Biotechnology_transgenic plants.pptx

2. Genetically modified organisms : plants

3. Genetically Modified Organisms (GMOs)
• Organisms which are modified at genetic level by genetic engineering methods.
• Plants/animals/microorganisms
• In case of plants - Genetically modified crops (GM crops)/transgenic plants.

4. Discussion on…….
• What is the need of GM crops?
• What do people do in past without genetic engineering approaches?
• What is the limitations of traditional ways?
• What is transgenic approach?
• What is the methodology to create transgenic plants?
• What are the applications of transgenics plants?
• What are the ethical concerns related to GM crops?

5. What is the need of Genetically Modified
Crops (GM crops)
• To increase the yield
• To enhance nutritional value
• To provide protection against viruses, pests and other infectious agents.
• To make them resistant to herbicides
• To make them tolerant against environmental stresses.
• Increase post-harvest shelf life.
• To produce such as pharmaceutical agents, biofuels, and other industrially
useful goods, as well as for bioremediation.
• In research – to study action of genes during development and other biological
processes.

6. Traditional Approach
• For thousand of years, we have been cultivating new varieties of plants for
desirable traits via selective breeding.
• Here we choose plants with particular characteristics and hope to have
progenies with desirable traits.
• It is also known as artificial selection.

7. Traditional Approach - example
Selective breeding powerpoint (slideshare.net)

8. Limitation of traditional approach
• Time-consuming (it takes several years…..).
• Unpredictable outcome, sometimes undesirable traits also appear.
• High cost.
• Sometimes hybrid are more prone to diseases and other stresses.

9. Transgenic approach….
• A genetically modified plant with desirable gene from different species
(unrelated one) is known as transgenic plant. The phenomenon is called as
transgenesis.
• Sometimes the desirable gene belongs to same or closely related species, the
phenomenon is called as cisgenesis.
• The foreign gene/inserted sequence is known as transgene.

10. How can we introduce foreign DNA into
plants?
• Agrobacterium-mediated Gene transfer
• Physical methods (such as Gene Gun and others)

11. Agrobacterium Tumefaciens
• Rod-shaped, gram negative soil bacteria
• Scientific name - Rhizobium radiobacter
• In 1907, Smith and Townsend postulated that it is a causative agent for crown
gall formation in plants. It Infects over 140 species of dicot plants.

13. Agrobacterium Tumefaciens – Ti plasmid
• Tumor is due to transfer, integration and expression of genes of specific segment of its plasmid
(called T-DNA region/Transfer DNA region) into plant cell genome.
• Plasmid is known Ti (Tumor-inducing) plasmid. Ti plasmid is generally present in most strains of
the bacteria. It is app 200Kb in size.

14. Agrobacterium Tumefaciens : Ti plasmid
organization
• T-DNA region : genes for biosynthesis of plant hormones
o Auxin (iaaM and iaa H) and cytokinin
o opine gene
(Opines are unique condensation products of either an amino acid and a keto acid or an amino acid
and a sugar, for example octopine is product of arginine and pyruvic acid/nopaline is product of
arginine and alpha-ketoglutaraldehyde/Argopine is a bicyclic derivatives of glutamic acid. It acts as
unique source of carbon and nitrogen specifically for Agrobacterium.
o Surrounded by right border and left border : set of 24bp sequence present on either side, critical for
T-DNA transfer and integration
• Virulence region : critical for infection, transfer of T-DNA, integration of T-DNA etc. Nine vir-gene
operons have been identified.
• Ori
• Opine metabolism : involved in uptake and metabolism of opines.

15. Agrobacterium Tumefaciens : Ti plasmid
organization

16. Agrobacterium Tumefaciens
Infection occurs by following steps:
• 1. Wounded plants release phenolic compounds such as acetosyringotone and
hydroxyacetosyringotone.
• 2. bacteria gets attracted to these compounds. Molecules get recognized by
bacterial receptors.
• 3. Activation of different vir genes and attachment of bacteria to the plant cells
• 4. Some vir gene products process ssT-DNA
• 5. formation of T-complex with vir gene products
• 6. T-DNA transfer
• 7. Nuclear import of T-DNA complex
• 8. Integration of T-DNA in the plant genome
• 9. Expression of bacterial genes and synthesis of bacterial proteins

17. agrobacterium tumefaciens infection to plants - Google Search

18. Can we tweak natural transformation by
Agrobacterium Tumefaciens
(591) Agrobacterium Mediated Transformation - YouTube

19. Can we tweak natural transformation by
Agrobacterium Tumefaciens
• Yes, need to insert desirable sequences (encoding for gene of
interest.
but there are certain limitations……
• Large size of plasmids
• Unnecessary genes such as plant hormones genes, opine genes,
opine metabolism genes.
• Does not have E.coli ori
(591) Agrobacterium Mediated Transformation - YouTube

20. Modified Ti plasmid – transfer gene of interest
to plants
• Need to insert desirable sequences (encoding for gene of interest)
• Marker gene
• Need to reduce the size as smaller plasmids are preferred for recombinant work.
• Unnecessary genes such as plant hormones genes, opine genes, opine metabolism genes could
be removed.
• An origin of DNA replication that allows the plasmid to replicate in E. coli.
• The right border sequence of the T-DNA region. This region is absolutely required for T-DNA
integration into plant cell DNA, although most cloning vectors include both a right and a left
border sequence.
• A polylinker (multiple cloning site) to facilitate insertion of the cloned gene into the region
between T-DNA border sequences.
• A “killer” gene encoding a toxin downstream from the left border to prevent unwanted vector
DNA past the left border from being incorporated into transgenic plants. If this incorporation
occurs, and the killer gene is present, the transformed cells will not survive

21. Modified Ti plasmid – Desired DNA sequences
• Gene of interest (gene with insecticidal nature, gene that can provide
resistance against herbicides, viruses, stresses etc.)
• Gene can be from related species or foreign species.
• Plant (eukaryotic) transcriptional regulation signals, including both a
promoter and a termination–polyadenylation sequence, to ensure
that it is efficiently expressed in transformed plant cells.

22. Marker Gene
 A marker gene is a gene used to confirm whether inserted nucleic acid sequence
has been successfully inserted into an organism's DNA
 such as neomycin phosphotransferase, that confers kanamycin resistance on
transformed plant cells.
 Selectable marker gene/non-selectable marker gene or reporter gene

23. Marker Gene

24. Marker Gene

25.  Unnecessary genes such as plant hormones genes, opine genes, opine
metabolism genes could be removed.
 Nine vir gene complex or vir operon (app 30Kb in size) – can be removed.
Reduction of size

26. Artificial Ti Plasmid

27. How the infection, transfer and integration of
desired gene in artificial Ti Plasmid will happen
without vir gene complex

28. Approaches to transfer Gene of interest via Modified Ti
Plasmid
• Modified Ti plasmids lack vir genes, they cannot by themselves effect the
transfer and integration of the T-DNA region into recipient plant cells.
• Two different approaches have been used
A binary Vector system
Cointegrate Vector approach

29. A Binary Vector System
• Here two vectors are being used. One is modified Ti Plasmid containing gene of
interest, ori either broad-host range or E. coli and A. tumefaciens origins of
DNA replication (example of shuttle vectors). This plasmid is also called as
pBIN19. (now-a-days, further smaller version i.e. mini binary vector/pCB301 otr
its derivatives are being used).
• The other plasmid is helper plasmid, a modified (defective or disarmed) Ti
plasmid that contains a complete set of vir genes but lacks portions, or all, of
the T-region.

30. A Binary Vector System
• All the cloning steps are being done in E.coli.
• Finally modified Ti plasmid with gene of interest is transformed to the recipient
A. tumefaciens strain that carries a helper plasmid. Then plants are infected
with this agrobacterium.
• With this system, the defective Ti plasmid synthesizes the vir gene products
that mobilize the T-DNA region of the binary cloning vector and ensures its
integration into plant genome.

31. Ti-binary vector system for Agrobacterium -mediated plant transformation. A binary vector, in which a
target gene and plant selection marker gene are cloned between the two border sequences (RB and LB), is
transformed into A. tumefaciens harboring a disarmed Ti-plasmid without the T-DNA region. Plant cells
are infected by the transformed A. tumefaciens and then the target gene and marker gene are transferred
into a plant chromosome by the vir genes on Ti-plasmid
Ti-binary vector system for Agrobacterium -mediated plant... | Download Scientific Diagram (researchgate.net)

32. Cointegrate Vector System
• The cointegrate vector system, the cloning (cointegrate) vector has a plant selectable marker
gene, the target gene, the right border, an E. coli origin of DNA replication, and a bacterial
selectable marker gene.
• The cointegrate vector is transformed to Agrobacterium that carries a disarmed Ti plasmid.
• The cointegrate vector recombines with a modified (disarmed) Ti plasmid that lacks both the
tumor-producing genes and the right border of the T-DNA within A. tumefaciens, and the entire
cloning vector becomes integrated into the disarmed Ti plasmid to form a recombinant Ti
plasmid.
• The cointegrate cloning vector and the disarmed helper Ti plasmid both carry homologous DNA
sequences that provide a shared site for in vivo homologous recombination; normally these
sequences lie inside the T-DNA region. Following recombination, the cloning vector becomes
part of the disarmed Ti plasmid, which provides the vir genes necessary for the transfer of the
T-DNA to the host plant cells.
• The only way that this cloning vector can be maintained in A. tumefaciens is as part of a
cointegrate structure. In this cointegrated configuration the genetically engineered T-DNA
region can be transferred to plant cells Glick and Pasternak

33. Cointegrate Vector System
Glick and Pasternak

34. https://www.youtube.com/watch?v=yesNHd9h8k0

35. Advantages of Agrobacterium-Mediated Gene
Transfer
• Effective in several plant species both dicots as well as monocots.
• High transformation efficiency
• better stability of transgene
• Comparatively less expensive as minimal equipment and facility required.
• Better quality of transgenic plants in terms of fertility and stability.
• Large amount of DNA can be transferred.

36. Limitations of Agrobacterium-Mediated Gene
Transfer
• Only applicable in case of dicot plants. Monocots plants including the world’s
major cereal crops (rice, wheat, and corn) are not readily transformed by A.
tumefaciens. (However, by refining and carefully controlling conditions,
protocols have been devised for the transformation of corn and rice by A.
tumefaciens carrying Ti plasmid vectors)
• Time-consuming

41. Applications of transgenic plants
• Transgenic plants with beneficial traits (brainkart.com)
• Transgenic plants: Types, benefits, public concerns and future –
ScienceDirect
• Ti plasmid (slideshare.net)
• Gene manipulation in plants: 2.2 Using A. tumefaciens to genetically
modify plant cells - OpenLearn - Open University - S250_1