2. Course title:- Seed Quality Testing
Topic :- Testing Of GM Seed And trait purity
Submitted by
Name :- Kartik S. Madankar
Reg.No :- 2017A76M
Submitted to
DR. V.S Mor
3. What are GM seed?
According to ISTA: Genetically modified seed are seed in
which the genetic material (DNA) has been altered in a way
that does not occur naturally by mating or natural
recombination.
It allows selected individual genes to be transferred
from one organism into another.
4. The object of testing for seeds of genetically
modified organisms (GMOs) is to give
guidelines to detect, quantify or confirm the
presence of GMO seeds in seed lots.
These guidelines can be applied to testing
adventitious presence (AP) of genetically
modified organisms (GMOs) and GMO trait
purity testing.
Objective:
6. Quantitative
Semi-quantitative
(subsampling)
Approach Qualitative
Working
sample 1-few
seedbulks
n. seedgroups
(subsamples)
individual
seeds
Result TraitPurity
Quantitative
or Adventitious Presence
Possible Workflows toQuantitation of Adventious presence or Trait Purity
2nd International Workshop of GMO-analysisNetworking
ELISA↑ or rt PCR↓
Method ep PCR↑ or immunoassay ↓
- - + + + + + ++++ +
non-GM
Substrate+
herbicide
GM
Bioassay
7. Testing approaches
1 DNA-based methods
General principles of DNA-based testing
End-point qualitative PCR
Real-time PCR
Other technologies
2 Protein-based methods
General principles of protein-based testing
Lateral flow strip test
Enzyme-linked immunosorbent assay
3 Bioassays
General principles of bioassays
Scoring of GMO presence
8. ELISA TEST:
Microwell plate ispre-coated with antibody of
trait of interest.
Protein isremoved from seedsor leaves and
transferred to plate. Antigen binds to wells.
After several stepsof adding chemicals and
washing material from plate, color develops in
wells.
Plate isread visually or with spectrophotometer.
10. Lateral flow strips:
Seedsor leaves are ground, water or buffer
added, and material mixed.
Liquid containing protein istransferred to reaction
tubes.
“Pre-coated” strip isplaced in tube.
After 3-5 minutes(typically), strip isremoved from
tube and examined for development of lines.
11. Get required number of seeds. Grind seeds Add water & mix
Insert strip
Results
12. What is Electrophoresis?
Migration of a charged particle througha
medium(agarose,polyacrylamide, starch)under
the influence of an electrical field.
Proteinsand NucleicAcid Separations
Molecular Weight
Electrical Charge
Biological or Chemical Properties
Combinationof theabove methods
13. IEF gel with 96 seeds (no parent seed)
Missing band
GeneticPurity – ProteinElectrophoresis
14. Polymerase Chain Reaction
Fingerprint of variety tested
Increases the amount of DNA being checked for
(if present, through use of primers).
Expensive equipment, but test is very precise
15. Three Major Steps in PCR
Denaturation (at 94o C): Paired strands separate
(single strands now accessible to primers).
Annealing (at 54o C): Excess primers added, cooling
allows double-strands to bind to primers.
Extension (at 72o C): Ideal working temperature for the
polymerase. Primers without exact match get loose
again, and don’t give an extension of the fragment.
16. Calculation and expression of results
The calculation and expression of results depend on the testing objectives,
testing methods and the associated units of measurement.
Different units:
• a) % in number of seeds: the estimate of the percentage of GM seeds in the
seed lot. In addition to individual testing, the percentage in number of seeds
is the unit to be used when a group testing approach is chosen;
• b) % in mass of seeds: the estimate of the percentage of GMO content by
mass. This unit should be used when a standard curve is prepared using
certified reference material certified by % mass (g/kg).
• c) % DNA copies: the estimate of the percentage of GMO content by number of
copies. This unit should be used when a standard curve is prepared using
certified reference material certified by % DNA copies.
All these three units are acceptable for preparing ISTA Certificates for
reporting results by accredited laboratories.