1. An Ecological Perspective (BIOL 346) Talk Seven: GMOs and the Environment

2. Why GMOs? “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. Indeed why? Hunger, starvation, and malnutrition are endemic in many parts of the world today. Rapid increases in the world population have intensified these problems! ALL of the food we eat comes either directly or indirectly from plants. Can’t just grow more plants, land for cultivation has geographic limits –Also, can destroy ecosystems!

4. Figure 9.1 Indeed why? The Earth is currently experiencing the most population increase in Human history. 2.5 billion in 1955 to 7 billion in 2012 At current rate, will double within 30 years! Fastest growing nations have growth rates at or above 4% - this will double the countries population every 17 years

5. Figure 11.13 Increasing crop yields 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

6. Figure 11.13 Increasing crop yields 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.

7. Figure 11.17 Chemical pest control 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

8. Figure 11.17 Chemical pest control 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

9. Figure 11.18 Chemical pest control 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.

10. 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

11. 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

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

13. 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.

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

15. 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

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

17. 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.

18. 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.

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

20. Meristems Injections 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! 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.

21. Particle Bombardment

22. Particle-Gun Bombardment 1.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 (CaCl 2 ). Particle Bombardment

23. 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. Particle Bombardment

24. 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 Particle Bombardment

25. Agrobacterium tumefaciens

26. Genetically modified crops 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 - maize As Agrobacterium tumefaciens do not naturally infect monocots

27. 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. Ti plasmids and the bacterial chromosome act in concert to transform the plant

28. 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. Ti plasmids and the bacterial chromosome act in concert to transform the plant

29. 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!!!

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

31. 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. The process

32. 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. The Process

33. 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. The Process

34. Overview of the Infection Process

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

36. 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

37. 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

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

39. 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-)

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

41. Organogenesis 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.

42. 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

43. Genetically modified crops Issues: Destroying ecosystems – tomatoes are now growing in the artic tundra with fish antifreeze in them! Destroying ecosystems – will the toxin now being produced by pest-resistance stains kill “friendly” insects such as butterflies. Altering nature – should we be swapping genes between species?

44. Genetically modified crops Issues: Vegetarians – what about those tomatoes? Religious dietary laws – anything from a pig? Cross-pollination – producing a super- weed

45. The End! Any Questions?