Transgenic Plants

Prepare tissue for transformation

Introduce DNA

Culture plant tissue• Develop shoots• Develop Roots

Field test the plants

• Leaf, germinating seed, immature embryos

• Tissue must be capable of developing into normal plants

• Locate genes for plant traits

• Introduction into cell

• Multiple sites, multiple years

How To Make A Transgenic Plant?

Introducing the Gene orDeveloping Transgenics

Steps

1. Create transformation cassette

2. Introduce and select for transformants

Locating Genes for Plant Traits

Identifying and locating genes for agriculturally important traits is the most limiting step.

Little is known about the specific genes required to enhance yield potential, improve stress tolerance, modify chemical properties of the harvested product, or otherwise affect plant characters. For a gene to be used as transgene, following must be known

How the gene is regulation in the cell

What other effects it might have on the plant, and

How it interacts with other genes active in the same biochemical pathway.

Designing Genes for Insertion

• Once a gene has been isolated and cloned (amplified in a bacterial vector), it must undergo several modifications before it can be effectively inserted into a plant.

Simplified representation of a constructed transgene, containing necessary components for successful integration and expression.

Contains

1. Gene of interest

The coding region and its controlling elements

2. Selectable marker

Distinguishes transformed/untransformed plants

3. Insertion sequences

Aids Agrobacterium insertion

Transformation Cassettes

• Two major delivery methods

• Agrobacterium

• Gene Gun

Tissue culturerequired to generatetransgenic plants

Delivering the Gene to the Plant

Transforming Plants

• Agrobacterium method

– Use bacterial strain Agrobacterium tumificiens (soil-dwelling bacteria that has the ability to infect plant cells with a piece of its DNA)

– Successful transformation in tomato, cotton, tobacco

– Preferable to the gene gun, because of the greater frequency of single-site insertions of the foreign DNA, making it easier to monitor.

• Gene Gun method

(microprojectile bombardment or biolistics).

– Useful in transforming monocot species like corn and rice.

Agrobacterium mediated infection

• Cell contains chromosome & a Ti (tumor-inducing) plasmid.

• The Ti plasmid contains

– a stretch of DNA termed T-DNA (~20 kb long) that is transferred to the plant cell in the infection process.

– a series of vir (virulence) genes that direct the infection process.

– A tumefaciens can only infect a plant through wounds. In response to wound signals, the vir genes of become activated and direct the transfer of the T-DNA from the Ti plasmid to the plant's chromosome.

– Transgene is inserted between the T-DNA border regions

Agroinoculation

Selection and Regeneration

• Selection of successfully transformed tissues.– Following the gene insertion process, plant tissues are

transferred to a selective medium containing an antibiotic or herbicide, depending on which selectable marker was used.

– Only plants expressing the selectable marker gene will survive, and it is assumed that these plants will also possess the transgene of interest.

When grown on selective media, only plant tissues that have successfully integrated the transgene construct will survive.

Regeneration of whole plants

• To obtain whole plants from transgenic tissues such as immature embryos, they are grown under controlled environmental conditions in a series of media containing nutrients and hormones.

• Once whole plants are generated and produce seed, evaluation of the progeny begins.

• This regeneration step has been a stumbling block in producing transgenic plants in many species, but specific varieties of most crops can now be transformed and regenerated.

The Next Test Is The Field

Non-transgenics

Transgenics

Herbicide Resistance

Some Achievements

1. Improved Nutritional Quality

– Rice with beta-carotene, a precursor to vitamin A

2. Insect Resistance

• Bacillus thuringiensis is a bacterium that is pathogenic for a number of insect pests.

• Its lethal effect is mediated by a protein toxin it produces.

• The toxin gene can be introduced directly into the genome of the plant where it is expressed and provides protection against insect pests of the plant.

3. Disease Resistance

• Genes that provide resistance against plant viruses have been successfully introduced into such crop plants as tobacco, tomatoes, and potatoes.

Next Generation of Ag Biotech Products

Golden Rice – increased Vitamin A content (but not without controversy)transgene = three pathway enzymes

Sunflower – white mold resistancetransgene = oxalate oxidase from wheat

Insect Resistance

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

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

Normal Transgenic

Disease Resistance

4. Herbicide Resistance. Genes for resistance to some of the newer herbicides have

been introduced into some crop plants and enable them to thrive even when exposed to the weed killer.

Effect of the herbicide bromoxynil on tobacco plants transformed with

a bacterial gene whose product breaks down bromoxynil.

Some Achievements Cont…

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

Herbicide resistant crops Now: soybean, corn, canola Coming: sugarbeet, lettuce, strawberry alfalfa, potato, wheat• transgene = modified phosphinothricin-N-

acetyltransferase

5. Salt Tolerance

• A large fraction of the world's irrigated crop land is so laden with salt that it cannot be used to grow most important crops.

• Transgenic tomatoes have been developed that grow well in saline soils. The transgene was a highly-expressed sodium/proton antiport pump that sequestered excess sodium in the vacuole of leaf cells.

There was no sodium buildup in the fruit.

6. Transgenes Encoding Antisense RNA (e.g. the Flavr Savr tomato) – Transgenic transcribes into an antisense RNA

complementary to the mRNA for an enzyme involved in ethylene production.

– These tomatoes make only 10% of the normal amount of the enzyme.

– The goal of this work was to provide supermarket tomatoes with something closer to the appearance and taste of tomatoes harvested when ripe.

Some Achievements Cont…

7. "Terminator" Genes

• This term is used (by opponents of the practice) for transgenes introduced into crop plants to make them produce sterile seeds.

8. Biopharmaceuticals • The genes for proteins to be used in human (and

animal) medicine can be inserted into plants and expressed by them.

• Advantages: – Glycoproteins can be made (bacteria like E. coli cannot

do this). – Virtually unlimited amounts can be grown in the field

rather than in expensive fermentation tanks. – There is no danger from using mammalian cells and

tissue culture medium that might be contaminated with infectious agents.

– Purification is often easier.

Some Achievements Cont…

• Some of the proteins that are being produced by transgenic crop plants: – human growth hormone– humanized antibodies against such infectious agents as

• HIV • respiratory syncytial virus (RSV) • herpes simplex virus, HSV

9. Edible Vaccines• 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

Some Achievements Cont…

Controversies• The introduction of transgenic plants into agriculture has been

vigorously opposed by some. There are a number of issues that worry the opponents.

• Examples:

– A gene for herbicide resistance in, e.g. corn, escaping into a weed species could make control of the weed far more difficult.

– The gene for Bt toxin expressed in pollen might endanger pollinators like honeybees.

– To date, field studies on Bt cotton and maize (corn) show that the numbers of some non-target insects are reduced somewhat but not as much as in fields treated with insecticides.

– Another worry is the inadvertent mixing of transgenic crops with non-transgenic food crops. Although this has occurred periodically, there is absolutely no evidence of a threat to human health.

• Despite the controversies, farmers are embracing transgenic crops.

Future Developments in Transgenic Technology

More efficient transformation, that is, a higher

percentage of plant cells will successfully incorporate the

transgene.

Better marker genes to replace the use of antibiotic

resistance genes.

Better control of gene expression through more specific

promoters, so that the inserted gene will be active only

when and where needed.

Transfer of multi-gene DNA fragments to modify more

complex traits.