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

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. A lso 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. What are Transgenic Plants ?

3. Global area (Million H ectares) 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 R ates (%) for Principal Biotech Crops 2016 ( M illion H ectares) 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 t umefaciens 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 : T L (14 kb ) and T R ( 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 vi r genes. Has origin of replication. Contains a region enabling conjugative transfer. Has genes for the catabolism of opines.

11. Genetic map of o ctopine 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

16. 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.

17. 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. D elivery 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

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

19. 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.

20. 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 . F oreign 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 . S uccessfully 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.

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

22. Advantages : i. Gene transfer can be efficiently done in organized tissues. ii. Different species of plants can be used to develop transgenic plants. Limitations : 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.

23. 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. T he 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.

24. 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

25. 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

26. 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

27. 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.

28. 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

29. 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.

30. CHEMICAL METHODS POLYETHYLENE GLYCOL (PEG)-MEDIATED TRANSFER: Polyethylene glycol (PEG), in the presence of divalent cations (using Ca 2+ ), 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.

31. 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.

32. 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.

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

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

35. STAGES IN DEVELOPMENT OF A TRANSGENIC CROP

36. 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:- Recombinant DNA A dvisory Committee (RDAC) Institutional Biosafety Committee (IBSC) Review Committee on Genetic Manipulation (RCGM) Genetic Engineering Appraisal Committee ( GEAC ) State Biotechnology Co-ordination Committees ( SBCC) District Level Committees ( DLC)

37. 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 S tepwise regulatory procedures for the development and commercialization of transgenic crops

38. 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.

39. 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.

40. 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. T he members of the DLC were government officials who were involved in the areas of agriculture , pollution control and health.

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

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

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

44. 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

45. Impact on Non-target O rganisms 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

46. 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.

47. Moratorium on Bt Brinjal

48. Public Acceptance- Transgenic Mustard

49. Thank You