Plant Biotechnology and GMOs

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Presentation transcript:

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

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.”

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

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

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

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

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

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

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

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 http://www.oercommons.org/courses/detecting-genetically-modified-food-by-pcr/

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.”

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

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

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

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

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.

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)

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.

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

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

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

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.

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.

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

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!

Particle Bombardment 26

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

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

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

Agrobacterium tumefaciens

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!!!

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

Overview of the Infection Process

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

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

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

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

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

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

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

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

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

Agrobacterium can be used to transfer DNA into plants 43

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

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

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

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

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

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

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

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

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

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

Overview of the Infection Process

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

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

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

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

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-)

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

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.

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.

The End! Any Questions?