Chapter One

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Introduction
 Chapter One: Introduction

Introduction

Man first learned about the domestication of plant about 9000 B.C. Since then people
began to alter the habitats of different crop species. For thousands of years farmers
and plant breeders have been changing crop plants to improve characteristics such as
size, resistance to disease and taste. Plants which grow well, have a higher yield or
taste better are selected and bred from. Some of the plant improvement techniques
which are widely used by the scientist include in vitro pollination, somaclonal
variation, somatic hybridization, anther culture, embryo culture etc.

Prior 1950s before the discovery of DNA double helix by Watson and Crick,
conventional plant modification technique that is plant breeding was not subject to a
great deal of regulation. Seed certification standards ensure the purity and quality of
seeds, but little attention has been paid to the possible food safety or environmental
impacts of new plant varieties derived from conventional breeding. Several problems
of conventional breeding system are,

(1) Conventional plant breeding always requires progeny large enough to recover
forms that recombine the desired features from the parents of a cross.

(2) Recombination results from random segregation due to chromosome reassortment

and from crossingover during gamete formation at meiosis. The gametes, pollen and
egg cells, fuse randomly to form fertilized eggs, or zygotes, that develop into seeds.

(3) The two parent organisms usually have to be related in some way.

(4) However, the progeny of this first cross inherit a mix of genes from both parent
plants and so both positive and negative traits may be inherited.

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(5) The process called ‘back-crossing’ takes place over a number of generations,
which usually means a number of years, until the progeny have all the desirable
traits and none of the negative ones of the original two parent plants

After 1950 modern biotechnology is the latest stage in the development of plant
breeding technology. Crick and Watson's discovery of DNA's double helix structure
in the 1950s held the key to cracking the genetic code which determines how all
living things work. The tools of the biotechnologist, developed as a result, have
further increased the speed and precision of plant breeding techniques and widened
the choice of characters for selection. Now if a cell to be transformed permanently,
only a foreign nucleotide sequence has to be introduced in such a way that alien
genetic information should be able to express in the transformed cells. Availability of
restriction endonucleases (which cut duplex DNA molecules into discrete fragments),
development of the DNA-DNA hybridization technique and marker genes for
selection of transformed cells have now enabled incorporation of exogenous DNA
into cells of Monera, fungi, animals, and higher plants.

Modern biotechnology allows crop improvement to take place at the level of

individual genes. Techniques such as genetic modification enable plant breeders to
identify the single gene responsible for a particular trait, and insert, delete or modify
it in a plant variety. This extends the range of characters at the breeder's disposal, and
enables specific genes to be expressed in a crop plant without the uptake of unwanted
characteristics which must then be selected out.

These modern techniques of crop improvement that is biotechnology involves the

transfer of genetic information from one organism into another, to affect the
production of recombinant products or to generate an organism or crop with new
recombinant properties which is termed as transgenic crops.

Scientists were first successful of doing such things using a soil born bacterium
Agrobacterium. Approximately a century ago, in 1907, Smith and Townsend

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discovered that plant tumors are caused by bacteria. In the 1940 it became evident
that these tumors grow even in the absence of bacteria, and that a tumor-inducing
principle (TIP) must be present in the plant. In the 1970s, TIP was identified as part of
the plasmid DNA that is termed as T-DNA .The T-DNA is coated with a number of
proteins that protect the DNA against degradation in the plant. The aim of the TI-
plasmid is to stimulate tumor growth in the plant in order to promote its own survival
and replication. Tumor growth is stimulated by the synthesis of cytokines and growth
factors such as Auxin, Opine and Cytokinin.

In research labs this principle of natural gene transfer is used as a tool in molecular
biology: Plasmid DNA can be modified to act as a shuttle vector to transfer DNA into
other organisms without the risk of tumor formation. For this purpose, tumor genes
are deleted from the original plasmid and replaced by components that are necessary
for the expression: a promoter, the gene of interest, a terminator and a selection
marker. The use of a selection marker gene is crucial to detect the incorporation of
plasmids as it encodes an enzyme that is able to degrade antibiotics (e.g. ampicillin or
kanamycin). For the generation of a transgenic plant, the plasmid is transferred into a
plant cell where it integrates into the genome. After selection and cultivation of
positive clones, the foreign DNA is expressed in all cells of the transgenic plant

The first targeted transfer of foreign DNA into a plant was performed in 1983, and
since then, gene technology in plants has been commercialized. To date, genetically
modified plants represent a high percentage of food crops grown in the USA and
Canada as well as all over the world.

During the last decades, a tremendous progress has been made in the development of
transgenic plants using the various techniques of genetic engineering. The plants, in
which a functional foreign gene has been incorporated by any biotechnological
methods that generally are not present in the plant, are called transgenic plants. As per
estimates recorded in 2002, transgenic crops are cultivated world-wide on about 148
million acres (587 million hectares) land by about 5.5 million farmers. Transgenic
plants have many beneficial traits like insect resistance, herbicide tolerance, delayed
fruit ripening, improved oil quality, weed control etc.

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Some of the commercially grown transgenic plants in developed countries are:

“Roundup Ready” soybean, ‘Freedom II squash’, ‘High- lauric’ rapeseed (canola),
‘Flavr Savr’ and ‘Endless Summer’ tomatoes. During 1995, full registration was
granted to genetically engineered Bt gene containing insect resistant ‘New Leaf’
(potato), ‘Maximizer’ (corn), ‘BollGard’ (cotton) in USA.

Use of genetic modification (GM) technique has several advantages over

conventional breeding technique such as

(1) It involves changing the genes of a plant so that a new and better variety is
developed. It is done for the same reasons as conventional breeding. The key
difference is that instead of randomly mixing genes, which occurs as a result of a
sexual cross, a specific gene, which is associated with a desirable trait, is selected
and inserted directly into the new plant variety with the help of a vector. This can
save time and reduces the chance of undesirable traits in the new plant variety.

(2) Genetic modification also allows breeders to use genes from unrelated plants
and sometimes other organisms into a new variety. This means breeders can access
and use a wider choice of genetic diversity to develop new plant varieties. This is
possible because all genetic information is stored in DNA – which is the same
chemical in all organisms.

Although using plasmid as vectors for development of genetically modified plant

proved to be very important .there was still some problem there. That is although
Agrobacterium or other plasmid was very efficient to introduce a new gene in a
crop species or producing transgenic crops the problem occur when researcher
wanted to introduce a gene of larger size(>1-10).So researcher continue their search
for the vectors that would be able to introduce foreign gene of interest of larger size
with high efficiency .

In the mid 1980s scientist were successful in their search. At last they got the vector
that was able to introduce gene of interest of larger size. They discovered yeast
artificial chromosome (YAC). Cloning of megabase sized DNA fragments became
possible and library-based exploration of even the largest genomes appeared

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practicable. However, YACs have some serious drawbacks as cloning vectors .For
example, roughly 50% of YAC clones are chimeric or possess insert rearrangements,
tedious steps in library construction and low yields of YAC insert DNA. Recently a
new E.coli based system has been developed, the bacterial artificial chromosome
(BAC) system.

In the early 1990s, “bacterial artificial chromosomes” (BACs) emerged as an

alternative
to YACs. Though they can carry inserts approaching 500 kb in length, insert sizes
between 80 and 200 kb are more typical. In particular, BACs are relatively immune to
chimerism and insert rearrangements.

Another important vectors that can be used for production of transgenic crops are
viruses. The use of viruses as gene transformation vector is very recent (in the mid
1990s).Viruses was choose as a vector for gene transformation because of its high
efficiency and high replicating power. Gene transfer is obtained by infection of the
plant by the virus and is occurs via viral genome replication. Mostly used viruses are
bacteriophage that infects bacteria. Other viruses include caulimovirus, geminivirus
and RNA virus.

Although many hopes are associated with transgenic plant development, a number of
problems have yet to be solved, many of them has technical obstacles such as the low
expression level of the transgenes, the positioning and stability of the transgene etc.
needs to be improved . But there is also the societal problem of public perception of
transgenic plants, which generates critical discussion.

OBJECTIVES:

The objectives of my review is to

1. Study the current status of plant modification techniques

2. Study about improvement of different vectors and their efficiencies.