1. Transgenic Plants Genome editing
Genetically modified organisms (GMOs)
aka genetically engineered plants
Genome editing
CRISPR-Cas9, and beyond

2. Why create transgenic plants?
No naturally occurring genetic variation.
Too complicated to capitalize on naturally occurring genetic variation.
Validating function of a candidate gene (allele).
Examples of “classic” transgenics: 
Glyphosate herbicide resistance in many crops
Vitamin A in rice
Blue roses

3. What genes to transfer? One gene to a few genes - the CP4 ESPS example
Multiple genes - Golden Rice and the Applause rose
In principle, any gene, genes, sequences

4. Constructing transgenes The minimal requirements for the transgene are a promoter, coding region, and terminator
General structure:
5’---Promoter …..Coding region…….terminator---3’
Example: Bt gene with 35S promoter, nptII selectable marker, and Tnos terminator sequence
5’ --P35S…Bt…Tnos--//--P35S…nptII…Tnos---3’

5. Transgene promoter: DNA sequence controlling spatial and/or temporal level of transgene expression.
Constitutive
Inducible

6. Transgene coding region: DNA sequence encoding target protein.
EPSPS gene with Tnos terminator
Selectable marker gene (nptII)

7. Transgene coding region: DNA sequence encoding target protein.

8. Transgene termination sequences:  DNA sequence signaling end of the gene during transcription.
NosT, 3′ region (terminator) of the nopaline synthase gene
OcsT, 3′ region (terminator) of the octopine synthase gene

9. Selectable Marker:  Encodes a protein (enzyme) that allows the transformed cells to grow while the growth of the non-transformed cells is inhibited.
sul, resistance marker gene conferring resistance to antibiotic sulfadiazine in plants
bla, β-lactamase gene conferring resistance to ampicillin in bacteria
nptII, neomycin phosphotransferase II gene conferring resistance to kanamycin
pat, coding sequence of the basta resistance gene from Streptomyces hygroscopus
EPSPS gene with Tnos terminator
Selectable marker gene (nptII)

10. Reporter genes:  Genes that, upon expression in the transgenic plants, provide a clear indication that genetic transformation did occur, and indicate the location and the level of expression.  
More on reporter genes:
Beta Glucuronidase (GUS)
Green fluorescent protein (GFP)

11. Reporter genes:  Genes that, upon expression in the transgenic plants, provide a clear indication that genetic transformation did occur, and indicate the location and the level of expression.   
Green fluorescent protein (GFP)
More on the protein
Prospects for auto-luminescent plants

12. Transformation procedures
In order for a transgene to be inherited, it must be incorporated into the genome of a cell which will give rise to tissues which will be asexually propagated or to tissues which will undergo gametogenesis.
The two principal mechanisms for transforming tissues with a transgene
Biolistics = “Gene gun”
Agrobacterium tumefaciens = “Agro”

13. Transformation procedures
In order for a transgene to be inherited, it must be incorporated into the genome of a cell which will give rise to tissues which will be asexually propagated or to tissues which will undergo gametogenesis.
V v
Mitosis

14. The gene gun Micro projectile bombardment or the biolistic method
Small metal particles are coated with the transgene DNA
Particles are delivered to target tissues via an explosive force
“The Helios Gene Gun is a new way for in vivo transformation of cells or organisms…This gun uses Biolistic ® particle bombardment where DNA- or RNA-coated gold particles are loaded into the gun and you pull the trigger. A low pressure helium pulse delivers the coated gold particles into virtually any target cell or tissue. The particles carry the DNA so that you do not have to remove cells from tissue in order to transform the cells.”
$30K

15. The Agrobacterium method
Agrobacterium tumefaciens is a soil bacterium causing a root disease called crown gall
In the case of disease, A. tumefaciens invades the host plant and transfers a piece of its own DNA to the host genome
For transformation, A. tumefaciens has been engineered to carry and transfer transgenes and to not cause disease

18. sul, resistance marker gene conferring resistance to antibiotic sulfadiazine in plants
bla, β-lactamase gene conferring resistance to ampicillin in bacteria
nptII, neomycin phosphotransferase II gene conferring resistance to kanamycin
pat, coding sequence of the basta resistance gene from Streptomyces hygroscopus

19. Selection and regeneration: a plant with one copy of the transgene is a hemizygote (heterozygous for transgene)

20. Why create transgenic plants?
No naturally occurring genetic variation.
Examples of “classic” transgenics: 
Glyphosate herbicide resistance in many crops
Vitamin A in rice
Blue roses

22. CP4 EPSPS: The gene conferring resistance to glyphosate herbicide
The gene was found in Agrobacterium tumefaciens and transferred to various plants
Coincidentally, A. tumefaciens is also used for creating transgenic plants

23. Glyphosate mode of action
Glyphosate inhibits EPSPS enzyme - impeding the synthesis of aromatic amino acids
EPSPS
Proteins
CO2
H2O
NH3
aromatic
amino acids
CO2
H2O
NH3
glyphosate
aromatic
amino acids
EPSPS
Proteins

24. Roundup ReadyTM Crops
EPSPS
Proteins
CO2
H2O
NH3
aromatic
amino acids
glyphosate
CP4 EPSPS
"Two key elements needed for the development of commercially viable glyphosate-tolerant crops were:
a resistant target enzyme
sufficient expression of that enzyme within the transgenic plant”
Heck et al Crop Sci. 45: (2005).

25. "Golden Rice”
Creation of a biosynthetic pathway in rice using genes from daffodil and bacteria; portions of genes from pea, rice, and cauliflower mosaic virus

26. Golden Rice Vitamin A deficiency in humans a serious problem
Rice is a major food crop
Rice lacks provitamin A
Therefore, create a de novo pathway for Beta-carotene synthesis
Phytoene synthase from Narcissus pseudonarcissus
Phytoene desaturase from Erwinia uredovora
Endosperm-specific glutelin promoter from Oryza sativa
CaMV promoter from cauliflower mosaic virus
Transit peptide sequence from Pisum sativum

27. Suntory Applause Rose Why create transgenic plants?
Complicated to capitalize on naturally occurring genetic variation.
No naturally occurring genetic variation.
Validating function of a candidate gene (allele).

28. Suntory Applause Rose
What genes to transfer? Multiple genes - Golden Rice and the Applause rose

29. Suntory Applause Rose
Transformation, selection, regeneration, propagation

30. Transgenic plants Frequency Concerns Definitions Regulation
and detection

31. Concerns regarding transgenic plants
Cultural/ religious issues - e.g. animal genes in plants
Dietary concerns - e.g. allergies to novel proteins
Gene flow/escape – e.g. gene flow to weedy or wild relatives
Wasting precious genes one at a time - e.g. widespread use of single Bt genes could provide intense selection pressure for resistant insects, rendering the use of Bt spray ineffective 
Ownership - e.g. transgene technologies, and genes, are generally subject to intellectual property protection

32. What is and what is not a transgenic plant?

33. Regulation and detection of transgenic plants

34. Genome editing Alternatives to transgenics
No naturally occurring genetic variation.
Too complicated to capitalize on naturally occurring genetic variation.
Validating function of a candidate gene (allele).
Induced mutations
Cisgenics
Manipulating native genes through RNAi
Arctic apple, Innate potato
Genome editing

35. Before exploring the alternatives A brief review of mutation
Naturally occurring
Random
Rare
Usually deleterious
The source of genetic variation
Caused by errors in
DNA replication
DNA repair
Naturally occurring color variant in hops.
Shaun Townsend, OSU

36. Mutation The source of polymorphisms Induced
Ionizing radiation (e.g. gamma rays)
Random BUT can target specific types of changes (e.g. deletions)
Rare BUT can manipulate frequency
Usually deleterious
A potential source of genetic variation
Caused by errors in
DNA replication
DNA repair
Gamma ray induced sexual variant in hops.
Shaun Townsend, OSU

37. The source of polymorphisms
Mutation
The source of polymorphisms
Induced
Chemicals (e.g. ethyl methanesulfonate (EMS))
Random BUT can target specific types of changes, e.g. G:C to A:T
Rare BUT can manipulate frequency (e.g. dosage)
Usually deleterious
A potential source of genetic variation
Caused by errors in
DNA replication
DNA repair

38. The source of polymorphisms
Mutation
The source of polymorphisms
Caused by errors in
DNA replication
Caused by errors in
DNA repair in response to
errors during replication
DNA damage by mutagens

39. Induced mutations TILLING

40. Alternatives to transgenics
Cisgenics
“…cisgenic insertion of additional copies of native genes involved in growth regulation may provide tools to help modify plant architecture, expand the genetic variance in plant architecture available to breeders and accelerate transfer of alleles between difficult-to-cross species.”

41. Alternatives to transgenics
Gene silencing - Manipulating native genes through RNAi
The Nature video
Arctic apple, Innate potato

42. Gene silencing - Manipulating native genes through RNAi

43. Gene silencing - Manipulating native genes through RNAi
The how of non-browning apples

44. Alternatives to transgenics
Genome editing
CRISPR-Cas9, and beyond
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)
Cas9 (currently most widely-used enzyme)
The MIT video
The Nature video

45. CRISPR-Cas9 Clustered regularly interspaced palindromic repeats
The tools
Target sequence
Cas enzymes (CRISPR-associated nucleases;
(e.g. Cas9)
gRNA (Guide RNA)

46. CRISPR-Cas9

47. Genome editing – Implications and possibilities
Gene drives – The TED talk

48. Plant genome editing – Implications and possibilities

49. Comparing “- enics” and “edits”
DNA
Transcription
Translation and beyond
Wild type gene 1
ATGCTC
Add a gene 2
ATGCTC
+ transgene
No protein, no phenotype 3
ATGCTC
+ RNAi construct
Degrade wild type mRNA 4
ATCTC
+ CRISPR construct
Change wild type DNA code