tetras educare study notes vol 22 ptc part 1

8/10/2019 Tetras Educare Study Notes Vol 22 Ptc Part 1

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Tetras Educare

Study Handout forStudy Handout forStudy Handout forStudy Handout forLife Science StudentLife Science StudentLife Science StudentLife Science StudentPlant Tissue culture Part

Edited and written by:

SHANMUGAM V. M.Lecturer, JNC Autonomous,Lecturer, JNC Autonomous,Lecturer, JNC Autonomous,Lecturer, JNC Autonomous,

Dept. of BiotechnologyDept. of BiotechnologyDept. of BiotechnologyDept. of BiotechnologyHosur Road, BangaloreHosur Road, BangaloreHosur Road, BangaloreHosur Road, Bangalore !""#!""#!""#!""# .

Not for private circulation, Please mail your Queries and Suggestions to [email protected]


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Study handout for life science aspirants volumevolumevolumevolume

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 allparts 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 inplanta by employing molecular probes.


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Shanes quick N fact files Plant tissue culture part - 1

3Study handout for life science aspirants

History:

Though the tissue culture technique is latest one but history of this began

more than 2 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 nutrientmedium, 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 mediumunder 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!os 5.

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.


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Applications of Tissue Culture in Plants:

1. Micro propagation 7 The term represents the vegetative multiplication of

plants in artificial media under aseptic conditions from tissue0 organs of plantse.g. root tip, shoot tip, embryo, stem and callus etc.

. Production of disease free plants 7 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 breeding 7 8ith the help of tissue

culture in plants haploid embryos or haploid plants are raised by anther

culture technique.

. E bryo 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 utants 7 By adding chemical mutagens into the

medium for growing various traits, useful viable mutants can be produced.

3. "o aclonal variation 7 These are variations produced in the plantsregenerated from tissue cultures involving callus formation. ;itiations

appearing during tissue culture in plants are called somaclonal variation

#asic re$uire ents 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 thee#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


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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


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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 syntheticcompounds. =?@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 *-((+,

, > dichloropheno#yacetic acid * , > +, and > amino : , 2, 3 > trichloro : >

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(, -((, , > and picloram are used at concentrations

ranging from !.!!1 > 1!mgCl, picloram and , > are e#amples of au#ins used

primarily to induce and regulate somatic embryogenesis.


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,* 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 i=. &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 : 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, /itrusand 6pruce.


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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 , > 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.

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


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Plant cell culture edia:

/ulture media used for the cultivation of plant cells in vitro are composed of

three basic components7

1.


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appears pale and Hglassy" and is usually unsuitable for further culture+. sing 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.

)* Microele ents

'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.


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&ron is usually added as iron sulphate, although iron citrate can also be used.


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that ma$e visual screening of contamination easy. ?el strength is determined by the

concentration of divalent cations *'g J and /a J+. =oor gelling may occur if the

cationic concentrations are lower than m' or higher than 4m'.2* Co ple+ organic supple ents:

/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* Antibiotics 7

(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.


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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,


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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 .


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soft and brea$s apart easily. %riable callus provides the inoculum to form cell:

suspension cultures.


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&nindirect somatic embr!ogenesis , callus is first produced from the e#plant.


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6


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MICROPROPA ATIO!

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.

STA ES O" MICROPROPA ATIO!:

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


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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 There is not any universal culture

medium0 however modifications of 'urashige and 6$oog basal medium *'urashige and

6$oog, 193 + 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. 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 * !: 3!E+, higher the light intensities, e#posure to

the pathogens and autotrophic growth. =lants are $ept under high humidity for :

wee$s just after a transfer to a green house. Night intensities are gradually increased

during hardening.


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"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.

. 'ultiplication of disease free plants.

. 'ultiplication of se#ually derived sterile hybrids.

. ;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.


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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.

. =roduction cost is high.

. @equirement of s$ill in handling and maintenance.

. 6omaclonal variations may arise during in vitro culture.

2. ;itrification can be a problem in some species.

3. &t isver! e#pensive, and can have a labor cost of more than )!E

). ( monoculture is produced after 'icropropagation, leading to a lac$ of overalldisease 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.

1 . The major limitation in the use of 'icropropagation for many plants is the costof production


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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 Develop ent 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 nitrogenstrength solid medium *agar should not be used to solidify the medium as it


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contains inhibitory substances+ under dull light *1!!!lu# light intensity+ or in

dar$ for 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 atthe 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.

. &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 supplementedwith 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 *whichoften has to be induced by treatment with chemicals such as colchicine+ may be an


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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 *1 O14

hr0 2,!!!:1!,!!! lu# m + at 4P/ and dar$ness *1 :3 hr+ at P/, 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 aretransferred 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)


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/allus cultures, broadly spea$ing, fall into one of two categories7 compact or

friable . &ncompact 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.


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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 phaseduration depends mainly on inoculum size and growth phase of the culture from which

inoculum is ta$en. The log phase lasts about : cell generations, and the duration of

a cell generation *time ta$en for doubling of cell number+ may vary from : 4 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* + 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#imalgrowth 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


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tetras educare study notes vol 22 ptc part 1

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