Advancements in seed testing technologies

1. Department of Seed Science and Technology
Chaudhary Sarwan Kumar Himachal Pradesh Krishi
Vishvavidyalaya , Palampur
Speaker - Jeenia Thalyari
Roll no - A-2018-30-070

2. Topic : Advances in seed testing
technologies in major crops

3. Outline of the seminar
This presentation includes:
1. Introduction
2. History of seed testing
3. Concept of seed quality
4. Advances in genetic purity testing
5. Advances in physical purity testing
6. Advances in physiological quality testing
7. Advances in seed health testing
8. Conclusion

4. Introduction
“Subeejam Sukshetre Jayate Sampadayte"
• High quality seed is determined by its parent’s genetic makeup, physical
integrity , purity , its health and physiological quality.
• Farmers continue to adopt precision agricultural practices and depend
on the performance of every single seed put into the ground— the only
way to achieve that is by seed testing.
Every inch of the field counts, and seed testing plays a significant role.

5. Seed Technology encompasses:
1. Development of superior
varieties
2. Production
3. Evaluation
4. Processing
5. Storage
6. Testing
7. Certification/quality control
8. Marketing and distribution
9. Seed pathology
10. Seed entomology
11. Seed physiology
12. Seed ecology

6. Seed Testing
 Seed testing is the art and science of evaluating seed quality for
agricultural purposes (Copeland and McDonald, 2001).
 Seed tests tell us if a crop of seeds is worth collecting, if handling
procedures are correct, and how many potential seedlings are available
for regeneration.
• Seed quality in India is legally controlled by The Seeds Act, 1966.

8.  Seeds - basic, vital and crucial
input for crop production
 Quality control of seeds depends
on the different seed testing
protocols which determine the
genuineness of the cultivar.
Evaluates the planting value and
the authenticity of the certified lot.
Assess the seed quality attributes
of the seed lots which have to be
offered for sale.

9. History of seed testing
• Seedquality test - seed germination and purity.
• First official seed testing station by Frederick Nobbe
(1869) in Tharandt (Germany)
• 1876- Lab established in USA - Connecticut
Agriculture experiment station.
• Now such labsestablishedin all countries. Professor F. Nobbe

10. o 1931:Germination, Purity,Sanitary conditions,
Genuineness of variety,weight determinations,
determination of moisturecontent.
o 1966:SeedHealth Methods
o 1968:Tetrazoliumtest
o 2001:Vigourtests
o 2004:Performancebased
methodsforspecifiedtrait testing

11. Conventional methods Modern methods

12. 1. Destructive
•Tests are carried out to understand the seeds performance
or behaviour under different loads.
• Example Tetrazolium test and vigour tests

13. 2. Slow
•Most of the methods require time for completion.
•Example germination test and vigour tests.

14. 3. Require pre-treatment
Viability tests like Tetrazolium test require pre-treatments
/ pre-conditioning of some seeds.
Example wheat seeds are kept between moist towels or
directly immersed in water for 18 hrs at 20°C.
Soaked wheat seeds Stained seeds

15. 4. Less accuracy
• Germination- “The emergence and development of the
seedling to a stage where the aspect of its essential
structures indicates whether or not it is able to develop
further into a satisfactory plant under favourable conditions in
the soil” (ISTA 2004)
• There are potentially many different interpretations of this
definition. Some variability within the interpretation of the rules
can lead to differences in the classification of a seed as normal
or abnormal.

16. • Destructive
• Slow
• Require pre-
treatments
• Less accuracy
Non destructive
Quick
No pre-treatments
required
High accuracy

17. Seed quality
• Trueness to variety
• Presence of inert matter
• Seeds of other crops
• Weed seeds
• Germination percentage
• Vigour
• Appearance
• Freedom from diseases
- aspects of seed quality.
Seed quality describes the potential performance of a seed lot.

19. Genetic purity
 Genetic purity is defined as true to type plants / seeds conforming to the
characteristics of the variety as described by the breeders.
 It refers to the percentage of contamination by seeds or genetic material of
other varieties or species.

20. Methods to assess genetic purity
1. Morphological
2. Conventional grow out test
3. Chemical tests
4. Gel electrophoresis
5. Polymerase Chain reaction
6. Molecular markers(DNA)
(Basra, 2002)

21. Traditional approach to purity testing
Morphological traits
Seed morphology Examination of Seedlings
In field
In lab or greenhouse

22. CHEMOTAXONOMY
The use of biochemical methods to analyze various components of
seeds. Chemo taxonomists have recognized two groups of compounds
that are generally useful in classification of plant species:
1. Episemantic or secondary metabolites
(pigments or fatty acid etc.)
2. Semantides or sense carrying molecules
(Proteins, Nucleic Acids)
Methods of testing based on -
1. Analysis of secondary metabolites
2. Protein analysis
3. Nucleic acid analysis

23. These tests range from simple colour tests to complex
chromatographic separations of phenols, anthocyanin,
flavonoids and other compounds.
1. Phenol test- Wheat
2. Peroxidase test- Soybean
3. Potassium hydroxide – bleach test- Sorghum
4. Fluorescence test - Ryegrass
5. NaOH test- Wheat
6. Anthocyanin test- Rice, Soybean
1. Analysis of secondary metabolites

24. Proteins are the direct gene products, therefore the analysis of
seed, seedling proteins and enzymes is most successful and widely
used. Hence much attention has been focused on seed storage
proteins.
There are two primary methods:
1. Gel electrophoresis
2. Isoelectric focusing
2. Protein analysis

25. Gel electrophoresis
• The term gel electrophoresis refers to a technique used for
separation and analysis of DNA, RNA and proteins based
on their size and charge.
• The suffix phoresis means “migration” or “movement”,
while the prefix electro indicates the use of electricity as a
mean to separate molecules.

26. SDS-PAGE (Sodium dodecyl sulphate-
Polyacrylamide gel electrophoresis)
 Denaturing and reducing sodium dodecyl
sulphate (SDS)-PAGE- widely used
electrophoresis technique
 SDS-PAGE separates proteins primarily by
mass because the ionic detergent SDS
denatures and binds to proteins to make
them uniformly negatively charged.
 Thus, when current is applied, all SDS-
bound proteins in a sample will migrate
through the gel toward the positively
charged electrode.
 Proteins with less mass travel more quickly
through the gel than those with greater
mass - sieving effect of the gel matrix.

28. Isoelectric focusing(IEF)
Isoelectric focusing (IEF)- separates molecules by differences in
their isoelectric point (pI).
Performed on proteins in a gel- takes advantage of the fact - overall charge on the
molecule of interest is a function of the pH of its surroundings.
A protein that is in a pH region below its isoelectric point (pI) will be positively
charged and so will migrate toward the cathode (negatively charged electrode).
As it migrates through a gradient of increasing pH, the protein's overall charge
will decrease until the protein reaches the pH region that corresponds to its pI.
At this point - no net charge and migration ceases.
As a result, the proteins become focused into sharp stationary bands with each
protein positioned at a point in the pH gradient corresponding to its pI.

30. 3. Nucleic acid analysis
Polymerase chain reaction
 Polymerase chain reaction (PCR) is an efficient and cost effective
molecular tool to copy or amplify small segments of DNA or RNA.
 PCR was originally developed in 1983 by the American biochemist
Kary Mullis. He was awarded the Nobel Prize in Chemistry in 1993
for his pioneering work.

31. Procedure
1. Denaturation: In order to make a copy of the DNA, the strands are separated
using heat to break the bonds between complementary nucleotides.
 Temp/time= 95°C for 30 seconds
2. Annealing: To add a primer that will serve to flag down the building crew –the
polymerase- so it knows where to start copying.
• For this PCR primer is added -a small piece of DNA that dictates which part of the
double helix will be copied.
 Temp/time= 50°C for 30 seconds.
3. Extension: To add new complementary nucleotides. In PCR, Taq polymerase, a
thermostable polymerase is used instead of DNA polymerase.
• Taq polymerase adds the complementary nucleotides to the single-stranded DNA,
forming a new, duplicate double helix.
 Temp/time= 72°C for 60 seconds

33. DNA Markers
Grow out test
Based on the identification of
morphological characteristics at various
stages of plant growth.
Subject to influence by environmental
factors and is time-consuming.
DNA markers
Based on the genotype of the
hybrids eliminating environmental
variations .
Based on identification of genotype
specific profiles

34. SSR markers- Simple sequence repeats – Repeating Sequence of 2-5
nucleotides.
 Microsatellite markers can be used for fingerprinting of hybrids,
assessing variation within parental lines and testing the genetic purity of
hybrid seed lot.
 DNA markers systems, PCR based co-dominant SSRs (microsatellites)
are preferred for genotyping – reproducibility and abundance .

35. After isolation of DNA , total of 75 variable SSR primer pairs
distributed across the 10 chromosomes were used for PCR
amplification.
General procedure
The banding pattern through gel electrophoresis of all these hybrids
showed both the amplicons present in female as well as pollen
parent, thus confirming the genuine crossing and heterozygotic
condition of the hybrid.
The SSR markers identified had both female and male specific bands
and are useful in genetic purity testing.
1.
2.
3.

37. Physical purity
Cleanliness of seed from other
crop seeds, weed seeds, inert
matter, diseased seed and insect
damaged seed.
Physically pure seeds should have
uniform size, shape, weight and
appearance.
 Lack of this quality characters will
indirectly influence the field
establishment and planting value
of seed.

38. General method of purity separation
Place the sample on the purity work board after sieving / blowing
operations and separate the pure seeds.
After separation, we identify the pure seeds, each kind of weed
seeds, other crop seeds as to genus and species, inert matter -
names and number of each are recorded.
Seed blower Purity work board

39. 1. Image analysis
Image analysis is based on the extraction of data from a captured image for
characteristics like colour, size, shape of seed and seedlings and their subsequent
processing with the help of suitable computer software.
Speedy analysis, cost-effectiveness, automatic nature and user-friendly
environment for work are some important advantages .
Machine vision or computerized image analysis system is found to be very
convenient method for seed related studies as it is free from human errors.

40. 2.Ergovision inspection station
An improved ‘Microscopic Station’ that
incorporates advanced optical, ergonomic
and mechanical technologies to achieve fast
and accurate testing.
The main goals that were considered in
developing the new station were:
1.Accuracy, by providing the best
magnification, clarity and resolution for
each seed
2.Ergonomics, to reduce analyst’s fatigue
and discomfort
3.Productivity, to ensure fast and timely
results
- increased about 20-30% compared to the
traditional method.

42. Physiological quality
 It is the actual expression of seed in further generation /
multiplication.
• It comprises those intrinsic attributes of seeds which determine
their capacity to germinate and emerge rapidly and to produce a
uniform stand of vigorous plants under the range of field conditions
.

43. Germination and viability tests
1.Paper
2.Sand 3.Soil
a. Top paper
b. Between paper
c. Pleated paper
On Sand
Conventional methods- Germination

44. Conventional methods- Viability
1. Tetrazolium test- Common
2. Embryo excision test - Trees
3. Indoxyl acetate test - Soybean
4. Fast green test - Maize
5. Ferric chloride test - Legumes

45. Technological advancements
1. Micro-optrode technique (MOT)
2. 3-dimensional X-ray imaging
3. Hyperspectral and multispectral imaging

46. 1. Micro-optrode technique
Micro-optrode technique (MOT) - invented by Porterfield and co
workers to measure seed viability in a quick and non-invasive
manner by measuring the oxygen influxes of intact seeds on the
cell surface.
There are two basic approaches :–
1. Long-term monitoring of oxygen consumption during seed
germination in an open system.
2. Detecting oxygen consumption by seeds by measuring the
decrease in oxygen concentration in a closed chamber within
10 seconds to screen one seed.

47. Procedure
Seeds soaked in solution (0.1 mM CaCl2, 0.1 mM KCl, pH 6.0) - three hours, and
then transferred to a new solution to detect oxygen fluxes.
A seed fixed on a Petri dish with a plastic colloidal cloth and immersed in
measuring solution.
The measuring point (embryo/embryonic axis) located by microscope.
Oxygen fluxes of seeds measured using micro-optrode calibrated in solution with
known oxygen concentrations .
The oxygen fluxes recorded every 10 seconds and measured for at least 5
minutes.
Final data of oxygen fluxes and the images acquired and recorded in real-time
using the imFlux software.

49. 2. 3-dimensional X-ray imaging
High-resolution X-ray computed tomography (HRXCT) - important method
for non-destructive and non-invasive evaluation of seed internal structure.
How X-ray imaging works?
Three-dimensional X-ray imaging is based on the same principles as
conventional radiography.
1. X-rays produced by an X-ray generator are projected towards the object.
2. A certain amount of X-rays are absorbed by the object(seeds) depending
on its density and structural composition.
3. The X rays that pass through the object are captured behind the object
by a detector.

50. In computed tomography (CT) an X-ray source and its associated
detectors rotate around the object which itself moves around the
conical X-ray beam produced.

51. 3. Spectral imaging
 Spectral imaging uses multiple bands across the electromagnetic spectrum.
 An ordinary camera captures light across three wavelength bands in the visible spectrum- red,
green, and blue (RGB).
 Spectral imaging encompasses a wide variety of techniques that go beyond RGB and may use
the infrared, the visible spectrum, the ultraviolet, X-rays, or some combination of the above.
 It is possible to capture hundreds of wavelength bands for each pixel in an image.

52.  Multispectral imaging captures a small number of spectral bands, typically three
to fifteen, through the use of varying filters and illumination.
 A hyperspectral camera uses special hardware to capture hundreds of
wavelength bands for each pixel, which can be interpreted as a complete
spectrum.

53. Vigour tests
Seed vigour is an important quality parameter which needs to be
assessed to supplement germination and viability tests to gain insight
into the performance of a seed lot in the field or in storage.
 ISTA congress in 1977 adopted the definition of seed vigour as " the
sum total of those properties of the seed which determine the level
of activity and performance of the seed or seed lot during
germination and seedling emergence".

54. Conventional methods
1. Growth tests
2.Conductivity test
3. Brick gravel test
4.Paper piercing test
5. Accelerated aging test
6.Cold test
Vigour tests

55. Seed Vigour Imaging System
 Computerized image analysis, has made objective information
accessible in a relatively short period of time, with less human
interference.
Sako et al. (2001) developed an automated system for assessing the
vigour of lettuce seeds called the Seed Vigour Imaging System
(SVIS).
The process involves scanning the seedlings and then generating
vigour, growth and uniformity indexes by using certain softwares.

56. s

57. Chlorophyll fluorescence test
This method was first used by Jalink et al.(1998) on cabbage
seeds for the assessment of maturity and quality.
 It is based on the non-destructive measurement of
chlorophyll- a in individual seeds.
During maturation for the majority of seed species the amount
of chlorophyll in the seed and seed coat decreases, whereas
the quality of the seeds increases.
Therefore, the quality of these seeds is inversely related to
the amount of chlorophyll in them.

58.  This method uses the property of chlorophyll that it fluoresces
when it is excited at a certain wavelength.
 Using this property and a combination of a red laser to
excite the chlorophyll and narrow bandwidth filter to filter out the
fluorescence, the chlorophyll in seeds can be determined.

59. Ethanol breath analyser
 A fast and simple method to analyse
seed deterioration by measuring ethanol
production from partially imbibed seeds.
 The test, uses a commercial breath
analyser, measures ethanol produced by
seeds in case of mitochondrial damage.
 The analysis showed an inverse
correlation between ethanol production
and seed quality.

60. Seed health
Seed health is a measure of
freedom of seeds from
pathogens.
The presence or absence of
seed-borne pathogens can be
confirmed through the use of
seed health testing (Agrawal,
1995).
The term “seed health” includes
the incidence in the seed lot of
organisms like fungi, bacteria,
viruses, nematodes and insects.

61. Conventional methods of seed
health testing
Basic methods:
1. Direct Inspection
2. Washing Method
3. Incubation Methods
4. The whole Embryo Count Method
5. Blotter Methods
6. Agar Plate Test
7. Freezing Method
8. Growing Out test
9. Ordinary seedling symptom Test
Immunodiagnostic Methods:
1. Serology
2. Nucleic Acid based methods
Including PCR
3. Immunodiffusion Tests
4. Immunosorbent Electron
Microscopy (ISEM)

62. For insect pests
For plant pathogens
1. Alkali or glycerine method
4. New PCR techniques
3. ELISA
2. Latex flocculation test
1. Double diffusion test
Technological advancements

63. 1. Double diffusion test
Seed or part (embryo)
of it is ground to a fine
powder.
Triturate is transferred
to a well cut in a
diffusion media (agar
gel).
Antiserum specific
for a suspect virus
in a seed is placed
in separate well.
In time the virus particles
(antigen) and antibody
molecules diffuse towards
one another.
Since diffusion is in two
direction it is called
Double diffusion.
Diffusion confirms
the presence of
virus.

64. 2. Latex agglutination test
Grinded seed extracts are
placed in a pipette
Specified quantity of tagged
latex is added
Pipette is then oscillated
for about 15 minutes
Observed under
microscope
When pathogens are present in the
sample the latex suspension
becomes flocculated .
1.
2.
3.
4.
5.

65. 3. Enzyme Linked Immunosorbent
Assay(ELISA)
ELISA (Enzyme-linked immunosorbent assay) is a plate-based assay
technique designed for detecting and quantifying peptides, proteins,
antibodies and hormones.
In ELISA, an antigen is immobilized to a solid surface and then complexed
with an antibody that is linked to an enzyme.

67. The authors mentioned various common methods of detecting seed
borne pathogens along with some recent methods :
1. Bio-PCR
2. IMS-PCR
3. Real time PCR

68. 1. Bio-PCR
• Bio-PCR is culturing of bacterial cells to the detectable limit
prior to PCR. This in turn increases sensitivity and allow early
detection of bacterial pathogens.
• Different plant pathogens Ex. Pseudomonas syringae pv.
Phaseolicola, Acidovorax avenae spp. Avenae, Xanthomonas
oryzae pv. Oryzae can be detected by Bio-PCR method.

69. 2. IMS-PCR

70. 3. Real time PCR
This method consists of coupling DNA amplification with fluorescence
substances which can be easily measured, giving an indirect measurement
of DNA amplification.
Procedure-
1. The PCR is prepared as usual , and the reporter probe is added.
2. As the reaction commences, during the annealing stage of the PCR both
probe and primers anneal to the DNA target.
3. Polymerization of a new DNA strand is initiated from the primers, and
once the polymerase reaches the probe, its 5'-3'-exonuclease degrades
the probe, physically separating the fluorescent reporter, resulting in
an increase in fluorescence.
4. Fluorescence is detected and measured in a real-time PCR machine.

72. Detection of insects – Visual examination

73. 1. Seeds are taken in a beaker (100 mI capacity) and then 10% Sodium hydroxide
solution is poured in it until the seeds are submerged.
2. The seeds are boiled in Sodium hydroxide solution for 10 minutes.
3. After decanting solution, translucent seeds are washed with water and then
examined with the help of magnifying glass.
4. Seeds with visible internal infestation are separated, cut open to confirm the
presence of insect and counted to report the result in percentage (by number)

74. Conclusion
The main feature of the mentioned technologies is that most of
them are multi- purpose and therefore can be used for carrying
out more than one component of seed quality.
Spectral
imaging
Viability
Damage
detection
Seed Health
PCR
Genetic
purity
Seed health
3-D X-ray
imaging
Physical
purity
Viability
Seed health

75. Safeguarding seed quality is the first step towards a satisfying yield.
It is estimated that the direct contribution of quality seed alone to the
total production is about 15-20% depending upon the crop species.
The determination of seed quality by seed testing through advanced
technologies is the most important measure for better crop production.

76. Thank you!