PREPARED BY

Kishan R. Sanja
T.Y.B.sc.(Biotechnology)

GUIDED BY
Mr. 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)

1
 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 _______________

2
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.

3
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

4
8 Reference

5
 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.

6
Callus culture: a mass of disorganized, 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.

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.

7
 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.

8
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 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 &

9
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.

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

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 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)

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

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NUTRIENT MEDIUM

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.

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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.

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 A. APPLICATION IN AGRICULTURE

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.

LARGE SCALE
MULTIPLICATION GENETIC
CRYOPRESERVATION
VARIABILITY
OF GERM PLASMA

SOMATIC
BIOMASS HYBRIDS/CYBRIDS
ENERGY

DISEASES
PLANT TISSUE
FREE SYNTHETIC
CULTURE
PLANTS SEEDS

HAPLOIDS,
WIDE POLYPLOIDS,
HYBRIDIZATION TRIPLOIDS

CRYOPRESERVATION OF SECONDARY
GERM PLANTS CLONAL METABOLITES
PROPOGATION
PLANT IM PROVEMENT AND THROUGH TISSUE CULTURE
TECHNOLOGY

15
 2. Encapsulated seeds:

In these seeds, the gel acts as seed coat and

the artificial endosperm providing nutrient as in true seeds.
Water 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.

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3. Production of disease resistant plants:

(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.

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 EASTER LILY BULB FROM MARKET
HOT WATER TREATMENT

VIRUS INDEXING
REANSFER SURFACE STERILIZED
SEGMENTS
DIRECT ORGANOGENESIS
( 3 WEEKS)
TRANSFER EXPLANTS TO ROOTING MEDIUM

ROOT INITIATION
( 3 WEEKS)
ROOT ELONGATION

PLANT IN POTS
HARDENING , TRANSFER
INTO SOIL
VIRUS FREE PLANTS
(IN VITRO GENE BANK, PRODUCTION
OF HEALTHY SEEDS)

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 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

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

EAR HEAD OF BAJRA INFECTED

SCIEROSPORA GRAMINICOLA
CULTURE ON MS MEDIUM

DUAL CULTURES
ONE OF PLANT,SECOND OF PATHOGEN
EMBRYOGENESIS
EMBRYOIDS IN EMBRYOGENIC
CALLUS OF DUAL CULTURE
SELECT EMBRYOGENIC CALLUS
FROM PATHOGENIC FREE PORTION
EMBRYOGENIC CALLUS OF DUAL CULTURE

SHOOTING

PLANTLETS
ROOTING

COMPLETE PLANTLETS WITH

ROOTS AND SHOOTS
TRANSFER DISEASE FREE
PLANTS TO EARTHENWARE
DISEASE RESISTANT PLANTS IN POTS POTS AFTER HARDENING

PLANT CHALLENGING WITH PATHOGEN

FIELD PERFORMANCE

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.

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,

19
 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, it is important commercially,

20
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

21
 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 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,

22
 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

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

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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.
oPoor cutilcle development: epicuticular
wax needs to be formed.
oAbnormal stomatal development and
function.
 Either are not properly depressed.
 Or do not open and close properly.
oNon- 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.

24
 •Soil medium and container important.
•Acclimatization structures:
 Plastic covers.
 Humidity tent. Overhead mist.
 Fog system.

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.

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 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:

• 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).

26
 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.

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.

27
 FILTER AIR EXHAUST

CONDENSER

AIR INLET
MULTISOCKET LID MEDIUM INLET

FLAT JOINT

WATER JACKET

GLASS ROD

MAGNETIC STIRRER

SAMPLING OUTLET

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.

28
 Selection and culture of natural
Callus Product secreting strain
from the culture

Specific product

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

29
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.

SOURCE OF CELLS IMMOBILISATION METHOD

SUBSTRATES

Catharanthus roseus Alginate, agarose, Immobilised in

polyacrylamide, carrageenan 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.

30
• 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
procedure are applied to develop high
yielding cell lines.

31
 D. TRASGENIC PLANT

Introduction to Transgenic Plants

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

32
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,

• 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.

33
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 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.

34
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 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

35
 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. 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

36
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.
Link to illustrated discussion of Bacillus thuringiensis.

2. Disease Resistance.

37
 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

38
(and thus force the farmer to buy fresh seeds for the following
season rather than saving seeds from the current crop).
The process involves introducing three transgenes into the plant:
• A gene encoding a toxin which is lethal to developing
seeds but not to mature seeds or the plant. This gene is
normally inactive because of a stretch of DNA inserted
between it and its promoter.
• A gene encoding a recombinase — an enzyme that can
remove the spacer in the toxin gene thus allowing to be
expressed.
• A repressor gene whose protein product binds to the
promoter of the recombinase thus keeping it inactive.

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´ mRNA
3´ 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.

39
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.)

40
Antisense RNA also occurs naturally
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

41
  MOLECULAR FARMING FROM TRANSGENIC PLANTS
(TRANSGENIC PLANTS AS BIOREACTER)

In recent years, transgenic plants are used by

biotechnology industries as ‘bioreactor’ for manufacturing special

42
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,
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

43
 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.

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.

44
 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 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.

45
 LIGHT CHAIN GENE HEAVY CHAIN GENE

TRANSFER IN PLANT

TRANSGENIC PLANT TRANSGENIC PLANTS

CONTAINS LIGHT CHAIN-K CONTAINS HEAVY CHAIN-α

CROSSING
TRANSGENIC PLANT TRANSGENIC PLANT
CONTAINING MONOMERIC CONTAINING J-CHAIN
Ig(IgA)
CROSSING

TRANSGENIS PLANT TRANSGENIC PLANT

CONTAINS DIMERIC CONTAINING SECRETARY
(DigA) COMPONENT(SC)
CROSSING

TRANSGENIC PLANT CONTAINS

SECRETARY IgA(slgA)

OUTLINE FOR THE PRODUCTION OF SECRETARY

ANTIBODIES IN PLANTS

46
 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 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.

47
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 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

48
 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 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?

49
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 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.

50
 http://en.wikipedia.org/wiki/Transgenic_plant
 users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/TransgenicPl
ants.html
 www.colostate.edu/programs/lifesciences/TransgenicCrops/

51