VII. Application of Tissue Culture - Inseda

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Tissue

Culture


Booklet No.

430

Biotechnology: BTS

-

5

Contents

Preface

I.

Introduction

II.

History

III.

Composition and Material

IV.

Media Preparation

V.

Inoculation and Culturing

A.

Sterilization

B.

Isolation

C.

Growth and maintenance

VI.

Methods of Tissue Cu
lture Study

VII.

Application of Tissue Culture

A.

Application in agriculture

B.

Application in horticulture

C.

Application in industries

VIII.

Area Availability and Prime Institutions

IX.

Conclusion


Preface

The main thrust of the scientific investigations t
oday is to find an alternative to the
traditional technologies. The traditional methods are more time consuming and cannot be
applied in all aspects of improvement and development. Today biotechnology has become
handy in furthering the improvement and deve
lopment This booklet deals with tissue culture
techniques in biotechnology for improvement and development of agriculture. This booklet is
prepared for persons who want to know about tissue culture technology.


Dr.
K
.
T. Chandy,
Agricultural &Environme
ntal Education


I. Introduction


Term biotechnology is the science of applied biological processes. The most widely
accepted def
in
ition of biotechnology is the integrated use of biochemistry, microbiology and
engineering sciences in order to achieve technolo
gy (industrial) application of the capabilities of
micro
-
organisms, cultured tissue cells and parts thereof.


Tissue culture is increasingly applied to wide range of biotochnology ventures especially
to the micropropagation and genetic improvement of crop
plants. The techniques of protoplast
culture and fusion are of special significance for
improvement

of crops such as potato,
sugarcane, cassava and banana which were vegetatively propagated. Somatic cell fusions have
been successfully employed for the resy
nthesis of rapeseed (
Brassica napus
) by fusing
protoplasts of
Brassica oleracea

and
B. campestris
and for developing hybrids between
Medicago sativa
and
M. julcata
.


Tissue culture" technique has a lot of scope in improving the crop cultivars, forestry an
d
animal sciences. In developing high yielding hybrid, resistant to insect pests and diseases,
resistant to drought and to transfer any other desirable character into another organisms, the
tissue culture technique playa major role.


Having acquired greate
r depth in the knoledge of tissue cellular and genetic systems
operating in the biosphere man has developed biotechnologies which can be used in medicine,
industrial and in agriculture to enhance quality and quantity in .all spheres of production and
proce
ssing. Todays biotechnology has a long history as ancient as any other biological
sciences.


II. History


In the history of biotechnology several stages of development can be identified as given
in table 1 below.


Table 1
: Historical development of biotechn
ology Year Development


Sl.No

Year

Development

1

Before 6000
B.C

Yeast employed to produce bear and
wine

2

Before 3000
B.C

Providing bread with leaven,
fermentation of juices to alcoholic
beverages

3

34 d Century
B.C

Manufacture of beer in Sumeria,
Babylonia and Egypt

4

From
1150
A.D

Manufacture of ethanol

5

14
th

century

Vinegar manufacturing industry New
Orleans

6

After about
1650

Artificial cultivation of mushroom in
France

7

Before 1670

Copper mined with the help of micro
organisms

8

1677

Antony Van Leeuwenhoek first
ob
served the yeast cells

9

1748

Observations on the generation
composition and decomposition of
animal and vegetable substances

10

1818

Discovery of the fermentation
properties of yeasts by Erxleben

11

1857

Lois Pasteur described Lactic acid
fermentation

12

1869

F . Miescher isolated nucleic acid

13

1878

Joseph Lister reported lactic acid
fermentation and its bearing on
pathology

14

1881

Production of lactic acid from micro
organisms

15

1890

Alcohol first used to fuel motors

16

1890

S. Kitasato and E.

Von Behring
observed antibodies

17

1897

Eduard Buchner observed alcoholic
fermentation without yeast cells

18

1910

Establishment of large scale sewage
purification system

19

1915

German process for the manufacture
of backery yeast

20

1920

Production o
f citric acid in a surface
process

21

1928

Discovery of penicillin by Alexander
Flemming

22

1944

Large scale production of penicillin
discovery of Streptomycin

23

1946

Genetic recombination in
bacteriophage

24

1953

Double stranded nature of DNA
reveale
d

25

1962

Mining of Uranium with the help of
microbes

26

1971

Transfer of nif gene from Rhizobium to
Klebsiella

27

1973

First successful genetic engineering
experiment done

28

1975

Discovery of hybridoma technique for
production of monoclonal antibodie
s

29

1978

Restriction enzymes and their
application to molecular biology

30

1981

US approval for using monoclonal
antibodies in diagnosis

31

1982

Genetically engineered insulin
approved for use

32

1984

Animal interferons approved for use
against cattle

disease

33

Mid Eighties

Use of interferons for the treatment of
viral disease. Genetically engineered
human growth hormone approved for
treatment of dwarfism. Wide use of
monoclonal antibodies in diagnosis.
Introduction of genetically engineered
hepatit
is vaccine. Production of new
antibodies

34

Late Eighties

Use of interferon for treatment of hairy
cell leukemia. Raw materials obtained
from microbes for plastic industry.
Large scale production of industrial
chemicals using microbes

35

Nineties

Extra
ction of oil from the ground with
the help of genetically engineered
microbes. Extraction of metals from
factory waste. Wide use of
recombinant DNA technology in
medicine, human health, agriculture
and industry


Biotechnology is not new sc
ience rather it is fast growing field of science. It includes
branches like molecular biology, tissue culture, genetic engineering and plant pathology.
Biotechnology is developing in close collaboration with these branches of science and can
develop furthe
r only by means of an interdisciplinary approach between them.


During the last two decads plant cell, tissue and organ culture have developed rapidly
and become a major biotechnological tool in agriculture, horticulture, forestry and industry.
Those proble
ms which were not solved through the conventional techniques, are now have
being solved by these techniques; for example inter
-

and intra
-
specific crosses, micro
-
propagation, somaclonal variation, encapsulated seeds etc.


The theory behind tissue culture i
s that "each living cell, of a multicellular organisms, is
capable of independent development, when provided with suitable conditions." This term was
coined as "totipotency".


The concept of totipotency is 'important in tissue culture. Use of multicellular

organisms
in research, as biological units, is rather difficult, therefore, attempts to study an organism by
reducing to its constituent cells and subsequently the cultured cells as basic organisms are of
fundamental importance.


In plant tissue cultural
experiments, organs, plant tissue: and cells are isolated from the
plant and made to grow in different types of culturing vessels, viz., flasks, bottles, tubes and
watch glasses containing essential mineral salts, organic compounds such as amino acids and
vitamins and auxin or growth substances e.g. Indole Acetic Acid (1AA),Indole Butyric Acid (IBA),
2,4
-
D, cytokinins and gibberellines in various concentrations and combinations, Since the green
cells also become dependent on external supply of carbohydrates
, the culture medium must
also contain a readily metabolizable carbon sources, a sugar.


III. Composition and Material


Tissue culture can be maintained either in liquid medium or semi
-
solid medium in the
former, plant material is immersed in the medium eit
her partially or completely while in the latter
plant material is placed on the surface of the medium.


The principal components of most plant tissue culture media are inorganic nutrients
(macronutrients and micronutrients, carbon sources, organic suppleme
nts, growth relulators and
a gelling agent


1. Inorganic nutrients

A number of inorganic nutrients are required for normal growth of plants. Besides
carbon, hydrogen, oxygen, other elements essential for plant growth include nitrogen,
Phosphorucs, potassiu
m, calcium, sulphur, magnesium, iron, manganese, copper, boron, zinc
and molybdenum. Iron is added in the form of Ferric sulphate while nitrogen is furnished in the
form of nitrate and ammonia. Of all the mineral nutrients, nitrogen is responsible for the
most
pronounced effects on growth and differentiation of cultured tissues.


2. Organic nutrients

Cultured plants accomplish better growth when medium is supplemented with organic
nutrients such as amino acids and vitamins. The most commonly used vitamin is
thiamine
(vitamin B
2
). Other vitamins which improve the growth of cultured plants include nicotinic acid
acid pantothenate and pyridoxine.

Besides these boitin, folic aci
d and ammobenzoic acId are
added in various concentrations in to some of the media. In

order to promote growth of callus,
some complex semi
-
synthetic substances are added in the medium. They include yeast extract
(YE), coconut milk (CM), Casein Hydrolysate (CH), tomato juice (TJ) and malt extract (ME).


As a carbon source, sucrose is the mo
st preferred carbohydrate. Glucose, fructose,
maltose, galactose, mannos and lactose are other favourable sugars.


3. Growth Hormones

Growth hormones helps in better plant development. The most commonly used auxins
are IAA, IBA, NAA (naphthalene acetic acid
) and 2,4
-
D (dichlorophenoxyacetic acid). These
auxins promote cell division and root differentiation. Cytokinins are responsible for cell division
and shoot differentiation. BAP (benzyl
-
amino purine), and 2
-
isopentanyl adenine are most
widely used cytokin
ins. Among gibberellines, GA is commonly used.


4. Agar

Agar (a polysaccharide obtained from sea weeds) is used to solidify the medium. Agar is
commonly used at a concentration of 0.8
-
1% (W/v). Use of higher concentration of agar makes
the medium hard This
prevents the diffusion of nutrients into the tissues.


5. pH

The pH of the medium is adjusted between 5 to 5.8 by adding (0.1 N) NaOH or HO.
Usually a pH higher than six results in a fairly hard medium whereas a pH below five does not
allow satisfactory sol
idification of the medium.


IV. Media Preparation


The simplest method of preparing culture media is to use commercially available
powdered media. These media contain all the requisite nutrients. However, agar, sugar and
other supplements are added in the f
inally prepared media.


For the preparation of medium, the following steps are followed.

1. Appropriate quantities of agar
-
agar and sucrose are dissolved in distilled water.

2. Required quantities of stock solutions, growth hormones and other supplements a
re added

3. More distilled water is added to make the final volume of the medium.

4. pH of the medium is adjusted between 5
-
5.8 by adding 0.l N NaOH or 0.l N HCl.

5. The medium is poured in culture tubes, flasks or any other containers.

6. Culture vessels
are plugged with non
-
absorbent cotton wool wrapped in cheese clothes. This
will allow free gaseous exchange but inhibits microbial contamination.

7. Culture vessels are sterilized by autoclaving at 120°C (1.06 kg/cm2) for 15
-
20 minutes.

8. Culture medium i
s allowed to cool at room temperature and used or stored at 4°C.


V. Inoculation and Culturing


The process of inoculation consists of sterilization and isolation of single cells with which
the medium is inoculated. This is followed by careful culturing the

inoculum.


A. Sterilization


Depending upon the type of sterilization and material to be sterilized. There are four
means of
sterilization
.


1. Dry heat

G
lass wares (flasks, tubes, filters and pipettes) metal instruments and other articles
which do not get c
harred by high temperature are put in special containers or wrapped in thick
brown paper or thick aluminium foil and placed in drying oven and sterilized for a period of not
less than 4 hours at a temperature of 140
-
160°C and then taken to the transfer cha
mber.


2. Wet heat

In this process an autoclave operated with water vapour under pressure (steam pressure
of 151b/inch2) and at a temperature of 120°C applied for 20 minutes when the autoclave has
reached proper temperature mid the residual air enclosed ins
ide the autoclave has been
displaced by steam. "Wet" heat sterilization process is recommended for heat stable solutions.
Rubber items or other partly heat labile articles must not be autoclaved.


3. Filtration

Some protein material, vitamins and growth su
bstances are heat labile and cannot be
autoclaved and, therefore, they must be sterilized by filtration using ultrafilter at room
temperature, before being added to an agar medium. The solution of these substances are
added to the autoclaved medium with a
sterile pipette while the agar medium is cooling and still
in the sol state. In using this procedure the filter pore size is given utmost importance. A pore
-
size of 0.22 mm has been recommended in Millipore Catalogue and pure chasing guide
1978/79. All th
e stopcocks of the filtration unit are opened before it is wrapped in thick brown
paper and autoclaved. Media or its constituents can also be sterilized by passing them through
a pyrex sintered
-
glass sterilizing filter (porosity H5).


4. Sterilization by c
hemicals

Surgical blades, scalpel are not sterilized by dry heat, because the high temperature
makes the cutting edges dull. These articles as well as spatulas and forceps are usually
immersed in 80% V/v, ethyl alcohol until required, and sterilized during

use by frequent
immersion in alcohol and flaming. Before the aseptic removal of plant organ or tissue explant it
is necessary to surface sterilize the plant material. Surface sterilization may be accomplished by
using an appropriate surface sterilant. Gen
erally aqueous solutions of calcium or sodium
hypochlorite (Ca(OCl)
2

or NaOCl), which release chlorine as the active sterilant are used. Some
other chemicals which have also been used are H
2
O
2
, bromine water, silver nitrate and mercuric
chloride.


B. Isolat
ion of Single cells

Isolation of cells are done in several methods which are explained here.


1. From Plant Organs

The most suitable material for isolation of single cells is the leaf tissue since a more or
less homogeneous population of cells are there i
n the leaves. There are good for raising defined
and controlled large
-
scale cell cultures. From such intact plant organs (as leaf tissue) single cell
can be isolated using mechanical or enzymatic method.


a. Mechanical method

The procedure involves mild mac
eration of 10 g leaves in 40 m1 of the grinding medium
(20 u mol sucrose, 10 u mol MgCl
2
, 20u mol tris
-
HCI buffer with pH 7.8) with a mortar and
pestle. The homogenate is passed through two layers of tmuslin cloth and the cells thus
released are washed by
centrifugation at low speed using the same medium. Isolation of free
parenchymatous cells can also be achieved on a large scale by the mechanical method.


b. Enzymatic method

Isolation of single cells by the enzymatic method has been found convenient as i
t is
possible to obtain high yields from preparations of spongy parenchyma with minimum damage
or injury to the cells. This can be accomplished by providing osmotic protection to the cells.
while the enzyme macerozyme degrades the middle lamella and cell w
all of the
parenchymatous tissue. Applying the enyzmatic method to cereals has proven difficult since the
mesophyll cells of these plants are apparently elongated with a number of interlock
constrictions, thereby preventing their isolation.


2. From culture
d tissues

The most widely applied approach is to obtain a single cell system from cultured tissues.
Freshly cut pieces from surface
-
sterilized plant organs are simply placed on a nutrient medium
(solidified) consisting of a suitable proportion of auxins an
d cytokinins to initiate cultures.
Explants on such a medium exhibit callusing at the cut ends, which gradually extends to the
entire surface of the tissue. The callus is separated from an explant and transferred to a fresh
medium of the same composition t
o enable it to build up a mass of tissue. Repeated subculture
on an agar medium improves the friability of the callus, a pre
-
requisite for raising a fine cell
suspension in a liquid medium.


The pieces of undifferentiated and friable callus are transferred

in a continuously
agitated liquid medium dispensed in autoclaved flasks or other suitable vials. Agitation is done
by placing the culture flasks/vials on an orbital platform shaker or any other suitable device.
Movement of the culture medium exerts mild p
ressure on small pieces of tissue, breaking them
into free cells and small cell aggregates. Further, it augments the gaseous exchange between
the culture medium and the culture air, and also ensures uniform distribution of cells as well as
cell clumps in t
he medium.


c. Growth and maintenance

Cell suspensions are clonally maintained by the routine transfer (sub
-
culture) of cells in
the early stationary phase to a fresh medium. During the incubation period the biomass of the
suspension cultures increases due
to cell division and cell enlargement This continues for a
limited period since the viability of cells in suspension after the stationary phase de
-

creases
due to the exhaustion of some factors or the accumulation of toxic substances in the medium. At
this

stage an aliquot of the cell suspension with uniformly dispersed free cells and cell
aggregates is transferred to a fresh liquid medium of the : original composition.


The timing of subcultures is very important. The incubation period from cultures initia
tion
to the stationary phase is determined primarily by: (a) initial cell density, (b) duration of lag
phase and (c) growth rate of cell line. The cell density used to subculture is critical and depends
largely on the type of suspension culture to be maint
ained. Low initial cell densities will prolong
the lag phase and exponential phases of growth. While initiating new suspension culture it is
necessary to determine optimal cell density, proportionate to the volume of the culture medium,
in order to achieve

maximum growth.


At an initial cell density of 9
-
15 x 10
3

mI
-
1
, the cells will generally undergo an eightfold
increase in cell number before entering the stationary phase. Subcultures established with a
high inoculums rate (0.5
-
2.5 x 10
5

cells
ml
-
1
) sho
w an increase in cell number during the
incubation period to a range 1.4 x 106 mI
-
1

before entering the stationary phase.


The normal incubation time of stock cultures is 21
-
28 days between subcultures
although cloning may occur within 18
-
25 days. In cases

in which the cells are in a very active
state of division, the passage length may be reduced to 6
-
9 days. Cell cultures initiated at very
low cell densities will not grow unless the medium is enriched with the metabolites necessary to
grow single cells or

a small population of cells.


VI. Methods of Tissue Culture Study


Process of biotechnology in tissue culture may be broadly placed in four groups.


1. Somatic Cell Fusion

This is achieved by inducing two or more pro top lasts to fuse and the fusion produc
t is
nurtured to produce a hybrid plant. Though, this phenomenon is of common occurrence,
protoplasts of some plant species fail to fuse. Some scientists have shown the production of
hybrids that cannot be produced by the conventional hybridization methods

because of
-
sexual
or physical incompatibility among certain plants.


2. Genetic Engineering

Experiments are being conducted by some scientists to introduce neclei, chloroplasts,
viruses, DNA, mitochondria, plastids etc. keeping in view the capability of is
olated protoplast to
ingest "foreign" bodies by a process similar to the process of endocytosis often seen in cells of
protozoan and other animals.


3. Wall biosynthesis

Scientist have made use of isolated protoplast for the study of wall biosynthesis and
d
eposition because of the ability of cultured protoplasts to regenerate a cell wall rapidly.


4. Study of protoplast population

As the protoplasts can be manipulated in a manner similar to that of the micro
-
organism,
selection of mutant cell lines and the cl
oning of cell population is possible by using
microbiological methods.


VII. Application of Tissue Culture


In the recent years plant protoplast, cell and tissue cultures have become an important
tool for crop improvement, commercial production of natural c
ompounds and in the development
of forestry. Some of the areas of biotechnological application of cultured plant
protoplasts/cells/tissues are: (a) tissue culture applications in order to capitalise on the
totipotency of cells, (b) cells and protoplast cul
tures coupled 'with DNA vectors to overcome
problems caused by barriers to gene transfer through sexual means, (c) culture of plant cells for
the production of useful compounds, (d) extension and increase of deficiency of biological
nitrogen fixation, and
(e) transfer of genes for nitrogen fixation ability to non
-
fixing species.


Due to the manipulation of plant tissues in the laboratory this technique has been
referred to by some researchers, as a 'botanical laser' whose numerous uses are yet to be fully
u
nderstood.


A. Application in agriculture

The major areas of application of tissue culture are briefly highlighted here.

1. Improvement of hybrids

Development of cell fusion and hybridization techniques have solved the problem of
incompatibility of plants
and widened the scope of production of new varieties with in a short
time. For example breeders obtained somatic hybrid of wild and cultivated potatoes (
S.
tuberosum, S. chacoense
) and succeeded in the induction or organogenesis.


The somatic hybrid plant
inherited many characters viz., intermediate leaf morphology,
stomata, forms and colour of tubers prolonged flowering, large and fertile pollen grains, high
yield, resistance against Y
-
virus. However, haploid plant materials available as protoplast, cell
a
nd tissue culture systems are currently being evaluated for the use in transfer of foreign
genetic material to selected plant species by protoplast fusion, transformation, transduction and
organelle transfer.


2. Production of encapsulated seeds

The two 'te
chniques, somatic embryogenesis and organogenesis are the alternative
methods for regeneration of the whole plant from cultured tissue in vitro. Moreover, the recent
works on the wrapping an in vitro derived embryos in a seed coat ie. production of synthet
ic
seeds are important because it may improve agriculture due to its low cost.


3. Production of disease resistant plants

Many plant species, which propagate vegetatively are systematically infected by virus,
bacteria, fungi and nematodes. Their inoculum is

carried over several generations resulting in
continued adverse effect of productivity and quality of crops. In order to ensure highest possible
yield and quality, it is necessary to provide disease free stock plants to growers. Tissue culture
techniques
have solved this problem and minimized the time of biological testing.


Unless large scale population of pure inoculum of test pathogens are available, it is
difficult to persue the establishment of pathogenicity and crop loss
-
assessment as it is done in
f
ield conditions. Now it has become possible to carry out such experiments in laboratory within
short span of time by using tissue culture technologies. Scientists have discussed the following
advantages for the study of several aspects of host
-
pathogen int
eractions and responses. They
are:


a. ability to isolate host cells without wounding,

b. control of inoculum of pathogen and number of host cells, c. ability t'J change the nature of
host pathogen interaction

by altering the constituent of growth medium,

d
. presence of only one or a few major host cell types and

e. easy to apply and remove materials e.g.
labeled

precursors

form cultured cells.


4. Production of stress resistance plant

Biochemical mechanisms exist is Cu1turetl cells which determine the resis
tance to
biocide chemicals and provide the

theoretical promise for selection in vitro. For example: cell
suspension tolerant to 2,4
-
D when the cells were subcultured for six months in liquid medium
supplemented with increasing amount of herbicid
es. The cells were able to grow in one rnM
(milli mol) 2,4
-
D, while the control suspension was completely inhibited at O.3mM 2,4
-
D. It is
suggested that tolerance was the result of induction of enzymatic systems responsible for the
degradation of 2,4
-
D.


Us
es of protoplasts as a system to select cell lines tolerant to herbicides have not been
extensively explored. Protoplast

technology can be used to increase the possibility to obtain
monoclonal lines and offer the opportunity for intraspecific
\
transfer o
f cytoplasmic factor of
resistance to some type of

herbicide. A potential application of transfer of herbicides
tolerance
factor is the transplant of cytoplasmic organelles, for l example chloroplasts.


5. Trans
fer of nif gene to eukaryotes

Nit
rogen fixing ability, a genetic character, exists in
prokaryotic diazotrophs. However,
one of the major tasks is the transfer of this character to eukaryotes. In recent years, researches
are being done to solve this problem through tissue culture techniq
ues coupled with the
recombinants DNA tochnology .


Historically, nitrogen fixation by rhizobia was believed to occur through symbiosis. For
th
e

first time an excitement was caused in scientific community with the discovery by Holsten et
al. (1971). They o
btained active rhizobia in the absence of nodules, leghaemoglobin and
bacteroids which were appar
ently necessary in the intact

plan
ts. They established
Rhizo
bium
joponi
cum

.In cell suspension of soybean roots. Callus

induced from

root explants of soybean
on a specific medium was inoculated with R. joponicum. Later on it was microscopi
cally
observed that infection threads were formed by the bacte
ria which were present between
intracellular spaces. They multiplied inside cells.
Moreover, development of nitrogenase in
s
o
ybean callus
-

Rhizobium system growing on solid was also observed and it was also found
that only specific (isomorphic) cells of callus are vulnerable to infection by the bacteria.


In addition to improvement the ba
cterial strains and in
creased nodulation, it is necessary
to seek those genotypes with the efficient photosynthesis and improved partitioning of
car
bohydrates to nodules.


6. Futu
re

Prospects

In addition to work done successfully on nif gene transfer, th
ere are other important
genes which have been clones, for example, ( a) phaseolin and leghaemoglobin genes in
soyabean; (b) storage protein genes in soybean, (c) genes of ribulose bi
-
phosphate
carboxylase/oxygenase (Ru BP case) of pea, maize, wheat etc. S
uccess achieved on these
aspects would certainly promote in green revolution
. Moreover, improvement in pri
mary
productivity by conversion of C3 plants
into C4 ones through genetic engineering techniques
hopefully would in
crease the primary productivit
y.


B. Application in horticultu
re

and fo
re
stry

Tissue culture is applied in a numbe
r of cases in horticul
ture and forestry.


1. Micropropagation

Microprogation is a perfect alternative to asexual propaga
tion of important plants and
trees, because
of following rea
sons.


a. In this method only a small piece of tissue is needed to

generate millions of clonal plants a
year

b. This method provides a possible alternative method for developing resistance in many
species.

c. It solved problem of quarant
ine for introduction of art

disease in new areas and pro
vide a
mean for international
exhange of plant material.

d By this method multiplication can be made in any season.


Regeneration of plantlets from cultured plant cells and tissues has been achieved
in
many trees of high economic value. Many of the studies are aimed at large scale
micropropagation of important trees yielding fuel, pulp, tim
ber, oils or fruits. Therefore, clonal
forestry and horticultural are gaining an increasing recognition as an a
lternative for tree
improvement. However, strategies for transferring cultured plants in vitro to field conditions are
based on relatively higher priced horticultural species rather than agricultural and for
estry
species.


At planting out stage the plantl
ets fail to survive because
of sudden change in the
environment and invasion by soil
microbes. So the regenerates should be transferred first to
green house and then to field. The humidity should be con
trolled by covering the plants with
transpare
nt polythylene

sheets. Some important plants on which work is done are:


Acacia nilotica, Albizia lebback
, Aprocera, Azadirachta indica,
Bauhinia purpurea, Butea
monosperma, Dalbergia spp,

Dendrocalmus strictus, Eu
ca
lyptus spp, Ficus
religiosa, Morus
spp, Populus spp, Shorea robusta, Tectona grandis, Cryptome
ria japonica, Picea smithiana and
Pinus spp
(all gymnosperms).


2. In vitro establishment of

mycon
-
biza

Mycorrhizal fungi show highest specialization of parasit
ism. But major pro
blems with
them is their failure to grow on an artificial medium in laboratory. Therefore, establishment and
multiplication of mycorrhizal fungi on cultured tissue of the sam
e

host plant, if successfully
developed, may be a good tool for handling mycorrhiza
l fungi, production of high poten
tial
inoculum and their establishment in root systems of nursery plants in horticulture and forestry
and the plantation of mycor
rhiza infested seedlings into the field.


Many attempts have been made to establish vesicul
arbuscular mycorrhizal (V
AM) fungi
in axenic culture but unfortunately none of them got success. Moreover, m
i
corhizal fungi have
been cultured only on cortical tissues of roots which were seperated from the whole plant, as in
root organ cultures where i
t acted as food base. V
AM fungi have very high degree of
specialization for food base on root cortex.


C. Application in industries

Production of useful compounds by cultured plant cells has become a field of special
interest in various biotechnologi
cal pr
ogrammes. However, much attention has been paid on the
production of pharmaceutical and other secondary compounds such as essential oils, food
flavourings and colourings which

are used
-

by the fast food, ice cream and confectionary
indus
tries.


Th
is can be achieved by sele
ction of specific cells produc
ing high amount of desired
compounds and the development of a suitable medium. In general, secondary metabolites
produced by plant cell cultures are rather in small amount but strains of

cells produ
cing the
same are in greater amount than those
found in the intact plants have been isolated by clonal
selection.

Co
mmonly two methods are employed for the selection of specific cells: Single cell
cloning and cell aggregate cloning. The difficulties a
ssociated with isolation and culture of single
cells limit application of this method. The later may appear to be more time
-
taking but easier
than the first one.


Recently, in vitro production of high amount of useful compounds has increased with the
succes
s obtained so far in experimental studies. It is hoped that in near future, the indus
trial
production of such compounds by using these techniques would be possible.


Similarly, monogenic cultures of nematodes have been used for the study of
mechanisms of
action of nematicide. This technique can be used extensively in industry to
supply nema
todes for nematicide
s
c
re
a
n
ing programmes.


VIII. Area avail
ability and Prime Institutions


Tissue culture/biotechnology

in India is a fast developing
science and

there are a
reas it
can be employed. Table 2

presents such areas and the products in which tissue culture can be
applied. Table
3

enumerates the institutions which are conduct
ing tissue culture and other
biotechnological researches are being done on spec
ific crop.


Table
-
2 Area of biotechnology/ tissue culture

Sl.No

Area of interest

Products

1

Recombinant DNA
technology

Fine chemicals, enzymes (Genetic engineering)
vaccines, growth hormones antibiotics, interfero
n

2

Treatment and utilization
of biomaterials (biomass)

Single cell protein and mycoprotein, alcohols and
biofuels

3

Plant & animal cell
culture essential oils,
dyes

Fine chemicals (alkaloids, steroids) somatic
embryos encapsulated seeds, interferon,
mon
oclonal antibodies

4

Nitrogen fixation

Microbial inoculants (biofertilisers)

5

Biofuels (bioenergy)

Hydrogen (via photolysis) alcohols (from biomass)
methane (biogas) (from wastes and aquatic
weeds)

6

Enzymes (biocatalysts)

Fine chemicals, food processi
ng biosensor,
chemotherapy

7

Fermentation

Acids, enzymes, alcohols antibiotics, fine
chemicals, vitamins toxins (biopesticides)

8

Process engineering

Effluent water recycling product extraction, novel
reactor, harvesting



It is

to be noted that all the products mentioned in table 2 are industrially important which
table 3 highlights the impor
-

tance given to crop and agricultural improvement.


Table 3: Crop and availabil
ity of biotechnology/tissue culture


Sl.No

Classification

Name of the crop

Technology available at

1

Fruits

1. Citrus

NBRI Lucknow, University of Jodhpur,
Jodhpur



2.Banana

IIHR Bangalore, NCL Poona



3.Pineapple

BARC, Bombay



4.Pomegranate

NCL, Poon
a



5.Papaya

IARI, New Delhi

2

Species

6.Cardamom

NCL, Poona



7.Ginger

NCL, Poona



Turmeric

NCL, Poona, CPCRI, Kasargod

3

Ornamentals

9.Orchids

IIHR, Bangalore, NBRI , Lucknow



10.Carnation

Delhi University, Delhi



Gladiolus

NBRI, Lucknow; NCL,
Poona; IIHR,
Bangalore



Bougainvillea

NBRI Lucknow, IIHR Bangalore



Chrysanthemum

NBRI, Lucknow; NCL Poona



Ferns

Baroda University, Baroda

4

Forest trees

Teak

NCL Poona



Eucalyptus

NCL Poona, IISC, Bangalore



Sandalwood

BARC, Bombay; IISC, Ban
galore

5

Commercial
crops

Sugarcane

NCL, Poona; SBI Coimbatore

6

Medicinal
plants

Glycyrrhiza

GAU,Anand



Jojoba

NBRI, Lucknow



Dioscorea sp.

NBRI, Lucknow, RRL, Jammu


IX. Conclusion


A brief idea of tissue culture one of the methods employed in biotechnology along with
its scope and importance is giv
en in this booklet. Indeed it is a highly
specialized

skill which pre
supposes the knowledge of various biological sciences. Those who are desirous of getting more
information could consult table
3

to find out the specific institution which are doing resea
rch on a
specific crop.


%%%%%%%%