and A-PAGE) is the rapid, accurate and dependable technique . The technique is useful to ensure the
genetic purity of plant cultivars and their parental lines in commercial seed production of hybrids.
• SDS-PAGE and A-PAGE are most economical, simple and extensively used biochemical techniques for
analysis of cultivar identification. As seed storage proteins are largely independent of environmental fluctuation
, their profiling using SDS-PAGE technology is particularly considered as a consistent tool for economic
characterization of plant cultivars.

28. Importance of VarietalIdentification
 In a single crop species, there exists number of different varieties that differ on the basis of few unique
characters possessed by them. These varieties give uneven production and yield results when grown on field. To
solve this issue varietal identification comes into existence as it differentiates the varieties present in a single
crop species
 Identifying the different varieties gives chance to choose the variety with highest performance of yield and also
resistance to insect, pest and diseases.
 This increases the produce and economic status of the farmer and helps the farmer to select the best variety that
suits his soil and other available resources.
 It also indirectly contributes to development of national economy.
 Varietal identification paves way for further research and development of new varieties and hybrids by
identifying the reasons that impart peculiar and commercially important characteristics to the identified
varieties.
 It also helps in identifying the traits and characters possessed by wild varieties that can be incorporated in
domesticated varieties for obtaining maximum gain.
 Identified varieties help farmers to choose the variety best suited as per their available resources and conditions.