Testing Of GM Seed And trait purity

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:

5. Procedure

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.

9. Immunoassay-ELISA

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.

17. GMO
testing
Handbook
(in prep.)
2nd International Workshop of GMO-analysisNetworking