PREPARED BYKishan R. Sanja

T.Y.B.sc.(Biotechnology)

GUIDED BYMr. Ramchandra Suthar Sir

(Lecturer-P S Science College, Kadi)

T.Y.B.sc.(Biotechnology)Biotechnology Department

P.S. Science & H.D. Patel Arts College(Hemchandracharya North Gujarat University)

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P.S. Science & H.D. Patel Arts College, Kadi

(Affiliated with Hemchandracharya North Gujarat University)

CERTIFICATE

TO WHOMSOEVER IT MAY CONCERN

This is to certify that Mr. Kishan R. Sanja B.Sc.

Biotechnology Student of

T Y B.Sc. (Biotechnology) Class; Roll No. 281 has

satisfactorily completed her project work on ”Application

of Plant Tissue Culture” of the subject Biotechnology

during the academic year 2008/09 and submitted report on

_______________

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Head of Department Staff in Charge

Certified that this project-work is accepted and assessed on __________

ACKNOWLEDGEMENT

I here with a ton Thanks to college chief and Principal Head

Mr. Ajay Gor sir & acknowledging all my mentors for successful completion of my Project…

On the completion of this project report I am very much thankful to my faculty members of Pramukh Swami Science College, Kadi, and my guide faculty

Mr. Ramchandra Suthar who has always helped and guided me in every possible way during the making of this report.

I am also very much grateful to Biotechnology Department of my college for their guidance and encouragement during the collection of data for my report.

I am completely indebted to H.O.D. (Biotechnology), Minal Trivedi madam for showing trust in me and allowing me to complete my Project wishfully.

I would like to thank all my mentors during the project period for giving their precious time to me and helping me in completing my Assignment.

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I am very much thankful to all my family members and friends for helping me and encouraging me for wonderful task and active role in development of such project reports…

Kishan R. Sanja

Table of Contents

No. Topic of Content Page No.

1 Abstract

2 Introduction

3 Application in Agriculture

4 Application in Horticulture and Forestry

5 Application in Industry

6 Transgenic plants

7 Bioethics in Plant Genetic Engineering

8 Reference

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Abstract

It is the technique of growing plant cells, tissues and organs in artificial prepared nutrient medium static or liquid, sterile & environmentally supportive conditions

(In Vitro)

Production of haploid through distant crosses or using pollen, anther or ovary culture, followed by chromosome doubling, reduces this time to two generations. This represents a saving of 4-6 years. The other example is the transfer of a useful bacterial gene say, cry (crystal) gene from bacillus thuringiensis, into a plant containing & expressing this gene (transgenic plants). This can be achieved only by a combination of tissue & genetic engineering; none of the conventional breeding approaches can ever produce such a plant.

Principle involved in PTC is very simple & primarily an attempt, where by an explants can be to some extent freed from inter organ, inter cellular & inter tissue interactions & subject to direct experimental control.

Embryo Culture is the sterile isolation & growth of an immature or mature embryo in vitro, with the goal of obtaining a viable plant.

Organ Culture: In this method a particular organ is isolated and cultured in lab. Conditions in a chemically defined medium where they retain their characteristic structures and other features continue to grow as usual.e.g. Root tips, shoot tips, embryos, etc.

Callus culture: a mass of disorganized, mostly undifferentiated or undeveloped cell culture.

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Meristem culture: when a meristem is cultured in vitro, then it produces a small plant bearing 5 or 6 leaves. This0 obtained within a few weeks. Then

Protoplast culture: culture of plant protoplasts, i.e. cells devoid of their cell walls.

Plant tissue culture is the technique of growing plant cells, tissues and organs in artificial prepared nutrient medium static or liquid, sterile & environmentally supportive conditions (in vitro) to produce many new plants, each a clone of the original mother plant, over a very short period of time. The technique has developed around the concept that a cell is tot potent that has the capacity & ability to develop into whole organism.

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1. INTRODUCTION

Definition:

It is the technique of growing plant cells, tissues and organs in artificial prepared nutrient medium static or liquid, sterile & environmentally supportive conditions

(In vitro)

It has advanced the knowledge of fundamental botany, especially in the field of agriculture, horticulture, plant breeding, forestry, somatic cell hybridization, phytopathology & industrial production metabolites etc.

In commercial settings, tissue culture is often referred to as micro propagation, which is really only one form of a set of technique micro propagation refers to the production of whole plants from cell cultures derived from explants (the initially piece of tissue put into culture) of meristem cells.

Production of haploid through distant crosses or using pollen, anther or ovary culture, followed by chromosome doubling, reduces this time to two generations. This represents a saving of 4-6 years. The other example is the transfer of a useful bacterial gene say, cry (crystal) gene from bacillus thuringiensis, into a plant containing & expressing this gene (transgenic plants). This can be achieved only by a combination of tissue & genetic engineering; none of the conventional breeding approaches can ever produce such a plant.

The term tissue culture is commonly used in a very wide sense to include in vitro culture of plant cells, tissue as well as organs. But in a strict sense, tissue culture denotes the in vitro cultivation of plants cells in an unorganized mass, e.g., suspension cultures. But, in general, the term tissue culture is applied to both callus & suspension cultures, and cell culture is often used for callus as well. When organized structures like root tips, shoot tips, embryos, etc. are cultured in vitro to obtain their development as organized structure, it

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is called organ culture. Plant tissue culture is used in its broad sense to denote aseptic in vitro culture of plant cells, tissues & organs.

Tissue culture is a process that involves exposing plant tissue to a specific regimen of nutrients, hormones, and lights under sterile, in vitro conditions to produce many new plants, each a clone of the original mother plant, over a very short period of time. Tissue culture plants are characterized by disease free growth, a more fibrous, healthier root system, a bushier branching habit, and a higher survival rate.

PRINCIPLE :-

The technique has developed around the concept that a cell is tot potent that has the capacity & ability to develop into whole organism.

Principle involved in PTC is very simple & primarily an attempt, where by an explants can be to some extent freed from inter organ, inter cellular & inter tissue interactions & subject to direct experimental control.

Isolated cells from differentiated tissues are generally non-dividing & quiescent; to express totipotency the differentiated cell first undergoes dedifferentiation & then redifferentiation. The phenomenon of a mature cell reverting to a meristamic state & forming undifferentiated callus tissue is termed dedifferentiation, whereas the ability of a dedifferentiated cell to form a whole plant or plant organs is termed redifferentiation. Thus cell differentiation is the basic event of development in higher organisms & conveniently referred to as cytodifferentiation.

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THE GENERAL TECHNIQUE

The technique of in vitro cultivation of plant cells & organs is primarily devoted to solve two basic problems: (1) To keep the plant cells & organs free from, microbes & (2) To ensure the desired development in the cells and organs by providing suitable nutrient media and other environmental conditions.

LAB SPACE

In general space for the following is needed:

(1) Washing, drying &storage of vessels

(2) Preparation, sterilization & storage of media

(3) Aseptic handling of explants & cultures

(4) Maintenance of cultures &

(5) Observations of cultures

In a modest laboratory, provisions for activities 1& 2 can be made in single room (media room), while the remaining work can be done in the another room (culture room)

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LABORATORY EQUIPMENTS

Laminar Air Flow Autoclave Refrigerator Freezer Balances pH meter Water distillation unit Hot plate magnetic stirrer Ovens Water bath Hot plate/gas plate Microwave oven Centrifuge tabletop Vortex Shaker, gyratory with platform & clips for different size flasks Dissecting microscope Lab carts

NUTRIENT MEDIUM

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1. Inorganic nutrient: in addition to C, H and O, all nutrient media provide the 12 elements essential for plant growth viz., N, P, K, Ca, S, Mg(6 macronutrients needed in concentration>0.5 m mol l-1 or > 0.5mM).the different tissue culture media provide different concentration of the inorganic nutrient.

2. Vitamins: for optimum callus growth, inositol, thiamine pyridoxine and nicotinic acid of which thiamine is essential and the rest are promontory.

3. Carbon source: sucrose (20-50 g l) is the most used carbon source, other sugars like maltose, glactose, lactose, mannose & even starch, but these are rarely used. Other complex nutrients include casein hydrolysate (CH), coconut milk, corn milk, malt & yeast extract.

4. Growth regulators: in culture media involve (I) auxins e.g. IAA(indole-3-acetic acid ), IBA(Indole-3-butyric acid), NAA(naphthalene acetic acid), 2,4-D (2,4-dichlorophenoxy acetic acid), etc., are commonly used to support cell division & callus growth, somatic embryo, rooting, etc., (ii) cytokines like(kinetin , BAP(benzyl amino purine), zeatine, 2-ip(isopentyle adenine), TPZ (thiadiazuron) are employed to cell division ,regeneration of shoots, se induction & to enhance proliferation & growth of axillary buds.(iii) abscisic acid (ABA) promotes SE & shoot bud regeneration in many species. (iv) gibberellins promotes shoot elongation & SE germination

TYPES OF CULTURE

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Embryo Culture is the sterile isolation & growth of an immature or mature embryo in vitro, with the goal of obtaining a viable plant.

Organ Culture: In this method a perticular organ is isolated and cultured in lab. Conditions in a chemically defined medium where they retain their characteristic structures and other features continue to grow as usual.e.g. Root tips, shoot tips, embryos, etc.

Callus culture: a mass of disorganised, mostly undifferentiated or undeveloped cell culture.

Meristem culture: when a meristem is cultured in vitro, then it produces a small plant bearing 5 or 6 leaves. This0 obtained within a few weeks.

Then Protoplast culture: culture of plant protoplasts, i.e. cells devoid of their cell walls.

A.APPLICATION IN AGRICULTURE

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1. Improvement of hybrids:

Development of cell diffusion and hybridization techniques has solved the problem of incompatibility of plants and widened the scope of production of new varieties within a short time.

The best example of the application of another culture in crop breeding and improvement is the production of another culture derived rice and wheat varieties in China. About 50 varieties in rice and 20 in wheat have been developed by using this technique.

CRYOPRESERVATION OF GERM PLASMA

PLANT TISSUECULTURE

CLONAL PROPOGATION

SECONDARY METABOLITES

LARGE SCALE MULTIPLICATION GENETIC

VARIABILITY

BIOMASS ENERGY

WIDE HYBRIDIZATION

CRYOPRESERVATION OF GERM PLANTS

SOMATIC HYBRIDS/CYBRIDS

SYNTHETIC SEEDS

DISEASES FREE PLANTS

HAPLOIDS, POLYPLOIDS,TRIPLOIDS

PLANT IMPROVEMENT AND THROUGH TISSUE CULTURE TECHNOLOGY

2. Encapsulated seeds:

In these seeds, the gel acts as seed coat and the artificial endosperm providing nutrient as in true seeds. Water

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soluble gels must be used as the protective gel. Usually Na/Ca alginate is selected for encapsulation purpose because it is less toxic to embryos and easy to handle.

Methods of production:

1. Induce pseudoembryos from cell suspension culture.

2. Mix embryos well with 2 per cent Na- alginate.

3. Drop the embryos in a bath of calcium salt e.g. solution of Ca (NO3)2 for 30 minutes. This results in very quick complex formation at surfaces due to exchange of ions.

4. Sieve the bead through a nylon mesh; the solution can be recycled and

5. Test the growth vigor of beads by plating in sand or soil amended with pesticides.

3. Production of disease resistant plants:

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(A) Production of virus free plants: About 10 percent of viruses transmitted through seeds. In some cases, they are confined to seed coat (e.g. TMV) or internally seed borne. Moreover, viruses result in great loss, for example, potato leaf roll virus or potato virus X can cause up to 95 per cent reduction in tuber yield and potato virus X between 5 and 75 per cent depending on virus strain and host cultivar.

Tissue culture technique can be utilized for the production of virus free plants either through meristem culture or chemotherapy or selective chemotherapy of larger explants from donor plants or dormant or a combination of the two.

EASTER LILY BULB FROM MARKET

VIRUS INDEXING

DIRECT ORGANOGENESIS( 3 WEEKS)

ROOT INITIATION( 3 WEEKS)

PLANT IN POTS

VIRUS FREE PLANTS (IN VITRO GENE BANK, PRODUCTION

OF HEALTHY SEEDS)

HOT WATER TREATMENT

REANSFER SURFACE STERILIZED SEGMENTS

TRANSFER EXPLANTS TO ROOTING MEDIUM

ROOT ELONGATION

HARDENING , TRANSFER INTO SOIL

OUTLINE FOR PRODUCTION OF VIRUS FREE PLANTS OF EASTER LILY

(B) In vitro selection of cell lines for disease resistance in vitro, one of the important considerations is the selection of suitable type of culture e.g. callus tissue, suspension culture, isolated cells or protoplasts. However callus culture

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have been widely used for study of expression of race specific and non-host resistance, and offer several advantages over suspension culture, isolated cell or protoplasts. The advantages are:

Ease of initiation and maintenance of tissue in culture

Ability to add inoculums

The ability to follow the progress of infection and colonization of callus tissue

Phytoalex in accumulation can be determined in pathogen challenge tissue and related to the extent of colonization.

EAR HEAD OF BAJRA INFECTEDSCIEROSPORA GRAMINICOLA

DUAL CULTURES ONE OF PLANT,SECOND OF PATHOGEN

EMBRYOIDS IN EMBRYOGENIC CALLUS OF DUAL CULTURE

EMBRYOGENIC CALLUS

PLANTLETS

COMPLETE PLANTLETS WITHROOTS AND SHOOTS

DISEASE RESISTANT PLANTS IN POTS

FIELD PERFORMANCE

SELECT EMBRYOGENIC CALLUS FROM PATHOGENIC FREE PORTION OF DUAL CULTURE

SHOOTING

ROOTING

TRANSFER DISEASE FREE PLANTS TO EARTHENWARE POTS AFTER HARDENING

PLANT CHALLENGING WITH PATHOGEN

CULTURE ON MS MEDIUM

EMBRYOGENESIS

IN VITRO SELECTION OF A CELL LINE OF BAJRA RESISTANT TO DOWNY MIDEW

This technique can be extensively applied for mass rearing of nematodes in vitro and screening of resistant breeding materials and nematicides.

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As a result of host cell pathogen interactions, protoplasts of plant tissues are damaged due to secretions of toxins and enzymes. They are equivalent to thousand acres of growing plants in the field conditions.

Miller and Maxwell have described several advantages of tissue culture systems over the suspension cultures, isolated cells or protoplasts for the study of expression of race specific host resistance. These are:

The ease of initiation and maintenance of cultured tissue

The ability to add inoculums directly to callus so that the tissue culture medium would not be a direct source of nutrition for the pathogens, and

The ability to follow cytologically the progress of infection and colonization of callus tissue by the pathogens and the host response.

B. Applications in Horticulture and Forestry

Micropropagation

Micropropagation is a method of propagating plants using very small parts of plants that are grown in sterile culture. Micropropagation is not most likely the major use of tissue culture for molecular biologists or plant breeders. However,

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it is important commercially, and can be used to introduce several concepts that apply to all of tissue culture.

Stages of Micropropagation

Micropropagation is now typically divided into 5 stages. Stages 1-4 were originally proposed by Murashige; Debergh and Maene added Stage 0.

Stage 0: Preparative Stage: Donor Plant Selection and Preparation

Explant quality and responsiveness is influenced the physiological phytosanitary condition of the donor plants.

C. Donor (stock) plants indexed for pathogens.

D. Pathogen-free stock plants maintained in clean conditions (low humidity, drip irrigation).

E. Vigorous growth is encouraged, but not over-fertilization.

F. Donor plants may be pretreated in certain ways.

Stage 1: Establishment of Explant in Culture

Surface-sterilization – disinfestations: Must free Explant tissues of all contaminating microorganisms, but not cause phytotoxity.

Isolation of shoot tip under sterile conditions.

Medium - Must contain all components necessary to nourish Explant (medium composition) and to make

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the Explant perform as desired (PGRs).

Browning of the medium: Results from oxidation of phenolics leached out from cut surfaces of explants; often seen with adult woody species. Handled with anti-oxidants, frequent transfer.

Medium formulation is often standard, e.g. M&S, but more complex media may be necessary for smaller explants.

Medium may be semi-solid or liquid; there are advantages and disadvantages of each.

Environmental conditions

Light

Temperature

Relative humidity

Culture stabilization.

Stage 2: Multiplication Proliferation of Axillary Shoots

Repeated enhanced axillary shoot production.

Encouraged by cytokinin in the medium, alone or with a smaller amount of auxin. Amount of cytokinin and presence and amount of auxin must be determined empirically.

Shoots harvested and shoot clusters transferred to fresh medium at frequent, regular intervals.

Number of subcultures possible from the original culture varies with species/cultivar: reduction of growth, increase in mutations.

Stag3:Pretransplant(Rooting)

Adventitious rooting of shoots or shoot clusters in

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vitro.

Harvested shoots may be pretreated before rooting: prehardening, elongation, fulfilling dormancy requirements.

For root initiation in vitro, auxins are important.

More dilute medium, activated charcoal may be added.

Advantages of rooting after removal from culture

Reduced costs

Structurally and physiologically better

Damage to roots may occur during transplanting

Stage 4: Transfer to Natural Environment:

Acclimatization: Process by which physiologically and anatomically adjust from in vitro to ex vitro conditions.

Relatively slow process, may take weeks, starch reserves important.

Must adjust from high to lower relative humidity (e.g. from 98-99% to 20 - 60%): development of sufficient defenses to control water loss.

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o Poor cutilcle development: epicuticular wax needs to be formed.

o Abnormal stomatal development and function.

Either are not properly depressed.

Or do not open and close properly.

o Non- or poorly functional roots.

From clean vs. presence of pathogens.

Must adjust from low light to high light: from low photosynthetic competence (heterotrophic nutrition) to photosynthetic competence.

Poorly differentiated leaf structure.

Poorly developed chloroplasts.

Supplied carbohydrate source to independent carbon fixation.

There may be culture medium carryover effects.

Dormancy may need to be overcome.

Soil medium and container important.

Acclimatization structures:

Plastic covers.

Humidity tent. Overhead mist.

Fog system.

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Applications of Micropropagation

The obvious: clonal mass propagation of large numbers of uniform plants.

Micropropagation may allow faster production of plants that are slow to propagate in vivo.

It may decrease the time needed for bulk-up of new cultivars before they are introduced commercially.

Storage of germplasm, e.g. by cryopresevation.

C.APPLICATION IN INDUSTRIES

1. SECONDARY METABOLITES FROM CELL CULTURES: Plant cells cultured in vitro have been considered to potential source of specific secondary metabolites. Cell culture may contribute in at least four major ways to the production of natural products. These are as:

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a new route of synthesis to establish products e.g. codeine, quinine

A new route of synthesis to novel product from plants difficult to grow or establish e.g. thebain from papaver bracteatum.

A source of novel chemicals in their own right e.g. rutaculin from culture from rata.

As biotransformation systems either on their own or as part of a larger chemical process e.g. dixogin synthesis (Fowler 1983).

A.CELL SUSPENSION AND BIOTRANSFORMATION:

Bio transformation is a process through which functional group of organic compounds are modified by living cells. Biotransformation done by plant cell culture can be desirable when a given reaction is unique to a plant cell and the product of reaction has a high market value.

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Plants are used in number of ways for biotransformation purposes. The most basic procedure is to supply the cell suspension with the components that is to be transformed, and harvest the products from the culture medium after incubation in suitable conditions.

FILTER AIR EXHAUST

CONDENSER

AIR INLETMULTISOCKET LID

WATER JACKET

GLASS ROD

MAGNETIC STIRRER

SAMPLING OUTLET

MEDIUM INLET

FLAT JOINT

DIAGRAM OF V-FERMENTER USED FOR THE PRODUCTION OF PLANT METABOLITES

The useful natural products are synthesized through secondary metabolism; hence they are also known as secondary metabolites. Secondary metabolites include alkaloids, terpenoids, tannins, glycosides and saponins. Their chief applications are in pharmaceuticals, in food flavouring and perfumery.

During metabolism in growing cells, the secondary metabolites are either deposited in vacuoles or excreted from gland cells. Genotypes, physiological conditions as much as location within a given plant determine the formation of secondary metabolites.

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PROCEDURE OF PROCESS DESIGN AND PRODUCT RECOVERY FROM THE CULTURED PLANT CELLS

B.SECONDRY METABOLITES FROM IMMOBILISED PLANT CELLS

Large scale yield of secondary metabolites from cultured plant cells can be increased simply by changing the physiological and biochemical conditions from growth medium. But one of the methods of production on increased rate is the use of immobilised plant cells. The method of immobilised plant cells has been found very effective for the production of secondary metabolites as it provides a stable and uniform environment. The plant cells are immobilised in inert matrix and bathed in a medium which does not allow the cell division but keeps the cells in viable conditions for a long time: This obviate the need of further subculturing. There are two commonly used metods for immobilisation: (i) immobilisation of cells or subcellular organelles, and (ii) adsorption to an inert substrate such as glass beeds.Examples of cell immobilisation are given table.

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SOURCE OF CELLS IMMOBILISATION SUBSTRATES

METHOD

Catharanthus roseus Alginate, agarose, polyacrylamide, carrageenan

Immobilised in inert

substatum

Alginate and gelatin

Digitalis lanata Alginate Do

Morinda citrifolia Alginate Do

C. FACTORS AFFECTING PRODUCT YIELD: There are number of that are affecting high yielding cell lines.

Tissue origin genetic character : plant cells are genetically totipotent, therefore, proper environmental conditions should be given so that any cell may be included to produce any substance according to the characteristic of parent plant

Culture conditions : chemical composition of nutrient media influences the potential synthetic machinery and synthesis of secondary products. A balance should be maintained between the production of biomass and secondary products.

Selection and screening : cell clones from better strains are selected which is rather a difficult task. The more difficult work is to detect the very small amount of desired product present in single cell or small population of cells. To reach the goal mutagenic techniques as a selection

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procedure are applied to develop high yielding cell lines.

D. TRASGENIC PLANT

Introduction to Transgenic Plants

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Transgenic plants possess a gene or genes that have been transferred from a different species. Although DNA of another species can be integrated in a plant genome by natural processes, the term "transgenic plants" refers to plants created in a laboratory using recombinant DNA technology. The aim is to design plants with specific characteristics by artificial insertion of genes from other species or sometimes entirely different kingdoms.

Varieties containing genes of two distinct plant species are frequently created by classical breeders who deliberately force hybridization between distinct plant species when carrying out interspecific or intergeneric wide crosses with the intention of developing disease resistant crop varieties. Classical plant breeders use a number of in vitro techniques such as protoplast fusion, embryo rescue or mutagenesis to generate diversity and produce plants that would not exist in nature (see also Plant breeding, Heterosis, New Rice for Africa).

Such traditional techniques (used since about 1930 on) have never been controversial, or been given wide publicity except among professional biologists, and have allowed crop breeders to develop varieties of basic food crop, wheat in particular, which resist devastating plant diseases such as rusts. Hope is one such wheat variety bred by E. S. McFadden with a gene from a wild grass. Hope saved American wheat growers from devastating stem rust outbreaks in the 1930s.

Methods used in traditional breeding that generate plants with DNA from two species by non-recombinant methods are widely familiar to professional plant scientists, and serve important roles in securing a sustainable future for agriculture by protecting crops from pests and helping land and water to be used more efficiently.

The only plasmid that plant cells take up is the Ti (tumor-inducing) plasmid from the bacterium Agrobacteriam. The plasmid Transferred by this bacterium causes plants to form a gall.  There are several methods for introducing genes into plants, including

infecting plant cells with plasmids as vectors carrying the desired gene

Shooting microscopic pellets containing the gene directly into the cell.

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

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can be transformed in the laboratory with the desired gene Grown into mature plants with flowers.

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

SELECTABLE MARKER GENES AND THEIR USE IN TRANSFORMED PLANTS

When plant cells are transformed by any of the transformation methods as given earlier, it is necessary to isolate the transformed cells/tissue. However, it is possible to do now. There are certain selectable markers genes present in vectors that facilitate the

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selection process. In transformed cells the selectable marker genes are introduced through vector. The transformed cells are cultured on medium containing high amount of toxic level of substrates such as antibiotic, herbicides etc. For each marker gene there is one substrate. For a model transgenic system, tobacco is the most common plant that is found everywhere. The young explants such as leaf disks are aseptically cut into pieces. These pieces are transferred onto tissue regeneration medium supplemented with an antibiotic, kanamycin. For the transformed discs shoot grow directly. The cells which do not undergo transformation will doe due to kanamycin. Therefore, antibiotics and herbicides should be used carefully because even in low concentration many cells are damaged. When regeneration has accomplished, selection should be done thereafter. Besides, another difficulty associated with successful selection is the regeneration of shoots from transformed celli because the ex-plants may be heterogeneous and non-transformed cells could not be selected. Therefore, such methods should be used that can ensure escape of only few non transformed shoots from selection. However, it is ensured by using leaf discs as only the cells which are in direct contact of medium containing antibiotic/herbicide will undergo regeneration.

In addition, there is an alternative procedure when there will be no selection process imposed on cells/shoot that develop from ex-plants. In this method, samples of tissue from regenerated shoot are taken; the samples are tested for expression of a marker gene. There are a number of marker genes which are commonly described as reporter genes or scoreable genes or screenable genes. Some of the reporter genes which are commonly used in plat transformation are: cat, Gus, lux, etc.

1. Chloramphenicol acetyl transfer (CAT) gene : The cat gene is not used as a selectable but as reporter gene. It was the first isolated from the bacterium E.coli but it is absent in higher plants and mammals. In transformed cells, its presence can be detected by assaying the enzyme CAT on P-chloramphenicol mixed growth medium. Therefore the enzyme uses the acetyl Co-A-chloramphenicol-p as substrate and transfer acetyl CoA to

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chloramphenicol converting the latter into acetyl chloramphenicol which is detected autoradiographically.

2. Neomycin phosphotransferase gene: This gene confers resistance again kanamycin detoxifies it by phosphorylation. It encodes enzyme. Presence of this gene in transformed tissue can be detected by selecting them on kanamycin supplemented medium. However, if this gene has adverse effect on expression of desirable gene, its expression can be improved by using an alternative approach.

3. Luciferase gene (lux gene): The lux gene is found in glow-warm, firefly and bacteria that secrete the enzyme Luciferase. Due to secretion of these enzymes the glow-warm becomes luminescent in dark. The lux gene has been transferred into tobacco through Ti-plasmid of agrobacteriam. Consequently lux gene containing bioluminescent tobacco plants were produced. Similarly a green fluorescent protein (GFP) isolated from jellyfish, aequorea Victoria is used as reporter gene or tag in a wide variety organisms. These act as visible marker for gene expression.

4. The ß-galactosidase gene (lacZ gene): The lacZ gene that encodes ß-galactosidase is a polylinker as it contains several restriction sites but maintains the proper reading frame. Most DNA fragments cloned into polylinker disrupt lacZ gene and abolish ß-galactosidase activity. When a foreign gene fused with lacZ gene is inserted into microbial cell, its presence and function can be detected. When the genetically engineered microbial/plant/animal cells contained a reporter gene is allowed to grow on medium containing a chemical X-gal, ß-galactosidase hydrolyses X-gal, and releases an insoluble blue dye shows the presence of foreign gene. If there appears no colour, it means the gene is disrupted.

Agricultural impact oftransgenic plants

Out crossing of transgenic plants not only poses potential environmental risks, it may also trouble farmers and food producers.

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Many countries have different legislations for transgenic and conventional plants as well as the derived food and feed, and consumers demand the freedom of choice to buy GM-derived or conventional products. Therefore, farmers and producers must separate both production chains. This requires coexistence measures on the field level as well as traceability measures throughout the whole food and feed processing chain. Research projects such as Co-Extra, SIGMEA and Tran container investigate how farmers can avoid out crossing and mixing of transgenic and non-transgenic crops, and how processors can ensure and verify the separation of both production chains.

Some Achievements

1. Insect Resistance .

Bacillus thuringiensis is a bacterium that is pathogenic for a number of insect pests. Its lethal effect is mediated by a protein toxin it produces. Through recombinant DNA methods, the toxin gene can be introduced directly into the genome of the plant where it is expressed and provides protection against insect pests of the plant.

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Link to illustrated discussion of Bacillus thuringiensis.

2. Disease Resistance.

Genes that provide resistance against plant viruses have been successfully introduced into such crop plants as tobacco, tomatoes, and potatoes.

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

3. Herbicide Resistance.

Questions have been raised about the safety — both to humans and to the environment — of some of the broad-leaved weed killers like 2,4-D. Alternatives are available, but they may damage the crop as well as the weeds growing in it. However, genes for resistance to some of the newer herbicides have been introduced into some crop plants and enable them to thrive even when exposed to the weed killer.

Effect of the herbicide bromoxynil on tobacco plants transformed with a bacterial gene whose product breaks down bromoxynil (top row) and control plants (bottom row). "Spray blank" plants were treated with the same spray mixture as the others except the bromoxynil was left out. (Courtesy of Cal gene, Davis, CA.)

4. Salt Tolerance.

A large fraction of the world's irrigated crop land is so laden with salt that it cannot be used to grow most important crops. [Discussion] However, researchers at the University of California Davis campus have created transgenic tomatoes that grew well in saline soils. The transgene was a highly-expressed sodium/proton antiport pump that sequestered excess sodium in the vacuole of leaf cells. There was no sodium buildup in the fruit.

5. "Terminator" Genes.

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

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A gene encoding a toxin which is lethal to developing seeds but not to mature seeds or the plant. This gene is normally inactive because of a stretch of DNA inserted between it and its promoter.

A gene encoding a recombinase — an enzyme that can remove the spacer in the toxin gene thus allowing to be expressed.

A repressor gene whose protein product binds to the promoter of the recombinase thus keeping it inactive.

6. Transgenes Encoding Antisense RNA.

Antisense RNA

Messenger RNA (mRNA) is single-stranded. Its sequence of nucleotides is called "sense" because it results in a gene product (protein). Normally, its unpaired nucleotides are "read" by transfer RNA anticodons as the ribosome proceeds to translate the message. (See mechanism of translation.)

However, RNA can form duplexes just as DNA does. All that is needed is a second strand of RNA whose sequence of bases is complementary to the first strand; e.g.,

5´   C A U G   3´     mRNA3´   G U A C   5´     Antisense RNA

The second strand is called the Antisense strand because its sequence of nucleotides is the complement of message sense. When mRNA forms a duplex with a complementary Antisense RNA sequence, translation is blocked.

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This may occur because the ribosome cannot gain access to the nucleotides in the

mRNA or Duplex RNA is quickly degraded by rib nucleases in the cell

(see RNAi below).With recombinant DNA methods, synthetic genes (DNA) encoding Antisense RNA molecules can be introduced into the organism.

Another example:

Right: Flower of a tobacco plant carrying a transgene whose transcript is Antisense to one of the mRNAs needed for normal flower pigmentation. Left: Flower of another transgenic plant that failed to have its normal pigmentation altered. (Courtesy of van der Krol, et. al., from Nature 333:866, 1988.)

Antisense RNA also occurs naturally

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Do cells contain genes that are naturally translated into Antisense RNA molecules capable of blocking the translation of other genes in the cell? Recently a few cases have been found, and these seem to represent another method of regulating gene expression. In both mice and humans, the gene for the insulin-like growth factor 2 receptor (Igf2r) that is inherited from the father synthesizes an Antisense RNA that appears to block synthesis of the mRNA for Igf2r. An inherited difference in the expression of a gene depending on whether it is inherited from the mother or the father is called genomic or parental imprinting.  

7. Biopharmaceuticals.

The genes for proteins to be used in human (and animal) medicine can be inserted into plants and expressed by them. Advantages:

Glycoproteins can be made (bacteria like E. coli cannot do this).

Virtually unlimited amounts can be grown in the field rather than in expensive fermentation tanks.

There is no danger from using mammalian cells and tissue culture medium that might be contaminated with infectious agents.

Purification is often easier. Corn is the most popular plant for these purposes, but tobacco, tomatoes, potatoes, and rice are also being used. Some of the proteins that are being produced by transgenic crop plants:

human growth hormone with the gene inserted into the chloroplast DNA of tobacco plants.

humanized antibodies against such infectious agents as o HIV o respiratory syncytial virus (RSV) o sperm (a possible contraceptive) o herpes simplex virus , HSV, the cause of "cold sores"

protein antigens to be used in vaccines

Other useful proteins like lysozyme and trypsin

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MOLECULAR FARMING FROM TRANSGENIC PLANTS (TRANSGENIC PLANTS AS BIOREACTER)

In recent years, transgenic plants are used by biotechnology industries as ‘bioreactor’ for manufacturing special chemicals and pharmaceutical compounds. Normally these chemicals are produced in lower amount.

Transgenic materials in the form of seed or fruit can be easily stored and transported from one place to another without fear for its degradation or damage.

In successful trials, transgenic plants have been found to produce monoclonal antibodies, functional antibodies phragments,

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proteins, vitamins and the polymer polyhydroxybutyrate (PHP). Some of examples have been discussed in this section.

1. Nutritional quality: It can be improved by introducing genes. They have been produced that are capable of synthesizing cyclodextrins, vitamins, amino acids etc. Consumption of such plants will help in improving the health of human body.

A. Cyclodextrins (CD): CD is cyclic oligosaccharides containing 6, 7 or 8 glucose molecules in α, ß or γ linkage respectively. It is used in pharmaceutical delivery system, flavor and odour enhancement and removal of undesirable compounds (e.g. caffeine) from food.

B. Vitamin A: It is required by all individuals as it is present in retina in eyes. Deficiency of vitamin A causes skin disorder and night blindness throughout the world 124 million children are the sufferers of vitamin A. Each year about 20 million new children are victimized due to deficiency of vitamin A. The transgenic rice was rich in pro vitamin A. Since the seeds of transgenic rice are yellow in colour due to pro-vitamin A, the rice is commonly known as golden rice.

C. Quality of seed protein : seeds are reservoir of all proteins, amino acids, oils etc. and used as food throughout the world. However, nutritional quality of legumes and cereals can be affected due to deficiency of certain essential amino acids such as lysine (in cereals like rice, wheat), methionine and tryptophan (in pulses e.g. pea). Following recombinant DNA technology improvement in quality of seed protein has been done. The two approaches were done for improvement in nutritional quality of seeds.

2. Immunotherapeutic drugs: For the first time Hiatt et al. (1989) produced antibodies in plants which could produce positive immunization. But the first report on production of edible vaccine appeared in 1990 in the patent application. In 1992, C.J Arntzen and co workers hepatitis B surface antigen in tobacco to produce immunologocally active ingredients via genetic engineering of plants.

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A. Edible vaccines: The plants are capable of producing vaccines in large quantity at low cost but the purification may require more cost. Therefore, attention has been paid to produce such antigens that stimulate mucosal immune system to produce secretary IgA (S-IgA) at mucosal surface such as gut and respiratory epithelia because of their effectiveness on sites as most of the pathogens invade these regions. For example, bacteria and viruses are transmitted via contaminated food or water and cause diseases such as diarrhea, whooping cough, etc.

Acute water diarrhea is caused by enteroxigenic Escherichia coli and Vibrio cholerae that colonize the small insetine and produce enteroxin. , A and B. Cholera toxin (CT) is very similar to E.coli toxin. The CT has two subunits.

A tobacco plant was produced that expressed CT-A or CT-B subunits of the toxin CT-A. CT-A produced in plant was not cleaved into CT-A1 and CT-A2 subunits which generally happens in epithelial cells. Similarly CT-B subunit when expressed into potato was processed in natural way, the pentameric form being the abundant form.

Similarly the rabies virus coat glycoprotein gene has been expressed in tomato plants. The value of vaccine can be improved by providing other adjuvant with either enhance the immunogenic potential or reduce degradation of the active ingredients by the micro-organisms of gut.

In transgenic tobacco plants the hepatitis B surface antigen (HBsAg) accumulates to 0.01% of soluble protein level. The HBsAg was recovered in virus like particles of 22 nm diameter which is known to be a prerequisite for better immonogenicity.

On of the alternative strategies of producing a plant-based vaccine is to infect the plants with recombinant virus carrying the desired antigen that is fused to viral coat protein.

B. Edible antibodies: transgenic plants are being looked upon as a source of antibodies also which can provide passive immunization by

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direst application. They provide as a tool for drug targeting. Moreover, the modified genes capable of expressing Fab fragments or scFV (single peptide chain where variable domains of heavy and light chains are covalently linked by a short flexible peptide) have also been expressed in bacteria and mammalian cells. Besides, the recent technology involving PCR and phage display allow clowning and screening of antibodies.

A hybride monocolonal antibody having constant region IgG and IgA fused ‘has been used successfully against human dental carriers caused by the bacterium, streptococcus mutans. The secretary antibody generated SIgA/G in transgenic tobacco and the original mouse IgG was compared. It is interesting to know that both had similar binding affinity to surface adhesion protein of S. mutans. SIgA/G survived for three days in the oral cavity, variers IgG survived only for one day. The plant antibody provided protection against the colonization of S.mutans for at least four months.

LIGHT CHAIN GENE HEAVY CHAIN GENE

TRANSGENIC PLANT CONTAINS LIGHT CHAIN-K

TRANSGENIC PLANTS CONTAINS HEAVY CHAIN-α

TRANSGENIC PLANT CONTAINING MONOMERIC Ig(IgA)

TRANSGENIC PLANT CONTAINING J-CHAIN

TRANSGENIC PLANT CONTAINING SECRETARY COMPONENT(SC)

TRANSGENIS PLANT CONTAINS DIMERIC (DigA)

TRANSGENIC PLANT CONTAINS SECRETARY IgA(slgA)

TRANSFER IN PLANT

CROSSING

CROSSING

CROSSING

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OUTLINE FOR THE PRODUCTION OF SECRETARY ANTIBODIES IN PLANTS

Application of Transgenic Plants

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

2. Specific genes have been transferred into plants to improve their agronomic and other features.

3. Genes for resistance to various biotic stresses have been engineered to generate transgenic plants resistant to insects, viruses, etc.

4. Several gene transfers have been aimed at improving the produce quality.

5. Transgenic plants are being used to produce novel biochemical like hirudin, etc. which are not produced by normal plants.

6. Transgenic plants can be used vaccines for immunization against pathogens; this is fast emerging as an important objective.

Through genetic engineering many desired genes can be introduced into a plant. At the moment the main transgenic crops grown

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worldwide possess herbicide tolerance, applied in soybean, corn and cotton, which include 77% of all GM crops grown worldwide, followed by insect resistance (Bt- crops) in the second position with 15% of all GM crops.

Many more applications of transgenic plants are known. Some of them have been commercialised but some are still at the research stage. Other attributes include disease resistances (viruses), stress tolerance (heat and salt tolerance), increasing the nutritional value (golden rice with increased provitamin A), delayed ripening (tomato), male sterility or a modified colour.

E.BIOETHICS IN PLANT GENETIC ENGINEERING

INTRODUCTION:

New variation arises in population due to mutation. However the frequency of variation depends in the rate of mutation. But in nature, the frequency of mutation is very low.

Cultivation of genetically modified (GM) crops by the farmer is increasing fast throughout the world. Hopefully the GM technology will support health care and industry and provide food, feedand fiber security at a global basis. However it should be used to increase the production of main staple food, increase the efficiency of production, reduce the environmental impact of agriculture and provide access to food for small scale farmers. The global community is facing the important challenges associated with public perception of transgenic crops. The major concerns about GM crops and GM food are given below.

1. The risk of transfer of allergies: there is fear for transferring allergens (usually glycoprotein) from GM food to human animals

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e.g. peanut and other nuts (GM food from peanut is now widely labeled, but what about GM crops (where there is no labeling).

2. Pollen transfer for GM plants : There is a risk of gene pollution i.e. transfers of transgenic of GM crop through pollen grains to related plant species and development of super-weeds. Will paste or herbicide resistance gene incorporated into GM crop be transferred into closely related plants and increase the ‘weediness’?

3. Effect of GM crop on non target and beneficial insects and microbes: there are many non-target beneficial microbes that harbour on plant surfaces. The insects to harbour on flowers.

4. Risk of change in fundamental vegetable nature of plant: transgenes from animal have been introduced into GM plants for molecular farming. There is risk of changing fundamental nature of vegetable.

5. Transfer of transgene from GM food to cathogenic microbes: antibiotic marker genes are used to identify and select the modified cell. Such cells grow on medium containing those antibiotics. Commonly, kanamycin and hydromyxin resistant genes are used in GM plant to confer resistance to these antibiotics, while ampicillin resistance marker gene is used for GM bacteria. If GM food containing antibiotic resistance marker gene is consumed by animals and humans, the transgene will transfer from GM food to micro flora of human and animals. Will their gut microbe be resistance to antibiotics?

6. Effect of GM crops on biodiversity and environment: The GM crop is not naturally evolved but they have been manipulated artificially. However, there is risk weather they pose harmful effect on biodiversity and overall impact on environment.

7. The GM crop may bring about the changes in evolutionary patterns: evolution is going on naturally. Plants adapt the fluctuations occurring in the environment through changes their genes and developing better races which one says the evolved

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races. Will transgene flow from GM crops to other non GM plants and results in alteration in non GM crops? Will non GM crop evolve through hybridization with GM crop?

Reference :-

A Textbook of Biotechnology By – R.C.Dubey

DNA-binding specificity of rice mariner-like transposases and

interactions with Stowaway MITEs, C. Feschotte et al, Nucleic Acids

Research 2005 33(7):2153-2165;

Vaeck, M., A. Reynaerts, H. Hofte, S. Jansens, M. De Beuckeleer, C.

Dean, M. Zabeau, M. Van Montagu & J. Leemans. 1987, Transgenic

plants protected from insect attack, Nature 328: 33-37.

Meagher, RB (2000). "Phytoremediation of toxic elemental and

organic pollutants". CURRENT OPINION IN PLANT BIOLOGY

Heong, KL, YH Chen, DE Johnson, GC Jahn, M Hossain, RS

Hamilton. 2005. Debate Over a GM Rice Trial in China. Letters.

Science, Vol 310, Issue 5746, 231-233 , 14 October 2005.

Huang, J., Ruifa Hu, Scott Rozelle, Carl Pray. 2005. Insect-Resistant

GM Rice in Farmers' Fields: Assessing Productivity and Health Effects

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in China. Science (29 April 2005) Vol. 308. no. 5722, pp. 688 – 690.

DOI: 10.1126/science.1108972

Syvanen, M. and Kado, C. I. Horizontal Gene Transfer. Second

Edition. Academic Press 2002.

Chrispeels, M.J. and Sadova, D.E. Plants, Genes, and Crop

Biotechnology. Second Edition. James and Bartlett 2003.

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