It is about recombinant DNA Technology and different Biotech crops developed as well safety measure to be taken with release of modified crops

1. Development of biotech crops, Biosafety
and Regulatory system in India
Presentation by
Sugandh chauhan

2. Terms to know
• Genetic modification involves altering the genes of an organism, be it a
plant, animal or microorganism. It allows the transfer of genes for specific
traits between species using laboratory techniques.
• Transgene is a foreign gene that has been transferred from one organism
to another
• Transgenic crops are those whose genome is altered by adding transgene
• Transgenesis is the phenomenon of introduction of exogenous DNA into
the genome to create and maintain a stable and heritable character.

3. Genetically modified crops (GM crops)
• With the rapid advances in biotechnology, a number of genetically modified
(GM) crops carrying novel traits have been developed.
• GM crops are developed by a process of genetic modification by which
selected individual genes are inserted from one organism into another to
enhance desirable characteristics or to suppress undesirable ones.
• GM crops are also known as genetically engineered or transgenic or
biotech crops.
• The first transgenic plant was developed in 1983 in tobacco in U.S.A.
• Commercial cultivation of transgenic crops started in the early 1990s.

5. Need of GM crops
• To improve the yield of crops
• To reduce dependance on pesticides
• To enhance resistance to weeds, pests and disease
• To reduce environmental stress
• To improve nutritional quality
• To improve taste and texture
• To provide additional health constituents

6. Area under GM crops is on the rise globally
• Globally, area under GM crops has
increased manifold from 17 lakh
hectares in 1996 to 1917 lakh
hectares in 2018.
• India has the world’s fifth largest
cultivated area under GM crops.
• In India, the area under Bt-Cotton has
increased from 0.29 lakh hectares in
2002-03 to 117.47 lakh hectares in
2019-20, according to Directorate of
Economics and Statistics.
• Major companies interested in GM
crops in India include Monsanto India,
Mahyco.

7. History of Bacillus thuringiensis (Bt)
• Discovered in 1901 by Ishiwataria a cause of sotto disease that was killing
silkworms and named it Bacillus sotto.
• In 1911, Berliner isolated this bacterium from dead Mediterranean flour
moth in Thuringia, Germany, and named it Bt.
• In 1915, Berliner reported the existence of a parasporal body, or crystalline
inclusion (called crystal) close to the endospore within Bt spore.
• In 1956, it was found that the main insecticidal activity against lepidopteran
insects was due to the parasporal crystal.
• In 1956, Bt was used commercially in the USA.
• In the 1980s, the use of Bt increased worldwide when insects became
increasingly resistant to the chemical insecticides.

8. Bacillus thuringiensis (Bt)
• Bacillus thuringiensis (Bt) is a naturally occurring soilborne gram-positive,
rod-shaped, sporulating bacterium found worldwide.
• Bt strains produce three types of insecticidal toxins
• Crystal (cry) toxins
• Cytolytic (cyt) toxins
• Vegetative insecticidal
proteins (Vip)

9. Crystal Toxins (δ-endotoxins)
• The most widely known δ-
endotoxins, include Cry and Cyt
toxins.
• The Cry toxin (cry from crystal),
insecticidal crystal protein is a
protein formed during sporulation
in Bt strains and aggregate to form
crystals.
• Some strains of Bt, produce
another toxic crystal, named
cytolytic protein or Cyt toxin.

10. Mode of action of Bt
1. Ingestion
2. Solubilization and proteolytic activation
3. Binding to target site
4. Formation of toxic lesions

12. Development of transgenic crops
• Isolation of the gene of interest
• Insertion of the gene into a transfer vector and plant
transformation
• Selection and regeneration of the modified plant cells into whole
plants
• Lab analysis and safety testing
• Approval by Government agencies
• Commercialization

13. Methods of gene transfer
• The methods of foreign gene (DNA) transfer in crop plants are-
• Electroporation
• Gene transfer by polyethylene glycol (PEG)
• Gene transfer through microinjection
• Agrobacterium mediated gene transfer
• Biolistics

14. Electroporation
• Short pulse of high voltage are applied to protoplasts which make
temporary pores in plasma membrane to increase their permeability and
facilitate the uptake of foreign gene.
Gene transfer by PEG
• It is applicable for protoplast only. The chemical used is polyethylene
glycol. It stimulates endocytosis and thereby causing the uptake of DNA.

15. Microinjection
• DNA is introduced into the
cytoplasm or nucleus by using
microinjection.
• Mainly used for transformation
of large animal cells.

16. Agrobacterium mediated gene transfer
• Agrobacterium tumefaciens is a soil borne gram negative bacterium which infects
wounded plant tissues and induces the formation of a plant tumor called crown gall
disease.
• The ability to cause crown gall disease is associated with the presence of Ti plasmid
within the bacterial cell.
• A segment of Ti plasmid known as T-DNA is transferred from bacteria into host where it
gets integrated into the host genome.
• T-DNA carries the gene that involve in the biosynthesis of hormones auxin and cytokinin
and plant metabolites opines and agropines.
• Agrobacterium has proved to be an incredible useful tool for the integration of genes into
plants.

18. Biolistics
• It is a physical method first
described by Stanford and co-
workers in 1984.
• Also known as Gene gun
method/particle
bombardment/microprojectiles.
• Gene gun is a device that fires
DNA into target cells.
• DNA to be transformed is coated
onto microscopic beads made of
either gold or tungsten.

20. Bt-cotton
• Bt cotton is genetically modified cotton crop that expresses an insecticidal
protein whose gene has been derived from a soil bacterium called Bacillus
thuringiensis.
• The transgenic cotton is of two types
(1) bollgard (confers resistance to bollworms).
(2) Roundup ready cotton (confers resistance to herbicides).
• Bt cotton is the only genetically modified (GM) crop that has been
approved for commercial cultivation in 2002 by the Government of India.

21. Bt cotton Cry protein Year Company
Bollgard cotton Cry 1 Ac 1996 Monsanto
Bollgard II cotton Cry 2 Ab 2003 Monsanto
Wide strike
cotton
Cry 1 Ac and Cry
1 F
2004 Dow Agro-
sciences

22. Need of Bt-cotton
• Cotton is a long duration crop and is attacked by
large number of insect pests throughout its
growth and development.
• The three bollworms, American bollworm , Pink
bollworm and the Spotted bollworms, are major
pests and cause serious threat to cotton
production.
• About 9400 M tonnes of insecticides worth Rs
747 crores were used only for bollworm control in
2001.
• Cotton bolls are highly vulnerable to insects.

24. Commercial Release of Different Bt-Cotton
Events in India, 2002-2014

25. Benefits of Bt-cotton
• Resistance to bollworms
• Yield improvement
• Reduce use of pesticides
• Reduce cost of cultivation
• More profit

26. Bt-brinjal
• Bt Brinjal is the first genetically modified vegetable crop in India,
developed by the Maharashtra Hybrid Seed Company Ltd., (Mahyco) in
2000.
• Bt brinjal incorporates the cry1Ac gene from the soil
bacterium Bacillus thuringiensis (Bt) expressing insecticidal protein to
confer resistance against FSB.
• The company used a DNA construct containing the cry1Ac gene, a CaMV
35S promoter and the selectable marker genes nptII and aad, to transform
young cotyledons of brinjal plants.

27. Cont.
• In India, the Genetic Engineering Appraisal Committee (GEAC) approved
its commercialization in 2009 following field trials and safety evaluations.
However, the seeds have not yet been released to farmers.
• The National Center on Plant Biotechnology (NRCPB) has developed Bt
brinjal varieties expressing the cryFa1 gene.
• The Indian Institute of Horticultural Research (IIHR) is also developing Bt
brinjal using the cry1Ab gene.

28. Need of Bt-brinjal
• Brinjal or eggplant (Solanum
melongena) is vulnerable to many
diseases caused by insects, pests,
fungi, bacteria and viruses.
• Production is affected by the brinjal
fruit and shoot borer (FSB,
Leucinodes orbonalis) and other
fruit borer insects Helicoverpa
arimegera.

29. Benefits of Bt-brinjal

30. Bt-corn
• Bt corn is a GM crop developed in
1996 that helps reduce pesticide
use against the European Corn
Borer, a pesky caterpillar that eats
the crop.
• Bt corn incorporates
the cry1Ab gene from Bt
expressing insecticidal protein to
confer resistance against corn
borers.

32. GM-Mustard
• Dhara Mustard Hybrid-11 or DMH-11 is a
genetically modified variety of mustard developed
by the Delhi University’s Centre for Genetic
Manipulation of Crop Plants in 2002.
• Hybridised mustard DMH-11 developed using
“barnase / barstar” technology. It is Herbicide
Tolerant (HT) crop.
• 2016: GEAC gave green signal to GM Mustard for
field trial, but SC stayed the order.
• GM mustard Dhara is pending for commercial
release

33. Golden Rice
• Golden Rice was engineered from normal rice by Ingo Potrykus and Peter Beyer in 1990
to help improve human health.
• Rice in its milled form is without vitamin A and its carotenoid precursors.
• Millions of rice consumers who depend on rice for a large proportion of their calories
suffer from vitamin A deficiency.
• Golden Rice has an engineered multi-gene β-carotene pathway in its genome. This
pathway produces beta-carotene, precursor of vitamin A
• The genes phytoene synthase (psy) from Narcissus pseudonarcissus, phytoene
desaturase (crt1) from Erwinia uredovora and lycopene cyclase (lcy) have been
introduced into the rice, driven by the endosperm specific glutelin promoter (Gt1).

34. β-carotene Pathway

36. How to make Golden Rice

37. Transgenic Tomato “Flavr Savr”
• The FLAVR SAVR tomato was the first
GM food crop to be marketed
commercially following FDA approval
in 1994,
• It was developed by Calgene
company of California in 1992 through
the use of antisense RNA technology
to regulate the expression of the
enzyme polygalacturonase (PG) in
ripening tomato fruit.
• Flavr Savr tomato has longer shelf
life.

38. Antisense RNA Technology
• Antisense RNA is a single
stranded RNA that is
complementary to mRNA
strand transcribed within a
cell.
• Antisense RNA introduced
into a cell to inhibit
translation of a
complementary mRNA by
base pairing to it and
creating barrier to the
translation machinery.

39. Genes involved
1. pTOM5 encodes for phytoene synthase that promote lycopene synthesis
that gives red coloration
2. pTOM6 encodes for polygalacturanase (PG) that degrades the cell wall
resulting in fruit softening
3. pTOM encodes for ACC oxidase that catalyze ethylene formation which
triggers fruit ripening
• PG breakdown the polygalacturonic acid component of the cell wall of fruit
pericarp of tomato resulting in softening of fruit. It also makes it more prone to
bruises and decreases its shelf life.

40. Mechanism
1. Isolation of gene from tomato plant that encodes the enzyme PG
2. Synthesis of complementary DNA (cDNA)
3. Introduction of cDNA into a fresh tomato plant to produce transgenic
plant.
GM tomato plant has two type of genes-
• Gene for PG that produces sense mRNA
• Synthetic antisense PG gene that produces antisense mRNA

42. Stacking of Transgenes
• Introduction of more than one gene into crop plants simultaneously or
sequentially, called transgene stacking.
• It is an effective strategy for conferring higher and durable insect and
disease resistance in transgenic plants than single-gene technology.
• The first stack that gained regulatory approval in 1995 was a hybrid Cotton
stack produced by crossing Bt-cotton expressing cry1Ab gene and
Roundup ready cotton producing EPSPS enzyme conferring resistance to
herbicide glyphosate.

43. Methods for transgene stacking
The methods used can be separated into two main groups:
(1) simultaneous introduction methods
(2) sequential introduction methods
Simultaneous introduction methods
This method refer to the introduction of several genes in the same process
of genetic transformation. This technique is described as co-transformation
and can be of two types-
• co-transformation with single plasmid
• co-transformation with multiple plasmids

44. Cont.
• Co-transformation with a single
plasmid - introducing a single
genetic construction with all genes,
each with its own promoter and
terminator, in the same plasmid.
• In Co-transformation with
multiple plasmids, several
constructions are made, each with
one or a few genes, and each
construct is inserted into a
separate plasmid.

45. Sequential introduction methods
Re-transformation involves the
transformation of an already
transgenic plant to insert a new
characteristic.
Sexual crossing – plants containing
several transgenes produced by
crossing parents with different
transgenes.

47. Biosafety
• As more and more transgenic crops are released for field-testing and
commercialization, concerns have been expressed regarding potential
risks to both human health and environment.
• Biosafety describes the principles, procedures and policies to be adopted
to ensure the environmental and personal safety.
The major biosafety concerns falls into following categories:
• Bio-safety of human and animal health
• Ecological concerns
• Public attitude

48. Bio-safety of human and animal health
• Risk of toxicity, due to the nature of the product or the changes in the
metabolism and the composition of the organisms resulting from gene
transfer.
• Newer proteins in transgenic crops from the organisms, which have not
been consumed as foods, sometimes has the risk of these proteins
becoming allergens.
• Genes used for antibiotic resistance as selectable markers have also
raised concerns regarding the transfer of such genes to microorganisms
and thereby aggravate the health problems.

49. Ecological concerns
• Gene flow due to cross pollination for the traits involving resistance can
result in development of tolerant or resistant weeds that are difficult to
eradicate.
• GM crops could lead to erosion of biodiversity and pollute gene pools of
endangered plant species.
• Genetic erosion has occurred as the farmers have replaces the use of
traditional varieties with monocultures.
Public attitude
• Consumer response depends on perceptions about risks and benefits of
genetically modified foods. The media, individuals, scientists and
administrator and NGO have the responsibility to educate the people
about the benefits of GM foods.

50. Regulatory system in India
Regulatory approval of GM crops
• The rules governing the handling of genetically modified organisms
(GMOs) and products thereof were notified in 1989 under Environment
Protection Act 1986.
• Two government agencies, the Ministry of Environment and Forests
(MoEF) and the Department of Biotechnology (DBT), Ministry of Science
and Technology, are responsible for implementation of the regulations.

51. Cont.
• There are basically 6 authorities to handle different aspects of the
regulation.
1. Recombinant DNA Advisory Committee (RDAC)
2. Review Committee on Genetic Manipulation (RCGM)
3. Genetic Engineering Approval Committee (GEAC)
4. Institutional Biosafety Committees (IBSC)
5. State Biosafety Coordination Committees (SBCC)
6. District Level Committees (DLC).

52. Cont.
• RDAC is constituted by DBT to monitor the developments in biotechnology
at national and international levels. RDAC submits recommendations from
time to time that are suitable for implementation for upholding the safety
regulations in research and applications of GMOs and products thereof.
• IBSC - set-up at each institution for monitoring institute level research in
genetically modified organisms.
• RCGM - committee is constituted by DBT to review all ongoing projects
involving high risk and controlled field experiments.
• GEAC - set-up in the Ministry of Environment, Forests and Climate
Change to authorize large-scale trials and environmental release of GMO.

53. Cont.
• SBCC - set up in each state where research and application of GMOs are
contemplated, coordinate the activities related to GMOs in the state with
the central ministry. SBCCs have powers to inspect, investigate and to
take action in case of violations.
• DLC is constituted at district level to monitor the safety regulations in
installations engaged in the use of GMOs in research and application.

54. References
• H. Khan, Gene transfer technologies in plants: Roles in improving crops. Recent
Research in Science and Technology 2009, 1(3): 116–123 ISSN: 2076-5061
• S Kumar, A Misra et al. Bt Brinjal in India: A long way to go. GM crops, 2011
• CC Ceccon, A Caverzan, R Margis, JR Salvadori. Gene stacking as a strategy to confer
characteristics of agronomic importance in plants by genetic engineering. Ciência Rural,
Santa Maria, v.50:6, e20190207, 2020
• L Palma, D Muñoz, C Berry, J Murillo, P Caballero. Bacillus thuringiensis toxins:
an overview of their biocidal activity. Toxins 2014, 6, 3296-3325

55. THANK YOU