Download Biotechnology in Agriculture

Biotechnology in Agriculture
Essential Idea: Crops can be modified to increase yields and
to obtain novel products.

Transgenic Organisms
 Transgenic organisms are organisms that contain
genetic material from multiple organisms.
3
Transgenic Organisms
 Transgenic organisms produce
proteins that were not previously
a part of their proteome.
http://blogs.nature.com/spoonful/2011/09/new_model_organism_could_be_th.html
4
Genetic Modification
 Genetic modification is
used for a variety of
reasons.
 To increase profit.
 To overcome environmental
problems such as drought.
 To increase yield.
 To improve shelf life,
appearance, and to help
them travel better.
http://www.agri.gov.il/departments/5.aspx
5
Genetic Modification
 Genetically modified crop plants can be used to
produce novel products such as grape tomatoes,
seedless grapes, oranges, watermelon, etc...
http://depositphotos.com/15365227/stock-photo-collage-made-of-many-images.html
6
Bioinformatics
 Bioinformatics combines the fields of computer
science, statistical analysis, and mathematics with that
of biology and bio-engineering to analyze biodata.
 The field of bioinformatics plays a role in identifying
target genes that can be inserted into different
organisms.
7
Target Genes
 Once the gene of interest has been identified, it’s not
as simple as cutting it out and inserting it into another
organism.
 The target gene is linked to other sequences within the
genome that control its expression.
 Proximal and Distal Control Elements
 Promoters
 Termination Sequence
8
Open Reading Frame
 An open reading frame is a portion of DNA that can
code for protein.
 It is a continuous stretch of DNA that begins with a
start codon and ends with a stop codon.
http://en.wikipedia.org/wiki/Open_reading_frame#/media/File:Sampleorf.png
9
Gene Insertion
 Once the gene of interest has been identified, it needs
to be inserted into the genome of the target organism.
Restriction Enzymes
 Restriction enzymes are
enzymes that cut DNA
molecules at a limited
number of specific locations.
 In nature, these enzymes
help prevent a bacterial cell
from foreign DNA (from
phages and other
organisms).
 Many different restriction
enzymes have been
identified and isolated.
 Click to edit Master text styles
Restriction Enzymes
 Each restriction enzyme is
very specific and recognizes
a short DNA sequence
known as a restriction site.
 The DNA itself is cut at
specific sites within the DNA
strand.
 A bacterial cell will protect
its own DNA from its own
restriction enzymes by
addition of methyl (-CH3)
groups to A’s and C’s within
the sequences recognized
by these enzymes.
 Click to edit Master text styles
Restriction Enzymes
 REs recognize sequences 4-6 nucleotides in length.
 Many such sequences occur by chance throughout
the genome, thus a restriction enzyme will produce a
numerous amount of fragments (called restriction
fragments) when they are introduced to DNA.
Restriction Enzymes
 All copies of a particular DNA molecule always
produce the same DNA fragments when introduced
to the same restriction enzymes.
 Thus, a restriction enzyme cuts DNA in a reproducible
way.
Restriction Enzymes
 The most useful RE’s cleave DNA in a certain way and
produce sticky ends.
 We call them sticky ends because they combine with other
DNA fragments that have been cut by the same enzyme.
 These fragments usually hydrogen bond together and then
are joined permanently by DNA ligase which catalyzes the
formation of covalent bonds in the sugar-phosphate
backbones.
 This produces a stable, recombinant DNA molecule.
Restriction Enzymes
 Movie
16
Marker Genes
 To ensure the organism has taken up the gene of
interest, selectable marker genes are also inserted that
are easily detectable.
 Commonly used genes include antibiotic resistance and
herbicide resistant genes.
 These additional genes allow for the selection of plants
that have taken up the desired gene.
17
Marker Genes
 For instance, in the preparation of Bt corn lines, a gene
called BAR (or PAT) confers resistance to a herbicide
called Liberty.
 Bt corn produces a toxin called normally produced in
the bacterium Bacillus thuringiensis.
 This toxin acts as an insecticide alternative in an
attempt to prevent the corn borer from destroying
crops.
18
 The larvae of the corn
borer feed on the corn
while they are
developing into the
moth.
 Without some sort of
treatment, these insects
can destroy a crop.
http://en.wikipedia.org/wiki/European_corn_borer
 The Bt toxin is specific to
these organisms and isn’t
as broad spectrum as a
pesticide would be.
http://www.organicgardeninfo.com/european-corn-borer.html
19
Gene Insertion
 Generally, bacteria are used to rapidly
multiply the gene of interest prior to
insertion into the crop plant.
 The gene is also inserted with a
herbicide resistant gene and then
grown on selective media to indicate
successful uptake.
 When the plant cells survive on the
selective media, those cells are then
used to regenerate a plant that
contains the gene.
http://plantandsoil.unl.edu/pages/informationmodule.php?idinformationmodule=957885612&topicorder=7&maxto=8&minto=1
21
Marker Genes
 Many different types of genes can be inserted into plants this
way. Common genes include:
 Bt
 Drought resistance
 Round Up Ready plants (glyphosate resistance).
 Overproduction of nutrients (niacin in wheat, vitamin A in rice)
 Etc.
 As long as the copy of the gene gets inserted into the
appropriate spot (plant cell chromosome or chloroplast DNA) the
gene will be expressed resulting in the desired outcome.
22
Recombinant DNA Introduction
 Genes can be introduced into plants using a variety of
methods.
 Chemical methods include:
 Calcium chloride.
 Liposomes are artificially prepared vesicles that can
be made to contain DNA. The liposomes then bind
to the bacterial cell and deliver the contents.
23
Calcium Chloride
 Calcium chloride balances the charges between the
DNA and the cell membrane of the bacterium (both
are negative). This facilitates uptake of DNA from
the surroundings during the heat-shock step.
http://www.biochem.arizona.edu/classes/bioc471/pages/Lecture4/Lecture4.html
24
Calcium Chloride
 It is used in conjunction with heat shock which creates
a temperature difference between the inside and
outside of the cell which acts to sweep the DNA into
the cell through the pores created by the CaCl2.
http://www.biochem.arizona.edu/classes/bioc471/pages/Lecture4/Lecture4.html
25
Liposomes
 Liposomes are artificially
prepared vesicles that
can be made to contain
DNA.
 The liposomes then bind
to the bacterial cell and
deliver the contents
across an otherwise
impermeable membrane.
http://en.wikipedia.org/wiki/Liposome#/media/File:Liposome.jpg
26
Recombinant DNA Introduction
 Genes can be introduced into plants using a variety of
methods.
 Physical methods include:
 Electroporation: electric current
 Microinjection: small glass pipette
 Biolistics (gunshot): a gene gun
27
Electroporation
 Electroporation zaps the cells with an electrical pulse and
makes the membrane more porous--facilitating DNA
uptake.
http://medicalphysicsweb.org/cws/article/research/27152
28
Microinjection
 Microinjection makes
use of a very small
glass micropipette to
inject things (DNA)
into the cell.
http://www.groupflorence.co.uk/ivf/embriyoloji-laboratuvar/mikroenjeksiyon-icsi.html
29
Biolistics
 Biolistics makes use of a gene gun, whereby millions of DNA
coated metal particles are shot at target cells in an attempt
to transform them.
https://physics.ucsd.edu/~groisman/Gene%20guns.html
30
Recombinant DNA Introduction
 Common methods for introduction include using
viruses and bacteria to introduce genes into whole
plants, leaf disks, or protoplasts.
 Viral or bacterial/plasmid uptake would be an
example of using a vector to introduce DNA into
plant cells.
 Ti plasmid of A. tumefaciens and TMV
 Direct introduction of DNA into plant cells would
make use of protoplasts.
31
Recombinant DNA Introduction
 With whole plant introduction, the viral vector
containing the gene of interest, such as TMV, can be
inserted through a wound site in the plant with hopes
that it will take up and express the desired gene.
 A bacterial plasmid can also be used.
 A tumor inducing (Ti) plasmid of Agrobacterium
tumefaciens is a commonly used to introduce such
genes.
32
Agrobacterium tumefaciens
http://www.travismulthaupt.com/page1/styled-10/styled-17/styled-2/MicrobiologyandOrganismsinIndustry.ppt
33
Recombinant DNA Introduction
 Leaf disks from petunias or tobacco plants are
commonly used as well. Often times these leaf disks
are cultured on special media, immersed into a
medium containing the bacteria and plasmid (A.
tumefaceins + Ti), and then transferred to selective
media to obtain the desired cells.
 These cells can then be cultured and induced in a series
of steps to give rise to whole plants.
34
Recombinant DNA: Roundup
 There are a variety of Roundup® Ready plants grown
today: Corn, soybean, cotton.
 To engineer these plants took a lot of science.
 First, a mutant form of the gene that Roundup® targets
had to be found.
 Interestingly, all plants sprayed with Roundup® died, so
there were no natural survivors to cultivate and breed.
35
Recombinant DNA: Roundup
 Eventually a species of bacteria (Agrobacterium) was
found growing in the waste column at a factory that
made Roundup.
 The EPSP synthase enzyme from this bacterium was
almost completely insensitive to glyphosphate.
36
Recombinant DNA: Roundup
 This gene was
modified, cloned
and inserted into a
modified bacterial
plant vector (Ti
plasmid from A.
tumefaceins) for
insertion into the
plant.
37
Recombinant DNA: Roundup
 Once the plant cells took up
the plasmid, they were
placed into a variety of
selective media, root and
shoot inducing media, and
then planted in soil and
grown.
 Seeds from these plants
were then used to create
more plants and so on...
38
Recombinant DNA Introduction
 Another commonly used method of introducing foreign
DNA into a plant cell makes use of protoplasts.
 Protoplasts are cells with a partially or completely
removed cell wall.
 Enzymes are commonly used to degrade the cell wall
making direct DNA uptake possible.
39
Recombinant DNA Introduction
 The newly created cell can then, like the leaf disks, be
run through a series of steps to regenerate a whole
plant.
http://www.plantmethods.com/content/5/1/16/figure/F1
40
Uses of Viruses in Vaccine Production
 Researchers have long sought to use plants to produce
vaccines.
 Plants are preferred over bacteria because they possess
an effective eukaryotic protein synthesis pathway.
 Plants are also intrinsically free of mammalian
pathogens making them ideal for the production of
vaccines.
 Plants appear to hold lots of promise for vaccines.
41
Hepatitis B Vaccine
 Hepatitis B is a viral disease that attacks the liver of the
infected people.
 It is prevalent worldwide, but tends to affect the
poorest countries the most (250-300 million).
 Researchers have long looked for ways to produce the
vaccine as cheaply as possible.
 They have also looked for ways to effectively get this
vaccine to people in poor, remote areas.
42
Hepatitis B Vaccine
 Genetic engineering of the
Tobacco Mosaic Virus have
provided researchers with a way
to get the genes that produce
the Hepatitis B viral antigens
into the tobacco plant.
http://www.apsnet.org/edcenter/intropp/lessons/viruses/Pages/TobaccoMosaic.aspx
 This has provided some promise
in producing Hep-B vaccine in
bulk quantities very cheaply.
43
Plant Based Vaccines
 Advantages of producing vaccines using plants:
 Can grow plants locally reducing transportation
costs.
 Can create oral vaccines saving cost on purification
and administration of the vaccine.
 Little to no refrigeration needed.
 Large amounts of antigen production is possible.
44
Hepatitis B Vaccine
 Problems with having plants produce vaccines:
 Getting the plants to express the antigens in high
concentration has proved difficult.
 Preservation of the plant material may be difficult.
 Processing of the plant material may destroy the
antigen.
 The need to contain the spread of the transgenic
plants may prove difficult.
45
Hepatitis B Vaccine-How it’s Made
 In much the same way
that glyphosate
resistance was built
into soybean plants,
the genes from HBV
were inserted into the
tobacco plant using the
Ti plasmid of A.
tumefaceins.
46
Hepatitis B Vaccine-How it’s Made
 The genetic material that gets
inserted into the plasmid
contains enhancers,
polyadenylation signals, the
gene that codes for the HBV
antigen proteins, multiple
promoters, and a terminator
sequence.
 Collectively these make up
what is known as an
expression cassette.
http://www.nature.com/nbt/journal/v18/n11/fig_tab/nbt1100_1167_F2.html
47
Hepatitis B Vaccine-How it’s Made
 Once the gene(s) have been prepared, they have to be
taken up by the cell of interest.
 There are a variety of ways in which this can be done:
 Calcium chloride
 Liposomes
 Electroporation
 Microinjection
 Biolistics
48
Hepatitis B Vaccine-How it’s Made
 Once the plasmid has
gotten into the cell, it
transfers genetic
material to the plant
chromosome, and the
genes get expressed.
 The antigen proteins
then need to be
purified and packaged
for delivery.
49
The Amflora Potato
https://www.biotechnologie.de/BIO/Navigation/EN/root,did=109208.html
50
The Amflora Potato
 Potatoes naturally produce a
mixture of amylose and
amylopectin.
 The Amflora potato has been
genetically modified to produce
amylopectin only.
 Amylopectin is used in the paper
industry, the textile industry, and
in adhesives and construction
materials.
https://voer.edu.vn/m/organic-compounds-essential-to-human-functioning/ee6fc860
51
The Amflora Potato
 There are 3 types of potatoes grown: seed,
consumption, and starch potatoes.
 Seed potatoes are used for making more potatoes.
 Growing potatoes are for food.
 Starch potatoes are grown for industrial purposes. The
Amflora potato is a starch potato.
52
The Amflora Potato
 Amylopectin can be
separated from amylose
using normal potatoes, but
the procedure is labor
intensive, energy consuming,
and economically
unfavorable.
 Thus, it was useful to develop
a genetically modified potato-the Amflora potato.
http://www.biotechnologie.de/BIO/Navigation/EN/root,did=109208.html
53
The Amflora Potato
 The Amflora was engineered to shut down the pathway
that synthesizes amylose.
 To develop this plant, the same types of methods we’ve
been discussing were used.
 A. tumefaciens was modified to contain the genetic
material that disrupts amylose production, along with
the nptII gene. The nptII gene is an antibiotic resistance
gene that enables researchers to select for the cells
that have taken up the desired gene.
54
The Amflora Potato
 The A. tumefaciens bacterium is
introduced to small pieces of
potato in a petri dish where it
infects them.
 These cells are then grown on
selective media containing
kanamycin. Only those cells which
have taken up the modified DNA
(containing both the amylose
disrupter and the antibiotic
resistance gene-nptII) will grow.
www.plantsci.cam.ac.uk/Haseloff/SITEGRAPHICS/Agrotrans.GIF
55
The Amflora Potato
 A lot of research was done to show that this antibiotic
resistance gene would not cause problems.
 The gene doesn’t readily transfer from the plant to
bacteria.
 The risk of the antibiotic resistance gene getting into
medically relevant bacteria is, at best, very low.