topics in (nano) biotechnology dining with dna lecture 5 31st march, 2004 phd course

TOPICS IN (NANO) BIOTECHNOLOGY

Dining with DNALecture 5

31st March, 2004

PhD Course

GMO Foods - VERY controversial!

What are genetically modified foods?

• Also called genetically modified organisms (GMO).• Involves the insertion of DNA from one organism

into another OR modification of an organism’s DNA in order to achieve a desired trait.

+A

strawberry resistant to

frost

=

4 5

Arctic fish DNA strawberry

A. Totipotency

• Definition– Entire plant can be generated from a single,

non-reproductive cell– Single cells can be separated from leaf, stem or

root tissue using enzymes to digest pectin holding cells together (pectinase)

• Clones from cuttings in tissue culture– Asexual reproduction of plants can occur using

fragments of plants• Shoots or stems or leaves = EXPLANTS

– In tissue culture, cells divide from exposed cell a callus forms

• Callus = undifferentiated cluster of rapidly dividing cells

• Adventitious roots often form from callus

A. Totipotency

• Callus tissue regeneration– Callus tissue will develop if cells are grown with proper

balance of nutrients and plant hormones• Magenta boxes, sterile medium and transfer instruments• Murishigee and Skoog medium (MS medium) – Artificial

medium (agarose, nutrients and hormones)• Under influence of increased cytokinin, shoots will

differentiate• Transferred to increased auxins, roots will establish• Eventually transferred to soil entire plant with

reproductive structures (ovules, pollen)– Calluses can be split into many smaller pieces before

hormones are added to increase # of plants

A. Totipotency

B. DNA inserted into plants Transgenic plant

• Characteristics of transgenic plants– All cells in the plant are derived from one cell– All cells express the desired genetic information

• Why make transgenic plants?– Genes from distantly related plant families can be introduced

without need for breeding (some families of plants are incompatible)

– To improve crop hardiness and characteristics of final plant product

• Protein content• Ripening rate• Drought resistance…..

• Procedures for generating transgenic plants

– Microinjection• DNA constructs injected using fine glass pipettes in

combination with phase contrast microscopy

– Electroporation of protoplasts• Electric pulses of high field strength• Reversibly permeabilize cell membranes

– Electric discharge gun – Gold beads• Firing DNA-coated pellets using a modified .22 caliber gun


– “Whiskers” of silicon carbide – holes punched, DNA introduced

– Agrobacterium tumefaciens

– Viral vectors• Cauliflower mosaic virus vectors• Gemini virus vectors

– Liposome-mediated transformation of protoplasts• Artificial lipid vesicles = Liposomes

– Chemically-stimulated DNA uptake by protoplasts • Polyethylene glycol + CaCl2


– Protoplast fusion can also be used to fuse two different plant types together New Plant Varieties (hybrid plantlet)

• Fused cell acquires some of the characteristic of both genetic backgrounds and can be regenerated into a plant with some traits from both parental plants

– Fusigenic agents (polyethylene glycol) or electroporation used to fuse membranes

• Useful if species are sexually incompatible or cross with difficulty


• US commercially important plants that can be grown from single somatic (non-seed) cells– Asparagus– Cabbage– Citrus fruits– Carrots– Alfalfa– Millet– Tomatoes– Potatoes– Tobacco

• More than 30 different crop plants developed with rDNA techniques are being tested in field studies


C. Agrobacterium tumefaciens• Characteristics

– Plant parasite that causes Crown gall disease– Encodes a large (~250kbp) plasmid called Tumor-

inducing (Ti) plasmid• Portion of the Ti plasmid is transferred between

bacterial cells and plant cells T-DNA (Tumor DNA)

– T-DNA integrates stably into plant genome– T-DNA ss DNA fragment is converted to

dsDNA fragment by plant cell– Then integrated into plant genome

C. Agrobacterium tumefaciens

• How is T-DNA modified to allow genes of interest to be inserted?– In vitro modification of Ti plasmid

• T-DNA tumor causing genes are deleted and replaced with desirable genes (under proper regulatory control)

• Insertion genes are retained (vir genes)• Selectable marker gene added to track plant cells

successfully rendered transgenic [antibiotic resistance gene geneticin (G418) or hygromycin]

• Ti plasmid is reintroduced into A. tumefaciens• A. tumefaciens is co-cultured with plant leaf disks under

hormone conditions favoring callus development (undifferentiated)

• Antibacterial agents (e.g. chloramphenicol) added to kill A. tumefaciens

• G418 or hygromycin added to kill non-transgenic plant cells

• Surviving cells = transgenic plant cells


• Techniques to transform plant cells by A. tumefaciens

– Wounding and direct inoculation– Inoculation of explants in vitro– Transformation of leaf-disks– Co-cultivation of Agrobacterium with protoplasts


II. Examples of Crop Improvement Measures

A. Nitrogen fixation

• To enable plants to fix atmospheric N2 so that it can be converted into NH3, NO3

-, and NO2- providing a

nitrogen source for nucleic acid and amino acid synthesis– Thereby eliminating need to fertilize crops with nitrogen

• Exploit N2 fixation metabolic machinery of bacteria and fungi– Some live freely in soil and water – Some live in symbiosis

• Rhizobium spp. live in symbiosis with leguminous species of plants in root nodules (e.g. soy, peas, beans, alfalfa, clover)

B. Frost Resistance

• Ice-minus bacteria– Ice nucleation on plant surfaces caused by bacteria that aid in protein-

water coalescence forming ice crystals @ 0oC (320F)– Ice-minus Pseudomonas syringae

• Modified by removing genes responsible for crystal formation• Sprayed onto plants

– Displaces wild type strains– Protected to 23oF

» Dew freezes beyond this point– Extends growth season– First deliberate release experiment – Steven Lindow – 1987-

sprayed potatoes• Frost Ban

– Different strain of bacteria – Julie Lindemann led different project – 1987

– Strawberries in California

C. Resistance to biological agents• Anti-Insect Strategy – Insecticides

– From Bacillus thuringensis• Toxic crystals found during sporulation

• Alkaline protein degrades gut wall of lepidopteran larvae

– Corn borer caterpillars– Cotton bollworm caterpillars– Tobacco hornworm caterpillars– Gypsy moth larvae

• Sprayed onto plants – but will wash off

Specific Examples

• Monsanto Chemical Company – 1991 Trials– BT into cotton plants using A.

tumefaciens vector– Cottton bollworms protection in 6

loctions, 5 different states, consistent results– First crops – 1996

» Corn» Cotton» Seed potatoes» Soybean» Others

C. Resistance to biological agents

– Cloned BT toxin gene into a different bacterium that lives harmlessly in corn plants

» Pressure applied to introduce modified bacterium into seeds

» Corn stalks protected from corn borers– BT in poplar and white spruce caterpillar

resistance• BT-resistant strains are beginning to emerge in

some caterpillars


• Anti-Viral Strategy– TMV-coat protein inserted into tobacco and tomato plant cells using Ti

plasmid• Viral capsids inhibit viral replication of TMV when infected

– Grape fan-leaf virus (GFLV)• Causes yellowing and deformation of grape leaves• Transmitted in soil by nematodes• Viral capsid genes introduced into champagne grape vines using T

plasmid• Resistance to virus acquired

– Other trials using capsid proteins: potato leaf-roll virus, cantaloupe mosaic virus, rice strip virus

– Concerns that recombination events may lead to new plant virus strains


• Anti-Bacterial Strategies– Resistance to Xanthomonas oryzae (rice wilting)

• Conferred by cloning resistance genes from wild rice strains

• Anti-Worm Strategies (Animal pest)– Nematode resistance gene from wild beet plants

• To protect sugar beet


Resistance to herbicides• Glyphosate resistance

– Glyphosate = “Roundup”, “Tumbleweed” = Systemic herbicide

– Glyphosate inhibits EPSP synthase (S-enolpyruvlshikimate-3 phosphate – involved in chloroplast amino acid synthesis)

– Escherichia coli EPSP synthase = mutant form less sensitive to glyphosate

• Cloned via Ti plasmid into soybeans, tobacco, petunias– Increased crop yields of crops treated with herbicides

• Bromoxynil– = bromine-based herbicide– Bromoxynil resistant cotton

• Concern over movement of resistance genes into weeds making compounds useless

Resistance to herbicides

Examples

III. Bioengineered foods

A. US-FDA Regulations• 1978 transgenic bacteria produce human insulin

• 1983 First transgenic plant produced (tobacco with kanamycin resistance gene from bacteria)

• May 26, 1992– US-FDA declares no special regulation for genetically modified

food compared to foods generated by conventional means• No special testing• No mandatory labeling

• 1994 FDA approves release of first transgenic crop: Flavr Savr tomatoes that show delayed fruit softening

Dining with DNA

Creating a balance• So are GM foods a good or bad thing?• It depend on each individual case.• Consumers, the government and scientists should be responsible

for weighing the benefits against the costs.

Improved Nutrition

Resistance to disease

Reduced use of chemicals

Environmental risks

Health risks

Economic risks

www.enn.comwww.propanefl.com/ images/corn.jpgwww.columbia.edu/cu/ opg/images/dna.jpgwww.arctictravel.com/ GJOA/haven.htmlwww.foodsubs.com/ Fruitber.htmlwww2.utmb.edu/scccb/mouse/ images/microinjection.jpgss.jircas.affrc.go.jp/engpage/ jarq/32-4/hagio/fig4.htmwww.enn.comTransgenic pollen harms monarch larvae (Nature, Vol 399, No 6733, p 214, May

1999)GM corn poses little threat to monarch (Nature Biotechnology, Vol 17, p 1154, Dec

1999) www.vme.net/dvm/ARNHA/ monarch.htmlhttp://www.csa.com/hottopics/gmfood/overview.htmlwww.greenpeace.orgwww.biotechknowledge.monsanto.comhttp://www.inspection.gc.ca/english/ppc/biotech/labeti/response.shtml

Interesting sites

http://www.enn.com/

http://www.enn.com/

http://www.enn.com/

http://www.csa.com/hottopics/gmfood/overview.html

http://www.greenpeace.org/

http://www.biotechknowledge.monsanto.com/

topics in (nano) biotechnology dining with dna lecture 5 31st march, 2004 phd course

Documents

plants transgenic plant

totipotency slide

organisms dna

families of plants

dna lecture

insertion of dna

fragments of plants

pectinase slide