UNIT-II

Gene CloningIntroduction to gene cloning- Gene transfer methods in plants -Ti plasmid mediated gene transfer-physical methods of gene transfer-molecular markers and marker assisted selection.

INTRODUCTION TO GENE CLONING / G ENETIC ENGINEERING / GENE MANIPULATION / RECOMBINANT DNA TECHNOLOGY

Definition:Recombinant DNA technology (genetic engineering) is a set of techniques for recombining genes from different organisms and transferring this DNA into a cell where it can be expressed (made into a protein). Using Recombinant DNA technology, we can isolate and clone single copy of a gene or a DNA segment into an indefinite number of copies, all identical. These new combinations of genetic materials or Recombinant DNA '(rDNA)' molecules are introduced into host cells, where they propagate and multiply. The technique or methodology is called Recombinant DNA technology or "Genetic engineering" or gene cloning. The first recombinant DNA molecules were generated by Paul Berg, Herbert Boyer, Annie Chang, and Stanley Cohen in 1973.

Possible applications in agriculture

1. Solving agricultural problemsa. Development of environmental stress tolerant plants:Temperature, water, salinity, heavy metals and herbicide etc.b. Development of biological stress resistance: weeds, insects, bacteria, fungi and viruses etc.2. Nutritional value enhancement3. Shelf life extension4. Yield improvement

Requirements for genetic engineering

1) Gene of Interests2) Enzymes3) Vector

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4) Host (Bacterium/Plant/Animal/intact) 1) Gene of interests

Identification and isolation of gene of interest

Gene library:Gene library is a random collection of cloned fragments in a suitable vector that ideally includes all the genetic information of that species. This is sometimes called as shot gun collection. The gene libraries are made by two methods referred to as complementary DNA and genomic DNA libraries

Method #1: Synthesis of complementary DNA In living organism some genes are expressed all the time i.e. irrespective of the growth stage of plant part but some other genes are expressed during certain stage and in certain organs. C- DNA libraries are used when it is known that the gene of interest is expressed in a particular tissue or cell type. The m RNA from the specific tissue is collected and used for DNA making.

Steps in c-DNA making 1. Isolate mRNA from the cells( processed transcript not the original

one)

(Reverse transcriptase + d NTPs+ primer)

2. Copy of single stranded c DNA from the m RNA

(Rnase +DNA polymerase + d NTPs)

3. Double stranded c DNA

4. Insert into a plasmid vector

5. Transform bacteria with recombinant plasmid

6. Selection of transformed bacterial cells containing gene of interest

Processing of heterogeneous mRNA inside the Nucleus

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Hair pin loop: This loop is formed when the single stranded DNA is isolated from the m RNA and DNA hybrid This hair pin loop acts as a primer for the synthesis of second complementary strands of cDNAAfter the formation of second strand the loop is removed by S1 Nuclease enzyme.

Method #2: Restriction fragment analysis by Southern blotting gel electrophoresis

1. Extract and purify DNA from the gene bank or genomic libraries2. Cut DNA with restriction enzyme (e.g. Eco R1)3. Separate DNA fragments by gel electrophoresis

4. Transfer DNA from the fragile gel to a nylon sheet and heat to separate the double strands.5. Hybridize gene of interest with a radio-labeled DNA or mRNA probe and expose with film to locate gene6. Insert into a plasmid vector

7. Transform bacteria with recombinant plasmid

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8. Selection of transformed bacterial cells containing gene of interest

2. Enzymes used in recombinant DNA technologies

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A. Restriction endonuclease enzymes

History

1962:

Arber and Dussoix discovered that E. coli can restrict the growth of the bacteriophage by degrading, specifically, the phage DNA

1965:

Arber discovered that E. coli methylate their DNA and this paved a way to distinguish their own DNA from invading foreign DNA - won Nobel Prize in Medicine in 1978.

1968:

The first “restriction” endonuclease was discovered in E. coli, but it was not sequence specific.

1970:

The first DNA-specific restriction endonuclease (type II) was discovered from Haemophilus influenzae -- HindII

An endonuclease is an enzyme that can cleave the phosphodiester bonds of a nucleic acid at an internal site (as opposed to cleavage by an exonuclease, which can only remove nucleotides from one of the ends of a nucleic acid). Some endonucleases cut internal bonds of DNAs or RNAs randomly. However, restriction endonucleases cut both strands of a double stranded DNA only at specific restriction sites. There are many different restriction endonucleases, and each is highly specific for a restriction site, which usually consists of 4, 6, or 8 base pairs, with a few exceptions.

Bacteria use restriction endonucleases as defense mechanisms, for example, against viral invasion. The name "restriction" endonuclease was originally given to these highly specific nucleases because they "restrict" invasion by foreign DNAs, such as those of bacterial viruses.

Each type of bacterium tends to have its own restriction enzymes and specific recognition sites. Foreign DNA is effectively destroyed by being cut at

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the recognition sites. Bacteria protect their genomes by modifying bases in the restriction sites in their own DNA, usually by methylamines. Thus, each strain typically has both a restriction endonuclease and a DNA methylase with the same target specificities. Depending on the different modes of action, the restriction enzymes have been classified into three main classes- type I, type II, type III. Out of these, type II restriction enzymes are used in recombinant technology as they can be used in vitro to recognize and cleave with in specific DNA sequences usually consisting of 4-8 nucleotides. Strictly speaking, the palindrome-specific (palindrome sequence) restriction endonucleases should be called type II restriction endonucleases and the other types are not very useful for recombinant DNA technology, and are generally ignored. Difference between Type I and Type II restriction endonuclease enzymes is mentioned below.

Type I Type II

Enzymes are made up of non identical subunits

Enzymes are made up of two identical subunits

Molecular weight is 400000 daltons Molecular weight ranges from 20,000 to 100000 daltons

Has both endonuclease and methylase activtity

Has only endonuclease activtity

Cleavage site is 1000 bp away from the recognition sequence

Cutting at the recognition site

Nonspecific cutting Specific cutting at restriction site or recognition site

The enzyme protect its own DNA by methylation

Not protected

ATP, Mg and S-adenosyl methionine are cofactors

Only Mg is needed as cofactor

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Naming of restriction endonucleases:

Restriction endonucleases are named for the species and strain of bacteria they are derived from. The first letter is for the genus, the next two for the species designation, the fourth for the strain, and the Roman numerals that follow designate which enzyme from that strain. In addition, the first three letters, standing for genus and species are normally italicized. Thus, Eco RI is the first restriction endonuclease derived from E. coli strain RY13.

Restriction sites:

The restriction site is the part of or specific sequences of DNA that can be cleaved by the restriction enzymes. The restriction sites are palindrome (they have the same double-stranded DNA base sequence in both directions) for the type II restriction endonuclease enzymes. As an example, the widely used Eco RI enzyme recognizes the sequence GAATT (read 5' to 3'). The different restriction enzymes and their restriction sites are given below.

Sticky ends:

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In many (but not all) cases, the DNA strand is not cut at the center of the restriction site. Thus, for example, Eco RI cuts its GAATTC recognition site between the G and the first A on each of the DNA strands (G|AATTC). This staggered cutting pattern leaves an overhanging segment of 4 base pairs AATT attached to the new 5' ends created by the cuts on each of the strands of all DNA fragments generated by Eco RI. These short unpaired segments are capable of forming transient double helical structures that can hold cut ends together long enough for DNA ligase to reseal them. If fragments cut from two different DNA molecules by the same restriction endonuclease are mixed, the ligation process will sometimes join the fragments in new combinations. For example, an isolated gene with sticky ends may be joined to the sticky ends of a circular plasmid that has been opened by a single cut with the same enzyme. This will form a larger circular plasmid whose DNA sequence now includes the gene. As described in greater detail below, this is the basic process used to clone genes.

Making recombinant DNA

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Isoschizomers:

In certain cases, two or more different enzymes may recognize identical sites. Enzymes from different sources that recognize the same site and cut it either the same way or differently are called isoschizomers. Sma I and Xma I in the list above are an example of isoschizomers that cut the same site in different ways.

Matching sticky ends:

It is possible to have an overhang at the 5'-end or the 3'-end, or to cut straight across the middle of the recognition site, leaving blunt ends. Thus, for example, DNA cut with Eco RI will all have an AATT overhang at the 5'-end of each strand, whereas DNA cut with Kpn I, will have a GTAC overhang at the 3'- end of each strand, and DNA cut with Sma I will have blunt ends. In order to use sticky ends for joining recombinant DNA molecules, it is necessary to have the same type of overhang on both fragments. When two fragments with compatible sticky ends encounter each other, the single-stranded overhangs will base pair in an antiparallel fashion to hold the fragments together long enough for DNA ligase to form new covalent phosphodiester linkages. Thus, it is easy to join pairs of fragments cut with the same enzyme. However, it is not possible to join a 5'- overhang and a 3'- overhang, even if the cut site sequences are identical, as in the case of the

The enzyme EcoRI cutting DNA at its recognition sequence

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isoschizomers Acc65 I and Kpn I, whose cut sites are G|GTACC and GGATC|C, respectively.

Regeneration of cut sites:

Ligating together two fragments that have been cut with the same restriction endonuclease generates a new cut site for that enzyme. This makes it easy to recover a cloned sequence by cutting it out of the vector with the same restriction endonuclease that was originally used to prepare it for cloning. In certain cases, two different restriction endonucleases may generate identical sticky ends even though their cut sites are not identical. A good example of this is Bam HI, whose cut site is G|GATCC, and Bgl I, whose cut site is A|GATCT. Both generate 5'- overhangs of GATC, which allows DNA fragments generated by these two enzymes to be ligated readily. However, the resulting double stranded link is no longer a palindrome and cannot be cut with either of the enzymes.

B. DNA ligases

These enzymes form phosphodiester bonds between the adjacent molecules and, covalently links two individual fragments of double stranded DNA. The most commonly used enzyme for cloning experiment is T4 DNA ligase.

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c. Alkaline phosphatase

The enzyme alkaline phosphatase (AP) removes the phosphate group from the 5’ end of a DNA molecule leaving a free 5’ hydroxyl group hence it is used to prevent unwanted self-ligation (circularization of vector DNA after cutting by the enzyme) vector DNA molecules in cloning procedures. This enzyme is isolated from bacteria or calf intestine.

3. Vectors for gene cloning

Vectors are the vehicles used to carry a foreign DNA sequence into a given host cell. For the successful cloning of gene of interest a vector should have the following characteristics.

a) Origin of replication,

b) A selectable marker to select the transformed host cells containing the vector from among those which do not have the vector,

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c) One unique restriction endonuclease recognition site (many restriction sites for many endonuclease enzymes but each enzyme will have single restriction site) and to enable foreign DNA to be inserted into the vector in order to generate a recombinant DNA molecule and,

d) It should be relatively small in size

e) The vector should not have pathogenecity

g) Must be present in multiple copies.

All the vectors (Plasmid, Phage, Yeast and Bacterial Artificial chromosomes) used for gene cloning purpose may not be having these above mentioned characteristics, so that to have a desirable vector, necessary modification is done in the natural structure of the vectors to have enhanced expression of gene of interest. The vectors obtained in this way are called derived vectors.

Plasmids

Plasmids are defined as autonomous elements, whose genomes exist in the cell as extra chromosomal units. They are self replicating, circular duplex DNA molecules present in number of copies in a bacterial cell, yeast cell or in organelles in eukaryotic cells. These naturally occurring plasmids have been modified to serve as vectors in the laboratory. Plasmid can carry 10-15 kbp of DNA (Gene of interest).

Plasmid based/ derived vectors

1) pBR322

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One of the most commonly used plasmid vector in gene cloning is pBR322 (Named after Bolivar and Rodriguez who constructed this). It was constructed after several alterations and modifications in earlier cloning vectors and derived from E. coli plasmid ColE1. It is 4,362 bp DNA with genes for resistance against two antibiotics- tetracycline and ampicillin.

2) pUC

pUC vectors are derived plasmid vectors. The plasmids belonging to pUC series of vectors are 2,700 bp long with ampicillin resistance gene, the origin of replication derived from pBR322 and the lac Z gene derived from E.coli. When the DNA fragments are cloned in this region of pUC, the lac gene is inactivated.

3) Yeast plasmid

Special vectors used to introduce DNA segments in yeast cells or a eukaryotic cell is being used to develop genetically engineered yeast cells. E.g. YIp or yeast integrative plasmids which allows transformation by crossing over and have no origin of replication. YEp or yeast episomal plasmids carry 2 micron DNA sequence including the origin of replication and rep gene. These vectors have been widely used to study yeast genome.

Bacteriophages

Bacteriophages are viruses that infect bacterial cells by injecting their DNA into these cells. They are used as vectors because they have a linear single or double stranded DNA molecule, which generate two fragments after breakage. These are later joined with foreign DNA to generate chimeric (recombinant) phage particle. The injected DNA is selectively replicated and expressed in the host cell resulting in a number of phages which burst out of the cell (lytic pathway) and further infect the neighbouring cells. E.g. M13, Lambda. Phage vector can carry 10-25 kbp of DNA. (Gene of interest)

Cosmids

Cosmids are constructed by combining certain feature of plasmids and the ‘cos’ sites of (specific DNA sequences) phage lambda. I.e. cos site of phage lambda is inserted into plasmid particles. The advantage of the using cosmids for cloning is that its efficiency is high to produce a complete genomic library

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of 10(6)-10(7) clones from only 1 microgrm of insert DNA. Cosmids vectors can carry 25-45 kbp of DNA. (Gene of interest)

Phagemids

Phagemids are prepared artificially by combining features of phages with plasmids. E.g. pBluescript II KS is derived from pUC19 and is 2961 bp long.

Plant viruses

A number of plant viruses can also be used as vectors for introducing foreign genes into cells and/or for gene amplification in host cells.

Plant viruses like Gemini viruses, cauliflower mosaic virus or CaMV and tobacco mosaic virus /TMV) are three groups of viruses that have been used as vectors for cloning of DNA segments. CaMV has a double stranded DNA molecule, 8kbp in size which infects the members of Cruciferae family. Gemini viruses are a group of single stranded DNA plant viruses infecting cassava, maize and other cereals.

Artificial chromosome (YAC and MAC) vectors

YAC vectors have linear double stranded DNA. YACs or Yeast Artificial Chromosomes are used as vectors to clone DNA fragments of more than 1Mb in size. These long molecules of DNA can be cloned in yeast by ligating them to vector sequences that allow their propagation as linear artificial chromosomes. These long DNA molecules can be generated and allow construction of comprehensive libraries in microbial hosts. A lot of work is going on to create mammalian artificial chromosomes (MACs) following the isolation of mammalian telomeres and centromere. However YACs have a low cloning efficiency (1000 clones/microgm) DNA as against 106-107 clones/microgm DNA for cosmids) and also it is difficult to recover large amount of pure insert DNA from individual clones.

BAC Vectors

BACs or Bacterial Artificial Chromosomes are vectors based on the natural, extra-chromosomal plasmid of E.coli- the fertility or F-plasmid, and are being used in genome sequencing projects. A BAC vector contains genes for replication and maintenance of the F-factor, a selectable marker and cloning sites and can accommodate up to 300-350 kb of foreign DNA.

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Shuttle vectors

Vectors containing two types of origin of replication which helps them to exist in both eukaryotic cell as well as E.coli (prokaryotic cells) are known as shuttle vectors.

Co integrate vector and Binary vectors

Both vectors are useful in Agrobacterium mediated gene transfer. Although historically the Co integrate vector first vector system to be developed, these vectors are less widely used. In this system, a recombined vector is constructed from a Ti plasmid and a small plasmid containing a gene of interest between two T-DNA borders.

Binary vector

Binary vectors are the major vector system used in Agrobacterium-mediated gene transfer. The binary vector system comprises two independent and complementing vectors: one vector having a T-region and the gene of interest and the other vector having a vir region.

Retriever vectors

A class of vectors which are used to retrieve specific genes from the normal chromosome of an organism like yeast through recombination. They are very useful in isolation of genes from yeast for molecular experiments like sequencing.

4. Host

The host for the gene cloning may be a bacteria or plant cell. To produce vector mediated transgenic plants, first the recombinant DNA is inserted into the Agrobacterium and then the agrobacteium is allowed to infect the plant cells.

Gene transfer methods in plants

Gene transfer methods in plants are of two types viz. Vector-mediated and the direct transfer of gene in to plant cell.

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1. Vector Mediated Gene Transfer a.Agrobacterium mediated transformation b.Viral mediated transformation 2. Vectorless or Direct DNA Transfer a) Particle gun/Particle bombardment/Biolistic / Micrproprojectile b) Microinjection c) Electroporation d) Chemical mediated gene transfers e Liposome mediated gene transfer or Lipofections f) Laser mediated gene transfer g) Pollen tube mediated h) Silicon fires mediated i) Inflorescence mediated

I. Ti plasmid mediated gene transfer or Vector-mediated or indirect gene transfer or Agrobacterium tumefaciens mediated

Agrobacterium tumefacien mediated plant transformation method is widely used among the various vectors based methods in plant transformation. This bacterium causes crown gall disease in plants in the Rosaceae family. Cells in the crown gall show abnormal cell division and cell elongation that leads to the formation of tumor like growth at the junction of plant root and shoot portion. Cells in the crown gall can be grown in artificial medium without the addition of growth hormones such as auxin and cytokinin.They also produces the unusual amino acids or sugar derivatives such as opines. (Nopalin, octopine, agropine and succinomopin)

Species of agrobacterium

Agrobacterium tumefaciens-Causes crown gall disease in dicots

Agrobacterium rhizogenes- Causes hairy root disease- monocots

Agrobacterium radiobacter- Avirulent strain

First two species Agrobacterium tumefaciens Agrobacterium rhizogenes are virulent and causes disease because of the presence of the plasmid namely Ti plasmid and Ri plasmid respectively.

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Crown gall symptom

Agrobacterium tumefaciens is known as “natural genetic engineer” of plants because these bacteria have natural ability to transfer T-DNA of their plasmids into plant genome upon infection of cells at the wound site and cause an unorganized growth of a cell mass known as crown gal in the plant.

Ti plasmid present in the Agrobacterium tumefaciens can be used as gene vectors for delivering useful foreign genes into target plant cells and tissues and thereby the production of transgenic plants for desired purposes. For that the oncogenes (tumor producing genes) of T DNA region is replaced with the gene of our interest. The process of removal of oncogenes from T DNA region is called disarming.

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Molecular structure of Ti plasmid

The Ti plasmid consisted of following parts.

i) T-DNA:

The T-DNA part of Ti plasmid contains oncogenes which are responsible for the tumor formation and opine synthesis. The oncogenes are flanked on both sides by border sequences called left and right border. These border sequences help in T-DNA transfer.

ii) Vir region:

This region is present outside the T-DNA part of Ti plasmid. Genes present in this region help in T-DNA transfer. There are 24 genes are present in this region and are arranged in 8 operons from A to H. All operons are involved in T-DNA transfer.

Functions of different operons of Vir region

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( For our convenience the different operons are arranged in alphabetical order here but the real arrangement is different from this order.)

Vir A:

It is the first operon to respond to the chemical substance (Acetosyringone) released by the wounded plant cells.

Vir B: Proteins of this operon are involved in the transfer of T-DNA to the plant cell by forming pores in the plant cell membrane. Among B operons, the B11 protein has ATPase activity and therefore provides energy for the transfer of T-DNA.

Vir C1 and C2:

Proteins of this operons act on the overdrive which is located near to the right border and thereby enhancing the transfer of T-DNA.

Vir D :( D1 and D2)

Proteins of this operon are involved release of the TDNA by nicking (Endonuclease activity) at the borders of TDNA. It is also involved in carrying of TDNA (having nuclear targeting sequence) by attaching itself (only D2 not D1) to the 5’ end of the TDNA to the plant cell. This attachment protects the oncogenes from other exonuclease enzymes activity.

Vir E2:

Proteins of this operon bind to the single stranded T-DNA and carry it to the plant nucleus.

Vir F:

Proteins of these operons are involved in T-DNA delivery.

Vir G:

Proteins of this operon are important for the transcriptional activation of other vir region operons. It is activated by the Vir A proteins.

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Vir H:

Proteins of these operons are involved in protecting the Agrobacterium from the plant compounds which inhibit the infection process.

ii) Ori: This region is meant for the replication of plasmid

iv) Tra: Helps in conjugation with nonvirulent bacterium

v) Opine catabolism: Used for the catabolism of opines

Role of bacterial chromosomal genes in TDNA oncogenes transfer

ChA and chB

Certain genes in the bacterial chromosome such as chA and chB code for exopolysaccharides important for attachment of bacterium to the plant.

Ch E:

This gene code for specific sugars that acts as a vir gene co inducer.

Steps in integration of T DNA into the plant cell

Agrobacteria attach to plant cell surfaces at wound sites.

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The signal binds to virA on the Agrobacterium membrane.

↓

VirA with signal bound activates virG

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Activated virG turns on other vir genes, including vir D and E.

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Vir D cuts at a specific site (Exonucleasae activity) in the left border and a similar sequence, the right border, delineate the T-DNA, the DNA that will be transferred from the bacterium to the plant cell

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Single stranded T-DNA is bound by vir E product as the DNA unwinds from the vir D cut site. Binding and unwinding stop at the right border.

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The T-DNA is transferred to the plant cell through the path created by the Vir B proteins and T-DNA integrates in nuclear DNA.

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T-DNA codes for proteins that produce hormones and opines. Hormones encourage growth of the transformed plant tissue (crown gall). Opines feed bacteria a carbon and nitrogen source.

(See the diagrams below)

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Steps in integration of T DNA into the plant cell (diagrammatic)

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Method of Ti plasmid mediated gene transfer in plants

Gene transfer using Ti plasmid into plant cell is done in two steps

Step; I Construction of recombinant Ti plasmid

Step: II Transformation and regeneration of plants

Construction of recombinant Ti plasmid

As such the Ti plasmid of Agrobacterium can not be used for gene transfer purpose because if it’s large size, presence of unwanted genes (which promote tumor formation) in the T DNA region and absence of unique restriction site. So some modification is done to the Ti plasmid structure to make it effective for plant transformation. It is achieved by the development of Co integrate and Binary vector systems.

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The simplest way to exploit Ti plasmid to genetically transform plants is just inserting the desired DNA sequence into the T-DNA region and then uses the Ti plasmid and A.tumefaciens to deliver and insert this gene into the genome of the susceptible plant cell. Though Ti plasmids are effective natural vectors they had certain limitations.

The phytohormone produced by transformed cells growing in culture prevents their regeneration into mature plants. Hence auxins and cytokinin genes must be removed from the Ti –plasmid derived cloning vector.

The opine synthesis gene must be removed as it may divert plant resources into opine production in transgenic plant.

Generally, Ti- plasmids are large in size (200-800 kb).For effective cloning, large segments of DNA that are not essential for cloning has to be removed.

As Ti plasmid does not replicate in E.coli,Ti-plasmid based vectors require an ori that can be used in E.coliTo overcome these constraints, Ti plasmid based vectors were organized with the following components:

A selectable marker gene that confers resistance to transformed plant cells. As these marker genes are of prokaryotic origin, it is necessary to put them under the eukaryotic control (plant) of post transcriptional regulation signals, including promoter and a termination- poly adenylation sequence, to ensure that it is efficiently expressed in transformed plant cells.

An origin of replication that allows the plasmid to replicate in E.coli.

The right border sequence of the T-DNA which is necessary for T-DNA integration into plant cell DNA.

A polylinker (MCS) to facilitate the insertion of cloned gene into the region between T-DNA border sequences.As these cloning vectors so organized lacked vir genes, they cannot effect the transfer and integration of T-DNA region into host plant cell. So two Ti-plasmid derived vector systems were developed. They include: 1. Binary vector system 2. Co-integrate vector system1. Binary vector system The binary vector system contains either E.coli or A.tumifaciens origins of DNA replication, i.e.an E.coli - A.tumifaciens shuttle vector or a single broad

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host range ori. In either case no vir genes are present on the binary cloning vector. All the cloning steps are carried out in E.coli before the vector is introduced into A.tumifaciens. The A.tumifaciens strain carries a modified (disarmed) Ti plasmid that contains a complete set of vir genes but lack portions of T-DNA region, so that this T-DNA cannot be transferred. With this system, the defective Ti plasmid synthesizes the vir gene products that mobilize the T-DNA region of the binary cloning vector plasmid. By providing the proteins encoded by the vir genes, the defective Ti plasmid acts as helper plasmid ,enabling the T-DNA from binary cloning vector to be inserted into the plant chromosomal DNA. Since transfer of T-DNA is initiated from the right border, the selectable marker which will eventually be used to detect the presence of the T-DNA inserted into the plant chromosomal DNA is placed next to the left border. If selectable marker is present adjacent to the right border, only small portion of T-DNA will be transferred resulting in a plant with only selectable marker and no gene of interest. Few binary vectors are developed with selectable markers one adjacent to the right and the other adjacent to the left border.

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Ti plasmid vector systems are often working as binary vectors

Virulence region

T DNA region removed

ori for A. tum

Gene of interest

Plant selectable marker

Bacterial selectable marker

ori for A. tumefaciensori for E.coli

HELPER plasmid

Disarmed Tiplasmid

DISADVANTAGE: Depending on the orientation, plasmids with two different origins of replication may be unstable in E. coli

ADVANTAGE: small vectors are used, which increases transfer efficiency from E. coli to Agrobacterium.

No intermolecular recombination is needed

2. Co-integrate vector system In the co integrate vector system the cloning vector has a plant selectable marker gene, the target gene, the right border, an E.coli origin of the DNA replication, and a bacterial selectable marker gene. The co-integrate vector recombines with the modified (disarmed) Ti plasmid that lacks both the tumor producing genes and the right border of the T-DNA within A.tumifaciens, and the entire cloning vector becomes integrated into the disarmed Ti plasmid to form a recombinant Ti plasmid. The co-integrate cloning vector and the disarmed helper Ti plasmid both carry homologous DNA sequence that provide shared sites for in vivo homologous recombination, normally these sequence lie inside the T-DNA region. Following recombination, the cloning vector becomes part of the disarmed Ti

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plasmid, which provides the vir genes necessary for the transfer of T-DNA to host plant cell. The only way that this cloning vector can be maintained in A.tumifaciens is a part of the co-integrated structure. In this co-integrated configuration genetically engineered T-DNA region can be transferred to plant cells.

II.Transformation and regeneration of plants

Naked plant cells or protoplasts or leaf disc are placed into a petridish and covered by a nutrient solution.

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Addition of Agrobacterium tumefaciens containing either the co integrate or binary vector to the nutrient solution (co cultivation)

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Incubation of all the contents for several days at 25-30oC.

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Plating of cells on nutrient agar with an appropriate antibiotic (carbenicillin) to kill the Agrobacterium.

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↓Selection of transformed cells by growing them on the selective media that contains kenamycin antibiotic (Kenamycin kill the non transformed cells and only the plant cells that have taken up the gene for antibiotic-resistance with

its foreign DNA will grow on this)

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Development of callus from the transformed cells

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Development of shoot

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Development of root

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Transgenic plant

Agro infection

Cereals are important as they are the major food crop for us. Under natural circumstances, Agrobacterium tumefaciens does not attack cereals therefore it cannot be used to modify the genome of these plants. However, it was observed that if the DNA of wheat dwarf virus is inserted into a Ti plasmid, the bacteria carrying Ti plasmids will attack wounded wheat plants. Similarly, bacteria carrying Ti plasmids with DNA from maize streak virus will attack wounded maize plants. This technique is called agroinfection which was first used in 1987. In this mature cereal plants are infected with plasmid-carrying bacteria. Transformed cells develop symptoms of the viral disease, and do not need to be identified by selection. The infection spreads from cell to cell until all the cells of the cereal plant have been transformed. Efforts are being made to use this technique to introduce foreign DNA into cereals.

Problems in producing monocot transgenic plants through agrobacterium mediated gene transfer

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As mentioned earlier the production of transgenic plants in monocotyledons was not so successful due to two reasons:

a) The Ti plasmids could not be used to transform monocots because monocots are not ordinarily infected by Agrobacterium, which is generally used to transform dicots.

b) The regeneration of plants from protoplasts or single cells which is generally used for transformation was not possible.

These limitations have been solved by using alternative and new methods of DNA uptake and regeneration protocols for crops like rice and maize. In rice (both in japonica and indica varieties, there have been successful production of transgenic plants. In Maize, a reporter gene for neomycin phosphotransferases (NPT II) associated with 35S promoter region of the cauliflower mosaic virus (CMV) was used for production of transgenic plants.

II) Direct gene transfer or Vectorless methods

(Refer H.S CHAWLA BOOK –INTRODUCTION OT PLANT BIOTECHNOLOGY)

a.) Particle gun/Particle bombardment/Biolistic /Micro projectile/G ene gun/Shot gun.

Monocot plants are often resistant to A. tumefaciens transformation due to inappropriate wound responses and hormonal differences between dicots and monocots. Biolistic devices have become much more sophisticated and are routinely used to create monocot transgenic plants such as rice, corn and sorghum plants. The micro projectile bombardment method was initially named as biolistics by its inventor Sanford (1988).

This instrument uses an evacuated sample chamber that positions the target seedling directly in the path of DNA-coated tungsten or gold particles traveling at a velocity of 1,100 mph.

Particle bombardment is simple both conceptually and in practice. In this method, typically, plasmid DNA is prepared by standard methods and precipitated onto tungsten or gold particles using CaCl2. Spermidine and PEG are included to protect the DNA during precipitation, and the particles are

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washed and suspended in ethanol before drying onto Mylar aluminized foil. The prepared Gold or tungsten particles (1-3 micrometers) called microparticles. These are fired against a retaining screen that allows the microprojectiles through, to strike the target tissue.

A pressurized hand-held gene gun can also be used to shoot DNA-coated gold particles directly into the meristematic tissue of plants growing in experimental fields.

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Advantages and disadvantages of particle bombardment

Particle bombardment is widely used because it circumvents two major limitations of the agrobacterium system.1) First, it is possible to achieve the transformation of any species and cell type by this method because DNA delivery is controlled entirely by physical rather than biological parameters . The range of plant species transformable by particle bombardment is therefore restricted only by the competence of cells for regeneration, and the technique is genotype independent and thus useful for the transformation of elite cultivars as well as model varieties. However, careful optimization is required to tailor the method for different species and different cell types and to achieve the highest efficiency transformation with the least cell damage. Important parameters include acceleration method, particle velocity (controlled by the discharge voltage and/or gas pressure), particle size, and the use of different materials (tungsten, gold). It has also been shown that, for some species and/or tissues, osmotic pretreatment prior to bombardment increases the transformation efficiency. This can be achieved by partial drying or the addition of osmoticum (mannitol and/or sorbitol) to the culture medium. In general, stable transformation by any direct DNA transfer method occurs at a much lower frequency than transient transformation.

2) Second, particle bombardment allows the stable and heritable introduction of many different genes at once using different plasmids, as these tend to concatemerize to form one DNA cluster that integrates at a single locus. Conversely, multiple transformations using the Agrobacterium system requires the cointegration of all the genes in the same T-DNA.

Disadvantages of the particle bombardment method

1) A potential disadvantage of the particle bombardment method is the cost of purchasing or hiring the bombardment device . However, a number of articles have been published providing instructions for the construction of alternative economical devices, such as the particle inflow gun based on flowing helium.

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2) Another disadvantage of particle bombardment is the tendency for DNA sequences introduced by this method to undergo complex rearrangements prior to or during integration. Transgene rearrangement is a pitfall of all direct DNA transfer methods but is perhaps more acute in particle bombardment because the forces involved may cause more DNA fragmentation than other methods and because bombarded plant cells may be induced to produce DNA degradation and repair enzymes in response to their injury. This may limit the usefulness of particle bombardment for the introduction of large DNA molecules. However, although an upper size limit has not been established, it has been possible to introduce YAC complementary DNA (cDNA) clones into plant cells by this method.

b. TRANSFORMATION OF PROTOPLASTS

Protoplasts are plant cells from which the cell wall has been enzymatically or mechanically removed. As with animal cells, the contents of the protoplast cytoplasm are enclosed in a cell membrane, and the transformation of protoplasts can be achieved using many of the procedures routinely used to transfect cultured animal cells. Two procedures that are commonly used to introduce DNA into protoplasts include the uptake of naked DNA mediated by polyethylene glycol and a divalent cation (either Ca2+ or Mg2+) and electroporation, although other agents such as lipofection have also been used.

Plant protoplasts can also be transformed using Agrobacterium. Following transformation, protoplasts are placed on selective medium and allowed to regenerate new cell walls. The cells then proliferate and form a callus from which embryos or shoots and roots can be regenerated with appropriate hormonal treatments. In principle, protoplasts from any plant species can be transformed, but the technology is limited by the ability of protoplasts to regenerate into whole plants, which is not possible for all species. Although economical and potentially a very powerful procedure, direct transformation of protoplasts is disadvantageous because of the long culture times involved. Not only does this mean that the transformation process itself is time consuming, but cells cultured for extensive periods either fail to regenerate or frequently regenerate plants that show full or partial sterility and other phenotypic abnormalities somaclonal variation. Protoplast preparation, maintenance, and transformation is a skilled technique, which, compared with Agrobacterium mediated transformation and particle

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bombardment procedures, requires a greater investment in training. Advances in other transformation techniques are making protoplast transformation obsolete.

3. OTHER DIRECT TRANSFORMATION METHODSAlthough the three( including the agrobacterium transformation) methods just discussed provide a route for the transformation of most plant species, there is value in the continuing exploration of novel transformation techniques. In some cases, it is possible to learn lessons from animal cell technology, where a number of alternative transformation systems have been established.

c. Microinjection of DNA into animal eggs and zygotes, for example, is a routine procedure for generating transgenic animals that is applicable to all animal species. Microinjecting DNA into plant cells, while laborious and technically challenging is advantageous in that it is the only current strategy available to study transformation events at a single-cell level. The injection of DNA into plant zygotes is being explored as a method for regenerating whole transgenic plants; however, it is currently highly inefficient. For example, isolated maize zygotes microinjected with DNA mimic embryonic development but abort at an early stage. Recently, the same technique has been applied to barley protoplasts, resulting in the recovery of numerous embryo-like structures but only two transgenic plants.

In 1986, Kurata et al. (57) devised a novel transformation strategy for animal cells in which DNA was taken up from solution after piercing the cell membrane with a finely focused laser beam . Although applicable to only a small number of cells, this technique achieved stable transformation efficiencies of 0.5%. A similar technique for plant transformation is available, although stable transformation has not been achieved.

d. Electroporation is used for the transformation of plant protoplasts but has been adapted for the transformation of walled plant cells in tissues. For example, maize explants have been transformed by electroporation either following partial enzymatic removal of the cell wall or without treatment. This technique has also been successfully applied to rice and sugarcane explants.

e. A novel approach for the transformation of maize cells is to mix cells with silicon carbide whiskers . These penetrate the cells, allowing DNA uptake

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presumably through transient pores. This method is cheap, reproducible, and very simple to perform. Although initially used only for maize transformation, the technique has now been applied to other cereals.

4 TRANSFORMATIONS WITHOUT TISSUE CULTURE

Most of the transformation procedures discussed so far have one major disadvantage— they rely on tissue culture for the regeneration of whole plants. As discussed, the tissue culture requirement adds significantly to the time and cost of producing transgenic plants and is perhaps the most serious limitation to the technology because it restricts the range of species amenable to genetic manipulation. Methods that circumvent tissue culture all together —in planta transformation systems—are therefore highly desirable, although until recently they were available only for the model dicot Arabidopsis thaliana.

These techniques include seed transformation, in planta DNA inoculation, and flower dipping or infiltration, and all involve Agrobacterium-mediated transformation.

For seed transformation, Arabidopsis seeds are imbibed and co-cultivated with Agrobacterium tumefaciens and then germinated. This is convenient in Arabidopsis because a large number of seeds are produced (10,000 per plant) and the seeds are very small. The procedure is relatively inefficient and is not very reproducible, but because the plant is small it is relatively easy to screen for transformants. A more efficient procedure is to inoculate plants with bacteria after severing the apical shoots. The bacteria infect the wounded meristematic tissue and about 5% of shoots emerging from the wound site are transgenic.

Another method is to dip Arabidopsis flowers into bacterial suspension or vacuum infiltrate the bacteria into the flowers, at around the time of fertilization. This generates plants with transformed and wild-type sectors of vegetative tissue, and these give rise to transgenic progeny at a low frequency (5—10 offspring per plant). Again, the low transformation frequency is acceptable because of the large number of seeds and the ease of screening.

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In planta transformation techniques involving direct DNA transfer have also been developed. These techniques have been applied to a number of economically important crop plants, although they have yet to catch on because they have low reproducibility. These techniques have met with variable levels of success.

Generally, these techniques fall into three classes:

1. Introduction of DNA into flowers around the time of fertilization, followed by the recovery of transgenic seeds—this has been attempted in cotton and rice.

2. Imbibing seeds with a DNA solution—this has been used for soybean

3. Introducing DNA into pollen or mixing pollen with DNA before applying to stigmata—this has been attempted with rice and tobacco (73).

MOLECULAR MARKERS/ MOLECULAR BREEDING

What is marker?

In plant breeding marker is defined as sign or flag or tag for the identification of characters or traits of plant. The identification and the inheritance of a character are possible because the marker and the trait area closely linked and inherit together.

Basic properties of a marker

Heritable Polymorphic

Co dominant or Dominant

Polymorphisms:

Different morphological form of expression of particular trait or character due to base composition at particular locus/loci in different plants is called as polymorphism. This variable expression of particular trait is occurred due to mutations (insertions, deletions of nucleotides etc.). For e.g. Flower color is

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controlled by single gene but this gene will have more than one form of expression in different plants.

Heritable:

Transfer of trait or character from one generation to subsequent generation is called as heritability

Dominance:

The character which is expressed the in the first generation plants is called as dominance.

Co dominance

Expression of one or more genes together without any dominace effect is called as co dominance.

Major types of markers

1. Morphological (Classical or Visible) Markers

2. Biochemical Markers

3. DNA or Molecular Markers

Morphological markers

In conventional breeding methods, the plant selection is done based on the phenotypic appearance i.e. visible characters expressed morphologically in the phenotype are used for identification, selection and to study the inheritance of a character. Markers which are themselves phenotypic traits are called morphological (classical or visible) markers. Morphological (classical or visible) markers may be qualitative e.g. flower colour (particular plant variety posses particular flower colour) or quantitative in nature e.g. plant height and yield etc. Morphological markers could be either dominant or recessive. Both the heterozygous and the homozygous dominant form of a particular gene will have the same expression and both cannot be distinguished in the phenotypic expression because only the expression of

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sdominant allele of a gene or the suppression of recessive form of gene. In case of recessive trait, the homozygous form of gene only is expressed. study of inheritance of a particular character based on phenotypic expression is time consuming process, because the breeder has to wait till the plant comes to maturity (in case of flower color and yield) and tedious (selfing, crossing and handling of large population in the segregating population) process. The plant breeder has to wait till the maturity of the pant. All the genes present in plant are not expressed morphologically (limited expression or less in number of gene expression) and also the specific characters are expressed at specific developmental stage of plant. One character cannot be clearly distinguished from others and no stable inheritance because of role of environment on the morphological characters. The morphological markers are also iinfluenced by the genetic background of ancestors and rarely the markers may produce lethal effects.

Biochemical markers: (Isozymes or allozymes)

The proteins produced by gene expression can be used as markers in plant breeding programmes.

1. When enzymes of similar function are produced at different loci/gene

2. Isozymes show heterozygous banding patterns depends on the complexity of the number of enzyme sub-units present,

3. Isozymes segregate according to Mendelian genetics with co-dominance in heterozygotes

Isozymes: The different molecular forms of the same enzyme.

Limitations of biochemical Markers

Multiple molecular forms of an enzyme. Products of gene expression.

Influenced by the environment.

Some times genetic control is very complex.

Possible interaction between genetic loci.

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Cannot be a stable marker unless the isozyme has some direct role with the trait.

Highly conserved among the closely relatives.

Need better understanding on the protein biochemistry and physiology

Caution

It is always advisable to use genetically well defined isozymes as markers. In other words, it would be advisable for the investigators to follow the ontogeny of isozymes which are known to be regulated by specific genes. Check for reproducibility.

Molecular Markers or DNA Markers

Molecular markers are specific fragments of DNA that can be identified within the whole genome. The markers are found at specific locations of the genome. They are used to ‘flag’ the position of a particular gene of interest or and also to study the inheritance of a particular character because the molecular marker is usually stay linked with the characteristics of interest. Because of the linkage, both the marker gene and the gene of interest inherit together. Since the marker indicates the presence of the desired character, individuals can be selected on the basis of the presence of molecular marker in the plant. Molecular markers are generated by different techniques. The molecular markers generated provide a true representation of the genetic make up at the DNA level and genetic map of a particular plant can be obtained using molecular markers. Screening methods for the molecular markers should be efficient, reproducible and easy and cost effective to carry out in plant breeding programme.

In conventional breeding methods, closeness of the marker to the gene of interest or the distance between two genes is known by assessing the products of crossing over between two parents.

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.

Properties of DNA Markers

1. Abundance, i.e. more in numbers.2. Ubiquitous

3. Highly polymorphic I e. all the variant form of a gene can be known.

4. Stable inheritance

5. No environmental influence

6. Not affected by developmental stage of organism

7. Co- dominant or dominant

8. Arise from different DNA mutations such as point mutations, substitution mutations, insertions or deletions and errors in replication-tandemly repeated DNA

9. They do not represent target genes themselves

10. They act as ‘Signs’ or ‘Flags for the presence of a trait

11. Located to close proximity to genes of study – ‘Gene tags’

12. Do not affect the trait – located near or linked to genes controlling the trait.

Types of DNA markers

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1. Nucleic acid hybridization based

Restriction Fragment Length Polymorphism (RFLP)

2. Polymerase chain reaction (PCR)-based

Randomly Amplified Polymorphic DNA (RAPD) Amplified Fragment Length Polymorphism (AFLP)

Microsatellites or Simple Sequence Repeats (SSR)

SNP- Single nucleotide polymorphism

3. Others

Sequence Tagged Sites (STS) Sequence Characterized Amplified Regions (SCAR)

Variable Number Tandem Repeats (VNTR)

Inter Simple Sequence Repeats (ISSR)

(1) Based on Nucleic acid (DNA) hybridization

The Restriction fragment length polymorphism (RFLP) was the very first technology employed for the detection of polymorphism based on the DNA sequence differences. RFLP is mainly based on the altered restriction enzyme sites, as a result of mutations and recombination of genomic DNA. The procedure involves the isolation of genomic DNA and it’s digestion by restriction enzymes. The fragments are separated by electrophoresis and finally hybridized by incubating with and labeled probes.

Restriction fragment analysis detects DNA differences that affect restriction sites

• Restriction fragment analysis indirectly detects certain differences in DNA nucleotide sequences.

• After treating long DNA molecules with a restriction enzyme, the fragments can be separated by size via gel electrophoresis.

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• This produces a series of bands that are characteristic of the starting molecule and that restriction enzyme.

• The separated fragments can be recovered undamaged from gels, providing pure samples of individual fragments.

• We can use restriction fragment analysis to compare two different DNA molecules representing, for example, different alleles.

• Because the two alleles must differ slightly in DNA sequence, they may differ in one or more restriction sites.

• If they do differ in restriction sites, each will produce different-sized fragments when digested by the same restriction enzyme.

• In gel electrophoresis, the restriction fragments from the two alleles will produce different band patterns, allowing us to distinguish the two alleles.

• Restriction fragment analysis is sensitive enough to distinguish between two alleles of a gene that differ by only base pair in a restriction site.

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• Gel electrophoresis combined with nucleic acid hybridization allows analyses to be conducted on the whole genome, not just cloned and purified genes.

• Although electrophoresis will yield too many bands to distinguish individually, we can use nucleic acid hybridization with a specific probe to label discrete bands that derive from our gene of interest.

• The radioactive label on the single-stranded probe can be detected by autoradiography, identifying the fragments that we are interested in.

• We can tie together several molecular techniques to compare DNA samples from three individuals.

• We start by adding the restriction enzyme to each of the three samples to produce restriction fragments.

• We then separate the fragments by gel electrophoresis.

• Southern blotting (Southern hybridization) allows us to transfer the DNA fragments from the gel to a sheet of nitrocellulose paper, still separated by size.

• This also denatures the DNA fragments.

• Bathing this sheet in a solution containing our probe allows the probe to attach by base-pairing (hybridize) to the DNA sequence of interest and we can visualize bands containing the label with autoradiography

• For our three individuals, the results of these steps show that individual III has a different restriction pattern than individuals I or II.

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• Southern blotting can be used to examine differences in noncoding DNA as well.

• Differences in DNA sequence on homologous chromosomes that produce different restriction fragment patterns are scattered abundantly throughout genomes, including the human genome.

• These restriction fragment length polymorphisms (RFLPs) can serve as a genetic marker for a particular location (locus) in the genome.

• A given RFLP marker frequently occurs in numerous variants in a population.

• RFLPs are detected and analyzed by Southern blotting, frequently using the entire genome as the DNA starting material.

• These techniques will detect RFLPs in noncoding or coding DNA.

• Because RFLP markers are inherited in a Mendelian fashion, they can serve as genetic markers for making linkage maps.

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• The frequency with which two RFPL markers - or a RFLP marker and a certain allele for a gene - are inherited together is a measure of the closeness of the two loci on a chromosome.

(2)Molecular markers based on PCR amplification.

A) Random Amplified Fragment Length Polymorphism

Polymerase chain reaction (PCR) is a novel technique for the amplification of selected regions of DNA. The most important advantage is that even a minute quantity of DNA can be amplified and the PCR- based molecular markers require only a small quantity of DNA to start with. Random amplified polymorphic DNA (RAPD) markers use PCR. For amplification purpose the DNA is isolated from the genome and is denatured. The template molecules are annealed with primers and amplified by PCR. The amplified products are separated on electrophoresis and identified. Based on the nucleotide alterations in the genome, the polymorphisms of amplified DNA sequences differ which can be identified as bands on gel electrophoresis.

The polymerase chain reaction (PCR) clones DNA entirely in vitro

• Template - the DNA to be amplified• Primers - 2 short specific pieces of DNA whose sequence flanks

the target sequence

Forward

Reverse

• Nucleotides - dATP, dCTP, dGTP, dTTP

• Magnesium chloride - enzyme cofactor

• Buffer - maintains pH & contains salt

• Taq DNA polymerase – thermophillic enzyme from hot springs

• Heat (94oC) to denature DNA strands

• Cool (59oC) to anneal primers to template

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• Warm (72oC) to activate Taq polymerase, which extends primers and replicates DNA

• Repeat 40 cycles

• DNA cloning is the best method for preparing large quantities of a particular gene or other DNA sequence.

• When the source of DNA is scanty or impure, the polymerase chain reaction (PCR) is quicker and more selective.

• This technique can quickly amplify any piece of DNA without using cells.

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• The DNA is incubated in a test tube with special DNA polymerase, a supply of nucleotides, and short pieces of single-stranded DNA as a primer.

• PCR can make billions of copies of a targeted DNA segment in a few hours.

• This is faster than cloning via recombinant bacteria.

• In PCR, a three-step cycle: heating, cooling, and replication, brings about a chain reaction that produces an exponentially growing population of DNA molecules.

• The key to easy PCR automation was the discovery of an unusual DNA polymerase, isolated from bacteria living in hot springs, which can withstand the heat needed to separate the DNA strands at the start of each cycle.

• PCR is very specific.

• By their complementarity to sequences bracketing the targeted sequence, the primers determine the DNA sequence that is amplified.

• PCR can make many copies of a specific gene before cloning in cells, simplifying the task of finding a clone with that gene.

• PCR is so specific and powerful that only minute amounts of DNA need be present in the starting material.

• Occasional errors during PCR replication impose limits to the number of good copies that can be made when large amounts of a gene are needed

• Devised in 1985, PCR has had a major impact on biological research and technology.

• PCR has amplified DNA from a variety of sources:

• fragments of ancient DNA from a 40,000-year-old frozen wooly mammoth,

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• DNA from tiny amount of blood or semen found at the scenes of violent crimes,

• DNA from single embryonic cells for rapid prenatal diagnosis of genetic disorders,

• DNA of viral genes from cells infected with difficult-to-detect viruses such as HIV.

B) Amplified Fragment Length Polymorphism

Amplified fragment length polymorphism (AFLP) is a novel technique involving a combination of RFLP and RAPD. AFLP is based on the principle of generation of DNA fragments using restriction enzymes and oligonucleotide adaptors (or linkers), and their amplification by PCR.

(3)Others

Microsatellites or VNTRS

Microsatellites are the tandemly repeated multiple copies of mono-, di-, tri-, and tetra nucleotide motifs. In some instances, there are unique flanking sequences present in the repeat sequences. Primers are designed for such flanking sequences to detect the sequence tagged microsatellites (STMS) which is done by PCR.

Applications of molecular markers in genetics and biotechnology

1. Genome mapping2. Gene mapping & Gene tagging (Oligogenes)

3. QTL mapping (Polygenes)

4. Marker Aided Selection

5. Genetic Diversity Analysis

6. Genetic Purity Analysis

Marker assisted selection or breeding

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Selection of trait or plant breeding material using either DNA marker alone or in conjunction with morphological marker (phenotypically expressed characters) is called as marker assisted selection or marker assisted breeding. In conventional plant breeding methods, the plants are selected on the basis of phenotypic expression of characters i.e. morphological characters alone. Morphological markers have their own limitations and make the conventional breeding method tedious and time consuming. But in marker assisted breeding the plants are selected using molecular markers along with the morphological markers to make the plant breeding method effective.

  Applications and benefits of MAS  Application Benefits

Varietal identificationDNA markers used to generate ‘fingerprint’ which can discriminate different varieties or hybrids.  This method can also be used to assess seed purity

Genetic diversity assessment

DNA marker data can be used to infer genetic relatedness of germplasm which can be applied to broaden the genetic base.  Allelic information can also be generated by targeting specific

Marker assisted backcrossing

Accelerates the backcrossing by efficient selection of a target gene and selecting against the donor parent (background selection).  Linkage drag can be minimized by selecting backcross lines with recombination events between the target locus and tightly linked flanking markers (recombinant selection).

Marker-assisted pyramiding

Process of combining several genes together into a single genotype.  Often used for combining disease resistance loci, this is difficult or impossible using conventional methods.

Early generation selection

Can be used in any breeding scheme such as the pedigree method to rapidly discard unwanted breeding lines.  This also reduces the number of lines to be tested which permits more efficient use of glasshouse or field space

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Combined MASSelection based on both phenotypic screening and marker data for improved genetic gain or to reduce screening costs for traits requiring expensive assays.

Off season nursery screening

Markers used as a replacement for phenotyping, which allows more generations to be screened per year

Advantages of MAS

Speed – DNA can be extracted from tissue from the first leaves or the cotyledons of a plant. Trait information can be discovered with markers prior to pollination allowing more informed crosses to be made.

Consistency – Markers remove the impact of environmental variation that often complicates phenotypic evaluation.

Biosafety – Using markers in screening for disease resistance means not having to introduce the pathogen into breeding populations. Particularly for livestock breeding this delivers a very important level of biosafety.

Efficiency – Screening progeny early in the process allows a breeder to drop “also-rans” from the program more quickly. Most breeding programs that use markers still evaluate the same number of plants in the field however the level of genetic quality is vastly increased because of the early-stage screening that has been carried.

Complex traits – Most multigenic or polygenic traits or quantitative characters (for e.g. yield) are very difficult to manage through conventional plant breeding. The statistical chance of getting the required allele at each of a number of loci is very low. Markers allow you to skew the odds in your favor.

END OF II UNIT

UNIT-III

Role of TransgenicOverall view of transgenic and their role in crop improvement-resistance to biotic stress- Insect - virus and disease resistance-resistance to abiotic stress-herbicide resistance-transgenics for quality

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parameters-increased vitamin A and zinc-manipulation for hormone biosynthesis

Overall view of transgenic and their role in crop improvement

What Are Transgenic Plants?

Transgenic crop plant contains a gene or genes which have been artificially inserted instead of the plant acquiring them through pollination. The inserted gene sequence (known as the transgene) may come from another unrelated plant, or from a completely different species: For e.g. transgenic Bt corn, for example, which produces its own insecticide, contains a gene from a bacterium. Plants containing transgenes are often called genetically modified or GM crops, although in reality all crops have been genetically modified from their original wild state by domestication, selection and controlled breeding over long periods of time.

Purpose of developing transgenic plants in India

1. To provide food security. This can be achieved by improving e the efficiency of production i.e. yield/hectare and bringing the marginalized land (which are saline or alkaline) for food production. This is essential to feed the people of developing countries i.e. 18% of the world population is in the developing world. The food security and access to food can be assured by developing plants resistant to pests, diseases and other abiotic factors such as cold, drought and salt. Changes in the patterns of global climate and alterations in use of land (conversion of land for nonagricultural purpose) can also be overcome by developing and cultivating transgenic plants.

2. To provide nutritional security . In addition to lack of food, deficiencies of micro-nutrients especially vitamin A, iodine and iron are widespread among the children of developing countries. This can be overcome by developing plant which provides different nutrients along with the food and may help to improve human health by addressing malnutrition and under-nutrition. GM technology has also shown its potential to address micronutrient deficiencies and thus reduce the national expenditure and resources required to implement the current supplementation programmes.

3. To make the environment free from pollution or reduce effect of modern agricultural practices on soil, water and on human health. These all can be

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overcome by adopting sustainable agriculture. Adoption of transgenic plants for cultivation purpose is one way of implementing sustainable agriculture so that the resources for agricultural production can be conserved and the agricultural production can be sustained for more food production in the future.

4. To provide employment to the rural people (650 million of the poorest people live in rural areas) whose business is mainly agriculture. 5. To overcome the post harvest losses of food materials by developing product with long shelf life through transgenic plants.

Risks associated with the GM food or transgenic plants. Biosafety measures should be given due importance to reduce or to avoid the risks associated with the transgenic crops. Some of the risk associated with the transgenic plants or GM foods have been mentioned below

Development of super weeds may be resulted due to the movement of transgene (transfer of pollen in case of cross pollinated crops.) from the transgenic crop (herbicide resistant transngenic variety) variety to other varieties and wild relatives (weeds).

Similarly, there is widespread apprehension that transgene products could be toxic or allergenic to humans and animals. For e.g. Transfer of antibiotic resistance genes may have some undesirable effect in human beings.

Transgenic crops (e.g. pest-resistant varieties) could have adverse impact on non-target organisms or beneficial insects.

Emergence of more virulent forms of pests and pathogens is also an important concern in case pest resistant transgenic.

Loss of diversity may occur due predominance growing of transgenic crops. The local cultivars and land races may become obsolete.

Concerning the need and the problems (risk associated) of transgenic plants, the following priorities and strategies can be adopted in developing transgenic plants for the country like India.

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1. Target crops for transgenic improvement

Crops that contribute the most to our food and nutritional security (e.g. rice, wheat, chickpea, pigeon pea, groundnut, mustard, tomato, peas, cauliflower, banana, etc.) should be accorded high priority.

Similarly, commercial crops such as sugarcane and cotton that provide rural employment and occupy large area also deserve high priority.

The international trade opportunities should be kept in mind while selecting crops for transgenic improvement.

2. Breeding behavior of the crop

Crop priority from the transgene movement angle will be vegetative propagated > self-pollinated > cross-pollinated to avoid the pollen transfer.

3. Presence of wild relatives of the crop

If the transgenic crops are grown in centers of crop origin/diversity; there is likelihood of transgene escape to the wild relatives. So transgenic crops should not be deployed in areas where the wild relatives growing in nature.

4. Consumption pattern of the crop

Priority-setting based on this factor alone will be non-edible crops > fodder crops > crops subjected to industrial processing and purification > crops eaten after cooking > crops consumed raw.

5. Target traits for modification

Currently, the transgenic approach is feasible to engineer traits that are controlled by one or a few major genes but the Quantitative traits like yield are not easily amenable to improvement through transformation. So transgenic crop development should focus on transferring quantitative traits to the transgenics.

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Further, traits that can be routinely modified via conventional breeding need should not be targeted for transformation.

Transgenic crop development could profit from an emphasis on engineering biotic and abiotic stress tolerance which are not successful in conventional plant breeding methods.

Nutritional enrichment via transgenic has become feasible and is receiving international support. So that nutritional enrichment of food should be given priority.

Shelf-life of vegetables and fruits is short in our tropical climate so, genetic engineering of crops for slow ripening should receive high priority.

Genetic engineering approach should be given priority in producing male sterile lines and bisexual plants (pollinators) for the seed production purpose which is tedious and time consuming in conventional breeding methods.

Priority should be given to the development of environmentally-safe transgenic crops.

Linking together genes for parthenocarpy and male sterility with genes for other traits (e.g. slow ripening, nutrient fortification) should be given priority.

Transgenic crop variety development using antisense expression

of gene sequences to develop viral resistant transgenics and strategies such as gene silencing to eliminate antinutritional compounds should be given high priority.

History of development of GM foods

1950: First regeneration of entire plants from an in vitro culture

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1973: Researchers develop the ability to isolate genes

1983:1st transgenic plant: antibiotic resistant tobacco

1985: GM plants resistant to insects, viruses, and bacteria are field tested for the first time 1990: First successful field trial of GM cotton

1994: Flavr-Savr tomato - 1st FDA approval for a food

1995: Monsanto’s Roundup Ready (Herbicide resistant variety) soybeans approved for sale in the United States.

Useful single gene traits that have been introduced into plants

Genes for pest resistance• Protease inhibitors• Bacillus thuringiensis insecticidal proteins• Lectins• Ribosome-inactivating proteins (RIPs)

Genes for f ungi resistance

• Chitinases and Beta-1,3-glucanases• RIPs• Thionins• Antifungal peptides

Improved post-harvest properties• Any poisonous protein can be detoxified by heating and rendered

safe e.g. lectins; inhibitors.• Ripening control• Cowpea trypsin inhibitor• Flavrsavr tomatoes contain antisense to polygalacturonase

(softens tomatoes by dissolving the cell wall).

List of approved transgenic higher plants using different methods

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Nicotiana tabacum (tobacco) Picea glauca (white spruce)

N. plumbaginifolia (wild tobacco)Avena sativa (oats)

Petunia hybrida (petunia) Zea mays (corn)

Lycopersicon esculentum (tomato)Triticum aestivum (wheat)

Solanum tuberosum (potato) Oryza sativa (rice)

Solanum melongena (eggplant) Secale cereale (rye)

Arabidopsis thaliana Dactylis glomerata (orchard grass)

Lactuca sativa (lettuce) Asparagus sp. (asparagus)

Apium graveolens (celery) Vitis vinifera (grape)

Helianthus annuus (sunflower) Carica papaya (papaya)

Linum usitatissimum (flax) Actinidia sp. (Kiwi)

Brassica napus (oilseed rape; canola)Fragaria sp. (strawberry)

Brassica oleracea (cauliflower) Ipomoea purpurea (morning glory)

Brassica rapa (syn. B. campestris)Ipomoea batatas (sweet potato)

Gossypium hirsutum (cotton) Digitalis purpurea (foxglove)

Beta vulgaris (sugarbeet) Glycorrhiza glabra (licorice)

Glycine max (soybean) Armoracia sp. (horse radish)

Pisum sativum (pea) Daucus carota (carrot)

Chrysanthemum sp. (chrysanthemum)Cichorium intybus (chicory)

Rosa sp. (rose) Cucumis melo (muskmelon)

Populus sp. (poplar) Cucumis sativus (cucumber)

Malus sylvestris (apple) Lotus corniculatum (lotus)

Pyrus communis (pear) Medicago sativa (alfalfa)

Azadirachta indica (neem) Carnation (Dianthus caryophyllus)

Gossypium sp (Cotton) Squash( Citrullus sp)

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Chicory (Chicorium sp) Linum sp)

Melon

Traits that plant breeders would like in plants• Improved Agronomic properties• High primary productivity• High crop yield• High nutritional quality• Adaptation to inter-cropping• Nitrogen Fixation• Adaptation to mechanised farming• Insensitivity to photo-period• Cold / Frost resistance• Seed protection• Improved plant breeding• Drought resistance• Pest resistance• Virus resistance• Fungal resistance• Herbicide resistance1. Glufosinater herbicide2. Sethoxydimr herbicide3. Bromoxynilr herbicide4. Glyphosater herbicide5. Sulfonylurear herbicide• Improved nutritional properties• Elimination of toxic compounds• Modified fatty acid• Flower colour• Flower life• Oil production• Plastics• Digestibility proteins• Antibodies• Golden rice (gene from Chrysanthemum giving - converted to

vitamin A).• High starch potatoes

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• Delayed ripening• Pollen-specific promoter plus RNAse• Male-sterility

D evelopment of transgenic plants to biotic and abiotic stress

In the crop field, the crop plants face challenges from the biotic and abiotic factors. The biotic factors such as pests, bacteria, fungi, nematodes, virus and abiotic factors such as cold, drought, heat, salinity and flood cause considerable yield loss to the crop plants. In modern agriculture, the high yield was obtained by growing high input responsive varietal seeds. Indiscriminate application of inputs (Fertilizres, pesticides, herbicides and water) to achieve high production led to the undesirable environmental consequences such as pollution of water, land and also the contamination of food in addition to the increased cost of production to the farmer. Practicing of sustainable agricultural practices is the only solution to overcome the consequences of modern agriculture. In sustainable agriculture, we want to achieve the high production by optimally using the inputs for crop growth so that the damage to the environment can be reduced or minimimized. Growing of transgenic crops resistant to biotic and abiotic factors is one away of implementing the sustainable agriculture practices. To develop transgenic crops, first we have to isolate the resistant gene from the living organisms, then the resistant gene is linked with the promoters, terminator and marker genes to enhance the expression of gene (construction of transformation cassettes). Then the transformation cassettes are inserted in to the plant cells either via direct or indirect gene transfer methods. Then the whole transgenic plant is regenerated from the transformed plant cells. The developed transgenic plant in the lab condition is then transferred to the field condition and tested for the stability of resistance and other agronomic characters. If the performance is found satisfactory then it will be released as variety for commercial purpose.

Developing transgenic for biotic and abiotic factors

Prepare tissue for transformation↓

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Tissue must be capable of developing into normal plants (Leaf, germinating seed, and immature embryos)

↓

Introduce DNA into the plant cell either through Agrobacterium or other methods

↓

Culture plant tissue-development of shoot followed by root↓

Field test the plants in multiple sites and multiple years

I) Development of Transgenic plants resistance to pests

A major challenge for farmers has always been in protecting their crops from pathogens like insects, viruses and fungi. Pests are often kept at bay by spraying chemical pesticides on crops, some of which have been shown to have adverse affects on the environment. Biotechnology provides new and environmentally friendly ways of dealing with these pests.In some instances, crops can be developed that are naturally resistant to these pests. Some tomato plants, corn, cotton and others have been

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protected from insect pests by inserting a gene(Cry gene)from a soil bacterium called Bacillus thuringiensis (BT).The other sources of resistant genes are Protease inhibitors, Lectins and Ribosome-inactivating proteins (RIPs) etc. are also used to produce insect resistant plants. Mechanism of action of Bt gene "Bt" is short for Bacillus thuringiensis, a soil bacterium whose spores contain a crystalline (Cry) protein. The BT gene causes a protein (crystalline (Cry) protein) to be produced that is lethal to some leaf-eating insects like the tomato hornworm. When the insect eats the plant, it ingests the protein, and enzymes in its stomach convert the protein into a lethal toxin (delta-endotoxin) that causes paralysis and death (This toxin binds to and creates pores in the intestinal lining, resulting in ion imbalance, paralysis of the digestive system, and after a few days, insect death). Since the necessary enzymes are not found in any other insects (non targets) or animals, the protein is harmless to them. Different versions of the Cry genes, also known as "Bt genes", have been identified. They are effective against different orders of insects, or affect the insect gut in slightly different ways. A few examples are shown in the table below.

Cry gene designation Toxic to insect orders

CryIA(a), CryIA(b), CryIA(c) Lepidoptera

Cry1B, Cry1C, Cry1D Lepidoptera

Lepidoptera, Diptera

Coleoptera

Lepidoptera, Coleoptera

Some examples of Bt insect-resistant crops currently on the market include

Corn: primarily for control of European corn borer, but also corn earworm and Southwestern corn borer.

Cotton: for control of tobacco budworm and cotton bollworm

Potato : for control of Colorado potato beetle. Bt potato has been discontinued as a commercial product.

Others: Soybean, canola, sugar beet, lettuce, strawberry, alfalfa and wheat and turf grass

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Benefits of insect protected crops

1. Economics and production Increased income, convenience of the system, potential to have less spray equipments and better Insect control.

2 . Environmental Less Pesticide impact, more beneficial/predatory bugs, spiders, insects, potential to improve water quality and reduced energy use.

3. For small holder farmers Macro economic benefit, economic benefit, social benefit and reduced spraying

Will pests become resistant to Bt toxin and what is the solution?

Although BT genes have proven to be quite effective in the short term for protecting against crop insect damage, as well as reducing fungal contamination of corn there are concerns that widespread use of Bt varieties will accelerate development of resistance to Bt in the target pests. This could mean the loss of Bt as an effective, environmentally friendly insecticide. In response to these concerns, the U.S. Environmental Protection Agency has mandated measures to reduce the risk of resistance development. These measures depend on a combination of high dose of the Bt toxin and a planting of refuges. A refuge refers to an area planted to a non-Bt variety that is physically close to a field planted with a Bt variety, as shown in the diagram below. Beginning in 2000, the EPA requires that farmers growing Bt corn must plant at least 20% of their total corn acreage to a non-Bt variety. The rationale is that the few Bt-resistant insects surviving in the Bt field would likely mate with susceptible individuals that have matured in the non-Bt refuge. Thus, the insect genes (alleles) for resistance to Bt would be swamped by the susceptible alleles.

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Diagram of the BT refuge strategy, in which at least 20% of a farm's corn acreage must be planted to non-BT corn. R = resistant European corn borer adult; S = susceptible adult.

II ) Development of transgenic plants resistance to viral pathogens

Plant virus diseases cause severe constraints on the productivity of a wide range of economically important crops worldwide. In India the Green Revolution ushered in intensive agricultural practices and reduced varietal diversity, resulting in the emergence of viral diseases at an alarming pace in the cultivated crops. Some such diseases, which are especially relevant to India, along with their yield losses, are listed in Table 1.Table: 1

Disease Yield loss Virus group

Cassava Mosaic 18–25 Indian cassava mosaic virus

Begomovirus

Cotton Leaf curl 68–71 Cotton leaf curl

Begomovirus

Groundnut necrosis

> 80 Groundnut bud necrosis virus

Tospovirus

Mungbean, Black gram and Soybean

mosaic 21–70

Mungbean yellow mosaic virus

Begomovirus

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Pigeon pea Sterility Mosaic

> 80* Pigeonpea sterility mosaic virus

Tenuivirus

Potato Mosaic 85 Potato virus Y Potyvirus

tungro 10 Rice tungro badna

and rice tungro spherical viruses

Badnavirus and waika virus

Sunflower Necrosis 12–17 Sunflower necrosis virus

Ilarvirus

Tomato Leaf curl 40–100 Tomato leaf curl

Begomovirus

It is important to know the structure of plant viruses and traditional control measures before we apply the biotechnological tools to develop virus resistant plants.

Structure of most typical plant viruses

The vast majority of viruses that infect plants are RNA viruses. This means that the nucleic acid carried in the virus particle, the viral genome, is comprised of RNA. There are a small number of plant viruses contain the DNA as genetic material (including Cauliflower Mosaic Virus (CaMV) from which the 35S promoter was obtained). Plant viruses are grouped into families based on their physical structure and the organization of their genomes.

Plant viruses are fairly simple creatures. The virus particle is made up of a protein shell or coat which surrounds the RNA or DNA genome. The shell is made up of one or two types of proteins, and these proteins assemble to form the coat. These proteins are known as viral coat protein . The genomes (Either DNA or RNA) of plant viruses contain only a small number of genes. These encode for the coat protein, a replicase gene (a protein to aid in replication of the RNA genome), and movement protein (helps the virus move through the plant from the original site of infection and enlarges the small passage ways between cells (the plasmodesmata)) and perhaps a few others genes. Many viruses produce a protein that systemically from cell to cell.

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Life cycle of a typical plant virus

When a virus enters a cell, the RNA is released from the protein coat called disassembly. This RNA is then used as template to make RNA and proteins encoded by the virus. The virus uses the machinery of the plant cell to express the genetic information carried by the viral genome. This somehow leads to the symptoms of viral diseases. The genome of the virus is replicated and new coat proteins are synthesized. This leads to the assembly of new virus particles in the infected plant cells. The virus is transmitted to another healthy plant by mechanically (using the same pruning tool to cut the healthy and infected plants), physically (by touching of infected and healthy plants) and also by a biological vector, such as aphids or other insects that feed on an infected plant, ingest some virus particles, then move to a healthy plant and transmit the virus in the saliva when feeding. Some viruses are carried in the seed and so are passed from one generation to another with the seed.

Biotechnological strategies to control plant viruses

Strategies for the management of viral diseases normally include control of vector population using insecticides, use of virus-free propagating material, appropriate cultural practices and use of resistant cultivars. However, each of the above methods has its own drawback.

Rapid advances in the techniques of molecular biology have resulted in the cloning and sequence analysis of the genomic components of a number of plant viruses. A majority of plant viruses have a single-stranded positive sense RNA as the genome. However, some of the most important viruses in tropical countries like India have single-stranded and double-stranded DNA genomes and RNA genomes of ambisence polarity, i.e. genes oriented in both directions. There are mainly two approaches for developing genetically engineered resistance depending on the source of the genes used.

1) Pathogen Derived Resistance (PDR)The genes are for viral resistant transgenic plants obtained from the pathogenic viruses. For PDR, a part, or a complete viral gene is introduced into the plant, which, subsequently, interferes with one or more essential steps in the life cycle of the virus. This was first illustrated in tobacco by the group of Roger Beachy, who introduced the coat protein (CP) of tobacco mosaic virus (TMV) into tobacco and observed TMV resistance in the

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transgenic plants. The concept of PDR has generated lot of interest and today there are several host–virus systems in which it has been fully established.

Types Pathogen derived resistancea) Coat Protein Mediated Resistance b) Replicase mediated resistance or post-transcriptional gene silencing (PTGS).c) Satellite RNAd) Defective-interfering viral genomic components.

Coat protein

Cross protection is the basis of coat protein mediated resistance. In cross protection the plants are deliberately infected with a mild strain of a virus, which produces only mild symptoms. The plants that have been infected with the mild strain of the virus are in some way protected against a subsequent infection by a more severe strain of the virus. Remember that plants do not have an immune system similar to that of mammals, and this is not the same as a vaccination that you might have received as a child, but the outcome is similar.

The use of viral CP as a transgene for producing virus resistant plants is one of the most spectacular successes achieved in plant biotechnology. Numerous crops have been transformed to express viral CP and have been reported to show high levels of resistance in comparison to untransformed plants (Table 2 and 3). Powell-Abel et al. first reported resistance against TMV in transgenic tobacco expressing the TMV CP gene. The resistance was manifested as delayed appearance of symptoms as well as a reduced titre of virus in the infected transgenic plants, as compared to the controls. The resistance against TMV using TMV CP in tobacco was also reported to be effective against other tobamoviruses whose CP was closely related to that of TMV but not effective against viruses which were distantly related to TMV10. Transgenic potato, expressing the CP of potato virus X (PVX) also showed resistance against PVX11.Mechanism of action coat protein The stage of the viral life cycle at which the CPMR is effective has been shown to vary.1) In TMV, it is at the virus disassembly and in the long-distance transport stage.

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2) In the case of alfalfa mosaic virus (AMV), it is only at the disassembly stage3) In PVX, it is at multiple stages, including replication, cell-to-cell and systemic movement stages. 4) In tospoviruses, the stage affected is believed to be replication.

b) Replicase mediated resistance or post-transcriptional gene silencing (PTGS).Gene constructs of Rep genes that have been used for resistance include full-length, truncated or mutated genes. This type of resistance remains confined only to a narrow spectrum of viruses, the spectrum being narrower than that of CPMR. To make the resistance broad-based, it may be necessary to pyramid such genes from several dissimilar virus sources into the test plant genome.

Post - transcriptional gene silencing:

Post-transcriptional gene silencing (PTGS) is a specific RNA degradation mechanism of any organism that takes care of aberrant, unwanted excess or foreign RNA intracellular in a homology-dependent manner. This activity could be present constitutively in any living organism to help normal development or induced in response to cellular defense against pathogens. In this mechanism, the elicitor double-stranded RNA (ds RNA), commonly produced in plant during viral infection and this elicitor double-stranded RNA is degraded to 21–25 nucleotides , termed as small interfering RNA (siRNA), supposedly degrade the viral RNA molecules bearing homology with the elicitor RNA . This degradation process, initiating from a concerned cell having the elicitor RNA, spreads later within the entire organism in a systemic fashion. This process is generally regarded to have evolved as a plant defense mechanism against invading viruses containing either RNA or DNA genomes. This technique has been used to confer resistance to potato leaf roll virus(PLRV) in potato, resistance to barley yellow dwarf virus in oats, cucumber mosaic virus in tomato, rice tungro spherical virus in rice, and wheat streak mosaic virus in wheat.Satellite RNA Attempts have been made to ameliorate resistance viruses via satellite RNA because different degrees of virus resistance have been obtained with coat

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protein-mediated resistance. Satellite RNAs are a class of small (approximately 300 nucleotides) single stranded RNA that are dependent upon the helper virus for replication and virion packaging to cause infection elsewhere and also vice versa. Satellite RNA has been associated with different disease causing viruses and control the replication and symptom development. Thus it is possible to control the severity of symptom development by inserting the mild strain of disease causing virus along with the sat RNA so the plant would be protected from the severe strain of disease causing virus., Example: 1) Transgenic tobacco against TMV 2) Transgenic Pea resistant pea early browning virus 21,

II) Non-pathogen-derived resistanceNon-pathogen-derived resistance is based on utilizing host resistance genes and other genes responsible for adaptive host processes, elicited in response to pathogen attack, to obtain transgenic resistant to the virus. Non-pathogen-derived resistance in comparison to PDR-based approaches holds a better promise to achieve durable resistance. Ribosome-inactivating proteins (RIPs) are antiviral proteins that have been used to protect the plants against more than one virus. RIPs are strong inhibitors of protein synthesis and, depending on the plant species from which they originate; they have different levels of toxicity against different hosts. Poke weed antiviral protein (PAP) confers resistance to PVX and PVY in transgenic potatoes and PAPII confers resistance to TMV, PVX, and fungal infections in tobacco. On a more experimental scale are approaches to achieve virus resistance by using antibodies against the virus coat protein. Such antibodies can neutralize virus infection, presumably by interacting with newly synthesized coat protein and disrupting viral particle formation . Similar to RIPs, broad-spectrum antibodies might be used to protect plants against a wider range of viruses, as has been demonstrated for poty viruses.Mammalian adenylate synthase gene:In mammals virus infections are fought via induction of the interferon system. Interferon induces additional proteins that defend the animal directly against virus. This has been exploited to confer resistance to viral infection in plants. Interferon don’t themselves posses antiviral activity and they induce the synthesis of additional proteins that leads to inhibition of virus multiplication. One of these proteins is oligo adenylate synthase enzyme . This enzyme is activated by double stranded RNA and the remains of replication

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intermediaries. After activation the enzyme activates the latent endoribonuclease that degrade the viral RNA. Using his enzyme gene the transgenic plants resistant to potato virus X in potato plant and cucumber mosaic virus also in cucumber.( Ribozyme mediated resistance also used to develop vial resistant transgenic plants(Read H.S. CHALA BOOK for details)

Development of Fungal resistant transgenic plants: Fungal resistance can be conferred by activation of specific self-defense mechanisms in the plant. One of the mechanisms is the so-called hypersensitive response (HR), which enables plants to enclose the pathogen in the infected area by formation of necrotic lesions. HR induces many defense-related signal molecules such as salicylic acid, ethylene, and phytoalexin. HR is also characterized by an accumulation of pathogenesis-related (PR) proteins that include fungal cell wall-degrading enzymes, antimicrobial peptides, thionins, lipid-transfer proteins, and proteinase inhibitors. In rice, the introduction of chitinase and thaumatin-like protein led to increased resistance to sheath blight (Rhizoctonia solani). Enhanced resistance to the rice blast fungus Magnaporthe grisea was observed on constitutive expression of chitinase and defense-related protein genes in transgenic rice. Pathogenesis-related proteins from plants have been used to confer fungal resistance in alfalfa, cucumber, oil-seed rape, tomatoes, wheat, grape vine, and oranges.

Other antifungal genes of plant origin are genes for RIPs, genes for phytoalexins, and anthocyanin genes. An example of an antifungal gene from non plant sources which has been transferred to plants is the human lysozyme gene.

In the USA field trials have been performed with fungus-resistant wheat, barley, maize, soybean, potato. rice, bananas, and cotton using different antifungal proteins. Individual PR-proteins, however, have a narrow spectrum of antifungal activity, and must function collectively to provide modest but long-term resistance. Research is, therefore, currently focusing on genes from mycoparasitic fungi as a means of improving resistance to fungal pathogens. An endochitinase of the mycoparasitic fungus Trichoderma harzianum has been transferred to tobacco and potato, and has been reported to confer a high level and broad spectrum of resistance. When

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transferred to apple, however, the endochitinase of Trichoderma harzianum increased resistance to apple scab but also reduced plant growth. A similar approach has been taken by transferring to wheat an antifungal protein from a virus that persistently infects Ustilago maydis. Transgenic wheat plants had increased resistance against stinking mut ( Tilletia tritici). For a comprehensive survey of the different approaches used to achieve fungal resistance in transgenic plants see Tables 1 and 2 in Ref. [90].

Bacterial resistance Resistance to bacterial infections is not yet as well developed as virus and fungal resistance, partly because bacterial diseases are a major problem only in crop plants such as potato, tomato, rice, and some fruit trees. The most efficient form of protection is genetic resistance, which is based on single dominant or semidominant genes. These R genes usually confer race-specific resistance, and their effectiveness is based on their interaction with complementary pathogen avirulence (AV) genes in the pathogen, the so called gene-for gene interaction [91]. Resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae was achieved by transferring the resistance gene Xa21 from a wild rice species to the elite indica rice variety “IR72” [92]. In the same way, the resistance gene Bs2 from pepper was transferred to tomato, which then had resistance to bacterial spot disease [93].The tomato disease resistance gene Pto gives race-specific resistance to Pseudomonas ssyringae pv. tomato carrying the avrPto gene. By overexpressing Pto race-nonspecific resistance was observed in transgenic tomatoes [94].Resistance based on single dominant gene expression always bears the danger of early evolution of counter-resistance in the pathogen as a result of the emergence of strains that no longer express the specific avirulence gene product [93]. Therefore, new resistance genes are being investigated for use in pyramiding strategies (combination of resistance genes against the same pathogen, but with different targets). One example is the AP1 gene from sweet pepper which delays the hypersensitive response when expressed in transgenic rice plants and which can be used in combination with Xa21 or other resistance genes [95]. Similarly to fungal resistance, overexpression of PR proteins or transfer of PR protein genes from other sources has led to enhanced resistance against bacterial infections – expression of barley lipid transfer protein LTP2 resulted in enhanced tolerance to bacterial pathogens in transgenic tobacco plants [96]. In several plant species, bifunctional enzymes with lysozyme activity have been detected which are thought to be

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involved in defense against bacteria. After transfer of the bacteriophage T4 lysozyme gene, transgenic potatoes had reduced susceptibility toward Erwinia carotovora atroseptica infections [97]. Transfer of the human lysozyme gene to tobacco led to enhanced resistance against both fungal and bacterial diseases [98]. Plant defense responses also involve production of active oxygen species, for example hydrogen peroxide (H2O2). This mechanism was exploited by transferring a fungal gene encoding H2O2-generating glucose oxidase to potato plants. The transgenic potato tubers had strong resistance to Erwinia carotovora subsp. Carotovora infections, which cause bacterial soft rot disease, and enhanced resistance to potato late blight, caused by Phytophthora infestans [99].Insects produce antimicrobial peptides as a major defense response to pathogen attack. These include sarcotoxins, cecropins, and attacins. The last of these have been transferred to apples and pears which then had improved resistance to Erwinia amylovora, which causes fire blight [100, 101]. Sarcotoxins seem to confer greater anti-bacterial activity and a broader spectrum of resistance, as indicated by experiments with transgenic tobacco [65]. Similarly, the expression of synthetic antimicrobial peptide chimeras in transgenic tobacco led to broad-spectrum resistance against both bacterial and fungal pathogens [102]. Bacteria-resistant grapes, transformed with the antibiotic protein mangainin from toads, are being field.

( III) Development of transgenic plants resistance to Herbicides

Herbicide:

Herbicides are the group of heterogeneous compounds that are used to destroy the weeds present in the crop fields.

Different herbicides and their mode of action

Mode of action Chemical group Example

Inhibition of EPSP Synthase

Glycine Glyphosate

Inhibition of Aceto- lactate synthase

Sulfonylurea Urea Chlorsulfouron

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Inhibition of photosynthesis at photo system II

Triazine, Uracil and Phenylcarbomate

Atrazine

Inhibition of Glutamine synthetase

Phosphinic acid Glufosinate ammonium and Bialaphos

Purpose and b enefits of developing herbicide resistant plants.

The purpose of developing herbicide resistant plant is to save the crop from the broad spectrum herbicides which would kill otherwise the crop plant on application in addition to the killing of weeds.

Benefits

Economics and production

Increased income, reduced labor, convenience and simplicity, potential to have less spray equipments, better system of weed Control

Environmental

Less Pesticide impact, conservation tillage, potential to improve water quality and reduced energy use

Strategies for developing herbicide resistant plants

Strategey1: Over expression of target proteins (by overproducing the target protein)

Strategy 2: Mutation of the target proteins (making target molecules insensitive to herbicide)

Strategy 3: Detoxification of herbicide using single foreign gene

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Strategy 4: Enhanced plant detoxification

Over expression of target proteins (by overproducing the target protein)

Over expression of target proteins can be achieved by the integration of multiple copies of target gene or use of promoter plus translational enhancer to express the target gene products in surplus to partially or fully overcome the inhibition effect of herbicide on the target protein. EPSP is a protein which is a precursor for the many aromatic amino acids synthesis and without theses amino acids the plant cannot survive that leads to death of plants. Synthesis of EPSP is mediated by the enzyme EPSP synthase in plants.Glyphosate is herbicide which is applied to kill the weeds in the crop fields. Being a broad spectrum herbicide, the glyphosate not only kill the weeds but also crop plants. So that we need to protect the plants on the one hand and on the other hand the weeds have to be killed effectively.

The m RNA of the EPSP gene is obtained and from this m RNA the c-DNA is obtained and then the c-DNA is inserted into the modified Ti plasmid vector( Mon 546) which have provisions for strong promoter(CaMV-35s promoter) and terminator sequence (Nos terminator) . The recombinant Ti plasmid is introduced into Agro bacterium and allowed to infect the target plants cells for the development of herbicide resistant transgenic plants. Then the resistant plants are tested under field condition if the result obtained is satisfactory, then it is releasesd as a herbicide resistant variety.

Mutation of the target proteins (making target molecules insensitive to herbicide)

Mutation of target protein allows the protein to have same function but have different structure from the normal gene protein. So the applied herbicide will have no affinity to the altered gene protein and hence the protein is escaped or nontargetted by the herbicides. One commonly used herbicide is glyphosate (commercially available as Roundup and Tumbleweed). This herbicide inhibits the activity of the plant enzyme used to synthesize certain amino acids found in the chloroplast and essential to plant growth (the enzyme's name is 5~enolpyruvylshikimate~3-phosphate synthetase, or EPSPS). It happens that the common intestinal bacterium Escherichia coli contain genes (mutant EPSPS gene) that confer resistance against glyphosate activity by making the organism much less sensitive to glyphosate activity.

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The E. coli has a gene (mutant EPSPS gene) that encodes a mutant EPSPS enzyme. The enzyme is less sensitive to glyphosate than the normal EPSPS enzyme, and a plant with the mutant enzyme would be less sensitive than the one with the normal enzyme. To engineer glyphosate resistance into plants, DNA technologists begin with E. coli cells and isolate the mutant EPSPS gene. Then they attach the gene to the Ti plasmid of Agrobacterium tumefaciens and transfer the plasmids to the cells of plants such as tobacco and petunia. The plant cells now produce the mutant enzyme and develop resistance to glyphosate. When glyphosate is sprayed on the tobacco or petunia field during the growing season, it effectively eliminates the weeds but has a minimal effect on the plants because they can resist the herbicide's activity. The crop yield is thus increased.

Potato, Tomato, Tobacco and petunia and cotton

Diagrammatic explanation is given below

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Detoxification of herbicide using single foreign gene

This strategy aims to convert the more toxic form of herbicide into less toxic form

To remove the herbicide form the plant

Don’t need the mode of action of herbicide

Weed-infested soybean plot (left) and Roundup Ready® soybeans after Roundup treatment. Source: Monsanto

Consequences of growing herbicide resistant transgenic plants

As with other forms of genetic engineering, some controversy has arisen in the scientific community about the possible consequences of introducing gene-related herbicide resistance. Critics of the process suggest that herbicide-resistance genes might move naturally from crops to weeds (super

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weeds) rendering the herbicide ineffective. A 1997 report seemed to confirm these concerns. A consultative group representing a consortium of sixteen research centers acknowledged that such gene transfers could occur and urged those preparing the bioengineered crop to assess how long the gene will serve its intended role.

What measures should we take to avoid the development of super weeds?

Transgenics for quality parameters

1) FLAVR SAVR TOMATO

One of the first examples of a genetically altered food to reach market was the Flavr Savr tomato. Developed by the California firm Calgene, Inc., the tomato contained a gene that delays rot. The value of such a tomato was that it did not have to be picked hard and green for shipping because it decays slowly. The tomato could be left on the vine to ripen for several days longer and shipped without refrigeration (shipping without refrigeration also helps retain flavor). The tomato resisted rot for more than three weeks, or about twice as long as the conventional tomato.

The Flavr Savr tomato worked this way: tomato plants produce the enzyme polygalacturonase (or "PG"), a chemical substance that digests pectin in the cell walls of tomato plants, as Figure 10.12 displays. This digestion induces the normal decay ("rotting") of the fruit. Researchers at Calgene identified the gene that encodes PG, removed the gene from plant cells, and inserted it into a vector organism. Then they induced the vector to produce a complementary copy of the gene. Continuing the process, researchers isolated the gene copy and inserted it into fresh tomato plant cells. Here it encoded an mRNA molecule (an antisense molecule) that unites with and inactivates the normal mRNA molecule (the sense molecule) for PG production. With the normal mRNA inactivated, the plant could not produce enzyme and, therefore, pectin digestion did not occur. And without pectin digestion, rotting slowed considerably. This use of an antisense molecule is similar to that used in drug research. The antisense RNA technology can also be used to inhibit the synthesis of ethylene by producing antisense mRNA for AdoMet synthetase, ACC synthase and ACC oxidase enzymes.

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(Producing a rot-resistant tomato. (a) DNA technologists begin by isolating from the tomato plant the gene that encodes polygalacturonase (PG), the "rotting enzyme." (b) In normal tomato plants, this gene encodes normal mRNA (the "sense" molecule), which is translated to PG. (c) DNA technologists transfer the PG gene to a vector bacterium and (d) induce it to produce a complementary DNA molecule. It is a complementary copy of the PG gene. (e) The complementary gene encodes an mRNA molecule that complements the normal mRNA molecule. The complementary mRNA molecule has "antisense." (f) Now the complementary DNA is inserted to a fresh tomato plant. (g) Here the DNA encodes the antisense mRNA molecule. (h)The antisense mRNA unites with the sense mRNA molecule, and thereby neutralizes it. The message in the normal mRNA molecule is not translated, and PG is not produced. Without PG, rotting does not occur in the tomato.)

Ethylene biosynthesisMethionine

↓ AdoMet synthetase

↓

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SAM (S-Adenosylmethionine)↓

ACC synthase↓

ACC (1-Aminocyclopropane-1-carboxylic acid)↓

ACC oxidase↓↓

Ethylene

2) Development of Golden Rice

The second major plant biotechnology product is more recent and was developed to address the vitamin A deficiency problems prevalent throughout the world. This vitamin deficiency is very critical because it can cause blindness and affects the severity of many diseases including diarrhea and measles. This is a severe problem that affects more than 100 million children worldwide. A simple solution would be to distribute vitamins to the affected children. Unfortunately, many countries where the deficiency is chronic do not have the necessary infrastructure to deliver the vitamin tablets to the most needed.

The solution that is currently being promoted is to improve the vitamin content in widely-consumed, and readily available to the consumer. Transgenic rice plants were developed that contain elevated levels of the precursor to vitamin A. This GMO is called “Golden Rice” because of its color: it is yellow rather than white. It is yellow because β-carotene, a yellow precursor to vitamin A is abundant in the seed. Unlike the single-step Roundup Ready pathway, the β–carotene synthesis pathway involves multiple enzymes. This important vitamin A precursor cannot be synthesized in rice because it lacks four of the key enzymes. Therefore, the precursor is not made, and the plant contains white kernels.

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In a major feat of genetic engineering, scientists inserted a complete functioning -carotene biosynthetic pathway into the rice plant. They did this by inserting genes from daffodil the produce functioning versions of the first and last enzymes of the pathway. In addition, a single bacterial gene that provides the same function as the second and third enzymes of the pathway

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was also introduced. With a functioning pathway, the transgenic rice is able to produce the vitamin A precursor β-carotene. It is this product that gives "Golden Rice" its characteristic yellow color.

Golden rice contrasted with normal rice

Other innovative foods Canola oil is a vegetable oil extracted from canola seeds and used for such diverse purposes as detergents, face creams, and ice cream. Scientists have transformed the canola plant by introducing a gene from the California bay laurel tree that encodes the enzyme thioesterase. This enzyme synthesizes lauric acid, a fatty acid not normally found in the canola oil. The transgenic canola plant now produces specialty oil containing up to forty percent lauric acid (laurical). The oil has the low saturated fat content of olive oil and is attractive to health enthusiasts. Furthermore, it does not break down under heat and is favored by chefs, as well as homemakers.

Also anticipated is a biotech potato that resists color changes when peeled. In this vegetable, DNA technologists have eliminated the enzyme that promotes brown color changes by removing the responsible gene. It will be joined by a variety of yellow squash called Freedom 2. The vegetable looks like a normal squash, but it has been genetically engineered to resist two common plant viruses.

Already on the market is a mini-pepper about the size and shape of a jalapeno pepper but with a mild, sweet flavor. (Strictly speaking, this pepper is not a product of DNA technology-- it was produced by cell cloning from a mutant specimen.)

Although not strictly a form of agriculture, transgenic plants are being used to crank out increased amounts of vitamins, other organic substances, and

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minerals. One product of a transgenic plant is vitamin E. Scientists at the University of Nevada is coaxing Arabidopsis thaliana plants to increase their production of alpha-tocopherol, a useful form of the vitamin. The plant normally produces a compound close to alphatocopherol, but it cannot produce the enzyme that makes the final conversion. DNA technologists have located the gene that encodes the enzyme in a bacterium, then found its equivalent lying unexpressed in Arabidopsis, shown in Figure 10.14. They found a regulatory sequence that would activate the dormant gene and inserted it into the plant cells. The plant now produced a tenfold amount of vitamin E. With the theoretical basis for producing the vitamin in place, scientists could envision soy plants, already known for their production of vitamin E, as major suppliers for health conscious individuals.

Scientists have dubbed their emerging discipline nutritional genomics. Research studies are already underway to insert bacterial genes that encode carotenoid into rice plants and induce the latter to synthesize vitamin A. Other researchers are attempting to enhance the iron content of edible plants by enhancing their ability to absorb iron from the soil

And consumers can expect to see other bioengineered foods in the near future: Researchers from Auburn University have spliced trout genes into catfish so the latter will reach maturity months earlier; genes from a chicken are being spliced to potatoes to make the potato more protein rich; and "ice-minus" genes are being incorporated to ice cream and cake products to prevent ice crystal formation during freezing. Figure 10.15 depicts an artist's whimsical approach to biotech agricultural products.

To help allay the fears of consumers, the FDA has not given carte blanche to bioengineered foods. Rather, its new policy allows new foods to be treated as conventional foods as long as they meet three conditions: their nutritional value has not been lowered; they incorporate new substances (e. g., proteins or carbohydrates) that are already a part of the human diet; and they contain no new allergenic substances. So constructed, the new foods are considered fit for human consumption.Vegetable vaccines

Before leaving the topic of bioengineered foods, we will consider transgenic plants as carriers of vaccines. Plants are desirable carriers because they are inexpensive to store and readily accepted in developing countries, where

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vaccines are needed most. Researchers at New York's Boyce-Thompson Institute were among the first to splice microbial genes into plant cells. In 1997, a research group reported successful incorporation of genes from a strain of Escherichia coli into a potato plant. When chunks of the "veggie vaccine" were eaten by volunteers during trials at the University of Maryland, the gene-encoded protein provoked an antibody response against the protein. Unfortunately, the potatoes were uncooked, and they turned up the volunteers' noses as well.

To resolve this dilemma, the researchers tried cooked potatoes, but they observed a significant reduction in the amount of bacterial protein. Colleagues at Loma Linda University attempted to amplify the amount of bacterial protein produced by the potato and administer "booster" feedings, both with limited success.

Future research efforts will use more palatable plants such as tomatoes and bananas. Tomatoes are appealing vaccine carriers because of their short growing season and the wealth of experience DNA technologists have with transgenic tomatoes. Bananas have attracted interest because children like them (children are the main recipients of vaccines); the fruit grows in many tropical countries (where vaccines are essential to public health systems); and bananas do not have to be cooked before consumption. Indeed, a research group has recently reported successful incorporation of the E. coli genes to bananas (Figure 10.16)

Using transgenic vegetables have its unique problems: Investigators must address the problem of how to get the antibody-stimulating antigens through the acid and enzymes of the stomach and intestine, respectively. Because these antigens are proteins, they may combine with food proteins to produce allergic substances. And there is the problem of getting the antigens from the gastrointestinal tract to the blood where the immune reaction occurs. Despite these obstacles, DNA scientists see great hope for the new wave of transgenic vaccines. Their hope is accompanied by many years of laboratory tinkering and many clinical trials before the banana replaces the needle as a vehicle for vaccine delivery.

The final achievement we will mention occurred in 1998. Researchers from Agracetus Inc. proudly announced that a field in Wisconsin was home to a

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corn crop where human antibodies were being produced. The scientists had altered corn seeds with genes for anti-cancer antibodies, and the corn plants were now synthesizing the so-called plantibodies. Company scientists hoped that radioactive killer substances could be tagged to the antibodies for delivery to cancer cells. They emphasized the plants engineered with inexpensive and allergy-free characteristics of their astounding achievement. But, they genes to encode human cautioned, clinical trials have yet to begin.

END OF III UNIT

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