RECOMINANT DNA TECHNOLOGY GENETIC ENGINEERING (manipulation) provides technique for investigation and alteration of gene structure and

function. The alteration in gene structure is done with aim to produce the desired characteristics and functions. Molecular biology has lead directly to immensely powerful and potentially profitable techniques for genetic engineering. A great deal of effort is being directed into genetic engineering or manipulation of microorganisms to make them produce a range of valuable polypeptides such as insulin, blood clotting factor VIII, growth hormones which they would not normally produce and which are expensive to prepare by conventional biochemical methods. It is easy and cheap to grow microorganism on the large scale. There are very attractive as potential sources of these polypeptides. Genetic manipulation of plants and livestock should also permit the introduction of beneficial characteristics such as resistance to herbicides or diseases that could not be readily obtain by conventional breeding. The understanding of gene structure has made it possible to understand the ways in which call store and express genetic information, which can be manipulated according to needs. Gene manipulation requires cuttings and joining lengths of DNA in a precise way. This is alternately called recombinant DNA technology. The DNA technology is a part of molecular genetics. It involves the cutting of DNA into specific fragments and joining these fragments using various enzymes. During this process there is transfer of genetic material from one organism to other organism. Requirements: There are basic three requirements for this technology.
(1) Vehicle DNA- It is a DNA used to carry foreign DNA molecule and then transferring it

molecule into the most cell.

(2) Passenger DNA. It is a DNA molecule (foreign DNA) which is being transferred from

one organism to another organism with the help of vehicle DNA.

(3) Enzymes-There are biocatalysts required for cutting and joining of DNA molecule.

Steps principal involved in DNA technology

(1) DNA fragments coding for proteins of interest are synthesized either chemically or isolated

from an organism with the help of enzymes. These DNA fragment are called passenger DNA.
(2) These DNA fragments (passenger DNAS) are inserted into vehicle DNAs with the help of

enzymes restriction endonucleases and ligases etc.

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A DNA molecule obtained after combination of vehicle DNA and passenger DNA is called recombinant DNAs (r DNAs). (3) The r DNA molecules are than introduced into a host cell to replicate. Generally three methods are than used to introduce into r DNA to host cell.

(a) Transformation (b) Transduction (c) Conjugation.

(a) Transformation- It is a process by which a cell takes a naked DNA segment

the environment, incorporated it into its own chromosome DNA.

(b) Transduction- It is transfer of genetic material or r DNA from one organism

to another with the help of bacteriophage.

(c) Conjugation- The transfer of genetic material from donor to recipient by

direct contact. (4) In the recipient host cells, r DNA replicates indefinitely.

In this way genes are cloned to provide sufficient material for detailed analyses or for insertion into genome of a cell i.e., to be genetically engineer. The principal steps involved in gene cloning are shown below

TOOLS OF RECOMBINANT DNA TECHNOLOGY Various tools like enzymes, vectors and host organisms are required for carrying on the recombinant DNA technology.
A) Enzymes: Various types of enzymes required are: i)

Restriction enzymes: Restriction enzymes are popularly called Molecular Scissors. These are mainly classified as restriction endonucleases and restriction exonucleases. Restriction exonucleases: They remove nucleotides from the ends of DNA. These digest base pairs from 5 to 3 ends and act on single stranded DNA only.

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Restriction endonucleases: They act upon genetic material and cleave the double stranded DNA at any point except the ends, but they act on only one strand of duplex. All restriction endonucleases have specific recognition sequence within the DNA molecule. The sequences generally fall between four to six base pairs in length. The restriction recognition sequences are often known as Pallindromic sequences i.e., the two strands are identical when both in forward and backward direction, in this region read. Each individual enzyme recognizes only one specific sequence. However, the same specific sequence is recognized by two or more different enzymes. Such types of enzymes are called isochizomers. These enzymes are called Natures Pinking Shears (Saw tooth blades) because these occur naturally in bacteria as a chemical weapon against invading viruses and cut both strands of DNA when certain foreign nucleotides are introduced in the cell. Types of restriction endonucleases Restriction endonucleases can be divided into three types: Type I, Type II and Type III. Type I: The type I enzymes recognizes specific sequence but cut the DNA molecule far away from the recognition sequence. The cleavage takes place about 1000 base pairs away from recognition sit. Hence, the use of class I enzyme in genetic engineering is restricted. E.g. Eco K, Eco B etc. Type II: These are able to recognize specific sequence and cut the DNA molecule from at the recognition site only. Hence, this class of enzymes is the major choice in genetic engineering work. Because majority of type II restriction enzymes recognize tetra, penta and hexa and sometimes hepta nucleotide sequence. They require Mg2+ for restriction digestion. More than 350 different type II endonucleases with over 100 different recognition sequences are known. The classical example for class II type restriction enzyme is EcoR I. The first type II endonuclease discovered in 1960 was Hind II. Type III: These are intermediate between type I and type II enzymes. They cleave the DNA near to the recognition site. E.g Eco P1, EcoP15 etc. They recognize asymmetric target site and cleave the DNA duplex on one side of the recognition sequence up to 20 base pairs away.

ii)

Ligases- Though cutting of DNA very useful for DNA analyses. Its full potential is revealed only when fragments produced are joining together to give new of called recombinant DNA. This joining or ligation is achieved by use of DNA ligases enzymes. Two different DNA
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preparations are treated with some enzymes to give fragments with sticky ends. These, when two fragments are mixed, base pairing between sticky ends will result in coming together of fragments. All these pairings are temporary owing to weakness of H-bonding between few bases in the sticky ends .But they can be stabilize by use of DNA ligase which forms covalent bond between 5 Po43- gp at end of one strand and 3 OH group of adjacent strand. This reaction is driven by ATP and carried out at 10oc to lower K.E of molecules. So, reducing the changes of base pairs sticky ends separating before they have not stabilized by ligation. However long reaction times are needed to compensate for low activity of DNA ligase in the cold. SIGNIFICANCE OF RESTRICTION ENZYMES:
1. Restriction enzymes are used in biotechnology to cut DNA into smaller strands in order

to study fragment length differences among individuals (Restriction fragment length polymorphism-RFLP) or for gene cloning. 2. RFLP techniques have been used to determine that individuals have distinctive differences in gene sequences and restriction cleavage patterns in certain areas of the genome. 3. Knowledge of these unique areas is the basis for DNA fingerprinting. Each of these methods depends on the use of agarose gel electrophoresis for the separation of the DNA fragments. Cloning vectors- In principle, DNA is introduced into a suitable host call using bacteria like E.coli. Where it is replicated as cell grows and divides. However, replication will occur only if DNA contains a sequence i.e., recognized by cell as origin of replication. Most DNA samples do not contain such sequence therefore DNA has to be cloned has to attach to a carrier or vector DNA that does contain an origin of replication. Hence, vector is a DNA molecule that has the ability to replicate in an appropriate host cell and into which the DNA fragment (passenger DNA) is inserted for cloning. It is also called vehicle DNA. Properties of a good vector 1. It should be able to replicate. 2. The vector should be easy to isolate and purify.
3. It should be easily introduced into the host cells.

4. The vector should have suitable marker genes that allow easy detection or / and selection of the transformed host cells. 5. A vector should contain unique target sites for restriction endonucleases.
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6. It should have ability to accommodate foreign DNA of various sizes without damage to replicate functions. Number of vectors can be employed for this purpose;1) PLASMID-

Plasmids that are used this cloning have following properties;(1) They are small and size varies from 1Kb to over 25 KB. (2) They are present in high copy number e.g., An E.coli may contain up to 7 different kinds of plasmids. (3) They contain suitable marker/ markers e.g, Antibiotic resistant marker (4) They are not transmissible i.e. they are not transferred from one cell to another.
(5) They contain suitable restriction site that can be used for inserting DNA fragment for

cloning. (6) They are stably maintained during replication in the host i.e. during cell division they are segregated evenly in daughter cell. (7) They can replicate in suitable hosts.

Not being a part of the main genome, the plasmids can be easily isolated and transferred to another bacteria or organisms. These features make the plasmid suitable for use as vector or vehicle DNA. A number of plasmids have been isolated from different hosts and several has properties listed above and can be used as suitable cloning vector. These are simplest cloning vectors and most widely used for gene cloning. A plasmid integrated to a chromosome is called an Episome. One of most widely used plasmid is PBR-322. These are called Natures interpolers. Because they are known to move around freely in bacterial world and may pick up genes from one bacterium and transfer them to another, thus providing variability to the asexually multiplying bacteria.

2) BACTERIOPHAGE
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Bacteriophages are viruses that attack bacteria. Hence, are also called bacterial viruses. Phage contains a proteinaceous head and a long tail attached to the head. Several bacteriophages are used as cloning vectors, the most commonly used E.Coli phages being (lambda) and M13 phages. Plasmid vectors have to be introduced into bacterial cells, which are then cloned and selected for recovery of recombinant DNA. In contrast, the phage vectors are directly tested on an appropriate bacterial lawn (a continuous bacterial growth on an agar plate) where each phage particle forms a plaque (a clear bacteria free zone in the bacterial lawn). Phage vectors present two advantages over plasmid vectors.
i) They are more efficient than plasmids for cloning of large DNA fragments; the largest

cloned insert size in a vector is just over 24 KB, while that for plasmid vectors it is less than 15 Kb.
ii) It is easier to screen a large number of phage plaques than bacterial colonies for the

identification of recombinant vector. Specific DNA sequences are present in vector are known as cos sites. These enable the DNA to be packed. These vectors contain different restriction sites, antibiotic resistance markers and carry large DNA fragments. 3) COSMIDS It is a hybrid vector derived by cloning plasmids with cos sites of vector. These are similar to plasmids in many characters such as replication, gene coding, and cleavage sites. These are dissimilar to it because of presence of DNA fragment from vector. They have small lengths of about 5Kb. These were developed by Collins and Hohn in1978.

Cosmids are also used for cloning. This system takes advice of the properties of both the plasmids and system. A cosmid consist of a circular DNA molecule containing s plasmid origin of rep. (origin) which allow autonomous replication, a suitable antibiotic marker, cos site (these are cohesive ends of linear chromosome infection, the cos site is also required for ligating and pillaging. The sight amount of DNA into a read).And unique restriction site (e.g. E.Coli) that can also be used for inserting into DNA fragment. The cosmid can replicate like a plasmid. The strategy for using the cosmid as a cloning vehicle is illustrated below;

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The cosmid can be used to make gene banks by partial endonuclease restriction (e.g E.Coli) of chromosome that will result in fairly large DNA fragments. These fragments will be mixed with E.Coli restriction cosmid and joined between 2 linear cosmid molecule .When the proper size DNA fragment (these can be up to 35 K.b long ) is inserted between 2 cosmid molecule. The distance between the resulting two cos sites will determine whether the recombinant DNA can be properly packaged into head to form like particle. When these particle are used to infect E.Coli. The recombinant cosmid DNA will be injected into the host and replicated by use of plasmid on present on the molecule proper selection technology will allow.The detection of specific recombinant DNA molecule in the clones.

4) PHASMIDS The insertion of plasmid into vector generates a phage genome containing at side and one or more plasmid molecules. These new genetic combinations are called as phasmids.

PASSENGER DNA It is DNA which is transferred from one organism into another by combining itself with the vector DNA. The three types of DNAs are used as passenger DNA.
i) Complementary DNA (cDNA)

True copy of an mRNA molecule is known as cDNA.It is synthesized on RNA template with the help of reverse transcriptase enzyme. The DNA strand is isolated from the hybrid RNA-DNA complex with the use of alkaline phosphatase enzyme. A cDNA strand is then synthesizes on the isolated single stranded DNA template with the help of DNA polymerase enzymes. The DNA duplex thus formed can be joined to vector DNA for introduction to a host cell. ii) Synthetic DNA It is synthesizes artificially with the help of DNA polymerases on DNA template. In 1965, Hargobind Khorana and His associates produce the first artificial DNA without using a DNA template. iii) Random DNA
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It refers to small fragments produced by breaking a chromosome of an organism with the help of restriction endonucleases.

Significance of transgenic animals: 1. Increased growth of livestock and fish

2. Increased and improved wool production in sheep

3. Transfection in molecular farming Application of biotechnology in the field of industry 1. In the area of food production a) To produce dairy products i) ii) iii) iv) v) Milk is pasteurized at 73oC for 15 sec to kill most of the micro-organisms. Lactobacillus bacteria is used to prepare cheese from milk. Enzyme rennin is added to separate milk into solid curd and liquid whey. Ripening of cheese is done by specific microorganisms. It gives flavor to cheese. Yoghurt is prepared from the pasteurized milk with much of the fat removed.For this, special strain of yoghurt making lactic acid bacteria(lactobacillus bulgaricus and Streptococcus

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thermophilus) are added to milk to convert lactose to lactic acid and mixture is incubated at around 45oc for five hours. b) Production of single cell proteins Single cell proteins are referred to any microbial mass produced by uni- and multicellular microorganisms and can be used as food or feed additives. SCP contains about 45-55% protein, although those of certain bacteria the protein content is as high as 80%. So, SCP is protein rich powder, so is used as food for human being or feed animals such as chicken and calves. c) Production of mycoprotein Mycoprotein if produced from fungus Fuserium graminearum which is grown on the carbon source containing ammonia in a fermenter in aerobic conditions at 30oC and pH 6. The threads like strands of mycoprotein can be flavored resemble to chicken meat. It also contains only about half of the fat content of the neat so is suitable for the human consumption. 2. In the field of transformation. 3. Biotransformations involve enzymatic modification of organic compounds into recoverable products. i) ii) d) e) Production of enzymes Production of steroids

Biotechnological extraction of metals. Microbial biotechnology and food production. PLANT TRANSFORMATION GMO's are produced by transforming the organisms. Transformation is the heritable change in a cell or organism brought about by the uptake and establishment of introduced DNA. There are two predominant procedures of transforming genes in organisms: the "Gene Gun" method and the Agrobacterium method. Agrobacterium-mediated Plant Transformation Process Agrobacterium tumefaciens is a widespread naturally occurring soil bacterium that causes crown gall, and has the ability to introduce new genetic material into the plant cell. The genetic material that is introduced is called T DNA (transferred DNA) which is located on a Ti plasmid. A Ti plasmid is a circular piece of DNA found in almost all bacteria.
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This natural ability to alter the plants genetic makeup was the foundation of plant transformation using Agrobacterium. Currently, Agrobacterium-mediated transformation is the most commonly used method for plant genetic engineering. One of the reasons for this is the large number of plants that are susceptible to this bacterium. Initially it was believed that this Agrobacterium only infects dicotyledonous plants, broadleaf plants, soybeans and tomatoes, for many years, but it was later established that it can also be used for transformation of monocotyledonous plants such as rice. During transformation, several components of the Ti plasmid enable effective transfer of the genes of interest into the plant cells, these include:

T-DNA border sequences, which demarcate the DNA segment (T-DNA) to be transferred into the plant genome Vir genes (virulence genes), which are required for transferring the T-DNA region to the plant but are not themselves transferred, and Modified T-DNA region where the genes that cause crown gall formation are removed and replaced with the genes of interest.

The Agrobacterium-mediated transformation process involves a number of steps: (a) Isolation of the genes of interest from the source organism; (b) Insertion of the transgene into the Ti-plasmid; c) Introduction of the T-DNA-containing-plasmid into Agrobacterium; d) Mixture of the transformed Agrobacterium with plant cells to allow transfer of T-DNA into plant chromosome; (f) Regeneration of the transformed cells into genetically modified (GM) plants; and (g) Testing for trait performance or transgene expression at lab, greenhouse and field level. In this method, the tumor inducing (Ti) region is removed from the T-DNA (transfer DNA) and replaced with the desired gene and a marker, which is then inserted into the organism. The marker is used to find the organism which has successfully taken up the desired gene. Tissues of the organism are then transferred to a medium containing an antibiotic or herbicide, depending on which marker was used. The Agrobacterium present is also killed by the antibiotic. Only tissues expressing the marker will survive and possess the gene of interest. Thus, subsequent steps in the process will only use these surviving plants. In order to obtain whole plants from these tissues, they are grown under controlled environmental conditions in tissue culture. This is a process of a series of media, each containing nutrients and hormones. Once the plants are grown and produce seed, the evaluation of the progeny process begins. This process entails
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selection of the seeds with the desired traits and then retesting and growing to make sure that the entire process has been completed successfully with the desired results.

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.
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Figure: Illustrates Agrobacterium-mediated plant transformation

Advantages And Disadvantages Of Agrobacterium As A Gene Transfer Tool Advantages

The overall advantages of using Agrobacterium-mediated transformation over other transformation methods are: 1. Technically simple 2. Yields relatively uncomplicated insertion events (low copy number, minimal rearrangements) 3. Unlimited size of foreign DNA 4. efficient (for most plants) 5. adaptable to different cell types, culture procedures (protoplasts, tissue sections, non-culture methods) 6. Transformants are mitotically and meiotically stable 7. Intact and stable integration of the transgene (newly introduced gene) into the plant genome Disadvantages 1. Host range is limited: not all plants may be susceptible to Agrobacterium 2. With susceptible plants, accessible culture/regeneration systems must be adaptable to Agrobacterium-mediated gene transfer In general, the Agrobacterium method is considered preferable to the gene gun, because of a greater frequency of single-site insertions of the foreign DNA, which allows for easier monitoring.

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Description Of Agrobacterium Method

Direct gene transfer

The term direct gene transfer is used to discriminate between methods of plant transformation that rely on the use of Agrobacterium (indirect methods) and those do not (direct methods). There are several methods for direct gene transformation such as electroporation, PEG mediated transformation, cationic carriers and biolistic.
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Plant Transformation Using Particle Bombardment During early 1990s, it was shown that DNA delivery to plant cells is also possible, when heavy microparticles (tungsten or gold) coated with the DNA of interest are accelerated to a very high initial velocity (1,400 ft per sec). These microprojectiles, each normally 1-3m in diameter, are carried by a 'macroprojectile' or the 'bullet' and are accelerated into living plant cells (target cells can be pollen, cultured cells, cells in differentiated tissues and meristems) so that they can penetrate cell walls of intact tissue. The acceleration is achieved either by an explosive charge (cordite explosion) or by using shock waves initiated by a high-voltage electric discharge.The Particle bombardment device, also known as the gene gun, was developed to enable penetration of the cell wall so that genetic material containing a gene of interest can be transferred into the cell. Today the gene gun is used for genetic transformation of many organisms to introduce a diverse range of desirable traits. Particle bombardment (biolistic) is a physical method widely used for gene transfer into plants. mammals fungi and bacteria. In this method, 1-2m tungsten or gold particles, coated with the DNA to be used for transformation, are accelerated to velocities, which enable their entry into plant cells/nuclei. Particle acceleration is achieved by using a device, which varies considerably in design and function. The most successful devices accelerate particles in one of the two ways: (1) by using pressurised helium gas or (2) by the electrostatic energy released by a droplet of water exposed to a high voltage. The earlier devices used blank cartridges in a modified firing mechanism to provide the energy for particle acceleration; this is the reason for the name particle gun to this approach. The method is also called biolistic or ballistic method of DNA delivery. This technique has general applicability to plant species and can be used to deliver DNA into virtually all tissues. More importantly, it can be used to transform shoot apical meristems, leaf blades, immature and mature embryos, mature pollen, and root and shoot sections etc. Meristematic cells show higher transformation frequency than nondividing cells. The main components of a helium pressure device are, gas acceleration tube, rupture disc., stopping screen, macrocarrier carrying particles coated with DNA, and target cells. These components are enclosed in a chamber to enable the creation of partial vacuum, which facilitates particle acceleration and reduces damage to plant cells. After creation of partial vacuum sufficiently pressurised helium gas is released in the acceleration tube to break the rapture disc. This generates helium shock waves, which accelerate the macroprojectile to which DNA coated microprojectiles are attached. The macroprojectile is stopped by a stopping screen, and the
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microprojectiles pass through this screen and become embedded in the cells kept about 10 mm below the stopping screen. Helium is preferable to air since it is lighter and offers certain advantages. Generally a 1000 psi (pounds per square inch) of helium pressure is used for acceleration. The macrocarrier or macroprojectile is a 2.5 cm diameter 0.06 mm thick plastic membrane, which is used only once. The microprojectiles, micropartic1es or microcarriers vary in diameter from 0.5 to 2.0 m; the average size of 1.0 11m is commonly used. Tungsten particles are cheaper, but are of irregular shape and size, may be toxic to certain cell types and show surface oxidation which may lead to precipitation of DNA.

Plant transformation using particle bombardment follows the same outline as Agrobacteriummediated method. The steps taken include: 1) Isolate the genes of interest from the source organism; 2) Develop a functional transgenic construct including the gene of interest; promoters to drive expression; codon modification, if needed to increase successful protein production; and marker genes to facilitate tracking of the introduced genes in the host plant; 3) Incorporate into a useful plasmid; 4) Introduce the transgenes into plant cells; 5) Regenerate the plants cells; and 6) Test trait performance or gene expression at lab, greenhouse and field level. The particle bombardment method starts with coating tungsten or gold particles (microprojectiles) with plasmid DNA. The coated particles are coated on a macro-projectile, which is accelerated with air pressure and shot into plant tissue on a petri plate, as shown in Figure 1. A perforated plate is used to stop the macro-projectile, while allowing the microprojectiles to pass through to the cells on the other side. As the microprojectiles enter the cells, the transgenes are released from the particle surface and may incorporate into the chromosomal DNA of the cells. Selectable markers are used to identify the cells that take up the transgene. The transformed plant cells are then regenerated into whole plants using tissue culture. Particle bombardment also plays an important role in the transformation of organelles such as chloroplasts, which enables engineering of organelle-encoded herbicide or pesticide resistances in crop plants and to study photosynthetic processes. Limitations to the particle bombardment method relative to Agrobacterium-mediated transformation include frequent integration of multiple copies of the transgene at a single insertion site, rearrangement of the inserted genes, and incorporation of the transgene at multiple insertion sites. These multiple copies can be linked to silencing of the transgene in subsequent progeny (Yao et al., 2006). Figure 2 and 3 show the different types of gene guns that are currently used in plant transformation.
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Figure 1: Diagrammatic illustration of gene transfer using Gene Gun method Source: http://www.artsci.wustl.edu/~anthro/blurb/fg8.t.gif

Figure 2 Standard Gene Gun

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Figure 3 Helios Gene Gun

Transgenic plants using the above technique have been obtained in many cases including soybean, tobacco, maize, rice, wheat, etc. Transient expression of genes transferred in cells by this method has also been observed in onion, maize, rice and wheat. During 1990s, there was no other gene transfer approach, which met with' so much of enthusiasm. Consequently considerable investment was also made in experimentation and manpower for development of this technique. The advantages of this method over microinjection include the following: (i} thousands of particles are accelerated at the same time, causing multiple hits resulting in transfer of genes into many cells simultaneously; (ii) since intact cells can be used, some of the difficulties encountered with the use of protoplasts are automatically circumvented; (iii) the method is universal in its application, so that cell type, size and shape or the presence/absence of cell walls do not significantly alter its effectiveness. In view of this, particle bombardment method using microprojectiles was used in a variety of plant species, particularly the cereals. The method was also used successfully for transfer of genes into the plastid genome. However, the emphasis on 'the use of particle gun decreased towards the end of 20th century, due to the success achieved in cereals with Agrobacterium mediated gene transfer.

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Advantages of transgenic plants

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. 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.

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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

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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. [Discussion] 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.

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7. Transgenes Encoding Antisense RNA.

These are discussed in a separate page. Link to it.

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

Application OR Significance of transgenic plants:

1. These can tolerate adverse conditions, such as drought, high temperature frost, salinity etc.
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2. These can produce pharmaceutically important compounds such as human insulin,

interferons, hormones, blood clotting factors etc.

3. 1. They have proved to be extremely valuable tools in studies on plant molecular biology, regulation of gene action, identification of regulatory/promotary sequences, etc. 2. Specific genes have been transferred into plants to improve their agronomic and other features. 3. Genes for resistance to various biotic stresses have been engineered to generate transgenic plants resistant to insects, viruses, etc. 4. Several gene transfers have been aimed at improving the produce quality. 5. Transgenic plants are being used to produce novel biochemicals like hirudin, etc. which are not produced by normal plants. 6. Transgenic plants can be used vaccines for immunization against pathogens; this is fast emerging as an important objective.

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.

To date, field studies on Bt cotton and maize (corn) show that the numbers of some nontarget insects are reduced somewhat but not as much as in fields treated with insecticides. Another worry is the inadvertent mixing of transgenic crops with nontransgenic food crops. Although this has occurred periodically, there is absolutely no evidence of a threat to human health. Despite the controversies, farmers around the world are embracing transgenic crops. Currently in the United States over 80% of the corn, soybeans, and cotton grown are genetically modified (GM) principally to provide

resistance to the herbicide glyphosate ("Roundup Ready") thus making it practical to spray the crop with glyphosate to kill weeds without harming the crop; resistance to insect attack (by expressing the toxin of Bacillus thuringiensis).

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What are the Advantages and Disadvantages of Transgenic Crops? The use of transgenic crops has been an issue for many years. Many concerns have been raised and these generally fall into two categories: 1.A concern about what affect genetically modified material could have on human health. For example, transgenic crops have been suggested to cause allergies in some people, although it is uncertain whether transgenic crops are the source of this reaction [3]. Furthermore the antibiotic resistance genes placed in these crops has been suggested to cause resistance to antibiotics leading to super bugs that cannot be killed with antibiotic treatments [3]. The idea of a population being uncomfortable with ingesting DNA that originated from another source, such as
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a virus or bacteria, must also be considered when thinking about producing transgenic crops. However, to date, there is no evidence of the DNA from transgenic crops being any different from the DNA ingested from conventional crops. 2. A concern about whether transgenic crops cause damage to the natural environment. One example includes pollen from transgenic corn, which has been suggested to kill the Monarch butterfly larvae. It has been shown that hybrid corn expresses a bacterial toxin in its pollen, which is then dispersed over 60 meters by wind. In this range, the corn pollen is deposited on other plants near cornfields where it can be ingested by non-target organisms including the monarch butterfly [5]. These butterflies have been found to eat less, have a slower growth rate and higher death rate [5]. A second example is the hybridization of crops with nearby weeds. This could cause these weeds to attain resistance to herbicides or other things that we have been trying to avoid for many years. Genes that provide resistance to viral disease or other traits allowing them to survive in their environment could end up benefiting weed populations around a crop field. This trait could make that population more difficult to control. To date, there has been little evidence to support this theory. On other side of the coin are the notions that support the use of transgenic crops. The potential benefits of which are quite obvious, including such things as increased yields (to feed a growing population), decreasing the use of pesticides (to save the environment and the cost of pesticides), and the production of novel crops (such as providing crops with increased nutritional value) [3]. Being able to retrofit any crop to our desires is a powerful concept, especially with the changing climates of today. Should We Use Transgenic Crops? In the end, the perceived advantages and disadvantages of transgenic crops must be married to each other to provide a crop that is environmentally sound and non-hazardous. Producers of transgenic crops and the agencies that study their effects are aware of this point. However, to date, there has been little evidence to support either case. More research is required in this field to determine the true safety of these plants and to decide whether they are safe for both the environment and for those who consume these products over the ages. At the least, most would agree that the potential advantage of producing crops that provide the human population with more and cheaper food makes transgenic technology a useful invention.

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Gene

Transfer

by

Particle

Bombardment

ABSTRACT Biolistic is a short term for biological ballistics; the biolistic process is one by which biological molecules, such as DNA and RNA, are accelerated (usually on microcarriers, termed microprojectiles) by gun powder, compressed gas or other means [1]. In this study, two particle bombardment system were used to directly transfer two construct which are pUC19 (control plasmid without P358-GUS) and pIG 121-HM (containing P358-GUS). The fist one is the Helios Gene Gun from Bio-Rad and the other is the Biolistic PDS-1000/He from Bio-Rad on Arabopsis thaliana and Nicotina tobacum respectively. INTRODUCTION MATERIALS AND METHODS Plant Material Arabopsis thaliana plants were grown in peat at 20oC with an 8 hour photoperiod. The 4 to 5 weeks old plants were used for gene delivery bombardment experiments and Nicotiana tabacum (tobacco) plants were grown at 23oC with a 16 hour photoperiod in vermiculite. When the first true leaf expanded, the plants were used for the bombardment.

Preparation of DNA-coated gold microcarriers for the Gene Gun system 5 mg gold microcarriers (diameter: 0.6 mm) and 10 ml of 0.05 M spermidine were vortexed for 5 s and sonicated for 10 s in an ultrasonic bath to break up gold clumps. Secondly, 10 mg plasmid
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DNA (pUC19 or pIG121 Hm) in 10 ml H2O is added. Then, 10 ml 1 M CaCl2 was added dropwise while vortexing the mixture at moderate rate. The mixture was incubated for 10 min at room temperature to precipitate the gold and DNA. After centrifugation for 15 s at 14,000 rpm, the pellet was washed 3 times in 0.1 ml absolute ethanol. The pellet was resuspended in 20 ml ethanol containing 0.1 mg/ml polyvinylpyrrolidone (PVP). The final volume was adjusted to 0.6 ml using the ethanol/PVP solution. The suspension was then used for the cartridge preparation as follows. Catridge preparation with the Tubing Prep Station for the Gene Gun system Firstly, the Gold-Coat tubing was first dried by purging with nitrogen for 15 min. Then, the suspension was drawn into the Gold-Coat tubing (5 in length) and placed into the Tubing Prep Station following, microcarriers are allowed to settle for 10 min. Then ethanol then was removed slowly. The Gold-Coat tubing was rotated and the particles were spread onto the inner surface of the tubing and subsequently dried with a flow of nitrogen. Any unevenly coated sections should be discarded before the remaining tubing was cut into 0.5 pieces with the Tubing Cutter. Eight cartridges were generated. Preparation of DNA-coated gold microcarriers for the PDS-1000/He system Stock suspension of microprojectiles was prepared by mixing 60 mg of 1.0-mm gold particles in 1,000 ml of absolute ethanol which can be stored at -20 oC. Then stock suspension was vortexed for 30 s and soon after 35 ml of the stock suspension was quickly removed and added to a 1.5 ml microcentrifuge tube to be microcentrifuged at high speed for 30 s. After removing ethanol with micropipette, pellet was resuspended in 1 ml water and microcentrifuged for 5 min. Then, microprojectiles were resuspended in 25 ml of DNA solution (pUC19 or pIG121-Hm) (1 mg/ ml). After that these reagents were added in written order and at the amount specified: 220 ml of water, 250 ml of 2.5 M CaCl2, and 50 ml of 0.1 M spermidine (Sigma Chemical S-0266 stock solution stored at 20 oC, diluted 14 ml to 1,000 ml with water). Later on, the solution was mixed thoroughly and vortexed on a vortex shaker for at least 10 min at 4 oC and microcentrifuged for 5 min and remove supernatant. Resuspension of DNA/microprojectile precipitate in 600 ml of absolute ethanol by pipetting up and down several times was the next step before another microcentrifugation for 1 min. Removal of ethanol and resuspension of pellet in 36 ml of absolute ethanol by pipetting up and down until well dispersed then were done before loading to the macrocarrier sheet. Lastly, 10 ml of the suspension was pipetted as evenly as possible onto the center of a Mylar macrocarrier sheet. It is important to let the ethanol evaporate.

Each DNA/microprojectile suspension yields enough solution to coat 3 Mylar macrocarriers. Bombardment Conditions and Transient Gene Expression Intact leaves of Arabidopsis and tobacco plants were bombarded with the Gene Gun system. The helium pressure for Arabidopsis was 75psi
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Detached Arabidopsis leaves were bombarded with the PDS-1000/He unit. First, 1,100 psi rupture disk (that has been soaked with isopropanol) was placed in the unit. The distance between the rupture disk and macrocarrier was 8-10 mm and between macrocarrier and stopping screen is 1 cm. Each Petri dish containing the leaves was placed in the device 6 cm below the stopping screen. Then chamber was evacuated to 28-29 mm Hg before bombarding the target. Storage procedure After bombardment, the leaves were sprayed with water and placed in a growth chamber for 24 hours before assaying for the GUS enzymatic activities. The detached leaves were incubated in a moisturized container.

Histochemical GUS assay The leaves were submerged in 1 mM 5-bromo-4-chloro-indolyl-b-D-glucuronide (X-Gluc) in a buffered solution [100 mM Na-phosphate buffer (pH 7.0), 10 mM EDTA, 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6, 1.0 mM X-Gluc, 0,1% Triton X-100] and incubated in the dark for 16 hours at 37 oC. After staining, chlorophyll was removed with absolute ethanol in order to better visualize the blue spots. RESULTS:

Figure 1: Gene gun (pIG121 Hm)

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Figure 2: Gene gun (pUC19)

Figure 3: PDS-1000/He (pIG121 Hm)

Figure 4: PDS-1000/He (pUC19)

If expressed, -Glucoronidase enzyme uses X-Gluc and gives a blue colored and product thus indicating the succesfull transformation and expression. pIG121Hm carries the gene and
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promoter whereas, pUC19 does not (negative control). DISCUSSION Despite, Argobacterium based based transformation widely used; it is almost but exclusively restricted to dicotyledons [9] Since, monocotyledonous plants, which constitute some of the world's most important food crops, there is an alternative method is required. Microprojectile bombardment, which is non-specific, provides an ideal alternative approach and has been successfully employed to transform several of the major cereals, including barley maize wheat rice together with other monocotyledons such as tulip and orchids [13].Unlike the other direct transformation methods, the Microprojectile bombardment permits the delivery of DNA to the wide range of plants (review), making it the choice of method used in transformation.

There are several parameters that affect the efficiency of this method. These include the properties of microprojectile, tissue culture method of the plants, DNA construct and etc. Nature of Microprojectiles The two major microprojectiles or microcarriers are gold and tungsten particles. The size of the particle is important and should be optimized in such a way that can deliver significant amount of DNA without causing damage. Beside this the spherical shape is advantageous as it gives less damage to the cell. In general, tungsten particles have an irregular surface and are predisposed to agglomeration during the DNA coating procedure, whereas gold microprojectiles, which are nearly spherical, remain separated [10].Moreover, these particles should be inert materials otherwise they may cause toxicity and degradation of DNA in the cell. The gold particles are not toxic to any of the cells assessed by Sanford et al. [11] and did not catalytically attack DNA adsorbed to the surface of particles where as, tungsten is toxic to certain cell types and is also subject to surface oxidation, which affects DNA binding and degradation of adhered DNA[13]. Finally, the concentrations of these carriers are another parameter to be considered. If the concentration too Low, it will give sparse coverage over the target area while high concentrations of microprojectiles can result in agglomeration of the particles during the DNA coating procedure. Agglomerated particles can lead to excessive damage to plant tissues upon bombardment Adherence of DNA to microprojectiles No doubt that the efficient DNA coatings of microprojectiles are essential for optimal delivery to the target. In both two procedures CaCl2 was used to precipitate DNA with gold particles. Polyvinyllpyrrolidone (PVP) was used as an adhesive during the coating of DNA/microcarrier suspension to the walls of the Goldcoat tubing[bulletin], resulting increased evenly coating. And the spermidine which is a polyamide was used to increase static charges on plasmid GC rich
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regions hence increasing the coating efficiency. Sonification was done to disrupt any aggregated particles. Resuspension of DNA/microprojectile with ethanol permits the precipitate to dry as ethanol evaporates easily. Uneven precipitation of DNA and the aggregation of microprojectiles are the problems encountered during the coating of DNA/microprojectiles. Uneven precipitation is due to rapid process of precipitation itself. The vortexing procedure is commonly used while adding the calcium chloride drop wise to the DNA microprojectile mixture. Increasing the concentration of DNA used to coat the microprojectiles should, theoretically, increase the transformation frequency in a linear manner, until saturation of the microprojectiles is achieved. However, high concentrations of DNA have been shown to result in agglutination of the microprojectiles, which reduce transformation frequencies because of the large size and, effectively, the reduced number of particles [12]. Delivery of Microprojectiles into Target Tissues Accelerating power, distance between device and the target and the vacuum in PDS-100/He system are important factors for velocity and so for penetration. Most systems utilize a vacuum to reduce air drag on the microprojectiles and to reduce the risk of shock waves, from the overlying gas, damaging the tissue [11]. Vacuum mediates a uniform distribution of particles. Incorporation of Introduced DNA into the Recipient Genome Integration of the DNA construct into the chromosome results in stable expression of the transgene where as otherwise, it will give a transient expression or most probably nothing. In a clearer sentence, the gene expression is related to the ultimate location of microprojectiles within cells. Choice of DNA Construct
The choice is extremely dependent on the purpose of the experiment itself. In this study, the purpose was to observe the succession of the two different gene bombardment procedures by using a reporter enzyme GUS. The GUS enzyme is encoded by an E coli gene (gusA or udiA). GUS (-Glucoronidase) cleaves a colourless substrate, X-Gluc (5 bromo-4-chlom-3-indolyl-l~-D-glucoronic acid) into a product which, on oxidation, produces an indigo coloured dye. Hence, the transformed cells can be identified by their blue colouration [13]. The DNA construct pIG121Hm that was used contains this enzyme with a promoter called Cauliflower Mosaic Virus (CaMV) 35S promoter where as the DNA construct pUC19 does not contain the gene, used for negative control. The CaMV 35S promoter is very strong and seemed to be constitutive, meaning inducing the 31 | P a g e

expression of the downstream located coding region, in apparently all plant tissues [1] Configuration of the vector used for gene delivery may influence gene integration and expression as linear forms of plasmid resulted in higher levels of gene expression than supercoiled DNA [13]. Constructs with selectable marker which is used to select the transformed cells to get a new plant can be used with a transgene of interest. The selectable marker and transgene may have their own promoters one is constitutive and the other is development or tissue specific depends on the interest opening an amazing doors of the science for use of the humans. Choice and Preparation of Explants

In the experiment the leaves used were young allowing easy penetration for the bombardment.

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