TRANSGENIC PLANTS

SUBMITTED BY:

SOWMYA.S

UNDER THE GUIDANCE OF:

Dr. H.N. ASWATHARAM

AND

Dr. SALEEMULLAH KHAN

CONTENTS

1. Introduction2. Definition3. Making of Transgenic plants

Introduction to DNA. Locating genes for plant traits. Designing genes for insertion. Transformation. Selection and Regeneration. Future developments in transgenic

technology.4. Advantages and disadvantages of Transgenic plants5. Applications of Transgenic plants

INTRODUCTION

Conventionally the genetic variation necessary for crop improvement is generated through hybridization, mutagenesis and polyploidy. More recently, biotechnological approaches have become available for genetic variation. The most potent biotechnological approach for the transfer of specifically constructed gene assemblies through various transformation methods, constitutes Genetic Engineering. The plants obtained through genetic engineering contain a gene usually from an unrelated organism, such genes are called Transgenes and the plants containing transgenes are called TRANSGENIC PLANTS. This directed genetic engineering of plants requires that the genes of interest are available, that the gene be introduced into plant cells capable of regenerating into intact plants, and that the gene carries with it a selectable marker so that the transformed plant cells can be isolated from a large population of untransformed, normal cells. Finally, the transformed plant cell must retain its capacity to regenerate. Certain species such as tobacco and petunia regenerate plants quite easily, making transgenic plants readily obtainable.

The transgenic plants, usually normal in appearance and character, differ from the parent only with respect to the function and influence of the inserted gene.

DEFINITION

The plant obtained through genetic engineering contains a gene or gene usually from an unrelated organism such genes are called transgene and the plant containing transgene are known as transgenic plants.

HOW TO MAKE TRANSGENIC PLANTS

The following steps are involved in the making of a Transgenic plants:

1. Introduction to DNA.2. Locating genes for plant traits.3. Designing genes for insertion.4. Transformation.5. Selection and Regeneration.6. Future developments in transgenic technology.7. Plant breeding and testing.

1. INTRODUCTION TO DNA:The underlying reason that transgenic plants can be constructed is the universal presence of DNA (deoxyribonucleic acid) in the cells of all living organisms. This molecule stores the organism's genetic information and orchestrates the metabolic processes of life. Genetic information is specified by the sequence of four chemical bases (adenine, cytosine, guanine, and thymine) along the length of the DNA molecule. Genes are discrete segments of DNA that encode the information necessary for assembly of a specific protein. The proteins then function as enzymes to catalyze biochemical reactions, or as structural or storage units of a cell, to contribute to expression of a plant trait. The general sequence of events by which the information encoded in DNA is expressed in the form of proteins via an mRNA intermediary is shown in the diagram below.

The transcription and translation processes are controlled by a complex set of regulatory mechanisms, so that a particular protein is produced only when and where it is needed. Even species that are very different have similar mechanisms for converting the information in DNA into proteins; thus, a DNA segment from bacteria can be interpreted and translated into a functional protein when inserted into a plant.

Among the most important tools in the genetic engineer's tool kit are enzymes that perform specific functions on DNA. A restriction enzyme (EcoR1) which recognizes and cuts the DNA at a specific region of the DNA. Other enzymes known as ligases join the ends of two DNA fragments. These and other enzymes enable the manipulation and amplification of DNA, essential components in joining the DNA of two unrelated organisms.

2. LOCATING GENES FOR PLANT TRAITS:Identifying and locating genes for agriculturally important traits is currently the most limiting step in the transgenic process. We still know relatively little about the specific genes required to enhance yield potential, improve stress tolerance, modify chemical properties of the harvested product, or otherwise affect plant characters. Usually, identifying a single gene involved with a trait is not sufficient; scientists must understand how the gene is regulated, what other effects it might have on the plant, and how it interacts with other genes active in the same biochemical pathway. These efforts should result in identification of a large number of genes potentially useful for producing transgenic varieties.

3. DESIGNING GENES FOR INSERTION:Once a gene has been isolated, it must undergo several modifications before it can be effectively inserted into a plant.

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

a) A promoter sequence must be added for the gene to be correctly expressed (i.e., translated into a protein product). The promoter is the on/off switch that controls when and where in the plant the gene will be expressed. To date, most promoters in transgenic crop varieties have been "constitutive", i.e., causing gene expression throughout the life cycle of the plant in most tissues.

b) The most commonly used constitutive promoter is CaMV35S, from the cauliflower mosaic virus, which generally results in a high degree of expression in plants. Other promoters are more specific and respond to cues in the plant's internal or external environment. An example of a light-inducible promoter is the promoter from the cab gene, encoding the major chlorophyll a/b binding protein.

c) Sometimes, the cloned gene is modified to achieve greater expression in a plant. For example, the Bt gene for insect resistance is of bacterial origin and has a higher percentage of A-T nucleotide pairs compared to plants, which prefer G-C nucleotide pairs. In a clever modification, researchers substituted A-T nucleotides with G-C nucleotides in the Bt gene without significantly changing the amino acid sequence. The result was enhanced production of the gene product in plant cells.

d) The termination sequence signals to the cellular machinery that the end of the gene sequence has been reached.

e) A selectable marker gene is added to the gene "construct" in order to identify plant cells or tissues that have successfully integrated the transgene. This is necessary because achieving incorporation and expression of transgenes in plant cells is a rare event, occurring in just a few percent of the targeted tissues or cells. Selectable marker genes encode proteins that provide resistance to agents that are normally toxic to plants, such as antibiotics or herbicides. As explained below, only plant cells that have integrated the selectable marker gene will survive when grown on a medium containing the appropriate antibiotic or herbicide. As for other inserted genes, marker genes also require promoter and termination sequences for proper function.

4. TRANSFORMATION:

Transgenic plants can be produced by two methods:

1) Vector mediated gene transfer (Indirect gene transfer)a. Bacterial vectors. E.g. Agrobacteriumb. Viral vectors. E.g. E.coli

2) Direct gene transfera. Direct gene transfer to protoplasts

i. Chemical treatment (PEG)ii. Electrical treatment (Electroporation)

iii. DNA delivery (liposomes)b. Direct gene transfer to cells and tissues

i. Microprojectile bombardment (Biolistic or DNA particle gun)ii. Microinjection of DNA into cells and protoplasts

c. Macro injection of DNA into plants

d. DNA uptake into imbibing zygotic embryose. Transmission of pollenf. Fibre-mediated DNA delivery into plant cellsg. Laser-induced DNA delivery

INDIRECT GENE TRANSFERVectors are the DNA carriers into which foreign DNA or genes of interest are inserted to make a recombinant DNA. Vectors along with this foreign DNA’s are then introduced into appropriate host cells. Vectors are divided into two types:

a) Cloning vectors (used for making millions of copies of DNA segment)b) Expression vectors (used for expression of cloned gene to produce the product).

AGROBACTERIUM METHOD OF PLANT TRANSFORMATION:

Agrobacterium tumefaciens is a remarkable species of soil-dwelling bacteria. This bacteria possess a large plasmid (90 – 150 × 106 daltons) known as tumour inducing (Ti) plasmid which is responsible for causing Crown gall disease in higher plants.

Structure of Ti plasmid

A – T-DNA responsible for tumor formation

B – Responsible for replication

C – Origin of replication

D – Responsible for virulence

L – Left border

R – Right border

T-DNA

T-DNA has two borders and only the DNA between these borders is transported to the plant cell. Hence this DNA is known as transfer DNA. The right border is critical, T-DNA transfer must start at this end. T-DNA is transferred and integrated into host genome during the infection. It brings about physiological and morphological changes due to expression of genes located on T-DNA.

The different regions of T-DNA are:

i. An onc region, which consists of three genes (TMS 1, TMS 2 and TMR) is responsible for the biosynthesis of phytohormones IAA (an auxin) and isopentenyladenosine 5’ monophosphate (a cytokinin).

ii. An OS region, which is responsible for synthesis of an unusual aminoacid or sugar derivatives, collectively called as opines. These are low molecular weight nitrogen containing compounds e.g. octopine, nopaline, lysopine, histopine, etc.

iii. 25 bp direct repeat sequence, which is essential for T-DNA transfer acting only in cis-orientation.

iv. Vir-region – It is essential for transformation of cis or trans orientation. The vir region is organized into six operons, namely Vir A, Vir B, Vir C, Vir D, Vir E and Vir G.

Vir genes are required for T-DNA movement. Vir A is a product specific inner membrane protein that recognizes and is responsive to the plant phenolic compounds. Vir A transduces information most likely by protein phosphorylation to the product of Vir G.

Vir G: It can act as a transcriptional activator of itself and other loci of Vir gene. Vir C and Vir D: The products of Vir C and Vir D are involved in the generation and

processing of T-DNA. Vir B and Vir E: The products of Vir B and Vir E are involved in forming most of the

structure component that facilitates T-DNA movement.

Vir H: Products of Vir H may allow the bacteria to survive in the presence of bactericidal or bacteriostatic plant compounds during the infection.

T-DNA transfer process:An early event in the T-DNA transfer process is the nicking of Ti plasmid between 3 rd and 4th

base of the bottom strand of each 25bp repeat. The Vir D operon encodes an endonuclease that produces nicks in the border sequences i.e. in 25bp repeats. Then the initiation of DNA synthesis in the 5’-3’ direction takes place. This is a complex process. Once the T strand is generated, this DNA must traverse the bacterial cell membrane, bacterial cell wall, the plant cell wall and plant cell and nuclear membranes. Once it gets into the plant nucleus it finally integrate stably into the plant genome and thus causing the crown gall disease. Thus by placing foreign genes into the T-DNA region of Ti plasmid it is possible to achieve transformation.

T-DNA transfer process

The production of T-DNA copy (single-stranded) for transfer into plant cells.

Thus by placing foreign genes into the T-DNA region of Ti plasmid it is possible to achieve transformation.

Agrobacterium mediated gene transfer is achieved by two ways:

1) Co-culture with tissue explants2) In planta transformation

Co-culture with tissue explants:• The appropriate gene construct is inserted within the T- region of Ti plasmid; either co-integrate or a binary vector is used.• In case of many plants species small leaf disc are removed from surface sterilized leaves and used for co-cultivation.• In general the transgene constructs includes a selectable reporter gene e.g. neo gene.• The recombinant vector is placed in Agrobacterium, which is co-cultured with the plant cell or tissue, transformation take place in two days.• During the leaf disc -Agrobacterium co-culture, acetosyningone released by plant cells induces the vir gene which brings about the transfer of recombinant T-DNA into many of the plant cell. The T-DNA would become integrated into the plant genome, and the transgene would be expressed.• The transformed cell become resistant to kanamycin(due to expression of neo gene)

• After two days the leaf disc are transferred to regeneration medium containing appropriate concentration of kanamycin and carbenicillin.• Kanamycin allows only transformed allows only transformed plant cells to divide and regenerate shoots in about 3-4 weeks , while carbenicillin kills Agrobacterium cells. • The shoots are separated, rooted and finally transferred into soil.• Using this approach successful transformation has been obtained in barley, wheat, maize and rice.

In Planta transformation:• Imbibition (sorption) of Arabidopsis seeds in fresh cultures of Agrobacterium leads to stable integration of T-DNA in the Arabidopsis genome.• It appears that Agrobacterium cells enter the seedlings during germination are retained within the plants, and when flowers developed they transform either the zygote or the cells that gives rise to zygote.• Alternatively Arabidopsis plants about to flower are immersed in a fresh culture of Agrobacterium and partial vacuum is created to facilated entry of bacterial cells into the plant cell.

Agroinfection:• Introduction of a viral genome into plant cells by placing it within the T-DNA of a Ti plasmid and using the Agrobacterium containing this recombinant plasmid for co-culture with the plant cell is called agroinfection.• This strategy is used with such viruses which have to be transmitted by an insect vector for successful infection. e.g. geminiviruses.

DIRECT GENE TRANSFER

Introduction of DNA into plant cells without the involvement of a biological agent e.g. Agrobacterium and leading to stable transformation is known as direct gene transfer. The spontaneous uptake of DNA by plant cells is quite low. In many crops including cereals and legumes, tissue culture techniques for regeneration is not very successful. These limitations forced to look for alternative methods of gene transfer. The various methods of direct gene transfer are as follows:

DIRECT GENE TRANSFER TO PROTOPLASTS:

1. ELECTROPORATION:Introduction of DNA into cells by exposing them for very short period to high voltage electrical pulses which is thought to induce transient pores in the plasmalemma is called electroporation. There are two types of systems of electroporation:

i. Low voltage-long pulses methodii. High voltage-short pulses method

E.g. For tobacco mesophyll protoplasts, for low voltage-long pulses method, 300-400 V cm-1 for 10-50 ms (milliseconds) and for the high voltage-short pulses method, 1000-1500 V cm-1 for µs were given.

Plant protoplasts are suspended in a suitable ionic solution containing recombinant plasmid DNA. The electroporation mixture is then exposed to the chosen voltage-pulses combination for the desired number of cycles. Protoplasts are then cultured to obtain cell colonies and plants.

Generally low voltage-long pulses produce high rates of transient transformation, which high voltage-short pulses five high rates of stable transformation.

2. CHEMICAL METHODS:Certain chemicals e.g. PEG (polyethylene glycol), polyvinyl alcohol and calcium phosphate, enhance the uptake of DNA by plant protoplast.

A generalized approach for PEG-mediated DNA delivery is as follows.• Plant protoplasts are suspended in a transformation medium rich in Mg2+ ions.• Plasmid DNA containing the gene constructs is added to the protoplast suspension, following which PEG (upto 20% concentration) is added and pH adjusted to about 8.• The protoplast may be given a 5 min heat-shock at 4500C followed by transfer to ice just before the addition of DNA.• After a period of incubation, the PEG concentration is reduced while that of Ca2+ is enhanced, this enhances transformation frequency.• The treated protoplasts are finally cultured to regenerate cell wall and form callus colonies from which plants are generated.

3. LIPO INFECTION:Introduction of DNA into cells via liposomes is known as lipoinfection. Liposomes are small lipid vesicles, in which large number of plasmids are enclosed. They can be induced to fuse with protoplast using devices like PEG and therefore have been used for gene transfer. These enters the protoplasts due to endocytosis of liposomes.

Advantages of this technique:

1) Protection of DNA/ RNA from nuclease digestion. 2) Low cell toxicity 3) Stability and storage of nucleic acids due to encapsulation in liposome. 4) High degree of reproducibility.

This method can be used in the gene transfer for the production of transgenic animal. This technique has been successfully used to deliver DNA into the protoplasts of a number of plant species such as carrot, petunia and tobacco.

DIRECT GENE TRANSFER TO CELLS AND TISSUES:

1. MICROINJECTILE BOMBARDMENT (Biolistic or DNA particle gun):

In this method 1-2 µm tungsten or gold particles, coated with the DNA to be used for transformation, are accelerated to velocities which enable their entry into plant cells/nuclei.Particle acceleration is achieved by using a device which varies considerably in design and function.The most successful device accelerates particles in one of the two ways: 1) By using pressurized helium gas 2) By the electrostatics energy release by a droplet of water exposed to high

voltageThis method is also called as biolistic or ballistic method of DNA delivery.

2. MICROINJECTION OF DNA INTO CELLS AND PROTOPLASTS:

The DNA solution is injected directly inside the cell using capillary glass micropipettes with the help of micromanipulators of a microinjection assembly. It is easier to use protoplasts than cells since cell wall interferes with the process of microinjection. The protoplasts are usually immobilized with agarose or on a glass coated with polylysine or by holding them under suction by a micropipette.

It is necessary to introduce the DNA into the nucleus or the cytoplasm of the cell for high transformation rates. Therefore, it is the most successful with densely cytoplasmic non-vacuolated embryonic cells. Large vacuolated cells show very low transformation rates possibly because, the DNA gets delivered into the vacuole, and as a consequence is degraded.

MACROINJECTION OF DNA INTO PLANTS

The injection of plasmid DNA into the lumen of developing inflorescence using a hypodermic syringe is called as macroinjection. It is hypothesized that the DNA is taken up by micropores during some specific stage of their development. The approach is very simple and easy but the problem is frequency and consistency of stable transformants obtained.

In secale cereal, a marker gene was macroinjected into the stem below the immature floral meristem, so as to reach the sporogenous tissue leading to successful production of transgenic plants.

DNA UPTAKE INTO IMBIBING ZYGOTIC EMBRYOS

When dry isolated embryos of wheat, barley, rye, pea and bean (isolated by grinding seeds in carbon tetrachloride or cyclohexane) are imbibed in a DNA solution they take up the DNA and show the expression of marker gene.The imbibed seed/embryos are germinated on appropriate selective media to isolate the transform embryos.

TRANSMISSION OF POLLEN

Some reports have claimed gene transfer by soaking pollen grains in DNA solution prior to their use for pollination. However, these reports could not be sustained by other workers using cloned genes. The method is highly attractive in view of its simplicity and general applicability, but so far there is no definite evidence for a transgene being transferred by this method.

FIBER-MEDIATED DNA DELIVERY INTO PLANT CELLS

In this method DNA is delivered into the cell cytoplasm and nucleus by silicon carbide fibres of 0.6 µm diameter and 10-80µm length. It was shown that the fibres mediated the delivery of DNA into the cytoplasm and nuclei of cells in a manner similar to microinjection. This method was successful with both maize and tobacco suspension culture cell.

LASER-INDUCED DNA DELIVERY

Laser has been successfully used for high frequency (10-3) transfection of animal cells. Laser puncture transient holes in the cell membrane through which DNA may enter into the cell

cytoplasm. Lasers have been used to deliver DNA into plant cells, but there is no information on transient expression or stable integration.

5. SELECTION AND REGENERATION

Selection of successfully transformed tissues: Following the gene insertion process, plant tissues are transferred to a selective medium containing an antibiotic or herbicide, depending on which selectable marker was used. Only plants expressing the selectable marker gene will survive, as shown in the figure, and it is assumed that these plants will also possess the transgene of interest. Thus, subsequent steps in the process will only use these surviving plants.

Regeneration of whole plants: To obtain whole plants from transgenic tissues such as immature embryos, they are grown under controlled environmental conditions in a series of media containing nutrients and hormones, a process known as tissue culture. Once whole plants are generated and produce seed, evaluation of the progeny begins. This regeneration step has been a stumbling block in producing transgenic plants in many species, but specific varieties of most crops can now be transformed and regenerated.

6. FUTURE DEVELOPMENTS IN TRANSGENIC TECHNOLOGYNew techniques for producing transgenic plants will improve the efficiency of the process and will help resolve some of the environmental and health concerns. Among the expected changes are the following:

More efficient transformation, that is, a higher percentage of plant cells will successfully incorporate the transgene.

Better marker genes to replace the use of antibiotic resistance genes. Better control of gene expression through more specific promoters, so that the inserted gene will be active only when and where needed. Transfer of multi-gene DNA fragments to modify more complex traits.

7. PLANT BREEDING AND TESTING:Intrinsic to the production of transgenic plants is an extensive evaluation process to verify whether the inserted gene has been stably incorporated without detrimental effects to other plant functions, product quality, or the intended agroecosystem. Initial evaluation includes attention to:

• Activity of the introduced gene• Stable inheritance of the gene• Unintended effects on plant growth, yield, and quality

If a plant passes these tests, most likely it will not be used directly for crop production, but will be crossed with improved varieties of the crop. This is because only a few varieties of a given crop can be efficiently transformed, and these generally do not possess all the producer and consumer qualities required of modern cultivars. The initial cross to the improved variety must be followed by several cycles of repeated crosses to the improved parent, a process known as backcrossing. The goal is to recover as much of the improved parent's genome as possible, with the addition of the transgene from the transformed parent.The next step in the process is multi-location and multi-year evaluation trials in greenhouse and field environments to test the effects of the transgene and overall performance. This phase also includes evaluation of environmental effects and food safety.

ADVANTAGES AND DISADVANTAGES OF TRANSGENIC PLANTS

ADVANTAGES:• Transgenic plants are free from harmful viruses and other diseases.• Some seeds are difficult to obtain or germinate, so this method is a preferable alternative.• Seed production is often at the mercy of the weather for successful pollination. The process does not depend on pollination.

• The uniformity of c plants is often desired as in hedges, orchards, rows of trees in landscaped areas where each should appear identical, etc. Noncloned plants often may vary widely in rate of growth and appearance.• It particularly increased crop yields and crop quality because the best plants are cloned.• Transgenic plant is stable. • Fixing superior genotypes.• Uniformity of population.

DISADVANTAGES:• The major disadvantage is the lack of genetic diversity which means if the one is susceptible to a disease or pest, all the members of the clone would be affected.• The process of transgenic plant is also very expensive and technically difficult.• Advanced skills are required for the successful operation, organization and training of staff require for present problems when carrying out procedure on a large scale.• Family specific methods may be necessary to obtain optimum results from each species & varieties.

APPLICATIONS OF TRANSGENIC PLANTS

1. They have proved to be extremely valuable tool in studies on plant molecular biology, regulation of gene action, identification of regulatory/promoter sequence etc.

2. Transgenic plant also permit the analysis of metabolic pathways, studies of cis- and trans- acting factors in gene function and the elucidation of plant response to environmental stress etc.

3. Specific genes have been transferred into plants to improve their agroeconomic and other features.

a) Gene conferring resistance to abiotic stresses eg. Herbicides, have been transferred in crop plants which enables the use of biodegradable herbicides like glyphosate in otherwise susceptible to crops.

b) Genes for resistance to various biotic stress have been engineered to generate transgenic plants resistant to insects, virus etc.

Expression of some bacillus thuringiensis insecticidal toxin gene in plants.

Sr. no. Plants Genes % exp. Insecticidal1. Tobacco Cry1Ab, truncated 0.003-0.012 Yes2. Cotton Cry1Ab,PM 0.05-0.1 Yes3. Tomato,

TobaccoCry1Ab,truncated 0.3 Yes

c) Development of herbicide resistance plant :Cyanamide – resistant tobacco plants were produced when a cyanamide hydrates gene from the fungus Myrotheciu verrucaria was introduced. The enzyme encoded by this gene converts cyanamide to urea.

d) Development of virus, fungus, bacterial and stress resistance plant, delay senescence, tolerance to environmental stress, altered flower pigmentation, improved nutritional quality of seed proteins, increased shelf life of plants.

4. Several gene transfers have been aimed to improving the produce quality eg. Protein or lipid quality etc. Improved quality may also achieved by either suppression of or overproduction by endogenous genes.

5. Transgenic plants are aimed to produced novel biochemicals like interferon, insulin, immunoglobulin etc. or useful biopolymers like polyhydroxy-butyrate which are not produced by normal plants.these compounds are extracted from the plants and can be used as pharmaceutical or industrial substrate.

Sr. no. Proteins Disorders1. Insulin Diabetes2. DNase I Cystic fibrosis3. Interferons Hairy cell leukemia

6. Transgenic plants have been produced that express a gene encoding an antigenic protein from a pathogen. When mice were fed on such plant produce, they become immunized against the pathogen. Therefore use of transgenic plants as vaccines for immunization against pathogens is fast emerging as an important objective.

REFERENCES

1. Singh B.D.; “Biotechnology; 1st edition; 2003; kalyani publishers; 291-329 .

2. Veeresham Ciddi ; “medicinal plant biotechnology”; 1st edition; 2006; CBS publishers & distributors; 348-360.