Tetras Educare Study Notes Vol 22 PTC Part 1

Tetras Educare
Study Handout for Study Handout for Study Handout for Study Handout for
Life Science Students Life Science Students Life Science Students Life Science Students
Plant Tissue culture Part - 1

Edited and written by:

SHANMUGAM V. M.
Lecturer, JNC Autonomous, Lecturer, JNC Autonomous, Lecturer, JNC Autonomous, Lecturer, JNC Autonomous,
Dept. of Biotechnology Dept. of Biotechnology Dept. of Biotechnology Dept. of Biotechnology
Hosur Road, Bangalore Hosur Road, Bangalore Hosur Road, Bangalore Hosur Road, Bangalore !""# !""# !""# !""#.

Not for private circulation, Please mail your Queries and Suggestions to proinsilico@greenmail.net

Study handout for life science aspirants volume volume volume volume

2
Shanes quick N fact files

PLANT TISSUE CULTURE
Unit 1:
Introduction:
The conventional breeding methods are the most widely used for crop
improvement. But in certain situations, these methods have to be supplemented with
plant tissue culture techniques either to increase their efficiency or to be able to
achieve the objective, which is not possible through the conventional methods.
The earliest organized efforts to induce sustained growth of plant cells in
culture were made in the 19!"s. They were followed rapidly by the development of
aseptic techniques and comple# nutrient media. These advances, combined with the
discovery of plant growth regulators li$e au#ins and cyto$inins, and their profound
effects on morphogenesis, led to the regeneration of plants from cultured tissues.
%urther refinement of techniques made it possible to recover whole plants from
isolated single cells, through organogenesis as well as somatic embryogenesis.
Today, plant regeneration can be obtained from cell and tissue cultures of a
wide variety of plants, including most of the economically important species. &ndeed,
'icropropagation has become an increasingly important and successful industry in all
parts of the world. ( new phase in the development of plant cell and tissue culture
techniques started around 19)! with the regeneration of plants from cultured
protoplasts *and protoplast fused products+, anthers and microspores.
(t the same time, newly emerging recombinant ,-( technology or genetic
engineering provided a powerful new tool for the study of the molecular basis of plant
development, as well as their procedures were developed for the delivery and stable
integration of alien genes into the germplasm of plants. The synergism of plant cell
culture and molecular biology has led to remar$able advances in our understanding of
plant development and in the production of transgenic plants with valuable agronomic
characteristics.
'any such transgenic crops are now undergoing field trials, and are e#pected
to be commercially available before the end of this decade. .ence, plant tissue
culture has largely been integrated in biotechnology and permits the regeneration of
plants as clones and as trangenics. /allus and cell suspension cultures, the pride of
laboratories for years, are being phased out0 cell phenomena are being studied in
planta by employing molecular probes.

Shanes quick N fact files Plant tissue culture part - 1
3
Study handout for life science aspirants

History:
Though the tissue culture technique is latest one but history of this began
more than 112 years bac$ when first callus formation was done by Duhamel du
Monceau in 1)23. Haberlandt 1494 successfully cultured somatic cells of higher plants
in simple nutrient solutions. (lthough he was able to maintain the cells in nutrient
medium, the cell division was not recorded until much later. The first real success
was made by Nobecourd, Gautheret and White who successfully cultured cambium
tissue and maintained them for more than a year through 2 or 3 sub segments sub
cultures.
Definition and overview of plant tissue culture:
Plant tissue culture defined as the culturing of isolated or individual cells or
tissue fragments or protoplasts (plant cell without cell wall on a s!nthetic medium
under optimi"ed and controlled ph!sico#chemical and ph!siological conditions to
produce either a callus (unorgani"ed mass of replicating cells or whole plants or
embr!os5.
The successful culturing technique requires nutrient medium, aseptic
conditions and proper aeration of tissue. 6eparated cells or tissues or organs are
grown aseptically in suitable containers on a nutrient medium under controlled
conditions of temperature and light. -utrient media contains inorganic salts of
essential and non essential elements, vitamins, sucrose and amino acids li$e glycine
etc. 6ometimes hormones and mi#ers of substances as coconut water, yeast e#tract
and bean seed e#tract are also added in the medium. 6$oog and 'iller 192! discussed
the role of au#in and cyto$inins in shoot and root formation.

4

Applications of Tissue Culture in Plants:
1. Micro propagation7 The term represents the vegetative multiplication of
plants in artificial media under aseptic conditions from tissue0 organs of plants
e.g. root tip, shoot tip, embryo, stem and callus etc.
1. Production of disease free plants7 By using tissue culture in plants healthy
disease free plants of potato, sugarcane, sweet potato, and strawberry have
been produced.
. Androgenic haploids and their use in breeding7 8ith the help of tissue
culture in plants haploid embryos or haploid plants are raised by anther
culture technique.
9. Ebryo rescue for successful hybridi!ation: The hybrid embryos produced as
a result of inter:specific or inter:generic cross usually collapse due to
incompatibility. 6uch embryos are isolated from female plants and rescued by
growing them on synthetic medium.
2. Induction and selection of utants7 By adding chemical mutagens into the
medium for growing various traits, useful viable mutants can be produced.
3. "oaclonal variation7 These are variations produced in the plants
regenerated from tissue cultures involving callus formation. ;itiations
appearing during tissue culture in plants are called somaclonal variation
#asic re$uireents for plant tissue culture lab:
The prerequisites for organization and establishment of a tissue culture
laboratory depend mainly upon the aims and objectives of the e#perimenter. The
laboratory may be simple, moderate and elaborate depending on the purpose of the
set up, the specific research programme in mind and one which would permit the
e#planted tissue to be grown under a wide range of strictly controlled environmental
conditions.
( set up in its simplest form is one that ma$es use of a pressure coo$er instead
of an autoclave, a glove bo# *in place of laminar flow cabinet+, the simplest transfer
cabinet fitted with a germicidal tube, a monocular compound microscope for
observation, some test tubes and flas$s, chemicals and a shelf to maintain cultures, a
wash up area, space for media preparation and aseptic transfer and other basic

5

facilities. (ir conditioning of the laboratory is a must especially for a tropical country
to maintain constant temperature.
Basic amenities for preparation of media, media sterilization, dispensation and
storage, sterile chamber for tissue transfer *laminar air flow cabinet for inoculation+
to media contained in glass vials or <rlenmeyer flas$s, incubation of cultures under
controlled conditions of temperature and light and if possible, of humidity are primer
requisites. <ach of the activities requires a separate room.
=rovision of ample space for storage of chemicals, glassware, culture
containers etc., would facilitate storage under dust free conditions. <quipment such
as sources of compressed air vacuum, hot plate stirrers, p. meter, micro oven for
thawing, semi:microbalances *!.!!!1 > 13!.!g+ Bunsen burners with a glass cylinder
are a must. ( refrigerator and a deep freezer are other essential equipments for
storage of chemicals, media, stoc$ etc. (n independent place for microscopic
observations, recording and analysis of data is idea. =rovision for dish washing is
essential and a glasshouse where plants may have to be grown on potted soil, are
other items of importance that need special attention.

Plant horones or Plant growth regulators %P&'s( and their role in in vitro
propagation
=?@s e#ert dramatic effects at low concentrations *!.!!1 : 1!A'+. They
control and modulate the initiation and development of shoots and roots on e#plants
and embryos on semisolid or in liquid medium cultures. They also stimulate cell
division and e#pansion. 6ometimes, a tissue or an e#plant is autotrophic and can
produce its own supply of =?@s. Bsually =?@s must be supplied in the medium for
growth and development of the tissues or cells in culture.

The most important classes of the =?@6 used in tissue culture are the au#ins
and cyto$inins. The relative effects of au#in and cyto$inin ration on morphogenesis of

6

cultured tobacco tissues were demonstrated by 6$oog and 'iller *192)+ and still serve
as the basis for plant tissue culture manipulations today. 6ome of the =?@s used are
hormones *naturally synthesized by higher plants+ and others are synthetic
compounds. =?@s e#ert dramatic effects depending on the concentrations used, the
target tissue and their inherent activity even though they are used in very low
concentrations in the media *from !.1 to 1!! A'+. The concentrations of =?@s are
typically reported in mgCl or in A' units of concentration.
)* Au+ins: (u#ins play a role in many developmental processes, including cell
elongation and
swelling of tissue,
apical dominance,
adventitious root
formation and somatic
embryogenesis.
?enerally, when
concentration of au#in
is low, root initiation is
favored and when the
concentration is high,
callus formation
occurs. The most common synthetic au#ins used are 1: napthaleneacetic acid *-((+,
1, 9 > dichloropheno#yacetic acid *1, 9 > ,+, and 9 > amino : , 2, 3 > trichloro : 1 >
pyridinecarbo#ylic acid *picloram+. -aturally occurring indoleacetic acid *&((+ and
indolebutyric acid *&B(+ are also used. &B( was once considered synthetic, but has
also been found to occur naturally in many plants including olive and tobacco. Both
&(( and &B( are photosensitive so that stoc$ solutions must be stored in the dar$. &((
is the wea$est au#in and is typically used at concentrations between !.!1 > 1! mgCl.
more active au#ins such as &B(, -((, 1, 9 > , and picloram are used at concentrations
ranging from !.!!1 > 1!mgCl, picloram and 1, 9 > , are e#amples of au#ins used
primarily to induce and regulate somatic embryogenesis.

7

,* Cyto-inins
/yto$inins promote cell division and shoot initiation and growth in vitro. The
cyto$inins most commonly used
are Deatin, ,ihydrozeatin,
$inetin, benzyladenine,
thidiazuron and 1i=. &n higher
concentrations *1: 1!A'+, they
induce adventitious shoot
formation but inhibit root
formation. They promote
a#illary shoot formation by
opposing apical dominance
regulated by au#ins.
Benzyladenine has significantly
stronger cyto$inin activity than
the naturally occurring zeatin. .owever, a concentration of !.!2 > !.1 A'
thidiazuron, a diphenyl substituted urea, is more active than 9 : 1!A' B( but
thidiazuron may inhibit root formation, causing difficulties in plant regeneration.

.* &ibberllins %&A(
?( is less commonly used in plant tissue culture.
(? is the most often used, but it is very heat sensitive
*after autoclaving 9!E of the biological acitivity is lost+.
Typically, it is filter sterized and added to autoclaved
medium after it has cooled. ?ibberllins help to stimaulate
elongation of internodes and have proved to be necessary
for meritem growth for some species.

/* Abscissic Acid %A#A(
(B( is not normally considered an important
=?@ for tissue culture e#cept for somatic
embryogenesis and in the culture of some woody
plants. %or e#ample, it promotes maturation and
germination of somatic embryos of /araway, /itrus
and 6pruce.

8

0* Ethylene
&t is a gaseous naturally occurring, plant growth regulator and most commonly
associated with controlling fruit ripening in
climacteric fruits and it is used in plant culture is
not widespread. Frgan and callus cultures are able
to produce ethylene, a gaseous =?@. 6ince culture
vessels are almost entirely closed, ethylene can
sometimes accumulate. 'any plastic containers
stop contribute to ethylene content in the vessels. There are contrasted reports in
the literature concerning the role played by ethylene in vitro growth can be promoted
by ethylene. (t other times, addition of ethylene inhibitors results in better initiation
or growth.
%or e#amples, the ethylene inhibitors, particularly silver nitrate, are used to
enhance embrogenic cultures initiation in corn. .igh levels of 1, 9 > , can induce
ethylene formation.

Totipotency:
$otipotenc! is the potential or inherent capacit! of a plant cell or tissue to
develop into an entire plant if suitabl! stimulated.
Totipotency implies that all the information necessary for growth and
reproduction of the organism is contained in the cell. (lthough theoretically all plant
cells are totipotent the meristematic cells are best able to e#press it.

::::::::::::::::

9

Plant cell culture edia:
/ulture media used for the cultivation of plant cells in vitro are composed of
three basic components7
1. <ssential elements, or mineral ions, supplied as a comple# mi#ture of
salts0
1. (n organic supplement supplying vitamins andCor amino acids0 and
. ( source of fi#ed carbon0 usually supplied as the sugar sucrose.
%or practical purposes, the essential elements are further divided into the
following categories7
1. 'acroelements *or macronutrients+0
1. 'icroelements *or micronutrients+0 and
. (n iron source.
/omplete plant cell culture medium is usually made by combining several
different components, as outlined in Table below.
Media coponents
&t is useful to briefly consider some of the individual components of the stoc$
solutions.
)* Macroeleents
(s is implied by the name, the stoc$ solution supplies macroelements required
in large amounts for plant growth and development. -itrogen, phosphorus, potassium,
magnesium, calcium, and sulphur *and carbon, which is added separately+ are usually
regarded as macroelements. These elements usually comprise at least !.1E of the dry
weight of plants.
-itrogen is most commonly supplied as a mi#ture of nitrate ions *from G-F
+
and ammonium ions *from -.
9
-F
+. Theoretically, there is an advantage in supplying

nitrogen in the form of ammonium ions, as nitrogen must be in the reduced form to
be incorporated into macromolecules. -itrate ions therefore need to be reduced
before incorporation. .owever, at high concentrations, ammonium ions can be to#ic
to plant cell cultures and upta$e of ammonium ions from the medium cause"s
acidification of the medium. %or ammonium ions to be used as the sole nitrogen
source, the medium needs to be buffered. .igh concentrations of ammonium ions can
also cause culture problems by increasing the frequency of vitrification *the culture

10

appears pale and Hglassy" and is usually unsuitable for further culture+. Bsing a
mi#ture of nitrate and ammonium ions has the advantage of wea$ly buffering the
medium as the upta$e of nitrate ions causes F.I ions to be e#creted.
=hosphorus is usually supplied as the phosphate ion of ammonium, sodium, or
potassium salts. .igh concentrations of phosphate can lead to the precipitation of
medium elements as insoluble phosphates.

)* Microeleents
'icroelements are required in trace amounts for plant growth and
development, and have many and diverse roles. 'anganese, iodine, copper, cobalt,
boron, molybdenum, iron, and zinc usually comprise the microelements, although
other elements such as nic$el and aluminum are found frequently in some
formulations.

11

&ron is usually added as iron sulphate, although iron citrate can also be used.
<thylenediaminetetra:acetic acid *<,T(+ is usually used in conjunction with iron
sulphate. The <,T( comple#es with the iron to allow the slow and continuous release
of iron into the medium. Bn:comple#ed iron can precipitate out of the medium as
ferric o#ide.
.* 1rganic suppleents
Fnly two vitamins, thiamine *vitamin B1+ and myoinositol *considered a B
vitamin+, are considered essential for the culture of plant cells in vitro. .owever,
other vitamins are often added to plant cell culture media for historical reasons.
(mino acids are also commonly included in the organic supplement. The most
frequently used is glycine *arginine, asparagine, aspartic acid, alanine, glutamic acid,
glutamine, and proline are also used+, but in many cases its inclusion is not essential.
(mino acids provide a source of reduced nitrogen and, li$e ammonium ions0
upta$e causes acidification of the medium. /asein hydrolysate can be used as a
relatively cheap source of a mi# of amino acids.
/* Carbon source
6ucrose is cheap, easily available, readily assimilated, and relatively stable,
and is therefore the most commonly used carbon source. Fther carbohydrates *such as
glucose, maltose, galactose, and sorbitol+ can also be used and in specialized
circumstances may prove superior to sucrose.
0* &elling agents
'edia for plant cell culture in vitro can be used in either liquid or Hsolid"
forms, depending on the type of culture being grown. %or any culture types that
require the plant cells or tissues to be grown on the surface of the medium, it must
be solidified *more correctly termed gelled+.
(gar, produced from seaweed, is the most common type of gelling agent, and
is ideal for routine applications. .owever, because it is a natural product, the agar
quality can vary from supplier to supplier and from batch to batch. %or more
demanding applications, a range of purer *and in some cases, considerably more
e#pensive+ gelling agents are available.
=urified agar or agarose can be used, as can a variety of gellan gums *such as
=hytagel *sigma+ and ?elrite *'erc$++. ?ellan gums are polysaccharides obtained
from the bacterium Pseudomonas elodea. These gelling agents are polymers made up
of glucose, glucuronic acid and rhamnose units. They produce clear, colorless gels

12

that ma$e visual screening of contamination easy. ?el strength is determined by the
concentration of divalent cations *'g1J and /a1J+. =oor gelling may occur if the
cationic concentrations are lower than 9m' or higher than 4m'.
2* Cople+ organic suppleents:
/omple# additions such as banana powder and the liquid endosperm of the
coconut *$nown as coconut mil$ or coconut water+ are frequently added to some
media to improve growth. (lthough coconut water *2 > 1!E+ is thought to provide
growth regulators and other organic compounds, it is not $nown which components of
this supplement are effective in improving the growth of cultures. Because of this
uncertainty, some e#perts discourage the use of these undefined additions.
3* Activated charcoal:
The addition of activated charcoal *(/+ to culture media is reported to
stimulate growth and differentiation in orchids, carrot, ivy and tomato. =arado#ically,
its effect on tobacco, soybean and /amellia has proved inhibitory. &nhibition of
growth is attributed to the adsorption of phytohormones to (/, whereas stimulation
could be due to any one of the factors, namely adsorption of inhibitory compounds to
(/ and dar$ening of the medium. (/ is generally acid washed and neutralized before
its addition at concentrations of !.2:.!E to the culture medium. &t also helps to
reduce to#icity by removing to#ic compounds *e.g. phenols+ produced during the
culture and permits unhindered cell growth.
4* Antibiotics7
(ddition of antibiotics to culture media is generally avoided because their
presence in the medium retards the cell or tissue growth. .owever, some plant cells
have a systemic infection of microorganisms. To prevent the growth of these microbes
it is essential to enrich the media with antibiotics. 6treptomycin or $anamycin at low
concentration effectively control systemic infection and media supplemented with
these antibiotics do not adversely inhibit the growth of cell cultures.

13

CULTURE TYPES
/ultures are generally initiated from sterile pieces of a whole plant. These
pieces are termed explants, and may consist of pieces of organs, such as leaves or
roots, or may be specific cell types, such as pollen or endosperm. 'any features of
the e#plant are $nown to affect the efficiency of culture initiation. ?enerally,

14

younger, more rapidly growing tissue *or tissue at an early stage of development+ is
most effective.
Callus cultures:
%allus means an unorgani"ed or undifferentiated proliferative mass of cells
produced from isolated plant cells, tissues or organs when grown asepticall! on
artificial nutrient medium under controlled e&perimental conditions.
<#plants, when cultured on the appropriate medium, usually with both an
au#in and a cyto$inin, can give rise to an unorganized, growing, and dividing mass of
cells. &t is thought that any plant tissue can be used as an e#plant, if the correct
conditions are found. &n culture, this proliferation can be maintained more or less
indefinitely, provided that the callus is sub:cultured on to fresh medium periodically.
,uring callus formation, there is some degree of dedifferentiation *i.e. the
changes that occur during development and specialization are, to some e#tent,
reversed+, both in morphology *a callus is usually composed of unspecialized
parenchyma cells+ and metabolism. Fne major consequence of this dedifferentiation
is that most plant cultures lose the ability to photosynthesize. This has important
consequences for the culture of callus tissue, as the metabolic profile will probably
not match that of the donor plant. This necessitates the addition of other
componentsKsuch as vitamins and, most importantly, a carbon sourceKto the culture
medium, in addition to the usual mineral nutrients.
/allus culture is often performed in the dar$ *the lac$ of photosynthetic
capability being no drawbac$+ as light can encourage differentiation of the callus.
,uring longterm culture, the culture may lose the requirement for au#in andCor
cyto$inin. This process, $nown as habituation, is common in callus cultures from
some plant species *such as sugar beet+.
/allus cultures are e#tremely important in plant biotechnology. 'anipulation
of the au#in to cyto$inin ratio in the medium can lead to the development of shoots,
roots, or somatic embryos from which whole plants can subsequently be produced.
/allus cultures can also be used to initiate cell suspensions, which are used in a
variety of ways in plant transformation studies.
/allus cultures, broadly spea$ing, fall into one of two categories7 copact or
friable. &n compact callus, the cells are densely aggregated, whereas in friable
callus, the cells are only loosely associated with each other and the callus becomes

15

soft and brea$s apart easily. %riable callus provides the inoculum to form cell:
suspension cultures.
<#plants from some plant species or particular cell types tend not to form
friable callus, ma$ing it difficult to initiate cell suspension. The friability of the callus
can sometimes be improved by manipulating the medium components or by repeated
sub:culturing. The friability of the callus can also sometimes be improved by culturing
it on semi:solid medium *medium with a low concentration of gelling agent+.

1rganogenesis
6omatic embryogenesis relies on plant regeneration through a process
analogous to zygotic embryo germination. Frganogenesis relies on the production of
organs, either directly from an e#plant or from a callus culture. There are three
methods of plant regeneration via organogenesis.
The first two methods depend on adventitious organs arising either from a
callus culture or directly from an e#plant. (lternatively, the third method is by
a#illary bud formation and growth, which can also be used to regenerate whole plants
from some types of tissue culture. Frganogenesis relies on the inherent plasticity of
plant tissues, and is regulated by altering the components of the medium. &n
particular, it is the au#in to cyto$inin ratio of the medium that determines which
developmental pathway the regenerating tissue will ta$e.
&t is usual to induce shoot formation by increasing the cyto$inin to au#in ratio
of the culture medium. These shoots can then be rooted relatively simply.

"oatic ebryogenesis
&n somatic *ase#ual+ embryogenesis, embryo:li$e structures, which can develop
into whole plants in a way analogous to zygotic embryos, are formed from somatic
tissues. These somatic embryos can be produced either directl! or indirectl!.
&n direct somatic embr!ogenesis, the embryo is formed directly from a cell or
small group of cells without the production of an intervening callus. Though common
from some tissues *usually reproductive tissues such as the nucellus, styles, or
pollen+, direct somatic embryogenesis is generally rare in comparison with indirect
somatic embryogenesis.

16

&n indirect somatic embr!ogenesis, callus is first produced from the e#plant.
<mbryos can then be produced from the callus tissue or from a cell suspension
produced from that callus.
&n contrast, embryos developing from zygotes are called zygotic embryos or
often simply embryos, while those derived from pollen are $nown as pollen embryos
or androgenetic embryos.
By 199, somatic
embryogenesis was
reported from over 1!!
species. ,evelopmental
=attern of 6<s. 6<s
generally originates from
single cells, which divide
to form a group of
meristematic cells.
Bsually, this
multicellular group
becomes isolated by
brea$ing cytoplasmic
connections with the
other cells around it and
subsequently by
cutinization of the outer walls of this differentiating cell mass. The cells of
meristematic mass continue to divide to give rise to globular *round ball:shaped+,
heart:shaped, torpedo and cotyeledonary stages. &n general, the essential features of
6< development, especially after the globular stage, are comparable to those of
zygotic embryos.
6omatic embryos are bipolar structures in that they have a radicle and a
plumule. The radicular end is always oriented toward the centre of callus or cell
mass, while the plumular end always stic$s out from the cell mass. &n contrast, a
shoot bud is monopolar as it does not have a radicular end.
&n many 6<s, radicle is suppressed so that they often do not produce roots0 in
such cases, roots have to be regenerated from the shoots produced by germinating

17

6<s. 6<s often show abnormal developmental features, e.g., or more cotyledons,
bell shaped cotyledon, larger size etc.0 these problems are often overcome by the
presence of (B( or a suitable concentration of mannitol. &n some species, normal
loo$ing somatic embryos are formed, but they fail to germinate.
6omatic embryogenesis is influenced by several factors, e.g.
*1+ ?rowth @egulators
*1+ -itrogen source,
*+ <#plant,
*9+ ?enotype and
*2+ Fthers

6omatic embryogenesis usually proceeds in two distinct stages. &n the initial
stage *embryo initiation+, a high concentration of 1, 9:, is used. &n the second stage
*embryo production+, embryos are produced in a medium with no or very low levels of
1, 9:,. &n many systems, it has been found that somatic embryogenesis is improved by
supplying a source of reduced nitrogen, such as specific amino acids or casein
hydrolysate.


18

MICROPROPAATIO!
I5T'1D6CTI15:
Tissue culture techniques are becoming increasingly popular as alternative
means of plant vegetative propagation. This involves ase#ual methods of propagation
and its primary goal is crop improvement. The success of many in vitro selection and
genetic manipulation techniques in higher plants depends on the success of in vitro
plant regeneration. /rop breeding and r,-( technology require the widespread use
of reliable true to type propagation and better regeneration methods.
/lonal propagation in vitro is called L'icropropagation5. The word Lclone5
was first used by 8ebber for cultivated plants that were propagated vegetatively.
'icropropagation is the practice of rapidly multiplying stoc$ plant material to
produce a large number of progeny plants, using modern plant tissue culture methods.
'icropropagation is used to multiply novel plants, such as those that have been
genetically modified or bred through conventional plant breeding methods. &t is also
used to provide a sufficient number of plantlets for planting from a stoc$ plant which
does not produce seeds, or does not respond well to vegetative reproduction.
The significant advantage offered by aseptic methods of clonal propagation
*'icropropagation+ over the conventional methods is that in a relatively short span of
time and space, a large number of plants can be produced starting from a single
individual. &t has been estimated that a#illary bud proliferation approach typically
results in an average 1!:fold increase in shoot number per monthly culture passage.
&n a period of 3 months, it is feasible to obtain as many as 1,!!!,!!! propagules or
plants starting from a single e#plant.
STAES O" MICROPROPAATIO!:
There are four major stages of micropropagation. 'urashige proposed three *&
to &&&+ stages, ,ebergh and 'aene added stage H!M. /urrently we follow five stages
procedure *! to &;+.
6tage ! > 6election and preparation of the mother plant
6tage & : &nitiation of culture
6tage && : 'ultiplication
6tage &&& : @ooting

19

6tage &; : Transfer to soil C .ardening or acclimatization stage
Stage #:
Sele$tion and %aintenan$e o& sto$' plants &o( tissue $ultu(e initiation ) in this
stage stoc$ plant are grown under more hygienic conditions to reduce the ris$ of
contamination. selection of elite or mother plant and e#plant selection, their
sterilization and transfer to nutrient ,media for establishment, i.e. initiation of a
sterile culture of the e#plant.
Stage I:
Initiation and establish%ent o& asepti$ $ultu(e ) ;egetative parts or reproductive
parts are used for the propagation and shoot tip and au#iliary buds are often used for
this. &n this process e#plants are surface sterilized by treating it with disinfectant
solution such as <thyl alcohol, bromine water, mercuric chloride etc.
Establish%ent o& explant on app(op(iate %ediu% > There is not any universal culture
medium0 however modifications of 'urashige and 6$oog basal medium *'urashige and
6$oog, 1931+ are most frequently used.
Stage II
Multipli$ation o& shoots ) Fften media are not changed between stage & and stage &&,
but cyto$inin proportion is increased for stage && to produce numerous shoots.
Stage III
Rooting o& (egene(ated shoots ) &n this stage, shoots are transferred on a rooting
medium containing an high concentration of au#in. <longation of shoots prior to
rooting, rooting of shoots *individual or clumps+, and prehardening cultures are
carried out to increase the survival rate.
Stage I*
+a(dening ) this stage involves transfer of an in vitro plant to the soil after
acclimituization. (cclimatization is the process that plant must undergo in order to
ma$e the transition from the culture environment *high relative humidity of about 94
> 99E+, low light intensity, completely aseptic environment using surcease as carbon
source+ to the greenhouse or filed. &n order that the micropropagated plants survive
on transfer to the filed, they are gradually hardened to withstand the e#posure to the
stress of lower realative humidity *1!: 3!E+, higher the light intensities, e#posure to
the pathogens and autotrophic growth. =lants are $ept under high humidity for 1:
wee$s just after a transfer to a green house. Night intensities are gradually increased
during hardening.

20

"igu(e: "tages of Micropropagation
*it(i&i$ation:
;itrification or hyperhydricity is a common occurrence in micropropagated
shoots. &t describes the water soa$ed, translucent and glassy morphological
appearance, particularly of leaves and stems. ;itrification may occur due to several
reasons, namely, tightly closed culture vessels, increased levels of carbon dio#ide
inside the culture vessels, high levels of ethylene and water vapour. The low wa#
deposition in leaves may lead to a translucent appearance. &t is very difficult for
vitrified shoots to survive in the field.
Ad,antages o& Mi$(op(opagation
1. @apid multiplication of superior clones and maintenance of uniformity.
1. 'ultiplication of disease free plants.
. 'ultiplication of se#ually derived sterile hybrids.
9. ;ery small size e#plants can be used as a starting culture.
2. &ndependent of the season0 can be carried out through out the year.

21

3. 'icropropagation produces rooted plantlets ready for growth, saving time for
the grower when seeds or cuttings are slow to establish or grow.
). &t is the only viable method of regenerating genetically modified cells or cells
after protoplast fusion.
4. 'icropropagation often produces more robust plants, leading to accelerated
growth compared to similar plants produced by conventional methods : li$e
seeds or cuttings.
9. 6ome plants with very small seeds, including most orchids, are most reliably
grown from seed in sterile culture.
Li%itations o& Mi$(op(opagation
1. 6ophisticated facilities are required.
1. =roduction cost is high.
. @equirement of s$ill in handling and maintenance.
9. 6omaclonal variations may arise during in vitro culture.
2. ;itrification can be a problem in some species.
3. &t is ver! e#pensive, and can have a labor cost of more than )!E
). ( monoculture is produced after 'icropropagation, leading to a lac$ of overall
disease resilience, as all progeny plants may be vulnerable to the same
infections.
4. (n infected plant sample can produce infected progeny. This is uncommon if
the stoc$ plants are carefully screened and vetted to prevent culturing plants
infected with virus or fungus.
9. -ot all plants can be successfully tissue cultured, often because the proper
medium for growth is not $nown or the plants produce secondary metabolic
chemicals that stunt or $ill the e#plant.
1!. 6ometimes plants or cultivars do not come true to type after being tissue
cultured0 this is often dependent on the type of e#plant material utilized
during the initiation phase or the result of the age of the cell or propagule
line.
11. 6ome plants are very difficult to disinfest of fungal organisms.
11. The major limitation in the use of 'icropropagation for many plants is the cost
of production

22

1. 'echanization of the process could reduce labor costs, but has proven difficult
to achieve, despite active attempts to develop technological solutions.
Haploid or Anther or Microspore culture
.aploid plants may be obtained from pollen grains by placing anthers or
isolated pollen grains on a suitable culture medium0 this constitutes anther and pollen
culture, respectively. The anthers may be ta$en from plants grown in the field or in
pots, but ideally these plants should be grown under controlled temperature, light
and humidity0 the optimum conditions may differ from species to species. Fften, the
capacity for haploid production declines with the age of donor plants. %lower buds of
the appropriate developmented stage are collected, surface sterilized, and their
anthers are e#cised and placed horizontally on culture medium.
Pathways of Developent in Pollen &rains 7
The early divisions in responding pollen grains may occur in one of the
following four ways.
i. The unicleate pollen grain may divide symmetrically to yield two equal
daughter cells both of which undergo further divisions, e.g., ,atura inno#ia
*=athway &+.
ii. &n some other cases, e.g., -. tabacum, ,atura metel, barley, wheat, triticale0
chillies, etc., the unicleate pollen divides unequally *as it does in nature+. The
generative cell degenerates callusCembryo originates due to successive
divisions of the vegetative cell *=athway. &&+.
iii. But in few species, e.g., .yoscyamus niger, the pollen embryos originate from
the generative cell alone0 the vegetative cell either does not divide or divides
only to a limited e#tent forming a suspensor li$e structure *=athway &&&+.
iv. %inally, in some species, e.g. ,atura inno#ia, the uninucleate pollen grains
divide unequally, producing generative and vegetative cells, but both these
cells divide repeatedly to contribute to the developing embryoCcallus
*=athway &;+.
Two main approaches can be ta$en to produce cultures in vitro from haploid tissue.
1. The first method depends on using the anther as the e#plant. (nthers *somatic
tissue that surrounds and contains the pollen+ can be cultured on half nitrogen
strength solid medium *agar should not be used to solidify the medium as it

23

contains inhibitory substances+ under dull light *1!!!lu# light intensity+ or in
dar$ for 1 to three days of 14 hours of light and 3 hours of dar$ photoperiod
conditions at 4!2 humidity.
=ollen:derived embryos are subsequently produced via dehiscence of the
mature anthers. The dehiscence of the anther depends both on its isolation at
the correct stage and on the correct culture conditions. &n some species, the
reliance on natural dehiscence can be circumvented by cutting the wall of the
anther, although this does, of course, ta$e a considerable amount of time.
1. &n the second method, anthers can also be cultured in liquid medium, and
pollen released from the anthers can be induced to form embryos, although
the efficiency of plant regeneration is often very low. &mmature pollen can
also be e#tracted from developing anthers and cultured directly, although this
is a very time:consuming process.
Both methods have advantages and disadvantages. 6ome beneficial effects to
the culture are observed when anthers are used as the e#plant material. There is,
however, the danger that some of the embryos produced from anther culture will
originate from the somatic anther tissue rather than the haploid microspore cells. &f
isolated pollen is used there is no danger of mi#ed:embryo formation, but the
efficiency is low and the process is time:consuming.
&n microspore culture, the condition of the donor plant is of critical
importance, as is the timing of isolation. =retreatments, such as a cold treatment, are
often found to increase the efficiency. These pretreatments can be applied before
culture, or, in some species, after placing the anthers in culture.
=lant species can be divided into two groups, depending on whether they
require the addition of plant growth regulators to the medium for pollenCanther
culture0 those that do also often require organic supplements, such as amino acids.
'any of the cereals *rice, wheat, barley, and maize+ require medium supplemented
with plant growth regulators for pollen or anther culture.
@egeneration from microspore e#plants can be obtained by direct
embryogenesis, or via a callus stage and subsequent embryogenesis *indirect+.
.aploid tissue cultures can also be initiated from the female gametophyte *the
ovule+. &n some cases, this is a more efficient method than using pollen or anthers.
=lants obtained from haploid cultures may not be haploid. This can be a consequence
of chromosome doubling during the culture period. /hromosome doubling *which
often has to be induced by treatment with chemicals such as colchicine+ may be an

24

advantage, as in many cases haploid plants are not the desired outcome of
regeneration from haploid tissues. 6uch plants are often referred to as di)haploids,
because they contain two copies of the same haploid genome.

Cultu(e En,i(on%ent -
(nther cultures are generally maintained in alternating periods of light *11O14
hr0 2,!!!:1!,!!! lu# m1+ at 14P/ and dar$ness *11:3 hr+ at 11P/, but the optimum
conditions vary with the species. The walls of responsive anthers turn brown and after
:4 wee$s they burst open due to the developing callus or embryos. (fter the
seedlings *from embryos+ or shoots *from callus+ become :2 cm long, they are
transferred to a medium conducive to good root development. %inally, they are
transferred to soil in the same way as other in vitro regenerated plantlets.

"uspension cultures
'uspension culture or cell culture is a t!pe of culture in which single cells or
small aggregates of cells multipl! while suspended in agitated li(uid medium)
%ultures grow faster than callus culture)

25

/allus cultures, broadly spea$ing, fall into one of two categories7 compact or
friable. &n compact callus, the cells are densely aggregated, whereas in friable callus,
the cells are only loosely associated with each other and the callus becomes soft and
brea$s apart easily. %riable callus provides the inoculum to form cell:suspension
cultures.
<#plants from some plant species or particular cell types tend not to form
friable callus, ma$ing it difficult to initiate cell suspension. The friability of the callus
can sometimes be improved by manipulating the medium components or by repeated
sub:culturing. The friability of the callus can also sometimes be improved by culturing
it on semi:solid medium *medium with a low concentration of gelling agent+.
8hen friable callus is placed into a liquid medium *usually the same
composition as the solid medium used for the callus culture+ and then agitated, single
cells andCor small clumps of cells are released into the medium. Bnder the correct
conditions, these released cells continue to grow and divide, eventually producing a
cell:suspension culture. ( relatively large inoculum should be used when initiating cell
suspensions so that the released cell numbers build up quic$ly. The inoculum should
not be too large though, as to#ic products released from damaged or stressed cells
can build up to lethal levels. Narge cell clumps can be removed during subculture of
the cell suspension.
Niquid cultures must be constantly agitated, generally by a gyratory sha$er at
1!!:12! rpm *revolution per minute+, to facilitate aeration and dissociation of cell
clumps into smaller pieces. 6uspension cultures grow much faster than callus cultures,
need to be subcultured about every wee$, allow a more accurate determination of
the nutritional requirements of cells and are amenable to scaling up for a large scale
production of cells and even somatic embryos *6<s+. The suspension cultures are
broadly grouped as7
*1+ batch cultures,
*1+ continuous cultures, and
*+ immobilized cell cultures.

#atch Cultures 8
&n a batch culture the same medium and all the cells produced are retained in
the culture vessel, e.g., culture flas$s *1!!:12! ml+, fermenters *variable size+, etc.
The cell number or biomass of a batch culture e#hibits a typical sigmoidal curve,

26

having a lag phase during which the cell number remains unchanged, followed by a
logarithmic *log+ phase when there is a rapid increase in cell number and finally
ending in a stationary phase during which cell number does not change. The lag phase
duration depends mainly on inoculum size and growth phase of the culture from which
inoculum is ta$en. The log phase lasts about :9 cell generations, and the duration of
a cell generation *time ta$en for doubling of cell number+ may vary from 11:94 hr
mainly depending on the plant species.
The stationary phase is forced on the culture by depletion of the nutrients and
possibly due to an accumulation of cellular wastes. &f the culture is $ept in stationary
phase for a prolonged period the cells may die. Batch cultures are maintained by sub:
culturing. They are used for initiation of cell suspensions, which may be used for
cloning, cell selection or as seed cultures for scaling up or for continuous cultures.
They are, however, unsuitable for studies on cell growth and metabolism because
there is a constant change in cell density and nutritional status of the medium. But
batch cultures are much more convenient than continuous cultures and, as a result,
are routinely used.
Continuous Cultures 8
&n a continuous culture, the cell population is maintained in a steady state by
regularly replacing a portion of the used or spent medium by fresh medium. 6uch
culture systems are of either *1+ closed or*1+ open type. &n a closed continuous
culture, cells are separated from the used medium ta$en out for replacement, and
added bac$ to the culture so that cell biomass $eeps on increasing. &n contrast, both
cells and the used medium are ta$en out from open continuous cultures and replaced
by equal volume of fresh medium.
The replacement volume is so adjusted that cultures remain at sub:ma#imal
growth indefinitely. The open cultures are of either turbidostat or chemostat types. &n
a turbidostat, cells are allowed to grow up to a pre:selected turbidity *usually
measured as F,+ when a predetermined volume of the culture is replaced by fresh
normal culture medium.
But in a chemostat, a chosen nutrient is $ept in a concentration so that it is
depleted very rapidly to become growth limiting, while other nutrients are still in
concentrations higher than required. &n such a situation, any addition of the growth

27

limiting nutrient is reflected in cell growth. /hemostats are ideal for the
determination of effects of individual nutrients on cell growth and metabolism.

Iobili!ed Cell Cultures 8
=lant cells and cell groups may be encapsulated in a suitable material, e.g.,
agarose, calcium alginate, etc., or entrapped in membranes or stainless steel screens.
The gel beads containing cells may be pac$ed in a suitable column or, alternatively,
cells may be pac$ed in a column of a membrane or wire cloth. Niquid medium is
continuously run through the column to provide nutrients and aeration to cells.
&mmobilization of cells changes their cellular physiology in comparison to suspension
culture cells0 this offers several advantages for their use in biochemical production,
but they are usually not used for other studies.

Tetras Educare Study Notes Vol 22 PTC Part 1

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Tetras Educare Study Notes Vol 22 PTC Part 1

Uploaded by

Copyright:

Available Formats

Tetras Educare

+. Theoretically, there is an advantage in supplying

You might also like