Term Paper

On
TRANSGENIC
PLANTS
SUBMITTED TO: SUBMITTED BY:

Mr. HARSH SAHIL MIDDHA

LECTURER in BIOTECHNOLOGY B.TECH BIOTECH

SECTION-B

ROLL NO.-18

REG.NO.-1040070112

L.P.U.
ACKNOWLEDGEMENT

This is to acknowledge all those without whom my project would have

not been completed. I would like to thanks my Biotech lecturer, Mr.
Harsh, as he had been of great help while making this term paper. He
devoted his precious time towards this term paper. He also provided a
great knowledge regarding this, which has been of great importance in its
fulfillment.

Although wide range of information in this term paper has been taken
from the internet, books and magazines are also consulted.
 INTRODUCTION
Conventionally, the genetic variation necessary for crop improvement is generated
through hybridization, mutagenesis and polyploidy. More recently, biotechnological
approaches have become available for creating genetic variation. Culture of plant
cells in vitro generates a considerable amount of genetic variation, called somaclonal
variation, from which several useful variants have been isolated; some of these
variants have been released as commercial varieties. Fusion of protoplasts of selected
plant species constitutes somatic hybridization, which often yields hybrid plants
between sexually incompatible. The somatic hybrids are either symmetric or
asymmetric; both provide an opportunity for gene introgression from a related species
into a crop but so far we have few commercially successful examples. Anther culture
yields haploid plants; chromosome doubling of such plants generates homozygous
lines in a very short time. Several commercial varieties have been developed,
particularly in China, by combining anther culture with the conventional breeding
programmes.

However, the most potent biotechnological approach is the transfer of specifically

constructed gene assemblies through various transformation techniques; this
constitutes genetic engineering. The plants obtained through genetic engineering
contain a gene or usually from an unrelated organism; such genes are called
transgenes, and the plants containing transgenes are known as transgenic plants. The
first transgenic plant was produced in 1983, when a tobacoo line expressing
Kanamycin resistance was produced. Soon transgenic crop varieties resistant to
herbicides, insects or viruses or expressing male sterility, delayed ripening or slow
fruit softening were developed. ‘Flavr Savr’ tomato was the first transgenic variety to
reach the market; fruits of this variety remain fresh for a prolonged period. In 1996,
the area under transgenic varieties was -3 million hectares, when had increased to
over 34 million hectares by 1998 in merely two years.
 Transgenic Plants

Progress is being made on several fronts to introduce new traits into plants using
recombinant DNA technology.

The genetic manipulation of plants has been going on since the dawn of agriculture,
but until recently this has required the slow and tedious process of cross-breeding
varieties. Genetic engineering promises to speed the process and broaden the scope of
what can be done.

Making transgenic plants

There are several methods for introducing genes into plants, including

• infecting plant cells with plasmids as vectors carrying the desired gene
• shooting microscopic pellets containing the gene directly into the cell.

In contrast to animals, there is no real distinction between somatic cells and germline
cells. Somatic tissues of plants, e.g., root cells grown in culture,

• can be transformed in the laboratory with the desired gene

• grown into mature plants with flowers.

If all goes well, the transgene will be incorporated into the pollen and eggs and passed
on to the next generation.

In this respect, it is easier to produce transgenic plants than transgenic animals.

Transgenic Plants can be cultured in the laboratory in nutritive agar,
regenerated from single cells.

Genetic modifications can be made in somatic cells, and the modified

somatic cell can develop into an adult plant that is of single-cell origin.
That's a pretty recent advance in genetics, of course. One of the best
examples of the gradual domestication of plants through genetics is that
of corn.
Introduction of DNA into Plant cell

Well, one problem with working with plants is that they have a cell wall -
how do you get a recombinant plasmid to cross that barrier?

Some strategies are microinjection of single cells, electroporation of cells

grown without a cell wall (protoplasts), biolistics, and Agrobacterium-
mediated transfer.

One very common way of introducing DNA into plant cells is through
DNA coated particles (e.g. gold 1 micron particles) that are literally shot
through the cell wall.

This is sometimes called "biolistics" - a cross between biology and

ballistics. Another term for the device is a "gene gun"
 How does it work? There are a variety of different engineering strategies,
but one way is to accelerate the particles using a pulse of helium.

Bt Crops
Here is an important example of the use of biotechnology in plant crops.

Bacillus thuringiensis has been used for decades as a "natural" insecticide, and is
often used by organic farmers. The bacterium synthesizes a protein (a procrystal),
that when consumed by larvae of certain insects is activated in the gut of the insect,
and becomes a pore in the membrane of the intestinal epithelium. The bacterial
crystals are biodegradable (in sunlight) and are not hazardous to humans. In Bt crops,
the same protein is expressed within the tissues of the plant, so application of the
bacteria or the purified crystal is not necessary.
Constitutive promoters lead to expression of Bt protoxin in all tissues, including root
and pollen. This is both an advantage (in range of effectiveness) and a
disadvantage(in rate of biodegradation)

Risks & Benefits

Ecological concerns include increased risk of invasiveness and volunteerism of the
crop, hybridization (intra and interspecies), effects on non-target organisms and
management of resistance. There are also concerns about product toxicity and
allergenicity.

Intraspecific hybridization

Saving seed from prior years' harvest could help modified strains persist. Wind-
pollination of grains such as corn can also spread traits within a species. Organic
farmers could find that their crops were being pollinated by GM pollen.

Interspecific hybridization

Some crops have weedy close relatives, and transfer of fitness-enhancing

characteristics could be a problem. A weed could become more invasive and
competitive, not only in the agricultural field but within the natural ecosystem. For
example, alfalfa and rice have close relatives in the wild. The domestic and wild
species must share some sexual compatibility and have sufficient chromosome
homology so that a viable hybrid can be formed.

An herbicide-resistant weed would not be predicted to have much of a gain in fitness

in the natural ecosystem. On the other hand, an insect-resistant weed could have a
significant increase in fitness, because there are plenty of insect pests in the wild.

Examples of Transgenic Plants

1.Golden Rice

2.BioPharm
 3.Bt Corn

Bio-pharming

• Bio-pharming is the production of pharmaceutical proteins in genetically

engineered plants.
• Pharmaceuticals can be made in plants at a significantly reduced cost compared to
current production methods.
• Major concerns with bio-pharming are that food or feed crops may become
contaminated with pharmaceutical products, and that the products may have
negative effects on natural ecosystems.
• Bio-pharm crops are regulated by two federal agencies (USDA and FDA) and by
state departments of agriculture.

Manufacturing pharmaceutical products in crops has been one of the promised

benefits of plant genetic engineering for the past 20 years. This use of biotechnology,
sometimes known as “pharming,” “bio-pharming,” or “molecular farming,” has
migrated from speculation to the testing phase in fields and greenhouses across the
country. Bio-pharming promises more plentiful and cheaper supplies of
pharmaceutical drugs, including vaccines for infectious diseases and therapeutic
proteins for treatment of such things as cancer and heart disease. “Plant-made
pharmaceuticals” (PMPs) are produced by genetically engineering plants to produce
specific compounds, generally proteins, which are extracted and purified after harvest.
As used here, the terms bio-pharming and PMP do not include naturally occurring
plant products or nutritionally enhanced foods.

Some Achievements
1. Improved Nutritional Quality

Milled rice is the staple food for a large fraction of the world's human population.
Milling rice removes the husk and any beta-carotene it contained. Beta-carotene is a
precursor to vitamin A, so it is not surprising that vitamin A deficiency is widespread,
especially in the countries of Southeast Asia.

The synthesis of beta-carotene requires a number of enzyme-catalyzed steps. In

January 2000, a group of European researchers reported that they had succeeded in
incorporating three transgenes into rice that enabled the plants to manufacture beta-
carotene in their endosperm.

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. Through recombinant DNA
methods, 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.

Tomato plants infected with

tobacco mosaic virus
(which attacks tomato plants
as well as tobacco). The
plants in the back row carry
an introduced gene
conferring resistance to the
virus. The resistant plants
produced three times as
much fruit as the sensitive
plants (front row) and the
same as control plants.
(Courtesy Monsanto
Company.)
4. Herbicide Resistance.

Questions have been raised about the safety — both to humans and to the
environment — of some of the broad-leaved weed killers like 2,4-D. Alternatives are
available, but they may damage the crop as well as the weeds growing in it. However,
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 (top row) and control
plants (bottom row). "Spray blank" plants were treated with the same spray
mixture as the others except the bromoxynil was left out. (Courtesy of Calgene,
Davis, CA.)

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.

However, researchers at the University of California Davis campus have created

transgenic tomatoes that grew 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. "Terminator" Genes

This term is used (by opponents of the practice) for transgenes introduced into crop
plants to make them produce sterile seeds (and thus force the farmer to buy fresh
seeds for the following season rather than saving seeds from the current crop).

The process involves introducing three transgenes into the plant:

• A gene encoding a toxin which is lethal to developing seeds but not to mature
seeds or the plant. This gene is normally inactive because of a stretch of DNA
inserted between it and its promoter.
• A gene encoding a recombinase — an enzyme that can remove the spacer in
the toxin gene thus allowing to be expressed.
• A repressor gene whose protein product binds to the promoter of the
recombinase thus keeping it inactive.
How they work ?
When the seeds are soaked (before their sale) in a solution of tetracycline

• synthesis of the repressor is blocked

• the recombinase gene becomes active
• the spacer is removed from the toxin gene and it can now be turned on.

Because the toxin does not harm the growing plant — only its developing seeds —
the crop can be grown normally except that its seeds are sterile.

The use of terminator genes has created much controversy:

• Farmers — especially those in developing countries — want to be able to save

some seed from their crop to plant the next season.
• Seed companies want to be able to be able to keep selling seed.

7. Transgenes Encoding Antisense RNA.

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.

Corn is the most popular plant for these purposes, but tobacco, tomatoes, potatoes,
and rice are also being used.

Some of the proteins that are being produced by transgenic crop plants:
 • human growth hormone with the gene inserted into the chloroplast DNA of
tobacco plants.
• humanized antibodies against such infectious agents as
o HIV
o respiratory syncytial virus (RSV)
o sperm (a possible contraceptive)
o herpes simplex virus, HSV, the cause of "cold sores"
• protein antigens to be used in vaccines
o An example: patient-specific antilymphoma (a cancer) vaccines. B-cell
lymphomas are clones of malignant B cells expressing on their surface
a unique antibody molecule. Making tobacco plants transgenic for the
RNA of the variable (unique) regions of this antibody enables them to
produce the corresponding protein. This can then be incorporated into
a vaccine in the hopes (early trials look promising) of boosting the
patient's immune system — especially the cell-mediated branch — to
combat the cancer.
• other useful proteins like lysozyme and trypsin

Controversies:-

The introduction of transgenic plants into agriculture has been vigorously opposed by
some. There are a number of issues that worry the opponents. One of them is the
potential risk of transgenes in commercial crops endangering native or nontarget
species.

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.
• Bibliography-
1. CONCEPTS IN BIOTECHNOLOGY written by B.D.SINGH
2. www.user.rcn.com/jkimball.ma.ultranet/biologypages/t/transgenicplants.html