1. Plant Biotechnology and GMOs
Chapter 14
(Plus other bits and bobs)

2. Plant Biotechnology
“For centuries, humankind has made improvements to crop plants through selective breeding and hybridization — the controlled pollination of plants.
Plant biotechnology is an extension of this traditional plant breeding with one very important difference —
plant biotechnology allows for the transfer of a greater variety of genetic information in a more precise, controlled manner.”

3. Increasing crop yields
Figure 11.13
To feed the increasing population we have to increase crop yields.
Fertilizers - are compounds to promote growth; usually applied either via the soil, for uptake by plant roots, or by uptake through leaves. Can be organic or inorganic
Have caused many problems!!
Algal blooms pollute lakes near areas of agriculture 3

4. Increasing crop yields
Figure 11.13
Algal blooms - a relatively rapid increase in the population of (usually) phytoplankton algae in an aquatic system.
Causes the death of fish and disruption to the whole ecosystem of the lake.
International regulations has led to a reduction in the occurrences of these blooms. 4

5. Chemical pest control Figure 11.17
Each year, 30% of crops are lost to insects and other crop pests.
The insects leave larva, which damage the plants further.
Fungi damage or kill a further 25% of crop plants each year.
Any substance that kills organisms that we consider undesirable are known as a pesticide.
An ideal pesticide would:-
Kill only the target species
Have no effect on the non-target species
Avoid the development of resistance
Breakdown to harmless compounds after a short time 5

6. Chemical pest control Figure 11.17
DDT was first developed in the 1930s
Very expensive, toxic to both harmful and beneficial species alike.
Over 400 insect species are now DDT resistant.
As with fertilizers, there are run-off problems.
Affects the food pyramid.
Persist in the environment 6

7. Chemical pest control Figure 11.18 DDT persists in the food chain.
It concentrates in fish and fish-eating birds.
Interfere with calcium metabolism, causing a thinning in the eggs laid by the birds – break before incubation is finished – decrease in population.
Although DDT is now banned, it is still used in some parts of the world. 7

8. Plant Biotechnology
The use of living cells to make products such as pharmaceuticals, foods, and beverages
The use of organisms such as bacteria to protect the environment
The use of DNA science for the production of products, diagnostics, and research

9. Genetically modified crops
All plant characteristics, such as size, texture, and sweetness, are determined on the genetic level.
Also:
The hardiness of crop plants.
Their drought resistance.
Rate of growth under different soil conditions.
Dependence on fertilizers.
Resistance to various pests and diseases.
Used to do this by selective breeding 9 9

10. Why would we want to modify an organism?
Better crop yield, especially under harsh conditions.
Herbicide or disease resistance
Nutrition or pharmaceuticals, vaccine delivery
“In 2010, approximately 89% of soy and 69% of corn grown in the U.S. were grown from Roundup Ready® seed.”
OER open education resources

11. Roundup Ready Gene
The glyphosate resistance gene protects food plants against the broad-spectrum herbicide Glyphosate - N-(phosphonomethyl) glycine [Roundup®], which efficiently kills invasive weeds in the field.  
The major advantages of the "Roundup Ready®” system include better weed control, reduction of crop injury, higher yield, and lower environmental impact than traditional weed control systems.
Notably, fields treated with Roundup® require less tilling; this preserves soil fertility by lessening soil run-off and oxidation.”

12. Glyphosate - N-(phosphonomethyl) glycine
An aminophosphonic analogue of the natural amino acid glycine.
It is absorbed through foliage and translocated to actively growing points. (Meristems!!!)
Mode of action is to inhibit an enzyme involved in the synthesis of the aromatic amino acids: 
tyrosine, 
tryptophan 
phenylalanine
Glyphosate
Glycine

13. Glyphosate - N-(phosphonomethyl) glycine
It does this by inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which catalyzes the reaction of shikimate-3-phosphate (S3P) and phosphoenol pyruvate to form 5-enolpyruvyl-shikimate-3-phosphate (ESP).
ESP subsequently dephosphorylated to chorismate, an essential precursor in plants for these  aromatic amino acids.
Glyphosate
Glycine

14. Roundup Ready Gene
Glyphosate functions by occupying the binding site of the phosphoenol pyruvate, mimicking an intermediate state of the enzyme substrates complex.
The "Roundup Ready®” system introduces a stable gene alteration which prevents Glyphosate binding and allowing the formation of the essential aromatic amino acids

15. Roundup Ready Gene
The shikimate pathway is not present in animals, which instead obtain aromatic amino acids from their diet.
Glyphosate has also been shown to inhibit other plant enzymes
Also has been found to affect animal enzymes.
The United States Environmental Protection Agency‎ considers glyphosate to be relatively low in toxicity, and without carcinogenic or teratogenic effects
However, some farm workers have reported chemical burns and contact skin burns

16. Environmental degradation
When glyphosate comes into contact with the soil, it can be rapidly bound to soil particles and be inactivated.
 Unbound glyphosate can be degraded by bacteria.
However, glyphosate has been shown to increase the infection rate of wheat by fusarium head blight in fields that have been treated with glyphosate.
In soils, half-lives vary from as little as 3 days at a site in Texas to 141 days at a site in Iowa.
In addition, the glyphosate metabolite amino methyl phosphonic acid has been shown to persist up to 2 years in Swedish forest soils.
Glyphosate absorption varies depending on the kind of soil.

17. Insect Resistance
B. thuringiensis (commonly known as 'Bt') is an insecticidal bacterium, marketed worldwide for control of many important plant pests - mainly caterpillars of the Lepidoptera (butterflies and moths) but also mosquito larvae, and simuliid blackflies that vector river blindness in Africa.
Bt products represent about 1% of the total ‘agrochemical’ market (fungicides, herbicides and insecticides)

18. Genetically modified crops
1992- The first commercially grown genetically modified food crop was a tomato - was made more resistant to rotting, by adding an anti-sense gene which interfered with the production of the enzyme polygalacturonase.
The enzyme polygalacturonase breaks down part of the plant cell wall, which is what happens when fruit begins to rot.

19. Genetically modified crops
Need to build in a:
Promoter
Stop signal
CODING SEQUENCE
INTRON
poly A signal
PROMOTER
ON/OFF Switch
Makes Protein
stop sign

20. Genetically modified crops
So to modify a plant:
Need to know the DNA sequence of the gene of interest
Need to put an easily identifiable maker gene near or next to the gene of interest
Have to insert both of these into the plant nuclear genome
Good screen process to find successful insertion

21. Building the Transgenes
ON/OFF Switch
Makes Protein
stop sign
CODING SEQUENCE
INTRON
poly A signal
PROMOTER
Plant Selectable
Marker Gene
Plasmid DNA
Construct
Plant Transgene
bacterial genes
antibiotic marker
replication origin

22. Cloning into a Plasmid
The plasmid carrying genes for antibiotic resistance, and a DNA strand, which contains the gene of interest, are both cut with the same restriction endonuclease.
The plasmid is opened up and the gene is freed from its parent DNA strand. They have complementary "sticky ends." The opened plasmid and the freed gene are mixed with DNA ligase, which reforms the two pieces as recombinant DNA.

23. Cloning into a Plasmid
Plasmids + copies of the DNA fragment produce quantities of recombinant DNA.
This recombinant DNA stew is allowed to transform a bacterial culture, which is then exposed to antibiotics.
All the cells except those which have been encoded by the plasmid DNA recombinant are killed, leaving a cell culture containing the desired recombinant DNA.

24. So, how do you get the DNA into the Plant?

25. Meristems Injections REMEMBER!!!!!!!
Tunica-Corpus model of the apical
meristem (growing tip). The epidermal
(L1) and subepidermal (L2) layers form
the outer layers called the tunica.
The inner L3 layer is called the corpus.
Cells in the L1 and L2 layers divide in
a sideways fashion which keeps these
layers distinct, while the L3 layer divides in a more random fashion.
REMEMBER!!!!!!!
The tissue in most plants consisting of undifferentiated cells (meristematic cells), found in zones of the plant where growth can take place.
Meristematic cells are analogous in function to stem cells in animals, are incompletely or not differentiated, and are capable of continued cellular division.
First method of DNA transfer to a plant.
Inject DNA into the tip containing the most undifferentiated cells – more chance of DNA being incorporated in plant Genome
Worked about 1 in 10,000 times!

26. Particle Bombardment 26

27. Particle Bombardment
Particle-Gun Bombardment
DNA- or RNA-coated gold/tungsten particles are loaded into the gun and you pull the trigger.
Selected DNA sticks to surface of metal pellets in a salt solution (CaCl2). 27

28. Particle Bombardment
2. A low pressure helium pulse delivers the coated gold/tungsten particles into virtually any target cell or tissue.
3. The particles carry the DNA  cells do not have to be removed from tissue in order to transform the cells
4. As the cells repair their injuries, they integrate their DNA into their genome, thus allowing for the host cell to transcribe and translate the transgene. 28

29. Particle Bombardment
The DNA sometimes was incorporated into the nuclear genome of the plant
Gene has to be incorporated into cell’s DNA where it will be transcribed
Also inserted gene must not break up some other necessary gene sequence 29

30. Agrobacterium tumefaciens

31. Overall process
Uses the natural infection mechanism of a plant pathogen
Agrobacterium tumefaciens naturally infects the wound sites in dicotyledonous plant causing the formation of the crown gall tumors.
Capable to transfer a particular DNA segment (T-DNA) of the tumor-inducing (Ti) plasmid into the nucleus of infected cells where it is integrated fully into the host genome and transcribed, causing the crown gall disease.
So the pathogen inserts the new DNA with great success!!!

32. Overall process
The vir region on the plasmid inserts DNA between the T-region into plant nuclear genome
Insert gene of interest and marker in the T-region by restriction enzymes – the pathogen will then “infect” the plant material
Works fantastically well with all dicot plant species
tomatoes, potatoes, cucumbers, etc
Does not work as well with monocot plant species - corn
As Agrobacterium tumefaciens do not naturally infect monocots

33. Overview of the Infection Process

34. Ti plasmids and the bacterial chromosome act in concert to transform the plant
1. Agrobacterium tumefaciens chromosomal genes: chvA, chvB, pscA required for initial binding of the bacterium to the plant cell and code for polysaccharide on bacterial cell surface.
2. Virulence region (vir) carried on pTi, but not in the transferred region (T-DNA). Genes code for proteins that prepare the T-DNA and the bacterium for transfer. 34

35. 3. T-DNA encodes genes for opine synthesis and for tumor production.
4. occ (opine catabolism) genes carried on the pTi and allows the bacterium to utilize opines as nutrient. 35

36. for transfer to the plant
Agrobacterium chromosomal DNA
pscA
chvA
chvB
T-DNA-inserts into plant genome
tra
bacterial conjugation
for transfer to the plant
pTi
vir genes
opine catabolism
oriV 36

37. Agrobacterium tumafaciens senses Acetosyringone via a 3-component-like system
3 components:
ChvE,
VirA,
VirG

38. 1. ChvE
periplasmic protein binds to sugars, arabinose, glucose
binds to VirA periplasmic domain
 amplifies the signal
Periplasmic domain
acetosyringone
ChvE
VirA
VirG
sugars
Transmitter
Inhibitory domain
receiver
DNA-binding

39. 2. VirA : Receptor kinase VirA ChvE VirG receiver sugars
Membrane protein five functional domains:
a) Periplasmic binds ChvE-sugar complex does NOT bind acetosyringone
b) Transmembrane domain
c) Linker region BINDS acetosyringone NOTE this is on the cytoplasmic side!
Periplasmic domain
acetosyringone
ChvE
VirA
VirG
sugars
Transmitter
Inhibitory domain
receiver
DNA-binding

40. 2. VirA : Receptor kinase VirA ChvE VirG receiver sugars
d) Transmitter domain (His) auto- phosphorylates and then transfers to the response regulator protein VirG
e) Inhibitory domain  will bleed off the phosphate from the His in the transmitter domain (to an Asp)
Periplasmic domain
acetosyringone
ChvE
VirA
VirG
sugars
Transmitter
Inhibitory domain
receiver
DNA-binding

41. 3. VirG : Response Regulator
Receiver domain that is phosphorylated on an Asp residue by the His on the transmitter domain of VirA
b) Activates the DNA binding domain to promote transcription from Vir-box continaing promoter sequences (on the Ti plasmid)
Periplasmic domain
acetosyringone
ChvE
VirA
VirG
sugars
Transmitter
Inhibitory domain
receiver
DNA-binding

42. VirA ChvE VirG receiver sugars Periplasmic domain acetosyringone
Transmitter
Inhibitory domain
receiver
DNA-binding

43. Agrobacterium can be used to transfer DNA into plants43

44. pTi-based vectors for plant transformation:
1. Shuttle vector is a small E. coli plasmid using for cloning the foreign gene and transferring to Agrobacterium.
2. Early shuttle vectors integrated into the T-DNA; still produced tumors.
pTi
Shuttle plasmid
conjugation
E. coli
Agrobacterium 44

45. MiniTi T-DNA based vector for plants
Disarmed vectors: do not produce tumors; can be used to regenerate normal plants containing the foreign gene.
1. Binary vector: the vir genes required for mobilization and transfer to the plant reside on a modified pTi.
2. consists of the right and left border sequences, a selectable marker (kanomycin resistance) and a polylinker for insertion of a foreign gene.
miniTi 45

46. MiniTi T-DNA based vector for plants
a binary vector system
T-DNA deleted
kanr
polylinker LB RB
modified Ti plasmid 1
bom 1
vir
ori
miniTi 2 2
oriV
bom = basis of mobilization 46

47. Transfer of miniTi from E. coli to Agrobacterium tumefaciens
kan resistance
pRK2013;
kan resistance
modified pTi
tra
bom
E. coli
contains tra genes
Agrobacteriumstr resistant
bom site for mobilization
ColE1 ori
Ti oriV
15A ori;
E. coli or Agrobact.
Triparental mating: 47

48. Steps in the mating 1-2: pRK2013; kan resistance miniTi;
E. coli 1 1
tra
bom
tra
contains tra genes 2 2
ColE1 ori
Helper plasmid (pRK2013) mobilizes itself into 2nd E. coli strain containing miniTi.
Triparental mating: 48

49. Helper plasmid mobilizes itself and the miniTi into Agrobacterium.
Steps in the mating 2-3:
E. coli
Agrobacterium
pRK2013
pRK2013
pTi
miniTi
miniTi 2 2
miniTi;
kan resistance 3 3
pRK2013 can not replicate.
Helper plasmid mobilizes itself and the miniTi into Agrobacterium. 49

50. Selection of Agrobacterium containing the miniTi on str r/kan plates
kan resistance
pRK2013;
kan resistance
modified pTi
tra
bom
str r
can not replicate
miniTi
pTi
str r
pRK2013
kanr
Agrobacterium
str resistant
plate on str and kan media 50

51. Summary
Agrobacteria are biological vectors for introduction of genes into plants.
Agrobacteria attach to plant cell surfaces at wound sites.
The plant releases wound signal compounds, such as acetosyringone.
The signal binds to virA on the Agrobacterium membrane.
VirA with signal bound activates virG. 51

52. Summary
Activated virG turns on other vir genes, including vir D and E.
vir D cuts at a specific site in the Ti plasmid (tumor-inducing), the left border. The left border and a similar sequence, the right border, delineate the T-DNA, the DNA that will be transferred from the bacterium to the plant cell
Single stranded T-DNA is bound by vir E product as the DNA unwinds from the vir D cut site. Binding and unwinding stop at the right border. 52

53. Summary
The T-DNA is transferred to the plant cell, where it integrates in nuclear DNA.
T-DNA codes for proteins that produce hormones and opines. Hormones encourage growth of the transformed plant tissue. Opines feed bacteria a carbon and nitrogen source. 53

54. Overview of the Infection Process

55. And then?....... Tissue culture
What is the last step?
Tissue culture
The basics!

56. What is Plant Tissue Culture?
Of all the terms which have been applied to the process,
"micropropagation" is the term which best conveys the
message of the tissue culture technique most widely in use today.
The prefix "micro" generally refers to the small size of the
tissue taken for propagation, but could equally refer to the
size of the plants which are produced as a result.
Relies on two plant hormones
Auxin
Cytokinin

57. Protoplast to Plant Callus: Induced by
2, 4 dichlorphenoxyacetic acid (2,4D)
Unorganized, growing mass of cells
Dedifferentiation of explant
Loosely arranged thinned walled, outgrowths
No predictable site of organization or differentiation

58. Protoplast to Plant 2, 4 dichlorphenoxyacetic acid (2,4D)
Stops synthesis of cellulose
Knocks out every other rosette
Makes b 1,3 linked glucose
Callose
Temporarily alters the cell wall

59. Auxin (indoleacetic acid)
Produced in apical and root meristems, young leaves, seeds in developing fruits
cell elongation and expansion
suppression of lateral bud growth
initiation of adventitious roots
stimulation of abscission (young fruits) or delay of abscission
hormone implicated in tropisms (photo-, gravi-, thigmo-)

60. Cytokinin (zeatin, ZR, IPA)
Produced in root meristems, young leaves, fruits, seeds
cell division factor
stimulates adventitious bud formation
delays senescence
promotes some stages of root development

61. Organogenesis Rule of thumb: Auxin/cytokinin 10:1-100:1 induces roots.
The formation of organs from a callus
Rule of thumb: Auxin/cytokinin 10:1-100:1 induces roots.
1:10-1:100 induces shoots
Intermediate ratios around 1:1 favor callus growth.

62. Transgenic Plants Serving Human Health Needs
Edible Vaccines
Transgenic Plants Serving Human Health Needs
Works like any vaccine
A transgenic plant with a pathogen protein gene is developed
Potato, banana, and tomato are targets
Humans eat the plant
The body produces antibodies against pathogen protein
Humans are “immunized” against the pathogen
Examples:
Diarrhea
Hepatitis B
Measles
Edible vaccines may be the most important and accepted biotech product. The principles are the same as those used for normal vaccines: a protein enters the body in some manner, and the human immune system produces antibodies against that protein. When the human is then exposed to the pathogen, the immune system is turned on and destroys the pathogen.

63. The End!
Any Questions?