Genetically modified crops are plants whose DNA has been altered using genetic engineering techniques. Common reasons for genetically modifying crops include making them resistant to herbicides or pests. There are two main techniques used to detect GM crops - detecting the genetic material (DNA) using PCR, and detecting proteins expressed by the new genes using immunoassays like ELISA or lateral flow strips. PCR is the most versatile technique for detecting GM content, while protein detection is useful for some raw commodities. Real-time PCR allows for quantifying the GM content in a sample.

1. GENETICALLY MODIFIED CROPS
AND THEIR DETECTION
TECHNIQUES
(SST-605)
Submitted to : Dr Chuni Lal Sharma
Submitted by : Paranjay Rohiwala
Amission No. : H-2019-40-D

2. GENETICALLY MODIFIED CROPS AND
THEIR DETECTION TECHNIQUES
What are Genetically Modified Crops
• A genetically modified organism (GMO) is any organism whose
genetic material has been altered using genetic engineering
techniques.
• GM is a technology that involves inserting DNA into the genome of
an organism. To produce a GM plant, new DNA is transferred into
plant cells. Usually, the cells are then grown in tissue culture where
they develop into plants. The seeds produced by these plants will
inherit the new DNA.

3. The characteristics of all living organisms are determined by their
genetic makeup and its interaction with the environment.
The genetic makeup of an organism is its genome, which in all
plants and animals is made of DNA.
The genome contains genes, regions of DNA that usually carry
the instructions for making proteins.
It is these proteins that give the plant its characteristics. For
example, the colour of flowers is determined by genes that carry
the instructions for making proteins involved in producing the
pigments that colour petals.

4. Genetic modification of plants involves adding a specific stretch of
DNA into the plant’s genome, giving it new or different
characteristics.
This could include changing the way the plant grows, or making it
resistant to a particular disease. The new DNA becomes part of the
GM plant’s genome which the seeds produced by these plants will
contain.

5. History of GMO's
In 1980, the first transgenic animal is created, a mouse.
• 1980s, transgenic plants are started to be created in china.
• 1988, first transgenic plants producing a pharmaceutical.
• 1995, GMO corn hits the market in the USA.
• 1996, Roundup Ready Soybeans hit market in USA.
• 1998, First GM labelling rules introduced to provide consumers
with information regarding the use of GM ingredients in food.
• Over the years, we have developed new and better ways to
manage and produce GMO crops.

6. • First crop introduced was Flavr Savr tomato in USA in 1995
• So far 20 crops approved for commercial cultivation in different
countries
• Only four crops being marketed commercially i.e., corn, cotton,
soybean and canola
• Commercial production initiated for papaya, squash, rice and
alfalfa in USA and other countries, Others are approved but not yet
being Marketed
• Major countries include USA, Canada, Japan, China, India, Brazil,
EU, Argentina, South Africa

7. Why to make transgenic crops?
Due to limitations of conventional breeding for attaining the
desirable traits use of recombinant DNA technology has been
taken advantage of and development of transgenics started

8. Transgenic Crops: Development Objectives
 Integrated pest management (IPM)
 Herbicide tolerance (HT)
 Nutritional enhancements
 Product quality improvement
 Increase in yield
 Stress tolerance (ST)
 Plant based pharmaceuticals

9. Development of GM Crop/Seed/Transgenic
Identifying gene(s)
• Giving a desired trait
• Make copies of the gene
• Transfer to plant tissue
• Regenerate plants
• Lab analysis and safety
testing
• Development of a variety
• Field tests
• Approval by Government
agencies
• Monitoring of safety

10. Produce Transgenic Plant

11. GM Seed-Pros
• Improved resistance to pests and diseases.
• Improved resistance to Herbicide
• Production of more nutritious staple crops
• Contribute to food security ,sustainability
• Contributing to the alleviation of poverty and hunger
• Increased crop productivity
• Stability of production
• Economic and social benefits

12. GM Seed- Cons
• Human health
• Environmental hazards
• Effects on Non-Target organisms and plants

13. HUMAN HEALTH
• Allergen and toxin
• Antibiotic resistance
• Unknown effects on human health
Environmental hazards
• Growing of GM crops may lead to monoculture
•The creation of pest or herbicide resistant GM crops could
result in superbugs or super weeds
• Effects on Non-Target organisms

14. Techniques Used to Detect a Transgenic Seed/Plant
Two different techniques:
1) Based on the detection of genetic material (DNA):
• For example by polymerase chain reaction (PCR). This
technique is most versatile for the detection of GM plants and
therefore preferably used and chosen for many applications
2) The alternative approach is detecting the newly expressed
protein(s) which most GM plants contain as a result of the
insertion of the new gene(s):
• Here specific antibodies are applied and used in lateral flow
strip tests or complex ELISA assays.

15.  As compared to PCR, protein techniques are more restricted in
their applicability but can be very useful for certain raw
commodities.
 DNA is relatively stable and is often still present in many
products, even after processing of the plant material.
 Therefore genetic modifications in plants are more easily and
reliably detected at the DNA level.

16. 1 DNA-Based Detection:
Targets the novel DNA sequences introduced into the crop genome.
Methods detect absence or presence of GM plant material in a
sample and can also measure the relative quantity (percentage) in a
tested sample.
i) Polymerase Chain Reaction:
• DNA-based testing for GM plants is commonly performed using
PCR,
• amplifying specifically a short segment of the targeted DNA.
• The design of specific primers depends on a knowledge of the
precise and comprehensive DNA sequence information of the
actually integrated DNA.

17. • PCR-based detection and quantitative measurement of the GM
content in a sample actually involves the use of two PCR systems
• One for determination of the inserted GM-derived DNA sequence
and
• Another system specific for an endogenous, plant-taxon specific
reference gene sequence.
• The latter is also thought to serve as a control for the quality and
quantity of the extracted DNA.

18. ii) Conventional Qualitative PCR
•Conventional PCR methods are mainly used for qualitative testing to
obtain yes/no answers concerning the presence of GM plant material.
•PCR products are analysed by agarose or polyacrylamide gel
electrophoresis and visualised using UV fluorescence with ethidium
bromide as fluorophor or by other means.
•It may be necessary to confirm GM-positive test results by further
analyses, either by restriction analyses, Southern hybridisation or
DNA sequencing.

19. iii) Quantitative Real-Time PCR
 The most preferred technique to quantify GM material in a sample
is real-time PCR.
 It allows the detection and measurement of increasing fluorescence
proportional to the amount of amplification products generated
during the PCR process.
 Of the various chemistries TaqMan fluorogenic probes are most
commonly applied in real-time PCR-based detection and
quantification of GM plant materials.
 Real-time PCR is mainly used for quantification purposes, but it is
increasingly utilised also for qualitative testing to screen or to
identify the GM event.

20. iv) Alternative DNA-Based Techniques
To check increasing GM plants, multi-targeted analysis are
necessary.
The DNA microarray technology, an option to parallelise the
multi-analyte detection of several PCR products in a single run.
Arrays consist of various oligonucleotide probes that are
immobilised on a glass support and used for screening of genetic
elements, for constructs and events including detection of plant
taxon-specific reference genes.
Approach based on multiplex PCR before hybridisation of the
PCR products to microarray and PCR is limited in its multiplexing
capacity.

21. Several alternatives are being tested for improvements in GMO
detection:
1. Loop-mediated isothermal amplification (LAMP)
2. Ligation-depended probe amplification (LPA)
3. SNPlex technology
4. Padlock probe ligation in combination with microarray detection
5. Nucleic acid sequence based amplification using transcription
techniques (NASBA) in combination with microarray detection.

22. 2) Protein-Based Detection:
 Detection of the novel proteins expressed by GM crops is based
almost exclusively on the application of immunoassay
technology.
 Several immunoassays are available for different traits present in
diverse GM plant crops and are used in a variety of applications,
including testing for unauthorised events and determining the
relative GM content.
 Immunoassays are based on the reaction of an antigen (e.g. the
GM-derived protein) with a specific antibody to give a antigen-
antibody complex that can be indirectly measured.
 The immunoassay formats commonly used for GM-protein
detection are the enzyme-linked immunosorbent assay (ELISA)
and the lateral flow device (LFD).

23. i) Lateral Flow Strip:
Lateral flow strip devices (LFD) are used for qualitative or semi-
quantitative detection of antigens and, in the case of novel GM
proteins, antibodies are used in the same sandwich immunoassay
format as in ELISA, except that the secondary antibody is labelled
with a coloured particle such as colloidal gold rather than an
enzyme as a means of generating a visible signal.
A typical LFD has linked simultaneously a second antibody on the
strip to provide visual control that the test has worked correctly.
LFDs are available for several traits, require low instrumentation
and allow rapid testing also in the field.
They are show to be sufficiently specific, but concerning
sensitivity only up to the 0.1% range is achievable.
LFD represent a useful tool to detect GM proteins in raw materials
such as seeds and leaves.

24. ii) Enzyme-Linked Immunosorbent Assay
Enzyme-linked immunosorbent assays (ELISAs) are commonly
96-well microplates with removable strips of 8–12 wells coated
with a primary antibody to capture a target antigen in the sample.
A secondary antibody, conjugated to an enzyme such as
horseradish peroxidase, is used to detect the presence of the bound
antigen, which results in a sandwich of the analyte between the
primary and secondary antibodies.

25. Detection Strategies
Detection of the presence of GM plants is an analytical process
involving several working steps.
It includes:
(i) the sampling step,
(ii) the extraction step for isolating DNA or protein fractions from
the ground material and
(iii) the final analysis for identification and/or quantification of GM
material.
The detection of GM plant DNA can be used for qualitative and for
quantitative testing. In quantitative PCR assays, the amount of the
specific target DNA present in the sample is estimated, whereas in
qualitative PCR tests the presence or absence of a specific GM
target sequence is determined.

26. A commonly applied strategy for testing the presence of GM plants
in seeds is to first perform screening tests with qualitative methods.
This is typically done with DNA-based PCR tests targeting the
genetic elements that are most frequently present in GM plants.
In the next working step the identification of the GM event is
performed by construct-specific or event-specific PCR methods,
followed by real-time PCR-based quantification of the relative
proportion of transgene DNA copy number versus the plant taxon-
specific DNA copies present in the analysed DNA sample.

27. Target sequences to be detected by analytical PCR methods include
sequences integrated in the GM event (screening, construct-
specific, event-specific), sequences for plant taxa-specific
reference genes and occasionally sequences from the donor
organisms in order to exclude false-positive results, e.g. possible
plant infections with cauliflower mosaic virus.

28. Screening
For the expression of newly integrated genes, GM plant developers
use a limited number of regulatory elements (promoters and
terminators).
Since these elements have been frequently used they are ideal
candidates for the screening of a large number of samples.
These are useful to assess whether or not a sample under
investigation is likely to contain GM-derived material.
To identify these elements Bruderer and Leitner (2003)
systematically surveyed which genetic components have been
introduced into GM crops at the worldwide level. E.g. 35S promoter
(P-35S) sequence from cauliflower mosaic virus (CaMV)

29. Identification
The next step in the work flow of analysing samples which reacted
positive in screening tests is the identification of the plant species
and the GM events which may be present.
If the results of the screening tests indicate the presence of several
different GM events, they must of course be first carefully
analysed as to which specific tests have to be performed next to
identify the GM plant with the most effective strategy.

30. Quantification
 For quantification of the GM plant material present in a sample,
real-time PCR assays are commonly employed to determine the
amount of sequence copies of the GM target versus the reference
gene target, which obviously is not generating a direct weight-to-
weight measurement.
 These assays use standard curves generated with a serial dilution
of DNA of known GM content and target sequence concentration.

31. Detection of Unauthorised/Unknown GMOs
 For GM plants not authorised for marketing as products, EU
regulations stipulate a zero tolerance.
 Examples of unauthorised GM products that have been identified
at the European market are GM papaya (‘SunUp’ events 55-1, 63-
1), several maize events (‘StarLink’ CBH-351, Bt10, ‘event 32’
DAS-59132-8, MIR604) and rice (LL601, LL62, ‘Bt63’).

32. Thank You