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.

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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

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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

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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

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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.

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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

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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

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Gene Insertion

Once the gene of interest has been identified, it needs to be inserted into the genome of the target organism.

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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.

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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.

Restriction Enzymes

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.

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

Restriction Enzymes

Movie

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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.

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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.

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http://en.wikipedia.org/wiki/European_corn_borer

http://www.organicgardeninfo.com/european-corn-borer.html

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.

The Bt toxin is specific to these organisms and isn’t as broad spectrum as a pesticide would be.

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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.

Gene Insertion

http://plantandsoil.unl.edu/pages/informationmodule.php?idinformationmodule=957885612&topicorder=7&maxto=8&minto=1

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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.

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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.

Recombinant DNA Introduction

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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

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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.

Calcium Chloride

http://www.biochem.arizona.edu/classes/bioc471/pages/Lecture4/Lecture4.html

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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

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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

Recombinant DNA Introduction

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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

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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

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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

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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.

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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.

Recombinant DNA Introduction

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Agrobacterium tumefaciens

http://www.travismulthaupt.com/page1/styled-10/styled-17/styled-2/MicrobiologyandOrganismsinIndustry.ppt

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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.

Recombinant DNA Introduction

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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.

Recombinant DNA: Roundup

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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.

Recombinant DNA: Roundup

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This gene was modified, cloned and inserted into a modified bacterial plant vector (Ti plasmid from A. tumefaceins) for insertion into the plant.

Recombinant DNA: Roundup

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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...

Recombinant DNA: Roundup

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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.

Recombinant DNA Introduction

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The newly created cell can then, like the leaf disks, be run through a series of steps to regenerate a whole plant.

Recombinant DNA Introduction

http://www.plantmethods.com/content/5/1/16/figure/F1

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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.

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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.

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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.

This has provided some promise in producing Hep-B vaccine in bulk quantities very cheaply.

Hepatitis B Vaccine

http://www.apsnet.org/edcenter/intropp/lessons/viruses/Pages/TobaccoMosaic.aspx

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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.

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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.

Hepatitis B Vaccine

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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.

Hepatitis B Vaccine-How it’s Made

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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.

Hepatitis B Vaccine-How it’s Made

http://www.nature.com/nbt/journal/v18/n11/fig_tab/nbt1100_1167_F2.html

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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

Hepatitis B Vaccine-How it’s Made

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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.

Hepatitis B Vaccine-How it’s Made

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The Amflora Potato

https://www.biotechnologie.de/BIO/Navigation/EN/root,did=109208.html

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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

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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.

The Amflora Potato

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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

The Amflora Potato

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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.

The Amflora Potato

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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.

The Amflora Potato

www.plantsci.cam.ac.uk/Haseloff/SITEGRAPHICS/Agrotrans.GIF

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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.

The Amflora Potato