Development, production and release of transgenic plants-Issues related to transgenics

1. DEVELOPMENT, PRODUCTION AND RELEASE
OF TRANSGENIC PLANTS-ISSUES RELATED TO
TRANSGENICS
Navaneetha Krishnan J
L-2016-A-18-D
School of Agricultural Biotechnology
Punjab Agricultural University

2.  Also known as genetically modified plants
 A transgenic crop plant contains a gene or genes which have been
artificially inserted instead of a plant acquiring them through
pollination.
 The inserted gene sequence (transgene) may come from another
unrelated plant, or completely different species.
 Throughout history all crops have been genetically modified from
their original wild state by domestication, selection, and control of
breeding over long periods of time.
 Genetic engineering speeds up the process and increases the
variety of genes which can be inserted into a particular plant.

3. Global area (Million Hectares) of Biotech crops, 1996-2016 by Country
and Mega-Countries
Source: ISAAA, 2016

4. Global Area of Biotech Crops, 2015 and 2016 by Crop (Million Hectares)
Global Area of Biotech Crops, 2015 and 2016 by Trait (Million Hectares)
Source: ISAAA, 2016

5. Global Adoption Rates (%) for Principal
Biotech Crops 2016 (Million Hectares)
Trait Distribution in Approved
Events, 1992-2016

6. Status of Biosafety Research Trails of Biotech Crops in India, 2016
Source: MOEF&CC, 2016, Analyzed by ISAAA, 2016

7. Crops and Traits under Field Testing by the Public Sector in 2016
Source: ISAAA, 2016

8. METHODS FOR PLANT GENE TRANSFER
INDIRECT METHODS
(VECTOR-BASED)
DIRECT
METHODS
(VECTOR-LESS )
• Agrobacterium
mediated
transfromation
IN PLANTA
TRANSFORMATION
Physical methods
• Particle
bombardment
• Electroporation
• Microinjection
• Liposome mediated
DNA transfer
• Silicon Carbide fibre
mediated DNA
transfer
Chemical method
• PEG-mediated DNA
transfer
• Floral Dip
• Vacuum
infiltration
• Agro injection

9.  A. tumefaciens is a gram-negative soil bacterium which naturally transforms
plant cells, resulting in crown gall (cancer) tumors
 Tumor formation is the result of the transfer, integration and expression of
genes on a specific segment of A. tumefaciens plasmid DNA called the T-DNA
(transferred DNA)
 The T-DNA resides on a large plasmid called the Ti (tumor inducing) plasmid
found in A.tumefaciens
INDIRECT DNA TRANSFER METHODS
Plant Transformation With The Ti Plasmid Of Agrobacterium tumefaciens
Image source : https://www.apsnet.org/edcenter/intropp/topics/Pages/PlantDiseaseDiagnosis.aspx

10. TI-PLASMID FEATURES
 Two strains of Ti-plasmid:
-Octopine strains- contains two T-DNA region: TL (14 kb)
and TR ( 7 kb)
-Nopaline strains- contain one T-DNA region(20 kb)
 Size is about 200 kb.
 Has a central role in crown-gall formation.
 Contains one or more T-DNA region that is integrated into the
genome of host plants.
 Contain a vir region ~ 40 kb at least 8~11 vir genes.
 Has origin of replication.
 Contains a region enabling conjugative transfer.
 Has genes for the catabolism of opines.

11. Genetic map of octopine strain Ti Plasmid
Image source: https://www.researchgate.net/figure/259477921_fig5_Fig-125-Genetic-map-of-octopine-type-Ti-
plasmid-Modi-fi-ed-from-Ream-2002-and-Ozcan-et

12. Overview of the Infection Process
Image source : https://www.slideshare.net/Dilippandya/agro2-61290451

13. AGROBACTERIUM-MEDIATED GENE TRANSFER
Major steps
• A DNA segment is constructed that contains a selectable marker and
a gene of interest to look like a T-DNA.
• The “T-DNA” should be inserted into an Agrobacterium cell so that
it can be mobilized by the vir genes.
• Selection of transformed plant cells that can be regenerated into
normal, fertile plants using marker genes.
Requirements
• A transfer cassette bounded by functioning borders.
• Ways to get this cassette into Agrobacterium.
• Disarmed Ti plasmids that retain functional vir genes.

14. BINARY VECTOR SYSTEM
Strategy:
1. Move T-DNA onto a separate, small plasmid.
2. Remove aux and cyt genes.
3. Insert selectable marker (kanamycin resistance) gene in T-DNA.
4. Vir genes are retained on a separate plasmid.
5. Put foreign gene between T-DNA borders.
6. Co-transform Agrobacterium with both plasmids.
7. Infect plant with the transformed bacteria.
Examples : pBIN19, pGreen series, pCAMBIA series etc.

15. Components of Binary vector system

18. Advantages
• Technically simple.
• Yields relatively uncomplicated insertion events (low copy number,
minimal rearrangements).
• Unlimited size of foreign DNA.
• Efficient (for most plants).
• Adaptable to different cell types, culture procedures (protoplasts,
tissue sections, “non-culture” methods).
• Transformants are mitotically and meiotically stable.
Disadvantages
• Host range is limited: not all plants may be susceptible to Agrobacterium.
• The cells that regenerate more efficiently are often difficult to transform,
e.g. embryonic cells lie in deep layers which are not easy targets for
Agrobacterium.

19. DIRECT DNA TRANSFER METHODS
1. ELECTROPORATION
 Involves the use of high field strength electrical impulses to
reversibly permeabilize the cell membranes for the uptake of
DNA.
 Delivery of DNA into intact plant cells and protoplasts.
 The plant material is incubated in a buffer solution containing
the desired foreign/target DNA, and subjected to high voltage
electrical impulses.
 Leads to formation of pores in the plasma membrane through
which DNA enters and gets integrated into the host cell genome.
 Electroporation has been successfully used for the production of
transgenic plants of many cereals e.g. rice, wheat, maize.
PYSICAL METHODS

20. Image source : https://www.slideshare.net/mohammedsami31508/plant-transformation-methods

21. Advantages :
i. This technique is simple, convenient and rapid, besides
being cost-effective.
ii. The transformed cells are at the same physiological state
after electroporation.
iii. Efficiency of transformation can be improved by optimising
the electrical field strength, and addition of spermidine.
Limitations :
i. Under normal conditions, the amount of DNA delivered into
plant cells is very low.
ii. Efficiency of electroporation is highly variable depending
on the plant material and the treatment conditions.
iii. Regeneration of plants is not very easy, particularly when
protoplasts are used.

22. 2. PARTICLE BOMBARDMENT (BIOLISTICS)
 The micro projectile bombardment method was initially named
as biolistics by its inventor Sanford (1988).
 Biolistics is a combination of biological and ballistics. There are
other names for this technique- particle gun, gene gun, bio
blaster.
 Foreign DNA containing the genes to be transferred is coated
onto the surface of minute gold or tungsten particles (1-3
micrometers) and bombarded onto the target tissue or cells
using a particle gun.
 Two types of plant tissue are commonly used for particle
bombardment- Primary explants and the proliferating embryonic
tissues.
 Successfully used for the transformation of many cereals, e.g.
rice, wheat, maize.
 A commercially produced particle bombardment apparatus
namely PDS-1000/HE is widely used these days.

23. Image source: http://www.oocities.org/eurekayao/bombgene.html

24. Advantages :
i. Gene transfer can be efficiently done in organized tissues.
ii. Different species of plants can be used to develop transgenic
plants.
Limitations :
i. The major complication is the production of high transgene copy
number. This may result in instability of transgene expression
due to gene silencing.
ii. The target tissue may often get damaged due to lack of
control of bombardment velocity.
iii. Sometimes, undesirable chimeric plants may be regenerated.

25. 3. MICROINJECTION:
 Microinjection is a direct physical method involving the
mechanical insertion of the desirable DNA into a target cell.
 The target cell may be the one identified from intact cells,
protoplasts, callus, embryos, meristems etc.
 The technique of microinjection involves the transfer of the
gene through a micropipette (0.5-10.0 pm tip) into the
cytoplasm/nucleus of a plant cell or protoplast.
 While the gene transfer is done, the recipient cells are kept
immobilized in agarose embedding, and held by a suction/
holding pipette.
 The transformed cell is cultured and grown to develop into a
transgenic plant.
 The major limitations of microinjection are that it is slow,
expensive, and has to be performed by trained and skilled
personnel.

26. Image source : http://moreonbiotechnology.blogspot.in/2008/06/genetic-engineering-techniques.html
https://www.wpiinc.com/blog/2013/04/29/application-notes/micro-injection-setup-101/
Suction
pipette
cell
Microinjection
pipette
Microinjection setup

27. 4. LIPOSOME-MEDIATED TRANSFORMATION:
 Liposomes are artificially created lipid vesicles containing a
phospholipid membrane.
 They are successfully used in mammalian cells for the delivery of
proteins, drugs etc.
 Liposomes carrying genes can be employed to fuse with
protoplasts to transfer the genes.
 Liposome-mediated transformation involves adhesion of
liposomes to the protoplast surface, its fusion at the site of
attachment and release of plasmids inside the cell

28. Image source : Pleyer U, Dannowski H. 2002. Delivery of genes via liposomes to corneal endothelial cells.
Drug News Perspect, 15(5): 283
Liposome-mediated Transformation

29. Advantages :
i. Being present in an encapsulated form of liposomes, DNA is
protected from environmental insults and damage.
ii. DNA is stable and can be stored for some time in liposomes prior to
transfer.
iii. Applicable to a wide range of plant cells.
iv. There is good reproducibility in the technique.
Limitations :
The major problem with liposome-mediated transformation is the
difficulty associated with the regeneration of plants from transformed
protoplasts.

30.  The silicon carbide fibres (SCF) are about 0.3-0.6 pm in
diameter and 10-100 pm in length. These fibres are capable of
penetrating the cell wall and plasma membrane, and thus can
deliver DNA into the cells.
 The DNA coated silicon carbide fibres are vortexed with plant
material (suspension culture, calluses).
 During the mixing, DNA adhering to the fibres enters the cells
and gets stably integrated with the host genome.
 The silicon carbide fibres with the trade name Whiskers are
available in the market.
5. SILICON CARBIDE FIBRE-MEDIATED TRANSFORMATION

31. Advantages
i. Direct delivery of DNA into intact walled cells. This avoids
the protoplast isolation.
ii. Procedure is simple and does not involve costly equipment.
Disadvantages
i. Silicon carbide fibres are carcinogenic and therefore have to
be carefully handled.
ii. The embryonic plant cells are hard and compact and are
resistant to SCF penetration.
 In recent years, some improvements have been made in SCF-
mediated transformation. This has helped in the transformation
of rice, wheat, maize and barley by using this technique.

32. CHEMICAL METHODS
POLYETHYLENE GLYCOL (PEG)-MEDIATED TRANSFER:
 Polyethylene glycol (PEG), in the presence of divalent cations
(using Ca2+), destabilizes the plasma membrane of protoplasts
and renders it permeable to naked DNA.
 DNA enters nucleus of the protoplasts and gets integrated with
the genome.
 The procedure involves the isolation of protoplasts and their
suspension, addition of plasmid DNA, followed by a slow addition
of 40% PEG-4000 (w/v) dissolved in mannitol and calcium nitrate
solution.
 As this mixture is incubated, protoplasts get transformed.

33. Limitations of PEG-mediated transformation:
i. The DNA is susceptible for degradation and rearrangement.
ii. Random integration of foreign DNA into genome may
result in undesirable traits.
iii. Regeneration of plants from transformed protoplasts is a
difficult task.

34. IN PLANTA TRANSFORMATION
 Plant transformation protocol that avoids the use of tissue
culture.
 The first "in-planta" method was described by Feldmann and
Marks in 1987 and consisted of the imbibition of seeds with
Agrobacterium.
 Major methods :
 Floral dip
 Vacuum infiltration
 Agro-injection
 These procedures offer two main advantages.
 Tissue culture and the resulting somaclonal variations are
avoided and only a short time is required in order to obtain
entire transformed individuals.
 However, the mean frequency of transformants in the progeny
of such inoculated plants is relatively low and very variable.

35. Floral dip method (Bent et al. 2000)

36. Image source :
http://www.genomine.co.kr/randd/e_Func_11.html#

37. STAGES IN DEVELOPMENT OF A TRANSGENIC CROP

38. REGULATION OF TRANSGENIC CROPS IN INDIA
 Rules for Manufacture, Use, Import, Export and Storage of
Hazardous Microorganisms (HMO)/Genetically Engineered
Organisms or Cells, 1989 under the EPA (1986) known as
‘Rules1989’.
 The regulatory agencies responsible for implementation of the
Rules 1989 are the Ministry of Environment, Forest and Climate
Change and Department of Biotechnology through the following
six competent authorities:-
1. Recombinant DNA Advisory Committee (RDAC)
2. Institutional Biosafety Committee (IBSC)
3. Review Committee on Genetic Manipulation (RCGM)
4. Genetic Engineering Appraisal Committee (GEAC)
5. State Biotechnology Co-ordination Committees (SBCC)
6. District Level Committees (DLC)

39. Applicant
GEAC
(MOEF&CC)
MEC
IBSC
RCGM
(DBT)
Commercial
Release
(MOEF&CC)
ICAR
RCGM FUNCTIONS
To note, approve and
recommend generation
of appropriate biosafety
data. Monitors BRL-I
trails.
IBSC FUNCTIONS
To note, approve,
recommend and seek
approval of RCGM.
GEAC FUNCTIONS
To approve for large
scale use, open release
in to environment.
Monitors BRL-II trails.
ICAR TRIALS
To generate complete
agronomic data and to
recommend for
commercial release of
GM crops.
MEC FUNCTIONS
Visit trial sites, analyze
data, inspect facilities,
recommend safe and
agronomically viable
transgenics to
RCGM/GEAC.
Note: MEC- Monitoring and Evaluation Committee
Stepwise regulatory procedures for the development and
commercialization of transgenic crops

40. RECOMBINANT DNA ADVISORY COMMITTEE (RDAC)
 Set up by DBT
 The RDAC is involved in reviewing the developments in
biotechnology both at national as well as international levels.
 Recommends safety regulations as per the indigenous
requirements of our country in recombinant research, use and
applications from time to time.
 The RDAC functions are of advisory nature.

41. STATE BIOTECHNOLOGY CO-ORDINATION COMMITTEES
(SBCC)
 Review and control safety measures adopted while handling
large scale use of genetically modified organisms in research,
developmental and industrial production activities.
 Monitor large scale release of genetically engineered products
with the environment, and oversee field applications and
experimental field trials.
 Provide information/data to RCGM upon surveillance of
approved projects, and in case of environmental releases, with
respect to safety, risks and accidents.
 The members of the SBCCs included representatives from the
state Ministries of Environment, Health, Agriculture, Industry
and Forests.

42. DISTRICT LEVEL COMMITTEES (DLC)
 The DLC could inspect any installations involving genetically
modified organisms and identify the sources of risks associated
with such installations and coordinate activities with a view to
meeting any emergency.
 The DLC was expected to submit regular reports to the relevant
SBCC and GEAC.
 The members of the DLC were government officials who were
involved in the areas of agriculture, pollution control and health.

43. ISSUES RELATED TO TRANSGENIC CROPS
Need for transgenics in developing countries!
Image Source :http://modernfarmer.com/2013/09/business-solutions-farmers-earning-2-day/

44. Gene Flow/Transgene Escape
Superweeds
Image source :
https://tandtperiod6.wikispaces.com/
Avoiding+super+weeds

45. Threat to Genetic Diversity
Image Source: http://www.mexicolore.co.uk/aztecs/you-contribute/amaizing

46. Impact on Human Health
Image source:http://www.occupyforanimals.net/study-reveals-that-safe-levels-of-monsantos-gm-corn-and-the-chemical-
herbicide-roundup-glyphosate-are-directly-linked-to-causing-cancerous-tumors.html

47. Impact on Non-target Organisms
Image source:
https://www.ag.ndsu.edu/piercecountyextension/livestock
https://www.optibacprobiotics.co.uk/blog/2015/06/soil-based-organisms-friend-or-foe
http://www.freepik.com/free-photo/bird-macro-pigeon-feather-nature-birds-walk-grey_667395.htm
http://xeriscapeaz.org/attracting_beneficial_insects.htm

48. Image source: http://seedfreedom.in/pest-resistant-bt-brinjal-comes-under-pest-attack-in-bangladesh/
Public Acceptance- Bt-Brinjal
Farmers protest during a public hearing on Bt Brinjal in Hyderabad.

49. Moratorium on Bt Brinjal

50. Public Acceptance- Transgenic Mustard