Applications of Plant Biotechnology in Agriculture

Crop Improvement 1. The following traits are potentially useful to plant genetic engineering: controlling insects, manipulating petal color, production of industrially important compounds, and plant growth in harsh conditions. B. Genetically Engineered Traits: The Big Six. 1. Herbicide Resistance a) Herbicides are a huge industry, with herbicide use quadrupling between 1966 and 1991, so plants that resist chemicals that kill them are a growing need. b) Critics claim that genetically engineered plants will lead to more chemical use, and possible development of weeds resistant to the chemicals.
A.

Applications of Plant Biotechnology

2

c) Glyphosate
i.

Resistance

Marketed under the name Roundup, glyphosate inhibits the enzyme EPSPS (S-enolpyruvlshikimate-3 phosphate involved in chloroplast amino acid synthesis), makes aromatic amino acids. ii. The gene encoding EPSPS has been transferred from glyphosate-resistant E. coli into plants, allowing plants to be resistant. Glyphosate = Roundup, Tumbleweed = Systemic herbicide 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

c)

Glufosinate Resistance
Glufosinate (the active ingredient being phosphinothricin) mimics the structure of the amino acid glutamine, which blocks the enzyme glutamate synthase. ii. Plants receive a gene from the bacterium Streptomyces that produce a protein that inactivates the herbicide.
i.

Herbicide Resistance (Glyphosate Tolerant 3 Crops)

Genetically engineered

Soybean

plants resistant to several herbicides (glyphosate) have been developed and are in commercial cultivation. Example involve Glyphosate Tolerant Soybean, Tobacco. Transgenic soybean has undergone a rapid expansion.

Tobacco

Mode Of Action Of Glyphosate

Glyphosate
4

specifically binds and blocks the activity of EPSPS

(enolpyruvateshikimate-3phosphate synthase).

enolpyruv ateshikima te-3phosphate synthase

EPSPS is an enzyme of

the aromatic amino acid biosynthetic pathway. Glyphosate inhibition of EPSPS prevents the plant from synthesizing
the aromatic amino acids essential for protein synthesis.

Applications of Plant Biotechnology

5

e) Bromoxynil

Resistance i. A gene encoding the enzyme bromoxynil nitrilase (BXN) is transferred from Klebsiella pneumoniae bacteria to plants. ii.Nitrilase inactivates the Bromoxynil before it kills the plant. f) Sulfonylurea. i. Kills plants by blocking an enzyme needed for synthesis of the amino acids valine, leucine, and isoleucine. ii.Resistance generated by mutating a gene in tobacco plants, and transferring the mutated gene into crop plants.

Applications of Plant Biotechnology

2.

Insect Resistance

The Bt toxin isolated from Bacillus thuringiensis has been used in plants. Monsanto Chemical Company 1991Trials BT into cotton plants using A. tumefaciens vector First crops 1996

Corn Cotton Seed potatoes Soybean Others

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

a)

BT in poplar and white spruce catepillar resistance

Plant protease inhibitors have been explored since the 1990s:
i.

ii.
iii. iv.

Naturally produced by plants, are produced in response to wounding. They inhibit insect digestive enzymes after insects ingest them, causing starvation. Tobacco, potato, and peas have been engineered to resist insects such as weevils that damage crops while they are in storage Results have not been as promising as with Bt toxin, because it is believed that insects evolved resistance to protease inhibitors.

Applications of Plant Biotechnology

7 Virus Resistance a) Chemicals are used to control the insect vectors of viruses, but controlling the disease itself is difficult because the disease spreads quickly. b) Plants may be engineered with genes for resistance to viruses, bacteria, and fungi. c) Virus-resistant plants have a viral protein coat gene that is overproduced, preventing the virus from reproducing in the host cell, because the plant shuts off the virus protein coat gene in response to the overproduction. 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
3.

INSECTICIDAL PROPERTIES OF Bt
Bacillus thuringiensis
8

(or Bt) is a Grampositive, soil-dwelling bacterium, which produces crystalline insecticidal proteins. Bt crystal protein gene added/transferred into plant genome. Expression of this gene results insect resistant crop.

Formation of toxin fragments by Cry protein

Mode of action of Bt toxin crystal

Advantages of Bt crops
10

The level of toxin expression is very high thus

delivering sufficient dosage to the pest. The toxin expression is contained within the plant system and hence only those insects that feed on the crop perish. The toxin expression replaces the use of synthetic pesticides in the environment.

Applications of Plant Biotechnology

11
e)

f)

g)

h)

Resistance genes for diseases such as fungal rust disease and tobacco mosaic virus have been isolated from plants and may be transferred to crop plants. Yellow Squash and Zucchini i. Seeds are available that are resistant to watermelon mottle virus, zucchini yellow mosaic virus, and cucumber mosaic virus. Potato. a) Monsanto developed potatoes resistant to potato leaf roll virus and potato virus X, which also contained a Bt toxin gene as a pesticide. b) hain restaurants do not use genetically engineered potatoes due to public pressures. Papaya. a) Varieties resistant to papaya ring spot virus have been developed.

Applications of Plant Biotechnology

12

4.

Altered Oil Content

a) Done

in plants by modifying an enzyme in the fatty acid synthesis pathway (oils are lipids, which fatty acids are a part of). b) Varieties of canola and soybean plants have been genetically engineered to produce oils with better cooking and nutritional properties. c) Genetically engineered plants may also be able to produce oils that are used in detergents, soaps, cosmetics, lubricants, and paints.
5.

Delayed Fruit Ripening

a) Allow

for crops, such as tomatoes, to have a higher shelf life. b) Tomatoes generally ripen and become soft during shipment to a store. c) Tomatoes are usually picked and sprayed with the plant hormone ethylene to induce ripening, although this does not improve taste.

Applications of Plant Biotechnology

13
d)

e)

Tomatoes have been engineered to produce less ethylene so they can develop more taste before ripening, and shipment to markets. What happened to the Flavr Savr tomato? i. Produced by Calgene by blocking the polygalacturonase (PG) gene, which is involved in spoilage. PG is an enzyme that breaks down pectin, which is found in plant cell walls. ii. Plants were transformed with the anti-sense PG gene, which is mRNA that base pair with mRNA that the plant produces, essentially blocking the gene from translation. iii.First genetically modified organism to be approved by the FDA, in 1994. iv.Tomatoes were delicate, did not grow well in Florida, and cost much more than regular tomatoes. v. Calgene was sold to Monsanto after Monsanto filed a patent-infringement lawsuit against Calgene, and the Flavr Savr tomato left the market.

Improved shelf-life (FlavrSavr 14 Tomato)

The FLAVRSAVR

tomato is the first genetically engineered whole food to be sold in commerce.

IMPROVED15 SHELF LIFE

POST TRANSCRIPTIONAL GENE SILENCING

Transgenic tomatoes are produced, which contain antisense construct of the gene encoding PG. Antisense RNA produced from the transgene could pair with the sense RNA transcript. Duplex RNA is target for degradation Base pairing could inhibit translation

The Flavr SavrTM Tomato

(First transgenic 16 Plant Product)

Applications of Plant Biotechnology

17

6.

Pollen Control
a)

b)

c)

Hybrid crops are created by crossing two distantly related varieties of the same crop plant. The method may generate plants with favorable traits, such as tall soybean plants that make more seeds and are resistant to environmental pressures. For success, plant pollination must be controlled. This is usually done by removing the male flower parts by hand before pollen is released. Also, sterilized plants have been genetically engineered with a gene from the bacteria Bacillus amyloliqueifaciens.

Applications of Plant Biotechnology

18

C.

Biotech Revolution: Cold and Drought Tolerance and WeatherGard Genes. 1. Plants such fruits are subject to frost damage at low temperatures, as well as from loss of water. They can be genetically engineered to resist these conditions, and increase crop yields as a result. 2. To resist cold weather, cold-regulated (COR) genes are also called antifreeze genes,, which encode proteins that protect plant cells from frost damage. 3. A transcription factor for a group of COR genes called CBF was patented as WeatherGard in 1997 by a group at Michigan State University. The genes also provide drought tolerance and tolerance to high-salt soils. 4. All major crop species, including corn, soybean, and rice contain CBF genes. 5. Genetically engineering plants with CBF genes survive temperatures as much as 4 to 50C lower than non-engineered plants.

Roundup Sensitive Plants

Shikimic acid + Phosphoenol pyruvate + Glyphosate Plant EPSP synthase

3-Enolpyruvyl shikimic acid-5-phosphate (EPSP)

Without amino acids, plant dies

X
X

Aromatic amino acids

Roundup Resistant Plants

Shikimic acid + Phosphoenol pyruvate + Glyphosate Bacterial EPSP synthase

RoundUp has no effect; enzyme is resistant to herbicide

3-enolpyruvyl shikimic acid-5-phosphate (EPSP)

With amino acids, plant lives

Aromatic amino acids

Applications of Plant Biotechnology

21

D.

Genetically Engineered Foods. 1. More than 60% of processed foods in the United States contain ingredients from genetically engineered organisms. 2. 12 different genetically engineered plants have been approved in the United States, with many variations of each plant, some approved and some not. 3. Soybeans. a) Soybean has been modified to be resistant to broad-spectrum herbicides. b) Scientists in 2003 removed an antigen from soybean called P34 that can cause a severe allergic response. 4. Corn a) Bt insect resistance is the most common use of engineered corn, but herbicide resistance is also a desired trait.

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

Why are transgenics important?

We can develop organisms that express a novel trait not normally found in the species

Extended shelf-life tomato (Flavr-Savr)

Herbicide resistant soybean (Roundup Ready)

Agriculture Transgenics On the Market

Insect resistant cotton Bt toxin kills the cotton boll worm transgene = Bt protein

Source: USDA

Insect resistant corn Bt toxin kills the European corn borer transgene = Bt protein

Normal

Transgenic

Herbicide resistant crops Now: soybean, corn, canola Coming: sugarbeet, lettuce, strawberry alfalfa, potato, wheat (2005?) transgene = modified EPSP synthase or phosphinothricin-N-acetyltransferase

Source: Monsanto

Virus resistance - papya resistant to papaya ringspot virus transgene = virus coat protein

VIRUS RESISTANCE(MALAYSIAN PAPAYA) 26

Occurs in nearly

every region where papaya is grown. In May 1992, PRSV (Papaya ringspot virus-type P) was discovered in Puna, the area where 95% of Hawaiis papaya was being grown.

MAIN FAILURE 27 TO CONTROL

There is no cure for infected

trees. They should be destroyed by cutting them as close to the ground as possible and immediately applying an herbicide such as concentrated Roundup to the cut stumps. Trees infected at a young stage almost never bear fruit, and the fruit of trees that do bear show characteristics ringspots, can be deformed, and generally have lower sugar contents.

Development of Transgenic Papaya

28

POST TRANSCRIPTIONAL GENE SILENCING

Base pairing between the

Vira l RN A

sense RNA transcript of the transgene and the negative strand of the viral RNA Antisense RNA produced from the transgene could pair with the viral RNA transcript.
Duplex RNA is target for

degradation . Base pairing could inhibit translation

Biotech chymosin; the enzyme used to curdle milk products transgene = genetically engineered enzyme
Source: Chr. Hansen

bST; bovin somatotropin; used to increase milk production transgene = genetically engineered enzyme

Source: Rent Mother Nature

Applications of Plant Biotechnology

30

5.

6.

7.

Products include corn oil, corn syrup, corn flour, baking powder, and alcohol. c) By 2002 about 32% of field corn in the United States was engineered. Canola. a) More than 60% of the crop in 2002 was genetically engineered; it is found in many processed foods, and is also a common cooking oil. Cotton. a) More than 71% of the cotton crop in 2002 was engineered. b) Engineered cottonseed oil is found in pastries, snack foods, fried foods, and peanut butter. Other Crops a) Other engineered plants include papaya, rice, tomato, sugar beet, and red heart chicory.
b)

Applications of Plant Biotechnology

31

E.

Nutritionally Enhanced PlantsGolden Rice: An International Effort. 1. More than one third of the worlds population relies on rice as a food staple, so rice is an attractive target for enhancement. 2. Golden Rice was genetically engineered to produce high levels of betacarotene, which is a precursor to vitamin A. Vitamin A is needed for proper eyesight. 3. Golden Rice was developed by Ingo Potrykus and Peter Beyer, and several agencies are attempting to distribute the rice worldwide. 4. Biotechnology company Syngenta, who owns the rights to Golden Rice, is exploring commercial opportunities in the United States and Japan. Monsanto will provide licenses to Golden Rice technology royalty-free. 5. Other enhanced crops include iron-enriched rice and tomatoes with three times the normal amount of beta-carotene

The Golden Rice Story

Vitamin A deficiency is a major health problem

Causes blindness Influences severity of diarrhea, measles

>100 million children suffer from the problem For many countries, the infrastructure doesnt exist to deliver vitamin pills

Improved vitamin A content in widely consumed crops an attractive alternative

-Carotene Pathway Problem in Plants

IPP Geranylgeranyl diphosphate
Phytoene synthase

Phytoene
Problem: Rice lacks these enzymes
Phytoene desaturase

-carotene desaturase

Lycopene
Lycopene-beta-cyclase
Normal Vitamin A Deficient Rice

-carotene (vitamin A precursor)

The Golden Rice Solution

-Carotene Pathway Genes Added IPP Geranylgeranyl diphosphate
Daffodil gene Phytoene synthase

Phytoene

Vitamin A Pathway is complete and functional

Phytoene desaturase Single bacterial gene; performs both functions

-carotene desaturase

Lycopene
Daffodil gene Golden Rice

Lycopene-beta-cyclase

-carotene (vitamin A precursor)

Applications of Plant Biotechnology

35
6.

Cause for Concern? The Case of StarLink Corn. a) StarLink corn had been approved for animal consumption, but in 2000 ended up in Taco Bell taco shells. The shells were immediately recalled. b) Aventis CropScience believed that precautions regarding the corn were in place, but some farmers did not know the corn was not for humans. c) Engineered and non-engineered corn was mixed in mills, contaminating food. d) StarLink contained two new genes: i. Resistance to butterfly and moth caterpillars by a modified Bt toxin gene called Cry9c. ii. Resistance to herbicides such as Basta and Liberty. e) StarLink was approved for animals because the Cry9c protein could be an allergen in humans because it was more stable to heat and in the stomach.

Applications of Plant Biotechnology

36

7.

Currently, no cases of allergic reactions have been reported, and the EPA ruled in 2001 that StarLink was not safe for humans. Cause for Concern? Genetically Engineered Foods and Public Concerns. a) The release of the Flavr Savr tomato generated much discussion over the potential risks of genetically engineered food: i. The primary public fear was that genetically engineering a plant may produce unexpected results, such as allergic reactions or even shock. ii. Genetically engineered food may also raise concerns about the selection of food if, for example, an apple has a gene from an animal. iii. The use of antibiotic resistance markers may possibly inactivate antibiotics, leading to scientists trying to find ways to remove markers from plants.
f)

Applications of Plant Biotechnology

37
iv.

v.

vi.

Another concern is that deleting genes may bring about side effects when ingested, such as secondary metabolites that may protect people from compounds that would normally be broken down by the plant. Uncharacterized DNA included along with the gene of interest may produce unexpected, harmful side effects in the plant. Crops may spread the trait to other plants through pollination, which may damage ecosystems. Male-sterile plants may deal with this problem.

Applications of Plant Biotechnology

38

F.

Molecular Farming 1. A new field where plants and animals are genetically engineered to produce important pharmaceuticals, vaccines, and other valuable compounds. 2. Plants may possibly be used as bioreactors to mass-produce chemicals that can accumulate within the cells until they are harvested. 3. Soybeans have been used to produce monoclonal antibodies with therapeutic value for the treatment of colon cancer. Clot-busting drugs can also be produced in rice, corn, and tobacco plants.

Applications of Plant Biotechnology

39

4.

5.

Plants have been engineered to produce human antibodies against HIV and Epicyte Pharmaceuticals has begun clinical trials with herpes antibodies produced in plants. The reasons that using plants may be more cost-effective than bacteria:
a)

b)
c) d) e)

Scale-up involves just planting seeds. Proteins are produced in high quantity. Foreign proteins will be biologically active. Foreign proteins stored in seeds are very stable. Contaminating pathogens are not likely to be present.

Applications of Plant Biotechnology

40
6.

Edible Vaccines a) People in developing countries have limited access to many vaccines. b) Making plants that produce vaccines may be useful for places where refrigeration is limited. c) Potatoes have been studied using a portion of the E. coli enterotoxin in mice and humans. d) Other candidates for edible vaccines include banana and tomato, and alfalfa, corn, and wheat are possible candidates for use in livestock. e) Edible vaccines may lead to the eradication of diseases such as hepatitis B and polio.

Applications of Plant Biotechnology

41
7.

Biopolymers and Plants a) Plant seeds may be a potential source for plastics that could be produced and easily extracted. b) A type of PHA (polyhydroxylalkanoate) polymer called poly (betahydroxybutyrate), or PHB, is produced in Arabidopsis, or mustard plant. c) PHB can be made in canola seeds by the transfer of three genes from the bacterium Alicaligenes eutrophus, which codes for enzymes in the PHB synthesis pathway. d) Monsanto produces a polymer called PHBV through Alicaligenes fermentation, which is sold under the name Biopol.

RAPID GROWTH & REDUCED REGENERATION TIME (ASPEN TREES)

42

Making paper requires

large amounts of natural cellulose from soft wood trees (Aspen). Genetic engineers have genetically modified Aspen to promote growth, so as to create a fast growing aspen that can supply our paper needs using considerably less land.

ALTERATION IN TREE SIZE OR FORM

43

Altered expression of a gene

involved in hormone synthesis can give wildly differing results

Over expression of GA

(gibberellins) 20-oxidase gives

faster growing trees both in height and diameter and longer wood fibers have greater cellulose content

These trees grow faster and

http://www.enn.com/news/enn-stories/1999/08/080999/trees_4724.asp

REDUCTION IN GENERATION TIME

44

Trees can take years to flower

Those over expressing the

can flower in as little as 7 months.

LEAFY (LFY) gene

This is of particular value in fruit bearing trees Also allows for rapid analysis mature traits